Search

ABSTRACT Pseudomonas aeruginosa is a highly problematic opportunistic pathogen that causes a range of different infections. Infections are commonly treated with β-lactam antibiotics, including cephalosporins, monobactams, penicillins, and carbapenems, with carbapenems regarded as antibiotics of last resort. Isolates of P. aeruginosa can contain horizontally acquired bla genes encoding β-lactamase enzymes, but the extent to which these contribute to β-lactam resistance in this species has not been systematically quantified. The overall aim of this research was to address this knowledge gap by quantifying the frequency of β-lactamase-encoding genes in P. aeruginosa and by determining the effects of β-lactamases on susceptibility of P. aeruginosa to β-lactams. Genome analysis showed that β-lactamase-encoding genes are present in 3% of P. aeruginosa but are enriched in carbapenem-resistant isolates (35%). To determine the substrate antibiotics, 10 β-lactamases were expressed from an integrative plasmid in the chromosome of P. aeruginosa reference strain PAO1. The β-lactamases reduced susceptibility to a variety of clinically used antibiotics, including carbapenems (meropenem, imipenem), penicillins (ticarcillin, piperacillin), cephalosporins (ceftazidime, cefepime), and a monobactam (aztreonam). Different enzymes acted on different β-lactams. β-lactamases encoded by the genomes of P. aeruginosa clinical isolates had similar effects to the enzymes expressed in strain PAO1. Genome engineering was used to delete β-lactamase-encoding genes from three carbapenem-resistant clinical isolates and increased susceptibility to substrate β-lactams. Our findings demonstrate that acquired β-lactamases play an important role in β-lactam resistance in P. aeruginosa, identifying substrate antibiotics for a range of enzymes and quantifying their contributions to resistance.IMPORTANCEPseudomonas aeruginosa is an extremely problematic pathogen, with isolates that are resistant to the carbapenem class of β-lactam antibiotics being in critical need of new therapies. Genes encoding β-lactamase enzymes that degrade β-lactam antibiotics can be present in P. aeruginosa, including carbapenem-resistant isolates. Here, we show that β-lactamase genes are over-represented in carbapenem-resistant isolates, indicating their key role in resistance. We also show that different β-lactamases alter susceptibility of P. aeruginosa to different β-lactam antibiotics and quantify the effects of selected enzymes on β-lactam susceptibility. This research significantly advances the understanding of the contributions of acquired β-lactamases to antibiotic resistance, including carbapenem resistance, in P. aeruginosa and by implication in other species. It has potential to expedite development of methods that use whole genome sequencing of infecting bacteria to inform antibiotic treatment, allowing more effective use of antibiotics, and facilitate the development of new antibiotics.

This study aimed to identify and evaluate the antimicrobial susceptibility profile of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales isolated from blood cultures of patients with suspected sepsis. This retrospective cross-sectional epidemiological study was performed for four years (2015 to 2018) by accessing the medical records of patients diagnosed with bloodstream infections in two tertiary public hospitals in Brazil. Culture results and susceptibility tests of bacterial strains isolated from blood samples were analyzed. During four years, 2,263 blood culture exams were performed, and Enterobacterales were isolated in 694 (30.7%) samples. The disk approximation test detected ESBL production in 171 (24.6%) of the 694 isolated Enterobacterales (p

ObjectivesTo evaluate the in vitro antimicrobial susceptibilities of Gram-positive and Gram-negative isolates from patients in Jordan between 2010 and 2021, through the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme.MethodsMedical centres in Jordan collected bacterial isolates from hospitalised patients with defined infection sources between 2010 and 2021 (no isolates collected in 2014). Antimicrobial susceptibility was interpreted using CLSI standards. FDA-approved breakpoints were applied for tigecycline. The identification of β-lactamase genes was performed for a proportion of isolates using multiplex PCR assays.ResultsMore than 92% of Acinetobacter baumannii collected were multidrug-resistant (MDR) and/or carbapenem-resistant (CR), and > 50% susceptibility was reported only to minocycline (62.2% among both MDR and CR isolates). Rates of MDR and CR Pseudomonas aeruginosa were 14.3% and 20.5%, respectively, and among all P. aeruginosa collected from adults, susceptibility to ceftazidime/avibactam was 95.3% and to ceftolozane/tazobactam was 88.4%. For Escherichia coli from adults and MDR E. coli, susceptibility to ceftazidime/avibactam, ceftolozane/tazobactam, imipenem, meropenem and meropenem/vaborbactam was 92.1%–98.7%. Susceptibility to tigecycline was > 94% among Klebsiella pneumoniae from adult, paediatric, and ICU patients (all ages). CTX-M-15 was the most frequently identified β-lactamase gene among E. coli and K. pneumoniae. Susceptibility to most antimicrobial agents was < 50% among K. pneumoniae carrying CTX-M-15, CTX-M-9-type, NDM-5, and/or OXA-48 β-lactamase genes. All S. aureus collected were susceptible to teicoplanin, vancomycin, daptomycin, linezolid and tigecycline, with 96.1% of S. aureus from adults were susceptible to ceftaroline. Overall, 58.8% of Staphylococcus aureus were MRSA.ConclusionThis study provides valuable information regarding antimicrobial susceptibility in Jordan between 2010 and 2021. Continued monitoring of in vitro antimicrobial susceptibility is critical in the fight against antimicrobial resistance.

ABSTRACT Pseudomonas aeruginosa encodes the beta-lactamase AmpC, which promotes resistance to beta-lactam antibiotics. Expression of ampC is induced by anhydro-muropeptides (AMPs) released from the peptidoglycan (PG) cell wall upon beta-lactam treatment. AmpC can also be induced via genetic inactivation of PG biogenesis factors such as the endopeptidase DacB that cleaves PG crosslinks. Mutants in dacB occur in beta-lactam-resistant clinical isolates of P. aeruginosa, but it has remained unclear why DacB inactivation promotes ampC induction. Similarly, the inactivation of lytic transglycosylase (LT) enzymes such as SltB1 that cut PG glycans has also been associated with ampC induction and beta-lactam resistance. Given that LT enzymes are capable of producing AMP products that serve as ampC inducers, this latter observation has been especially difficult to explain. Here, we show that ampC induction in sltB1 or dacB mutants requires another LT enzyme called MltG. In Escherichia coli, MltG has been implicated in the degradation of nascent PG strands produced upon beta-lactam treatment. Accordingly, in P. aeruginosa sltB1 and dacB mutants, we detected the MltG-dependent production of pentapeptide-containing AMP products that are signatures of nascent PG degradation. Our results therefore support a model in which SltB1 and DacB use their PG-cleaving activity to open space in the PG matrix for the insertion of new material. Thus, their inactivation mimics low-level beta-lactam treatment by reducing the efficiency of new PG insertion into the wall, causing the degradation of some nascent PG material by MltG to produce the ampC-inducing signal.IMPORTANCEInducible beta-lactamases like the ampC system of Pseudomonas aeruginosa are a common determinant of beta-lactam resistance among gram-negative bacteria. The regulation of ampC is elegantly tuned to detect defects in cell wall synthesis caused by beta-lactam drugs. Studies of mutations causing ampC induction in the absence of drug therefore promise to reveal new insights into the process of cell wall biogenesis in addition to aiding our understanding of how resistance to beta-lactam antibiotics arises in the clinic. In this study, the ampC induction phenotype for mutants lacking a glycan-cleaving enzyme or an enzyme that cuts cell wall crosslinks was used to uncover a potential role for these enzymes in making space in the wall matrix for the insertion of new material during cell growth.

Background To limit the use of antimicrobials without disincentivising the development of novel antimicrobials, there is interest in establishing innovative models that fund antimicrobials based on an evaluation of their value as opposed to the volumes used. The aim of this project was to evaluate the population-level health benefit of cefiderocol in the NHS in England, for the treatment of severe aerobic Gram-negative bacterial infections when used within its licensed indications. The results were used to inform the National Institute for Health and Care Excellence guidance in support of commercial discussions regarding contract value between the manufacturer and NHS England. Methods The health benefit of cefiderocol was first derived for a series of high-value clinical scenarios. These represented uses that were expected to have a significant impact on patients’ mortality risks and health-related quality of life. The clinical effectiveness of cefiderocol relative to its comparators was estimated by synthesising evidence on susceptibility of the pathogens of interest to the antimicrobials in a network meta-analysis. Patient-level costs and health outcomes of cefiderocol under various usage scenarios compared with alternative management strategies were quantified using decision modelling. Results were reported as incremental net health effects expressed in quality-adjusted life-years, which were scaled to 20-year population values using infection number forecasts based on data from Public Health England. The outcomes estimated for the high-value clinical scenarios were extrapolated to other expected uses for cefiderocol. Results Among Enterobacterales isolates with the metallo-beta-lactamase resistance mechanism, the base-case network meta-analysis found that cefiderocol was associated with a lower susceptibility relative to colistin (odds ratio 0.32, 95% credible intervals 0.04 to 2.47), but the result was not statistically significant. The other treatments were also associated with lower susceptibility than colistin, but the results were not statistically significant. In the metallo-beta-lactamase Pseudomonas aeruginosa base-case network meta-analysis, cefiderocol was associated with a lower susceptibility relative to colistin (odds ratio 0.44, 95% credible intervals 0.03 to 3.94), but the result was not statistically significant. The other treatments were associated with no susceptibility. In the base case, patient-level benefit of cefiderocol was between 0.02 and 0.15 quality-adjusted life-years, depending on the site of infection, the pathogen and the usage scenario. There was a high degree of uncertainty surrounding the benefits of cefiderocol across all subgroups. There was substantial uncertainty in the number of infections that are suitable for treatment with cefiderocol, so population-level results are presented for a range of scenarios for the current infection numbers, the expected increases in infections over time and rates of emergence of resistance. The population-level benefits varied substantially across the base-case scenarios, from 896 to 3559 quality-adjusted life-years over 20 years. Conclusion This work has provided quantitative estimates of the value of cefiderocol within its areas of expected usage within the NHS. Limitations Given existing evidence, the estimates of the value of cefiderocol are highly uncertain. Future work Future evaluations of antimicrobials would benefit from improvements to NHS data linkages; research to support appropriate synthesis of susceptibility studies; and application of routine data and decision modelling to assess enablement value. Study registration No registration of this study was undertaken. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment Policy Research Programme (NIHR award ref: NIHR135591), conducted through the Policy Research Unit in Economic Methods of Evaluation in Health and Social Care Interventions, PR-PRU-1217-20401, and is published in full in Health Technology Assessment; Vol. 28, No. 28. See the NIHR Funding and Awards website for further award information. Plain language summary This project tested new methods for estimating the value to the NHS of an antimicrobial, cefiderocol, so its manufacturer could be paid fairly even if very little drug is used in order to reduce the risk of bacteria becoming resistant to the product. Clinicians said that the greatest benefit of cefiderocol is when used for complicated urinary tract infections and pneumonia acquired within hospitals caused by two types of bacteria (called Enterobacterales and Pseudomonas aeruginosa), with a resistance mechanism called metallo-beta-lactamase. Because there were no relevant clinical trial data, we estimated how effective cefiderocol and alternative treatments were by doing a systematic literature review of studies that grew bacteria from infections in the laboratory and tested the drugs on them. We linked this to data estimating the long-term health and survival of patients. Some evidence was obtained by asking clinicians detailed questions about what they thought the effects would be based on their experience and the available evidence. We included the side effects of the alternative treatments, some of which can cause kidney damage. We estimated how many infections there would be in the UK, whether they would increase over time and how resistance to treatments may change over time. Clinicians told us that they would also use cefiderocol to treat intra-abdominal and bloodstream infections, and some infections caused by another bacteria called Stenotrophomonas. We estimated how many of these infections there would be, and assumed the same health benefits as for other types of infections. The total value to the NHS was calculated using these estimates. We also considered whether we had missed any additional elements of value. We estimated that the value to the NHS was £18–71 million over 20 years. This reflects the maximum the NHS could pay for use of cefiderocol if the health lost as a result of making these payments rather than funding other NHS services is not to exceed the health benefits of using this antimicrobial. However, these estimates are uncertain due to limitations with the evidence used to produce them and assumptions that had to be made. Scientific summary This section reproduces and adapts, with permission, content previously published as part of the study protocol (Harnan SE, Woods B, Schmitt L, Kearns B, Srivastava T, Scope A, et al. Protocol for the technology evaluation of cefiderocol for treating severe aerobic Gram-negative bacterial infections. Health Technol Assess 2021.) Background The National Institute for Health and Care Excellence (NICE), NHS England and NHS Improvement are currently undertaking a project to assess the feasibility of innovative models that pay for antimicrobials (AMs) based on an evaluation of their value to the NHS as opposed to the volumes used. This project involves evaluation of two AMs to inform commercial discussions regarding contract value for a period of up to 10 years. This report details the evaluation phase of this project for cefiderocol (Fetcroja), which is manufactured by Shionogi. Cefiderocol is an injectable siderophore cephalosporin and received a marketing authorisation in April 2020 for treating infections due to aerobic Gram-negative organisms in adults with limited treatment options. Aim and objectives The aim of this evaluation is to assess the value of cefiderocol to the NHS in England when used within its licensed indications. Specific objectives are: To identify two high-value clinical scenarios (HVCSs), within its broad licensed indications, for which cefiderocol is expected to have a significant impact on patients’ outcomes in terms of reducing mortality risks and improving health-related quality of life (HRQoL). To undertake an ‘evidence mapping’ exercise and relevant systematic literature reviews to characterise the available clinical effectiveness evidence for the use of cefiderocol in the HVCSs. To establish an appropriate decision-analytic model to quantify the costs and health benefits of the use of cefiderocol under various usage scenarios compared with usage scenarios involving other available AMs in the HVCSs. The decision-analytic model was required to estimate costs and health effects at both the individual level and the aggregate population level, providing population-level incremental net health effects (INHEs). Drawing on the systematic reviews and evidence synthesis, national-level data on healthcare-associated infections and other sources as needed, to identify evidence to populate the decision-analytic models. To use structured expert elicitation as necessary to supplement the available evidence to populate the decision-analytic models. To use available evidence and, where necessary, expert opinion quantitatively to extrapolate estimated population-level INHEs associated with cefiderocol in the HVCSs to other expected uses for the product within the product’s licensed indications. Expected usage and high-value clinical scenarios The licensed indication for cefiderocol is broad. In practice, to control the spread of resistance to cefiderocol and to preserve its long-term viability as an effective treatment option against highly resistant pathogens, cefiderocol may be used in a more restricted group of patients than permitted by its license. Quantifying the health and cost implications of using cefiderocol across anticipated NHS usage, even within this restricted population, remains challenging, as the use is expected in infections which differ in causative organism (pathogen, resistance mechanism), site of the infection, healthcare setting and other patient characteristics. This evaluation characterises the value of cefiderocol across its range of expected uses via a two-step approach. First, decision modelling is used quantitatively to assess the value of cefiderocol in a set of scenarios defined by features of the pathogen, site of infection, healthcare setting and other patient characteristics, considered to represent important uses of cefiderocol; referred to as the HVCSs. Second, rescaling is used to estimate how this evidence can be used to provide quantitative assessments of value in the overall population expected to receive cefiderocol, including patients who fall outside the HVCSs but who are relevant to determining the overall value of cefiderocol to the English NHS. The HVCSs were selected to reflect areas of clinical use where there is a current significant burden from resistant infections, and cefiderocol is expected to offer significant improvements over existing treatments in terms of efficacy and/or safety. The HVCSs were selected based on feedback from the manufacturer, clinical advisors to the Policy Research Unit in Economic Methods of Evaluation in Health and Social Care Interventions (EEPRU) and broader stakeholders involved in the NICE scoping process. The HVCSs focus on the following patient populations: Empiric setting (ES): patients with an infection strongly suspected to be caused by metallo-beta-lactamase (MBL)-producing Enterobacterales or MBL-producing Pseudomonas aeruginosa in patients with hospital-acquired pneumonia or ventilator-associated pneumonia (HAP/VAP). In this patient group, the pathogen, resistance mechanism and antibiotic susceptibility have not yet been established, but treatment is initiated immediately due to the severity of the infection. Microbiology-directed setting (MDS): patients with an infection confirmed to be caused by MBL Enterobacterales or MBL P. aeruginosa, where antibiotic susceptibility testing results are available, and where the site of infection is HAP/VAP or complicated urinary tract infection (cUTI). The resourcing for this project was equivalent to that of a diagnostic assessment review or multiple technology assessment for NICE, but the levels of analysis extend from the typical focus of those evaluations on a single type of patient for one indication and setting. In this evaluation, we also include population-level health effects now and over time, and across several indications and settings. The objective is to use appropriate analyses of the available evidence at every level, but the detail in those analyses is inevitably constrained by the time and resources available for the project. Clinical evidence Methods There are evidential challenges when evaluating the use of new or nearly new AMs to treat infections caused by multidrug-resistant (MDR) pathogens. Randomised controlled trials (RCTs) are of generally low relevance as they tend not to recruit patients with MDR pathogens. Therefore, relative treatment effects between the intervention and comparator cannot be generalised to MDR pathogens, as this may overestimate the efficacy of the comparator. It was anticipated that RCTs were unlikely to be the primary source of evidence. Therefore, a mapping review was undertaken to establish available evidence and ascertain which approach to estimating comparative effectiveness could inform an economic model. Reviews 1 and 2 considered RCTs and observational studies of cefiderocol in patients with HAP/VAP or cUTI infections caused by MBL Enterobacterales or P. aeruginosa or suspected to be caused by carbapenem-resistant Enterobacterales or P. aeruginosa. In Review 3, in vitro susceptibility studies were considered. These studies provide evidence on the proportion of isolates that are susceptible to treatments and comparators as an indication of relative efficacy. Susceptibility studies were considered eligible if they reported susceptibility for MBL Enterobacterales or P. aeruginosa isolates, from any infection site (clinical advisors indicated that infection site was not associated with susceptibility profile), and reported susceptibility to cefiderocol and at least one comparator as defined by the HVCSs (colistin, meropenem, tigecycline, aztreonam, fosfomycin, gentamicin, amikacin, tobramycin). Susceptibility studies test isolates in vitro to ascertain the minimum concentration of any given treatment that is needed to inhibit growth of the microbe [the minimum inhibitory concentration (MIC)]. If this is below the clinical breakpoint published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [or by the Clinical Laboratory Standards Institute (CLSI) from the USA], the isolate is considered susceptible, and is likely to respond to treatment in vivo. In the UK, the British Society for Antimicrobial Chemotherapy (BSAC) recommends use of EUCAST guidelines. Systematic searches across relevant databases [MEDLINE, EMBASE and Centre for Review and Dissemination (CRD) database] were conducted in March 2021. EEPRU also considered evidence submitted by Shionogi and Public Health England (PHE). After mapping, only Review 3 was pursued, since there was insufficient evidence from Reviews 1 and 2. Risk of bias assessment was performed using a bespoke tool developed for this evaluation. No studies of fosfomycin were identified. An additional rapid review was conducted to identify studies reporting the susceptibility of fosfomycin and at least one other HVCS comparator. A random-effects network meta-analysis (NMA) of susceptibility studies was conducted to allow a comprehensive synthesis of evidence on all relevant treatments. A different network was constructed for each subgroup of pathogens (MBL Enterobacterales and MBL P. aeruginosa), since susceptibility profiles were expected to differ. For each of these, a network was constructed using EUCAST breakpoints and another using CLSI breakpoints. The EUCAST networks were considered the main analysis, while the CLSI networks were considered in sensitivity analyses. Sensitivity analyses were also conducted to test assumptions around the inclusion of comparators in the network, to assess the impact of missed data on the analysis, and prioritising PHE data where possible as these are from English isolates and, therefore, have the highest relevance. Two additional reviews were conducted to provide evidence on the link between susceptibility and clinical outcomes (Review 4) and between susceptibility and long-term outcomes (Review 5) in the sites of interest. A further review (Review 6) was conducted to identify any important safety implications of cefiderocol. Results The reviews of RCT and observational studies identified insufficient evidence to inform an assessment of clinical effectiveness. One RCT did report subgroup data relating to MBLs, but the subgroup was small (n = 16 in the cefiderocol arm, n = 7 in the best available therapy arm) and was, therefore, not used due to the chance of baseline imbalances introducing bias. The key limitations of the observational studies that were identified included small numbers of MBL infections (range n = 2–17 patients), high levels of heterogeneity with respect to prognostic factors, and data not being reported for the sites of interest. Review 3 identified in vitro susceptibility studies in relatively large samples of MBL Enterobacterales or P. aeruginosa isolates obtained from a range of clinical sites of infection. Three studies (SIDERO CR, SIDERO WT and Johnston et al.) reporting data for cefiderocol and at least one comparator met the inclusion criteria and were synthesised. The separate review of fosfomycin identified 10 studies. In the MBL Enterobacterales EUCAST analysis, cefiderocol was associated with a lower susceptibility relative to colistin [odds ratio (OR) 0.32, 95% credible intervals (CrI) 0.04 to 2.47], but the result was not statistically significant. Fosfomycin had a similar OR (OR 0.34, 95% CrI 0.06 to 1.96) compared to colistin as cefiderocol. The remainder of the treatments were also associated with lower susceptibility than colistin, but the results were not statistically significant. In the MBL P. aeruginosa EUCAST analysis, cefiderocol was associated with a lower susceptibility relative to colistin (OR 0.44, 95% CrI 0.03 to 3.94), but the result was not statistically significant. The remainder of the treatments were associated with no susceptibility. Heterogeneity was high in both networks. In the sensitivity analyses using CLSI breakpoints, where the breakpoint for cefiderocol is higher, cefiderocol was associated with a higher susceptibility relative to colistin. However, the results were very uncertain in some of the NMAs due to sparse data and a large number of treatment arms with either zero or 100% susceptibility. Review 4 (link between susceptibility and clinical outcomes) identified two studies that reported mortality or hospital length of stay (LoS) conditional on susceptibility to empiric treatment and were selected for use in the model for the ES. No useful evidence relating to the MDS was identified. Review 5 (link between susceptibility and long-term clinical outcomes) did not identify any relevant literature, but an unpublished study (CARBAR) was submitted by Shionogi that contained useful data. Review 6 indicated that cefiderocol does not appear to increase the risk of acute kidney injury (AKI), Clostridium difficile, or any other serious adverse events, compared to non-toxic comparators (i.e. comparators other than colistin or an aminoglycoside). No study reported a comparison of cefiderocol exclusively to colistin or aminoglycosides. Event rates were generally very low or zero. Discussion of clinical evidence: There were limitations to the approach selected and analyses done. Key limitations include: susceptibility could be considered, at best, a surrogate outcome, but no pre-specified criteria for judging the suitability of the surrogate or the linking evidence were applied; linking data were limited and not specific to the pathogen-mechanism combination, and expert elicitation had to be relied upon to evidence the link in the MDS; uncertainties relating to the determination and application of breakpoints; and the NMAs results were associated with high levels of heterogeneity. Economic evidence Methods No published existing economic evaluations assessed the use of cefiderocol in the HVCSs or areas of expected usage. The manufacturer did not submit a cost-effectiveness model. A de novo decision-analytic model was developed to predict the cost and health consequences of introducing cefiderocol within the HVCSs. These were summarised as INHEs. These are estimates of the quality-adjusted life-years (QALYs) associated with introducing cefiderocol if it was supplied free of charge to the NHS, taking in to account both its health benefits and the health benefits of freeing up NHS resources (e.g. via reduced time in hospital) calculated using an estimate of health opportunity cost of £20,000/QALY. This means that for every £20,000 saved, 1 QALY of health can be generated within the NHS. The estimates of INHE will be used in subsequent negotiations to determine an appropriate payment level for cefiderocol. This quantitative analysis comprises three components: an assessment of the INHEs of introducing cefiderocol within the HVCSs at the patient level; an assessment of INHEs within the HVCSs at the population level; and an assessment of how population-level INHEs within the HVCSs might appropriately be rescaled to reflect expected usage across the English NHS. The patient-level component is structured similarly to decision models developed as part of other NICE processes and characterises the cost, mortality and morbidity consequences of introducing cefiderocol over a patient’s lifetime. Separate but related models are developed for the ES and MDS. In the ES, empiric treatment with cefiderocol is compared to: empiric treatment with a non-colistin/aminoglycoside-based treatment (MBL P. aeruginosa only); empiric treatment with colistin/aminoglycoside-based treatment (considered more toxic). In the MDS, we compare outcomes in the overall microbiology-directed cohort who receive tailored therapy with cefiderocol available as a treatment option, to outcomes when this cohort receive tailored therapy with existing AMs only. In the ES, patients are suspected to have a MDR infection, so it is necessary to model outcomes for both patients in whom this suspicion is confirmed and for those in whom this suspicion turns out to be incorrect. The probability of having the suspected pathogen/resistance mechanism is informed by Second-Generation Surveillance System (SGSS) national surveillance data supplied by PHE for this evaluation. The key driver of effectiveness is in vitro susceptibility as estimated via the evidence syntheses discussed above. Higher susceptibility reduces mortality and LoS in hospital. These relationships are based on a combination of evidence from the literature and structured expert elicitation. Colistin or aminoglycoside-based therapy is expected to be associated with higher rates of AKI than other agents (including cefiderocol), which has significant consequences for patients’ short- and long-term mortality, morbidity and costs. Safety differences between colistin or aminoglycoside-based therapy and other agents are, therefore, modelled using evidence from published systematic reviews. Lifetime costs, quality of life and mortality were predicted accounting for the highly comorbid nature of the patient population and the increased risk of chronic kidney disease (CKD) resulting from AKI. The population-level component uses a forecast-based approach to aggregate the patient-level predictions to the population level accounting for the size of, and growth over time in, the eligible patient population in England within each HVCS. This component also reflects how resistance is likely to develop to cefiderocol and existing AMs over time. Predictions of population-level INHE are presented for patients initiated on treatment over the next 20 years. Current numbers of patients within the HVCSs were based on evidence from SGSS. Future growth in the number of patients in the HVCSs was based on statistical forecasting models fitted to time-series data from a national reference laboratory data set supplied by PHE for this evaluation. A series of scenarios reflects the potential emergence of resistance to cefiderocol. We did not model changes in resistance to existing AMs over time due to the sparsity of evidence available to inform these forecasts. Predicted overall population-level INHEs corresponding to the expected use of cefiderocol in the English NHS were generated by rescaling the population-level INHEs from the HVCSs to reflect additional areas of expected usage. These included patients with bloodstream and intra-abdominal infections (BSIs and IAIs) known or suspected to be caused by MBL Enterobacterales or MBL P. aeruginosa, and patients with infections caused by Stenotrophomonas across a range of sites (HAP/VAP, cUTI, BSIs and IAIs). This rescaling was based on population size estimates from SGSS and the use of expert opinion to inform the similarity in patient-level INHEs between the patients falling within the HVCSs and these additional sites and pathogens of interest. The literature on the economic evaluation of AMs has described a range of elements of value associated with these products that are not relevant to evaluations of other healthcare interventions. We also, therefore, summarise the extent to which these elements of value are captured within the quantitative estimates and, where this has not been possible, whether they are likely to substantively modify the quantitative estimates of value presented. Results Patient-level INHEs in the HVCS For HAP/VAP patients treated empirically, the base-case INHEs (0.15 QALYs) are similar in patients with suspected MBL Enterobacterales, and those with suspected P. aeruginosa. The results were very sensitive to the proportion of patients who have MBL Enterobacterales, use of the CLSI breakpoints, AKI risk, AKI-related mortality and long-term survival (range across scenarios: 0.00–0.23 QALYs). For patients treated in the MDS, the base-case INHEs associated with cefiderocol are much higher for MBL P. aeruginosa (0.15 QALYs for HAP/VAP and cUTI) than MBL Enterobacterales (0.02 QALYs for HAP/VAP and cUTI) as the latter patient group has a higher probability of being susceptible to a non-colistin/aminoglycoside-based treatment, and hence not requiring treatment with cefiderocol. There is a large degree of uncertainty in the patient-level INHE in the MBL P. aeruginosa population (range across scenarios 0.01–0.25). This reflects uncertainties in the susceptibility to non-colistin/aminoglycoside-based treatments. Results in the MBL P. aeruginosa populations are subject to additional uncertainty due to limitations in the evidence base for the comparators (fosfomycin plus meropenem and fosfomycin plus colistin), as fosfomycin does not have an established breakpoint for P. aeruginosa, making links to clinical outcomes more tenuous. Furthermore, the available evidence to inform the relative susceptibility of fosfomycin was based on very small studies (n = 7 in the EUCAST and n = 20 in the CLSI networks). We were unable to select a base case for the population-level results. Population-level results are, therefore, presented for two different approaches to estimating infection numbers (based on different methods to classify infections from clinical specimen sites); two alternative approaches to forecasting increases in infections over time (based on whether observed trends are assumed to persist indefinitely or not); and three different trajectories with respect to resistance emergence to cefiderocol (1%, 5% and 10% at 20 years). Across these scenarios, population-level INHEs range from 896 to 3559 QALYs. The results are particularly sensitive to the assumption about which clinical specimen sites are indicative of HAP/VAP. A substantial part of the value of cefiderocol (20 to 38% depending on scenario) is generated among patients with Stenotrophomonas who were outside of the HVCSs considered by EEPRU. Departures from the base-case assumptions in the patient-level model also had substantial effects on the mean population-level INHEs. The population size estimates used to generate the estimates of population-level INHEs are subject to additional uncertainties relating to the completeness of the national data, whether all tested patients would fall within the HVCS population for empiric treatment, the potential double counting of samples from the same infectious episode and inherent uncertainties in forecasting population size over time. In addition, estimates of population-level INHEs were generated using a number of strong assumptions about how evidence can be generalised between settings. Namely, that patient-level INHE of cefiderocol in patients with BSI can be approximated based on outcomes in HAP/VAP patients, and that the patient-level INHE of cefiderocol in patients with IAI can be proxied by that in patients with cUTI. These assumptions were based on discussions with clinical experts. Based on the available evidence, we concluded that diversity value, transmission value and spectrum value were unlikely to be significant drivers of population-level INHEs for cefiderocol. Insurance value was captured for the HVCS, though the benefits of cefiderocol use in novel future outbreaks were not captured and remain highly uncertain. EEPRU considers that it is possible that cefiderocol use will facilitate additional or at least more prompt receipt of required treatments/procedures for certain groups but that the magnitude of these enablement benefits remains highly uncertain. Conclusion The quantitative assessment of value in this report indicates that cefiderocol is associated with a base-case population-level INHE across its areas of expected usage of 896 to 3559 QALYs over 20 years. T

Introduction: Plasmid-mediated class C ß-lactamase (pAmpC) is a member of broad-spectrum ß-lactamase that spreads worldwide. However, its prevalence was under-evaluated. Objective: To characterize the prevalence and distribution of pAmpC types in 294 cefoxitin (FOX) and third-generation cephalosporin (3GC) resistant Enterobacteriaceae collected in several regions of Thailand and Vietnam in 2018 and 2020. Methods: Multiplex Polymerase Chain Reaction (PCR) for pAmpC identification was utilized to investigate prevalence and diversification of pAmpC among 294 cefoxitin and third-generation cephalosporin resistant Enterobacteriaceae isolated from Thailand (n= 197) and Vietnam (n= 97). Results: The prevalence of pAmpC was 37.1 % found in second and third-generation cephalosporin resistant Enterobacteriaceae. CMY-2 like was prominent in Thailand and Vietnam; however, prevalence of CMY-2 was varied in each hospital. DHA contributed 25.7 %, ACT/MIR rate was dominant in Chiang Rai hospital, reached 100 % in Thanh Hoa Pediatrics hospital. Worrisome, 3.7 % - isolates carried two types of pAmpC. The incidence of pAmpC in Vietnam was significantly higher than those in Thailand. Conclusions: These findings provide evidence-based of highly spreading and diversified distribution of transferable AmpC among Enterobacteriaceae in two Asia-Pacific countries.

Weiqiang Xiao,1,&ast; Xiaokun Wang,1,&ast; Yuanye Qu,1 Mingyue Sun,1 Yanmin Chang,1 Wenjiao Li,1 Yong Shen,1,2 Xiufang Shi,3 Min Jing,1 Qingxia Xu1,4 1Department of Clinical Laboratory, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 2Institute of Biophysics, Chinese Academy of Sciences, Foshan, People’s Republic of China; 3School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, People’s Republic of China; 4Zhengzhou Key Laboratory for Diagnosis of Digestive System Tumor Markers, Zhengzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Qingxia Xu; Xiufang Shi, Email zlyyxuqingxia0575@zzu.edu.cn; xfshi@zzu.edu.cnBackground: Emergence of blaKPC and blaNDM co-harboring Klebsiella pneumoniae has escalated the threat of Carbapenem-resistant Klebsiella pneumoniae (CRKP) to healthcare. It remains unknown the prevalence and molecular characteristics of CRKP co-producing KPC and NDMs carbapenemases in Henan.Methods and Results: Twenty-seven CRKP strains isolated from different times were selected randomly in affiliated cancer hospital of Zhengzhou University from January 2019 to January 2021, among which one KPC-2 and NDM-5 positive CRKP named K9 was isolated from an abdominal pus sample of a 63-year-old male patient with leukemia. Sequencing of K9 determined that K9 belonged to ST11-KL47, which is resistant to antibiotics such as meropenem, ceftazidime-avibactam and tetracycline. K9 carried two different plasmids that contained blaNDM− 5 and blaKPC− 2. Both plasmids were shown to be novel hybrid plasmids and IS 26 played an important role in generation of two plasmids. Gene blaKPC-2 was flanked by the NTEKPC-Ib-like genetic structure (IS 26-ΔTn 3-ISKpn8-blaKPC-2-ISKpn6-IS 26) and was located on a conjugative IncFII/R/N type hybrid plasmid.Conclusion: The resistance gene blaNDM− 5 located on a region organized as IS 26-blaNDM– 5-ble-trpF-dsbD-ISCR1-sul1-aadA2-dfrA12-IntI1-IS 26 was carried by a phage-plasmid. We described a clinical CRKP co-producing KPC-2 and NDM-5 and emphasized an urgent need to control their further spread.Keywords: carbapenem-resistant Klebsiella pneumoniae, KPC-2, NDM-5, beta-lactamases, hybrid plasmid, IS 26

ABSTRACT The bla PAC-1 has been reported in Central Asia and Europe countries like Afghanistan and France in Aeromonas caviae and Pseudomonas aeruginosa strains from animals and patients, respectively. However, there is no record of bla PAC-1-carrying strain from the natural environment, and bla PAC-1-carrying Aeromonas has not been reported in the Asia Pacific. Here, we report the first known enviromental bla PAC-1-carrying Aeromonas enteropelogenes in the world from reservoir water in Singapore. We have performed a comprehensive genetic environment alignment and comparison of bla PAC-1 between our strain and other strains from different countries and sources and found the bla PAC-1 located on a highly conserved gene cluster. We suggest that environmental Aeromonas strains may act as a hidden reservoir involved in the circulating of bla PAC-1. The finding of conserved bla PAC-1 cluster also suggested the existence of multiple transmission pathways of bla PAC-1 in the Asia-Pacific region, involving multiple sources and different species.

Objective. To describe antimicrobial resistance profiles of Escherichia coli and Salmonella spp. isolated from chicken carcasses and the antimicrobials commonly used in animals in Ecuador and provide information on antimicrobial resistance patterns for implementing evidence-based corrective measures. Methods. Meat samples were collected from chicken carcasses in 199 slaughterhouses across Ecuador as part of a national pilot study for monitoring antimicrobial resistance in agricultural sources in 2019. Samples were tested for E. coli and Salmonella spp. Sensitivity to 10 critically important and three highly important antimicrobials (from a human health perspective) was assessed. The country report submitted to the World Organization for Animal Health was accessed to extract the quantity of antimicrobials produced or imported for use in animals. Results. Of 383 samples, E. coli was isolated from 148 (39%) and Salmonella spp. from 20 (5%) samples. Ninety percent of the isolates were resistant to at least one critically important antimicrobial. Resistance was highest to erythromycin (E. coli 76%; Salmonella spp. 85%) and tetracycline (E. coli 71%; Salmonella spp. 90%). Critically or highly important antimicrobials (colistin, tetracycline, trimethoprim/sulfamethoxazole) formed the bulk (87%) of antimicrobials used in animals as per the World Organization for Animal Health report. Conclusions. High prevalence of antimicrobial resistance in poultry in Ecuador calls for the development of guidelines and regulations on the use of antimicrobials and for engagement with livestock producers. The existing surveillance system needs to be strengthened to improve the monitoring of antimicrobial use and evolving resistance patterns.

Rongrong Li,1,2 Huaming Xu,3 Hao Tang,4 Jilu Shen,1,5 Yuanhong Xu1 1Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, People’s Republic of China; 2Department of Pathogen Biology and Provincial Laboratories of Pathogen Biology and Zoonoses, Anhui Medical University, Hefei, People’s Republic of China; 3The First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei, People’s Republic of China; 4Department of Clinical Laboratory, The Second Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of China; 5Anhui Public Health Clinical Center, Hefei, People’s Republic of ChinaCorrespondence: Yuanhong Xu, Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, People’s Republic of China, Tel +86 13505694447, Email xyhong1964@163.comBackground: Bloodstream infection (BSI) is a common type of infection frequently diagnosed in clinics. The emergence and spread of ESBLs-producing Escherichia coli (E. coli) has emerged as one of the biggest challenges in global community health.Methods: The production of ESBLs was determined by the composite disk diffusion method. The expression of the various resistance and virulence genes were detected by PCR and sequencing. Multi-locus sequence typing (MLST) and phylogenetic groups were used for the classification. The transfer of resistant plasmids was determined by conjugation assay. The statistical differences were analyzed using Statistical Product and Service Solutions (SPSS) version 23.0.Results: A total of 60 strains of ESBLs-producing E. coli were collected. The resistance genes that were identified included blaCTX-M, blaTEM, blaSHV, blaOXA-1 and mcr-1. The most common one was the blaCTX-M including blaCTX-M-27 (n = 16), blaCTX-M-14 (n = 15), blaCTX-M-15 (n = 11), blaCTX-M-55 (n = 14) and blaCTX-M-65 (n = 5). A total of 31 STs were detected, and the most abundant among which was ST131 (n = 16, 26.7%). Most of the E. coli (n = 46, 76.7%) belonged to the groups B2 and D. And some virulence genes were related to the classification of the E. coli. Among them, the detection rates of hek/hra, kpsMII and papGII-III in groups B2 and D were higher than those in groups A and B1. The detection rates of cnf1, iucC and papGII-III in ST131 were higher than those in non-ST131. And the distributions of hek/hra, iroN, iucC, kpsMII and papGII-III were related to the blaCTX-M subtypes. Finally, most bacterial (n = 32, 53.3%) resistance genes could be transferred between the bacteria by plasmids, especially IncFIB.Conclusion: ESBLs-producing E. coli in BSI exhibited had high resistance rates and carried a variety of virulence factors (VFs). This is necessary to strengthen the monitoring of ESBLs-producing isolates in the medical environment.Keywords: Escherichia coli, sepsis, beta-lactamases, blaCTX-M

Despite the presence of more than 100 different antibacterials in the therapeutic arsenal, beta-lactam antibiotics, in general, and the third-generation cephalosporins, in particular, remain the main option for the treatment of the most of infections in inpatients. At the same time, the widespread and oftentimes inappropriate use of the third-generation cephalosporins in Russian hospitals lead to the emergence and spread of antimicroabial resistance. The review covers the problems of antibiotic resistance to cephalosporins due to the production of beta-lactamases, the role of beta-lactamase inhibitors in overcoming this type of resistance, options for combinations of cephalosporins with beta-lactamase inhibitors, in vitro activity of cefotaxime/sulbactam and cefepime/sulbactam, the results of clinical studies, and the role of the above combinations in the treatment of infections in the hospital.

O estudo tem como objetivo verificar a capacidade de adesão e formação de biofilme de isolados de Salmonella spp. produtores de enzimas beta-lactamases de espectro estendido (ESBL), bem como a resistência de isolados produtores ou não de ESBL, frente às condições gástricas e intestinais simuladas in vitro. Foram utilizados 18 isolados de Salmonella spp. obtidos de cortes de frangos congelados produzidos no Estado do Paraná, Brasil. Os isolados produtores de ESBL foram avaliados de forma fenotípica e genotípica quanto a capacidade de adesão e formação de biofilme em lâminas de polipropileno. Além disso, todos os isolados foram submetidos a avaliação in vitro de resistência às condições gástricas e intestinais e após as simulações, realizadas contagens das células sobreviventes. Com relação à adesão e formação de biofilme, 13 isolados apresentaram essa capacidade. Quanto a tolerância às condições gástricas e intestinais, a quantificação de Salmonella spp. diminuiu (p0,05) na taxa de sobrevivência total. Os resultados demonstram que, apesar da diminuição da contagem de microrganismos conforme a passagem simulada pelos sucos gástricos e intestinais, a taxa de sobrevivência média total dos isolados de Salmonella spp. foi alta, o que permite inferir que os isolados possuem a capacidade de sobreviver às condições gastrointestinais simuladas, e ainda sim permaneceram em número capaz de causar infecção.

Background and objectives: colonization by extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae in Intensive Care Unit (ICU) patients is considered a risk factor for infections, and poses as a source of spreading these strains in hospital facilities. This study aimed to perform the genetic characterization of ESBL-producing K. pneumoniae isolates recovered from surveillance swabs in an ICU in northeastern Brazil. Methods: the isolates were recovered between 2018-2019 from the nasal, axillary, and rectal sites of 24 patients admitted to the ICU. Bacterial identification was performed by traditional biochemical tests. Antimicrobial susceptibility was assessed by disk diffusion, and ESBL phenotype was detected by double-disc synergy test. Polymerase chain reaction (PCR) for blaCTX-M, blaSHV, and blaTEM genes, PFGE, and MLST were carried out in representative isolates. Results: a total of 27 isolates were recovered from 18 patients (75%). The ESBL production was detected in 85% of isolates. Resistance to ciprofloxacin, sulfamethoxazole/trimethoprim and most of the β-lactams tested was recurrent, except for carbapenems. The blaSHV, blaTEM, and blaCTX-M genes were found in high frequency, and the CTX-M-(1, 2 and 9) groups were identified. Seven sequence types (ST11, ST14, ST17, ST395, ST709, ST855, and ST3827) were described, most of them considered high-risk. Conclusion: these findings emphasize the potential threat of well-established high-risk clones in an ICU, and highlight the importance of monitoring these clones to prevent infections.

Introdução/Objetivo: As carbapenemases presentes em cepas de Klebsiella pneumoniae de pacientes internados constituem uma grave problemática para o tratamento, especialmente os genes blaKPC-2, e o blaNDM. Diante do exposto o objetivo desse trabalho foi investigar a presença dos genes de resistência aos carbapenêmicos blaKPC, blaNDM, blaGES, blaVIM, blaIMP e blaOXA-48-LIKE em isolados clínicos de Klebsiella pneumoniae provenientes de pacientes de um hospital de Recife-PE. Métodos: Foram selecionadas 19 isolados clínicos resistentes aos carbapenêmicos por demanda espontânea, tanto de colonização (swab retal) quanto de sítios infecciosos de pacientes internados. O perfil de susceptibilidade antimicrobiana foi determinado através do equipamento automatizado BD PhoenixTM. Após a confirmação, foi realizada a extração do DNA total e realização da técnica de Reação em Cadeia da Polimerase para a identificação dos genes. Em seguida, foi realizado o sequenciamento dos amplicons. Resultados: Foram identificados os genes blaKPC e blaNDM. Em relação ao blaKPC, sua presença foi detectada em três cepas, sendo duas originárias de culturas de vigilância e uma de hemocultura. Quanto ao blaNDM, foram identificadas 15 cepas, sendo nove provenientes de culturas de vigilância e seis de sítios infecciosos. Foi observada a co-presença dos genes blaKPC e blaNDM em uma cepa isolada de cultura de vigilância. Das amostras analisadas, dez foram obtidas de culturas de vigilância e nove de sítios infecciosos, incluindo dois casos de sangue, dois de ponta de cateter, dois de urina, um de secreção de ferida, um de tecido e um de fragmento ósseo. Conclusão: Foi observada uma maior presença do gene blaNDM, independentemente da origem da amostra (infecção ou colonização), destacando a necessidade urgente de medidas de controle de infecções. Demonstrando a importância das culturas de vigilância, pois tais cepas estavam disseminadas no ambiente hospitalar, e caso não seja realizado o isolamento correto dos pacientes infectados por cepas que possuam os genes blaKPC e blaNDM, podem ocorrer surtos hospitalares ou mesmo a autoinfecção causada por tais cepas em casos de imunossupressão do paciente ou em falhas nos cuidados de higiene.

IntroductionFluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug-resistant (MDR) Enterobacterales (Ent) infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies.MethodsA case–control study of children (0–18 years) at three Chicago hospitals was performed. Cases had infections by FQ-susceptible, β-lactamase-producing (bla) Ent harboring a non- or low-level expression of PMFQR genes (PMFQS Ent). Controls had FQR infections due to bla Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay®) and PMFQR genes by PCR. We performed rep-PCR, MLST, and E. coli phylogenetic grouping. Whole genome sequencing was additionally performed on PMFQS Ent positive isolates. Demographics, comorbidities, and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed.ResultsOf 170 β-lactamase-producing Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). Eighty-five (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent was 4.3 and 6.2 years, respectively (p = NS). Of 23 PMFQS Ent, 56% were Klebsiella spp., and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes detected in PMFQS Ent were blaSHV ESBL (44%) and oqxAB (57%), and the corresponding genes detected in PMFQR Ent were blaCTX-M-1-group ESBL (79%) and aac(6’)-Ib-cr (83%). Whole genome sequencing of PMFQS Ent revealed the additional presence of mcr-9, a transferable polymyxin resistance gene, in 47% of isolates, along with multiple plasmids and mobile genetic elements propagating drug resistance. Multivariable regression analysis showed that children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6–22) and isolates containing multiple bla genes (OR 3.8, 95% CI 1.1–14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found.ConclusionsParadoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, and additional silent, yet transferrable antibiotic resistance genes such as mcr-9, affecting therapeutic options and suggesting the need to address infection prevention strategies to control spread. Control of PMFQS Ent infections will require validating community and healthcare-based sources and risk factors associated with acquisition.

Background Emergence of extended-spectrum beta-lactamases (ESBLs), AmpC β-lactamases, and metallo-β lactamases (MBL), and their co-existence among members of Enterobacteriaceae pose newer diagnostic and therapeutic challenges. The present study examines the ESBL, AmpC, and MBL production by various phenotypic methods and their co-occurrence among the multidrug-resistant (MDR) Enterobacteriaceae clinical isolates. Materials and Methods Four hundred non-repetitive Enterobacteriaceae clinical isolates were collected from the Central Referral Hospital, Sikkim. The isolates were used for identification and their antibiotic susceptibility tests were performed according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL was detected by double-disc synergy test (DDST) and phenotypic confirmatory disc-diffusion test (PCDDT), AmpC detection by AmpC E-test, and boronic acid disc diffusion (BD) test. MBL was detected using the imipenem–imipenem/EDTA disc and carba-NP tests. Results Around 76% were considered MDR. ESBL was seen in 58% and 50.4% based on DDST and phenotypic confirmation disc-diffusion test (PCDDT), respectively. AmpC was detected in 11.8% and 13.1% using a commercial E-test and boronic acid test, respectively. MBL were identified in 12.8% and 14.8% based on MBL imipenem-EDTA and carba-NP tests, respectively. Co-occurrence of ESBL and AmpC, ESBL and MBL, AmpC and MBL was seen in 5.2%, 11.5%, 1.3%, respectively, whereas a combination of these three β-lactamases was observed in only 0.3% of 304 MDR isolates. Conclusion The findings highlight a high prevalence of β-lactamases and their co-production among the Enterobacteriaceae, mainly in Klebsiella pneumoniae and Escherichia coli isolates. The study further highlights the necessity to identify the MDR β-lactamases stains for effective therapy in severe as well as mild bacterial infections, thereby enabling to reduce the risk of MDR in hospital and community settings.

IntroductionHospitals and wastewater are recognized hot spots for the selection and dissemination of antibiotic-resistant bacteria to the environment, but the total participation of hospitals in the spread of nosocomial pathogens to municipal wastewater treatment plants (WWTPs) and adjacent rivers had not previously been revealed.MethodsWe used a combination of culturing and whole-genome sequencing to explore the transmission routes of Escherichia coli from hospitalized patients suffering from urinary tract infections (UTI) via wastewater to the environment. Samples were collected in two periods in three locations (A, B, and C) and cultured on selective antibiotic-enhanced plates.ResultsIn total, 408 E. coli isolates were obtained from patients with UTI (n=81), raw hospital sewage (n=73), WWTPs inflow (n=96)/outflow (n=106), and river upstream (n=21)/downstream (n=31) of WWTPs. The majority of the isolates produced extended-spectrum beta-lactamase (ESBL), mainly CTX-M-15, and showed multidrug resistance (MDR) profiles. Seven carbapenemase-producing isolates with GES-5 or OXA-244 were obtained in two locations from wastewater and river samples. Isolates were assigned to 74 different sequence types (ST), with the predominance of ST131 (n=80) found in all sources including rivers. Extraintestinal pathogenic lineages frequently found in hospital sewage (ST10, ST38, and ST69) were also found in river water. Despite generally high genetic diversity, phylogenetic analysis of ST10, ST295, and ST744 showed highly related isolates (SNP 0-18) from different sources, providing the evidence for the transmission of resistant strains through WWTPs to surface waters.DiscussionResults of this study suggest that 1) UTI share a minor participation in hospitals wastewaters; 2) a high diversity of STs and phylogenetic groups in municipal wastewaters derive from the urban influence rather than hospitals; and 3) pathogenic lineages and bacteria with emerging resistance genotypes associated with hospitals spread into surface waters. Our study highlights the contribution of hospital and municipal wastewater to the transmission of ESBL- and carbapenemase-producing E. coli with MDR profiles to the environment.

Infections caused by extended-spectrum β-lactamase-producing Enterobacterales have increased rapidly and are mainly attributed to the production of CTX-M enzymes. This study evaluated the NG-Test® CTX-M MULTI lateral flow assay (CTX-M LFA) and the Rapid ESBL NP® test (ESBL NP test) for rapid detection of CTX-M-producing Enterobacterales directly in midstream urine (MSU) samples. Testing was performed on 277 clinical MSU samples in a hospital microbiology laboratory from November 2022 to January 2023; 60 of these samples (30 positive for ESBL producers and 30 positive for non-ESBL producers) were tested retrospectively after the identification and susceptibility results were obtained, and 217 samples were tested prospectively immediately after a Gram stain showing the presence of Gram-negative bacilli. The results were compared against phenotypic detection of ESBL and molecular testing as the reference methods. Overall, 67 of the 277 samples were culture-positive for ESBL-producing Enterobacterales. PCR for the blaCTX-M gene was positive for all ESBL-producing Enterobacterales isolates. All CTX-M LFA results were interpretable, while three of the ESBL NP test results were noninterpretable. The sensitivity of the CTX-M LFA (100%, 95% CI 94.6–100%) was higher than that of the ESBL NP test (86.6%, 95% CI 76.0–93.7%). Both tests had high specificities (CTX-M LFA, 99.1%, 95% CI 96.6–99.9% and ESBL NP test, 100%, 95% CI 98.2–100%). In conclusion, both the CTX-M LFA and the ESBL NP test can deliver rapid results that could improve antimicrobial stewardship for urinary tract infections.

The recommended therapy for severe infections caused by AmpC-inducible Enterobacterales (AmpC-E) typically involves cefepime or carbapenems. In an era of emerging resistance to these antimicrobials, we aim to assess the impact of third-generation cephalosporins (3GCs) vs. alternative antibiotics on clinical outcomes in bloodstream infections (BSIs) due to AmpC-E. We retrospectively included hospitalized adult patients with BSIs caused by 3GC-susceptible AmpC-E between 2012 and 2022, comparing the outcomes of 3GC and non-3GC definitive therapies. The primary outcome was overall treatment failure (OTF), encompassing 90-day all-cause mortality, 90-day reinfection, and 90-day readmission. Secondary outcomes comprised components of the OTF, in-hospital all-cause mortality, and length-of-stay. Within a total cohort of 353 patients, OTF occurred in 46.5% and 41.5% in the 3GC- and non-3GC-therapy groups, respectively (p = 0.36). The 3GC-therapy group exhibited a longer length-of-stay (38 vs. 21 days, p = 0.0003) and higher in-hospital mortality (23.3% vs. 13.4%, p = 0.019). However, the 90-day mortality, 90-day reinfection, and 90-day readmission were comparable between the therapy groups. Subgroup analyses involving high-risk AmpC-E and 3GC vs. standard-of-care yielded similar conclusions. Overall, our findings suggest that 3GC definitive therapy may not result in poorer clinical outcomes for the treatment of BSIs caused by AmpC-E.

Background: Carbapenem-resistant Acinetobacter baumannii is an opportunistic pathogen responsible for nosocomial infections and is one of the biggest global threats according to the World Health Organization (WHO), particularly causing substantial morbidity and mortality.Objectives: This study aimed at using computational approaches to screen meropenem and its analogs against OXA-23-positive Acinetobacter baumannii, analyzing the correlations between kinetic and phenotypic characteristics.Methods: A total of 5,450 compounds were screened using virtual screening workflow (HTVS, Glide-SP, and Glide-XP) to identify the best compounds based on their binding energy and interactions against OXA-23 and OXA-27 as they had phenotypic data available. Molecular dynamics simulation and density functional theory (DFT) studies were performed from the outcome of molecular docking analysis.Results: During simulations, meropenem and its analogs exhibited high-level stable interactions with Ser79, Ser126, Thr217, Trp219, and Arg259 of OXA-23. Meropenem displayed a CovDock energy of about −3.5 and −1.9 kcal mol-1 against OXA-23 and OXA-27, respectively. Among the 5,450 compounds, Pubchem_10645796, Pubchem_25224737, and ChEMBL_14 recorded CovDock energy between −6.0 and −9.0 kcal mol-1. Moreover, the infra-red (IR) spectrophotometric analysis revealed C=O and C-N atoms showing bands at 1,800 and 1,125 cm-1, respectively. These observed data are in congruence with the experimental observations.Conclusion: The identified compounds showed good agreement with the spectrophotometric analysis using DFT methods. In the earlier studies, meropenem’s MIC value was 32 μg mL−1 in OXA-23-positive isolate A2265 compared to the MIC of 1 μg mL−1 in ΔblaOXA-23 A2265. Comparing the CovDock energy and hydrogen-bonding interactions, the predicted results are in good agreement with the experimental data reported earlier. Our results highlight the importance of OXA-23 molecular docking studies and their compliance with the phenotypic results. It will help further in developing newer antibiotics for treating severe infections associated with carbapenem-resistant A. baumannii.

The presence of Shiga toxin-producing Escherichia coli (STEC) and resistance to beta-lactams in healthy sheep represents a potential public health risk. This study aimed to characterize STEC isolates in sheep feces for toxin production and resistance to beta lactam antibiotics. In the present study, among the 40 isolates, we found a predominance of subtype Stx1 (22/40), followed by subtype Stx1 + Stx2 (11/40), while the less prevalent group was Stx2 (7/40). Also, we found phenotypical resistance to beta-lactam antibiotics in 50% (20/40) of the strains analyzed, forming two groups, one with resistant isolates and the other with non-resistant isolates. The cytotoxicity of the isolates did not vary among the groups. In addition to having this characteristic, some multiresistant isolates produced significant amounts of toxins. This leads to the conclusion that the mechanisms of antimicrobial resistance via beta lactamases are present in sheep STEC and that the cytotoxicity of those isolates is variable regarding such resistance.

Background: Antimicrobial resistance is a global problem with enormous public health and economic impact. This study was carried out to get an overview of acquired antimicrobial resistance gene sequences in the genomes of Escherichia coli isolated from different food sources and the environment in Nigeria. Methods: To determine the acquired antimicrobial-resistant genes prevalence, genome assemblies of 272 isolates were analyzed In silico with KmerResistance 2.2 software. Results: A total of 107 antimicrobial resistance genes, which included genes that encode for 24 extended-spectrum beta-lactamases were detected. Potential multidrug resistance was found in 90% of the genomes analyzed. All strains analyzed contained at least one resistant gene sequence and had high similarity or homology (95% ID and above). Two strains harboured over 30 sequences of antimicrobial resistant genes, and in 24 strains over 20 genes were detected. Conclusion: The resistant genes found in all the genomes analyzed were acquired intra-species and not inter-species. This provides an opportunity for further studies of the orthologous nature of the genes detected and the data obtained can help monitor the epidemiology of E.coli resistant genes in the food and environment. DOI: 10.18502/jfqhc.8.4.8260

ABSTRACT As multidrug and pan-resistance among Enterobacterales continue to increase, there is an urgent need for more therapeutic options to treat these infections. New β-lactam and β-lactam inhibitor (BLI) combinations have a broad spectrum of activity, but those currently approved do not provide coverage against isolates harboring metallo-β-lactamases (MBL). Aztreonam (ATM) and avibactam (AVI) in combination (ATM/AVI; AVI at 4 μg/mL fixed concentration) provides a similarly broad range of activity while maintaining activity against MBL-producing isolates. The in vitro susceptibility testing of ATM/AVI by standard methods was evaluated during development. This study investigated the impact of nonstandard testing conditions on the activity of ATM/AVI as observed during broth microdilution testing as well as the equivalency between agar dilution and broth microdilution MIC values when testing a diverse panel of Enterobacterales (N = 201). Nonstandard test conditions evaluated included inoculum density, atmosphere of incubation, media pH, varied medium cation concentrations, incubation time, varied serum concentrations, testing in pooled urine instead of media, addition of blood to the media, and the presence of surfactant. Generally, apart from low pH and high inoculum density, nonstandard testing parameters did not affect ATM/AVI broth microdilution MIC values. Correlation of MIC values obtained by agar dilution and broth microdilution resulted in an essential agreement of 97.0% for all tested Enterobacterales. Variation of standard testing conditions had little impact on broth microdilution MIC values for ATM/AVI. The correlation between broth microdilution and agar dilution MICs suggests both methods are reliable for determination of ATM/AVI MIC values. IMPORTANCE Increasing antibiotic resistance and emergence of pan-resistant isolates threaten the ability to control infections and to provide many other medical interventions such as surgery and chemotherapy, among others. New therapies are required to control emerging resistance mechanisms, including the increase in metallo-β-lactamases. Some new antibiotic combinations provide coverage against highly resistant isolates but are unable to target organisms that produce metallo-β-lactamases. Aztreonam in combination with avibactam provides a broad spectrum of activity against highly resistant isolates that also targets metallo-β-lactamase-producing organisms. An important part of drug development is the ability for clinical labs to determine the susceptibility of isolates to the antimicrobial. This manuscript investigates the in vitro susceptibility testing of aztreonam/avibactam with nonstandard testing conditions and a correlation study between broth microdilution and agar dilution against clinical isolates encoding a variety of resistance mechanisms. Overall, aztreonam/avibactam was generally unaffected by changes in testing conditions and showed strong agar/broth correlation.

ABSTRACT Antibiotics disturb the intestinal bacterial microbiota, leading to gut dysbiosis and an increased risk for the overgrowth of opportunistic pathogens. It is not fully understood to what extent antibiotics affect the fungal fraction of the intestinal microbiota, the mycobiota. There is no report of the direct role of antibiotics in the overgrowth in healthy humans of the opportunistic pathogenic yeast Candida albicans. Here, we have explored the gut mycobiota of 22 healthy subjects before, during, and up to 6 months after a 3-day regimen of third-generation cephalosporins (3GCs). Using ITS1-targeted metagenomics, we highlighted the strong intra- and interindividual diversity of the healthy gut mycobiota. With a specific quantitative approach, we showed that C. albicans prevalence was much higher than previously reported, with all subjects but one being carriers of C. albicans, although with highly variable burdens. 3GCs significantly altered the mycobiota composition and the fungal load was increased both at short and long term. Both C. albicans relative and absolute abundances were increased but 3GCs did not reduce intersubject variability. Variations in C. albicans burden in response to 3GC treatment could be partly explained by changes in the levels of endogenous fecal β-lactamase activity, with subjects characterized by a high increase of β-lactamase activity displaying a lower increase of C. albicans levels. A same antibiotic treatment might thus affect differentially the gut mycobiota and C. albicans carriage, depending on the treated subject, suggesting a need to adjust the current risk factors for C. albicans overgrowth after a β-lactam treatment. IMPORTANCE Fungal infections are redoubtable healthcare-associated complications in immunocompromised patients. Particularly, the commensal intestinal yeast Candida albicans causes invasive infections in intensive care patients and is, therefore, associated with high mortality. These infections are preceded by an intestinal expansion of C. albicans before its translocation into the bloodstream. Antibiotics are a well-known risk factor for C. albicans overgrowth but the impact of antibiotic-induced dysbiosis on the human gut mycobiota—the fungal microbiota—and the understanding of the mechanisms involved in C. albicans overgrowth in humans are very limited. Our study shows that antibiotics increase the fungal proportion in the gut and disturb the fungal composition, especially C. albicans, in a subject-dependent manner. Indeed, variations across subjects in C. albicans burden in response to β-lactam treatment could be partly explained by changes in the levels of endogenous fecal β-lactamase activity. This highlighted a potential new key factor for C. albicans overgrowth. Thus, the significance of our research is in providing a better understanding of the factors behind C. albicans intestinal overgrowth, which might lead to new means to prevent life-threatening secondary infections.

Objective(s): Isolates producing Metallo-β-lactamase (MBL) have a significant impact on therapeutic and diagnostic layouts, plus their increased frequency has been reported globally. Determination of incidence of clinical isolates of Pseudomonas aeruginosa that are capable of producing MBL and AmpC-β-lactamases making them resistant to imipenem and cefoxitin. Materials and Methods: Out of 1159 collected samples of urine, wound swabs, blood, tissue, and pus, the isolation rate of P. aeruginosa in the period of March 2020 to February 2021 was 22.0% (255/1159). Bacterial strains that were resistant towards imipenem were further processed for detecting the β-lactamase group of genes followed by statistical analysis of risk factors done based on clinical sample, gender, plus department of sample collection. Results: The percentage of resistance against imipenem was found to be 53%. Out of 135 strains, phenotypic tests revealed MBLs incidence to be 61.5% by combination disc test and 81.5% by Modified Hodge test (MHT). Frequencies of blaIMP-1, blaVIM, blaSHV, blaTEM, and blaOXA genes were calculated to be 13%, 15%, 32%, 43%, and 21%, respectively. Co-expressions of blaMBLs (blaVIM and blaIMP-1) plus blaESBL (blaSHV, blaOXA, blaTEM) were detected using simplex and multiplex PCR. blaTEM, blaSHV, and blaOXA co-existed in 7.5% of clinical isolates. 5.5% of the isolates exhibited simultaneous expression of MBL/ESBL genes. 15% of the isolates resistant to cefoxitin were positive for the blaAmpC gene (17/114).Conclusion: This is a pioneer report from Pakistan that concomitantly presents expression of blaVIM and blaIMP-1 with blaTEM, blaOXA, blaSHV, and blaAmpC in isolates of P. aeruginosa.

Background: When animal manures are used, food products may include pathogenic bacteria, especially Escherichia coli. The major aim of the current study was to investigate TEM (blaTEM) and SHV beta-lactamase (blaSHV) genes in E. coli isolated from strawberry samples in Sanandaj, Iran. Methods: In this study, 150 strawberry samples were collected from farms (traditional), greenhouses, and packages in Sanandaj, Iran. E. coli contamination was done using routine culture methods. Then, isolates were investigated for Extended-Spectrum Beta-Lactamase (ESBL) production and blaTEM or blaSHV genes using phenotypic and genotypic methods, respectively. Results: The most susceptibility and resistance of E. coli to antibiotics were related to chloramphenicol and trimethoprim/sulfamethoxazole, respectively. Out of 21 isolates of E. coli, eight were resistant to ceftazidime and cefotaxime; from which, six isolates were ESBL-producer. Furthermore, Polymerase Chain Reaction (PCR) analysis of six ESBL- producing E. coli isolates showed that four isolates included blaTEM gene, while no isolates included blaSHV gene. Conclusion: In this study, multiple antibiotic resistance patterns were seen in E. coli isolates, especially ESBL patterns in E. coli isolated from strawberries produced in Iran. DOI: 10.18502/jfqhc.8.3.7199

Introduction: Antibiotic-resistance is a phenomenon by which bacteria manage to survive antimicrobial treatment; with incidence in intra and extra hospital environments such as: water sources, agricultural / livestock sector and fomites. Aim: To describe bacteria present in high circulation fomites in a central-western region of Colombia, with their phenotypic sensitivity profile and presence of genes beta-lactamases (TEM, OXA3 and SHV). Methodology: We isolate bacterial strains from banknotes, escalator handrails and ATM buttons. We evaluated its phenotypic sensitivity profile by minimal inhibitory concentration automated technique using Vitek 2® and presence of genes for beta-lactamases type TEM-full, OXA-3 and SHV-full by conventional PCR. Results: A total of 30 isolates were obtained; Acinetobacter baumannii complex, was the most common; banknotes were the form with the highest number of isolates and resistance. Of the total isolates, 53% carried at least one of the genes studied. Phenotypically, gram-negative bacteria were identified with resistance against: Imipinem, Piperacillin / Tazobactam, Colistin, Ceftazidime, Tigecycline and Ceftriaxone; Gram-positive bacteria with resistance to: Quinupristin / Dalfopristin, Minocycline, Tetracycline, Teicoplanin, Nitrofuratoin, Oxacillin, Clindamycin, Trimethropine-sulfamethoxazole, and Minocycline. Conclusion: Taking into account the circulation of strains with these resistances, it is important to educate the community to avoid the acquisition or spread of infections due to the inappropriate handling of this type of inanimate elements.

ABSTRACT The prevalence of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Escherichia coli has been increasing, with this spread driven by ESBL-encoding plasmids. However, the epidemiology of ESBL-disseminating plasmids remains understudied, obscuring the roles of individual plasmid lineages in ESBL spread. To address this, we performed an in-depth genomic investigation of 149 clinical ESBL-like E. coli isolates from a tertiary care hospital. We obtained high-quality assemblies for 446 plasmids, revealing an extensive map of plasmid sharing that crosses time, space, and bacterial sequence type boundaries. Through a sequence-based network, we identified specific plasmid lineages that are responsible for the dissemination of major ESBLs. Notably, we demonstrate that IncF plasmids separate into 2 distinct lineages that are enriched for different ESBLs and occupy distinct host ranges. Our work provides a detailed picture of plasmid-mediated spread of ESBLs, demonstrating the extensive sequence diversity within identified lineages, while highlighting the genetic elements that underlie the persistence of these plasmids within the clinical E. coli population. IMPORTANCE The increasing incidence of nosocomial infections with extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli represents a significant threat to public health, given the limited treatment options available for such infections. The rapid ESBL spread is suggested to be driven by localization of the resistance genes on conjugative plasmids. Here, we identify the contributions of different plasmid lineages in the nosocomial spread of ESBLs. We provide further support for plasmid-mediated spread of ESBLs but demonstrate that some ESBL genes rely on dissemination through plasmids more than the others. We identify key plasmid lineages that are enriched in major ESBL genes and highlight the encoded genetic elements that facilitate the transmission and stable maintenance of these plasmid groups within the clinical E. coli population. Overall, our work provides valuable insight into the dissemination of ESBLs through plasmids, furthering our understating of factors underlying the increased prevalence of these genes in nosocomial settings.

Beta-lactamase (β-lactamase)-producing Gram-negative bacteria (GNB) are of public health concern due to their resistance to routine antimicrobials. We investigated the antimicrobial resistance and occurrence of carbapenemases, extended-spectrum β-lactamases (ESBLs) and AmpCs among GNB from clinical sources. GNB were identified using matrix-assisted laser desorption/ionization time of flight–mass spectrometry (MALDITOF-MS). Antimicrobial susceptibility testing was performed via Kirby–Bauer disk diffusion and a microscan autoSCAN system. β-lactamase genes were determined via multiplex polymerase chain reactions. Of the 181 archived GNB analyzed, Escherichia coli and Klebsiella pneumoniae constituted 46% (n = 83) and 17% (n = 30), respectively. Resistance to ampicillin (51%), third-generation cephalosporins (21%), and ertapenem (21%) was observed among the isolates, with 44% being multi-drug resistant (MDR). β-lactamase genes such as AmpCs ((blaFOX-M (64%) and blaDHA-M and blaEDC-M (27%)), ESBLs ((blaCTX-M (81%), other β-lactamase genes blaTEM (73%) and blaSHV (27%)) and carbapenemase ((blaOXA-48 (60%) and blaNDM and blaKPC (40%)) were also detected. One K. pneumoniae co-harbored AmpC (blaFOX-M and blaEBC-M) and carbapenemase (blaKPC and blaOXA-48) genes. blaOXA-48 gene was detected in one carbapenem-resistant Acinetobacter baumannii. Overall, isolates were resistant to a wide range of antimicrobials including last-line treatment options. This underpins the need for continuous surveillance for effective management of infections caused by these pathogens in our settings.

Objective To explore the expression, clinical significance and molecular mechanism of miR-3934-5p in hepatocellular carcinoma (HCC). Methods HCC tissues and adjacent normal tissues were collected from 53 HCC patients diagnosed in our hospital from September 2013 to August 2014. Real-time quantitative PCR (RT-qPCR) was performed to detect the level of miR-3934-5p in the above tissues and human liver cell line L02 and HCC cell lines. The Cancer Genome Atlas (TCGA) database was used to analyze the expression of miR-3934-5p in normal liver tissues and HCC tissues; Chi-square test with Kaplan-Meier survival curve were applied to analyze the correlation of miR-3934-5p and clinicopathological characteristics and survival prognosis; RT-qPCR and Western blotting were adopted to detect transfection efficiency; CCK-8 assay, colony formation assay and EdU assay were employed respectively to evaluate the activity and proliferation of HCCLM3 cells; Spearman correlation analysis was applied to study the correlation between LACTB mRNA and miR-3934-5p expression in HCC tissues; TargetScan database was used to predict the downstream target genes of miR-3934-5p, and the prediction results was verified by luciferase reporter assay. Results MiR-3934-5p was highly expressed in the liver cancer tissues and cells (P < 0.05), and its high expression was significantly correlated with tumor size (P=0.040), tumor multiplicity (P=0.013), vascular invasion (P=0.006), and TNM stage (P=0.017); Compared to the low miR-3934-5p expression group, the HCC patients with high expression had poorer survival prognosis. CCK-8 assay, colony formation assay and EdU assay confirmed that knocking down miR-3934-5p notably inhibited the proliferation of HCCLM3 cells (P < 0.05). LACTB had been proved to be the direct target of miR-3934-5p, and mediated the biological effect of miR-3934-5p on HCC cells. Conclusion MiR-3934-5p promotes the proliferation of HCC cells by targeting tumor suppressor gene LACTB.

Purpose The purpose of this study was to investigate the clinical outcomes of non-carbapenem treatment for urinary tract infections (UTIs) caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) in young children. Methods We retrospectively reviewed the medical records of children under 2 years of age who were diagnosed and treated for UTIs caused by ESBL-producing E. coli from September 2014 to March 2020. Results Forty-three children under 2 years of age were treated with non-carbapenem antimicrobials for UTIs caused by ESBL-producing E. coli without bloodstream infections. The overall clinical and microbiological success rates for empirical antimicrobial treatment were 90.7% and 97.7%. Three of the patients (7.0%) experienced a relapse of UTI within a month. An in vitro susceptibility test showed that two patients were sensitive and one was resistant to the antimicrobial treatments. Furthermore, there were no significant differences in the time to defervescence, clinical success, microbiological success, and relapse rate between the susceptible (n=13) and non-susceptible groups (n=30). Conclusion In this study, the overall relapse rate of patients treated with non-carbapenem antimicrobials was 7.0%. The patients showed high success rates in the clinical and microbiological responses to the non-carbapenems regardless of the results of the in vitro antimicrobial susceptibility test. These results provide evidence that non-carbapenems may be viable alternative treatments for UTIs caused by ESBL-producing E. coli.

Objective. To evaluate frequency and intensity of cefazolin inoculum effect among methicillin-susceptible staphylococci isolated from patients with skin infections. Materials and Methods. A total of 80 methicillin susceptible isolates of Staphylococcus aureus were identified by cefoxitin disk-diffusion test and negative results of real-time PCR for mecA gene. Inoculum effect was measured by broth microdilution test with two inocula with concentrations of 5 × 105 CFU/mL and 5 × 107 CFU/mL. The disk-diffusion test with cefoxitin was also performed. Penicillin susceptibility was determined by disk-diffusion method. Beta-lactamase blaZ gene was identified by real-time PCR. Results. The frequency of cefazolin inoculum effect in tested isolates was 30% which is consistent with data from different countries. The MIC values for concentrated inoculum reached CLSI breakpoint for cefazolin resistance in 2.5% of isolates. The isolates with inoculum effect and those without it had the similar MIC values for cefazolin in broth microdilution test for standard inocula and similar diameters of inhibition zone in disk-diffusion test with cefazolin. Penicillin resistance was more frequent in inoculum effect-positive isolates. Beta-lactamase activity is considered as a main cause of cefazolin inoculum effect in staphylococci. The beta-lactamase blaZ gene was identified in the majority of isolates with cefazolin inoculum effect, but it was also prevalent among inoculum effect-negative isolates. Conclusions. Up to 30% of MSSA isolates from skin lesions in dermatological patients from SaintPetersburg are positive for cefazolin inoculum effect. Those isolates are usually characterized by penicillin resistance. Most of the cefazolin inoculum effect-positive isolates also carry beta-lactamase blaZ gene.

Background and Objectives: To provide data on the occurrence of classical K. pneumoniae (cKp) and hypervirulent Klebsiella pneumoniae (hvKp) strains harboring the gene encoding regulator of mucoid phenotype A (rmpA) and evaluated characteristics of virulence biomarkers, carbapenemase, extended-spectrum-β-lactamase (ESBL)-producing, and capsule serotypes among K. pneumoniae clinical isolates collected in the south of Iran. Materials and Methods: A total of 400 K. pneumoniae isolates were collected. First, the K. pneumoniae isolates were screened for rmpA gene by PCR, and then they were characterized for the presence of the virulence genes (pagO, iucA, iroB, luxR), capsular serotype genes (K1, K2, K5, K20, K54, and K57), carbapenemase (blaNDM, blaIMP, blaVIM, blaKPC, blaSPM, blaOXA-48, and blaOXA-181) and ESBL (blaCTX-M, blaSHV and blaTEM) genes. For all K. pneumoniae isolates phenotypic tests include of string test and disk diffusion test were performed. Results: In total, 16 (4%) hvKp-rmpA+ and 384 (96%) cKp were observed. Of hvKp-rmpA+ strains, 16 (100%) were carried pagO, iroB, and luxR genes, and 13 (81.3%) strains harbored iucA gene. The most prevalent capsular type genes were K1 (62%) and K2 (19%) in hvKp-rmpA+ strains. The incidence of blaSHV gene in hvKp and cKp was 94% (15/16) and 87.5% (336/384), respectively. The cKp isolates carried blaNDM (30/384; 7.8%) gene. Conclusion: Our data suggest that the incidence of hvKp was low. Also, hvKp-rmpA+ strains have less antibiotic resistance than cKp isolates. Serotypes K1 and K2, and blaSHV gene were strongly associated with hvKp-rmpA+.

New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the detection of β-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing Escherichia coli or Klebsiella pneumoniae complex. Selected targets were the β-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6′)-Ib, ANT(2′′)-I, and APH(3′)-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6′)-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the E. coli porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing E. coli or K. pneumoniae complex.

Abstract Background The distribution of β-lactam resistance genes in P. aeruginosa is often closely related to the distribution of certain high-risk international clones. We used whole-genome sequencing (WGS) to identify the predominant sequence types (ST) and β-lactamase genes in clinical isolates of multidrug-resistant (MDR)-P. aeruginosa from Qatar Methods Microbiological identification and susceptibility tests were performed by automated BD Phoenix™ system and manual Liofilchem MIC Test Strips. Results Among 75 MDR-P. aeruginosa isolates; the largest proportions of susceptibility were to ceftazidime-avibactam (n = 36, 48%), followed by ceftolozane-tazobactam (30, 40%), ceftazidime (n = 21, 28%) and aztreonam (n = 16, 21.3%). All isolates possessed Class C and/or Class D β-lactamases (n = 72, 96% each), while metallo-β-lactamases were detected in 20 (26.7%) isolates. Eight (40%) metallo-β-lactamase producers were susceptible to aztreonam and did not produce any concomitant extended-spectrum β-lactamases. High risk ST235 (n = 16, 21.3%), ST357 (n = 8, 10.7%), ST389 and ST1284 (6, 8% each) were most frequent. Nearly all ST235 isolates (15/16; 93.8%) were resistant to all tested β-lactams. Conclusion MDR-P. aeruginosa isolates from Qatar are highly resistant to antipseudomonal β-lactams. High-risk STs are predominant in Qatar and their associated MDR phenotypes are a cause for considerable concern.

Introduction: The data on members of the genera Proteus, Pseudomonas, Enterobacter, Citrobacter, and Staphylococcus as the etiologic agents of urinary tract infections (UTIs) is not much. This study investigated the frequency of various beta-lactamases in urine isolates of Proteus mirabilis, Enterobacter cloacae, and Citrobacter freundii in Gorgan, Golestan province. Methods: A total of 632 urine samples were collected from hospitalized patients in a teaching hospital. The samples were cultured on blood agar and Eosin Methylene blue agar and incubated overnight at 37°C. The cultures with a ≥105 CFU/mL bacterial count were defined as positive for UTI. Bacteria identification was performed using standard biochemical methods and the API20E enteric identification system. The antibiotic resistance pattern was determined by the Kirby-Bauer disk diffusion method, and a phenotypic confirmatory test was used for detecting ESBL, MBL, and AmpC beta-lactamases producers. Results: Out of 632 samples, 317 (50.1%) were positive for UTIs, and 27 (8.5%), 21 (6.6%), and 12 (3.7%) were positive for Enterobacter cloacae, Citrobacter freundii, and Proteus mirabilis isolates, respectively. All the isolates were sensitive to piperacillin-tazobactam and colistin. The prevalence of ESBL and AmpC beta-lactamases in P. mirabilis isolates was higher than the other isolates, but No MBL producers were detected. Conclusions: In this study, the high frequency of ESBL and AmpC beta-lactamases in P. mirabilis isolates may suggest an increasing trend in resistance to cephalosporins and monobactams, which could have a significant impact on the management and treatment of UTI caused by this organism. Therefore, continuous monitoring is required to control the spread of β-lactamase-producing isolates in different geographical areas.

Abstract Background Extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-PE) infections are frequent and highly impact cancer patients. We developed and validated a scoring system to identify cancer patients harboring ESBL-PE at the National Institute of Cancer of Colombia. Methods We retrospectively analyzed medical records of 1695 cancer patients. Derivation phase included 710 patients admitted between 2013 to 2015, ESBL-PE positive culture (n = 265) paired by month and hospitalization ward with Non-ESBL-PE (n = 445). A crude and weighted score was developed by conditional logistic regression. The model was evaluated in a Validation cohort (n = 985) with the same eligibility criteria between 2016 to 2017. Results The score was based on eight variables (reported with Odds Ratio and 95% confidence interval): Hospitalization ≥7 days (5.39 [2.46–11.80]), Hospitalization during the previous year (4, 87 [2.99–7.93]), immunosuppressive therapy during the previous 3 months (2.97 [1.44–6.08]), Neutropenia (1.90 [1.12–3.24]), Exposure to Betalactams during previous month (1.61 [1.06–2.42]), Invasive devices (1.51 [1.012–2.25]), Neoplasia in remission (2.78 [1.25–1.17]), No chemotherapy during the previous 3 months (1.90 [1.22–2.97]). The model demonstrated an acceptable discriminatory capacity in the Derivation phase, but poor in the Validation phase (Recipient Operating Characteristic Curve: 0.68 and 0.55 respectively). Conclusions Cancer patients have a high prevalence of risk factors for ESBL-PE infection. The scoring system did not adequately discriminate patients with ESBL-PE. In a high-risk population, other strategies should be sought to identify patients at risk of resistant ESBL-PE infection.

The purpose of this study was to assess the levels of antimicrobial susceptibility during a period of the last 20 years among Moraxella catarrhalis isolates and to evaluate their resistance mechanisms. Material/Methods: A total of 618 Bulgarian clinical significant isolates M.catarrhalis was collected during the period 1999–2018 from patients to assess the current levels of antimicrobial susceptibility and to evaluate the beta-lactam resistance mechanisms. The minimum inhibitory concentrations (MICs) by E-test were determined against: penicillin, ampicillin, amoxicillin+clavulanic acid, cefalexin, cefuroxime, cefixime, cefotaxime, levofloxacin, azithromycin, clarithromycin, tetracycline, sulfamethoxazole + trimethoprim. PCR examination of genes encoding beta-lactamases was done. Results: Almost all (98.88%) of the tested strains M. catarrhalis showed positive cefinase test and were demonstrated elevated MICs and a lack of susceptibility to penicillin, ampicillin and cefalexin. An increase of resistance to cephalosporin of second and third generations (cefixime) was found. Higher MIC values of cefuroxime and cefixime than these of cefotaxime were detected. PCRs revealed that bro-1 genes (94.11%) were more frequent than bro-2. Conclusions: The results of examined 20 years period showed that nearly all Bulgarian isolates of M. catarrhalis in the last years become resistant to penicillins and first generation cephalosporins via beta-lactamases, encoded predominantly by bro-1 gene. An increase of the resistance to members of second and third generation (cefixime) cephalosporin’s and slower to other antimicrobials was found. The rapid development of resistance in recent years to the most popular antimicrobial agents affects the recommendations regarding the initial approach to therapy.

Introduction: The emergence of extended-spectrum β-lactamase (ESBL) and carbapenem-resistant Enterobacteriaceae, especially Klebsiella pneumoniae isolates, has become a severe concern worldwide. This study aimed to determine the prevalence of blaVIM and blaNDM genes among K. pneumoniae isolates. Methods: One hundred-eighty-one K. pneumoniae isolates were obtained from different clinical specimens of patients hospitalized at Firoozgar hospital, Tehran, Iran. The isolates were identified by standard biochemical tests, and their identity was confirmed by Vitek 2 (bioMérieux, France), a fully automated system for bacterial identification. The isolates were subjected to antimicrobial susceptibility testing and screened for ESBL by double-disc synergy test (DDST) and modified Hodge test (MHT) for the detection of carbapenemase. PCR was also used to detect the presence of blaVIM and blaNDM resistance genes in the isolates. Results: The Vitek 2 system confirmed the biochemical test results. The highest and lowest rates of resistance to antibiotics belonged to cefepime (83.9%) and imipenem (55.2%). Eighty-six and 100 isolates showed to produce ESB and KPC by DDST and MHT, respectively. About 71% and 97% of the 100 isolates were positive for blaVIM and blaNDM genes, respectively. Conclusion: The high rate of ESBL- and KPC-producing K. pneumoniae isolates in our hospital setting revealed resistance to conventional antibiotics, which limit our options in choosing appropriate antimicrobials. Although the management of infections associated with these organisms is challenging, it is essential to control such strains to prevent the outbreak.

Patients with cancer have a higher risk of severe bacterial infections. This study aims to determine the frequency, susceptibility profiles, and resistance genes of bacterial species involved in bacteremia, as well as risk factors associated with mortality in cancer patients in Colombia. In this prospective multicenter cohort study of adult patients with cancer and bacteremia, susceptibility testing was performed and selected resistance genes were identified. A multivariate regression analysis was carried out for the identification of risk factors for mortality. In 195 patients, 206 microorganisms were isolated. Gram-negative bacteria were more frequently found, in 142 cases (68.9%): 67 Escherichia coli (32.5%), 36 Klebsiella pneumoniae (17.4%), and 21 Pseudomonas aeruginosa (10.1%), and 18 other Gram-negative isolates (8.7%). Staphylococcus aureus represented 12.4% (n = 25). Among the isolates, resistance to at least one antibiotic was identified in 63% of them. Genes coding for extended-spectrum beta-lactamases and carbapenemases, blaCTX-M and blaKPC, respectively, were commonly found. Mortality rate was 25.6% and it was lower in those with adequate empirical antibiotic treatment (22.0% vs. 45.2%, OR: 0.26, 95% CI: 0.1–0.63, in the multivariate model). In Colombia, in patients with cancer and bacteremia, bacteria have a high resistance profile to beta-lactams, with a high incidence of extended-spectrum beta-lactamases and carbapenemases. Adequate empirical treatment diminishes mortality, and empirical selection of treatment in this environment of high resistance is of key importance.

Bacteremia caused by extended-spectrum β-lactamases-producing Enterobacterales has increased rapidly and is mainly attributed to CTX-M enzymes. This study aimed to evaluate the NG-Test® CTX-M MULTI lateral flow assay (CTX-M LFA) for rapid detection of CTX-M producers in blood cultures (BCs) positive for Gram-negative bacilli in spiked and clinical BCs. Retrospective testing was performed on BC bottles spiked with a collection of well-characterized Enterobacterales isolates producing CTX-M (n = 15) and CTX-M-like (n = 27) β-lactamases. Prospective testing of clinical, non-duplicate BCs (n = 350) was performed in two hospital microbiology laboratories from April 2021 to March 2022 following detection of Gram-negative bacilli by microscopic examination. Results were compared against molecular testing as the reference. In the spiked BCs, the CTX-M LFA correctly detected all CTX-M producers including 5 isolates with hybrid CTX-M variants. However, false-positive results were observed for several CTX-M-like β-lactamases, including OXY-1-3, OXY-2-8, OXY-5-3, FONA-8, -9, -10, 11, 13 and SFO-1. In clinical BCs, the CTX-M LFA showed 100% (95% CI, 96.0–100%) sensitivity and 99.6% (97.9–100%) specificity. In conclusion, this study showed that rapid detection of CTX-M producers in BC broths can be reliably achieved using the CTX-M LFA, thus providing an opportunity for early optimization of antibiotics.

ABSTRACT β-Lactamase expression is the major mechanism of resistance to penicillins, cephalosporins, and carbapenems in the multidrug-resistant (MDR) bacterium Acinetobacter baumannii. In fact, stable high-level expression of at least one β-lactamase has been rapidly increasing and reported to occur in up to 98.5% of modern A. baumannii isolates recovered in the clinic. Moreover, the OXA-51 β-lactamase is universally present in the A. baumannii chromosome, suggesting it may have a cellular function beyond antibiotic resistance. However, the consequences associated with OXA β-lactamase overexpression on A. baumannii physiology are not well understood. Using peptidoglycan composition analysis, we show that overexpressing the OXA-23 β-lactamase in A. baumannii drives significant collateral changes with alterations consistent with increased amidase activity. Consequently, we predicted that these changes create new cellular vulnerabilities. As proof of principle, a small screen of random transposon insertions revealed three genes, where mutations resulted in a greater than 19-fold loss of viability when OXA-23 was overexpressed. The identified genes remained conditionally essential even when a catalytically inactive OXA-23 β-lactamase was overexpressed. In addition, we demonstrated a synergistic lethal relationship between OXA-23 overexpression and a CRISPR interference (CRISPRi) knockdown of the essential peptidoglycan synthesis enzyme MurA. Last, OXA-23 overexpression sensitized cells to two inhibitors of peptidoglycan synthesis, d-cycloserine and fosfomycin. Our results highlight the impact of OXA-23 hyperexpression on peptidoglycan integrity and reveal new genetic vulnerabilities, which may represent novel targets for antimicrobial agents specific to MDR A. baumannii and other OXA β-lactamase-overexpressing Enterobacteriaceae, while having no impact on the normal flora. IMPORTANCE Acinetobacter baumannii has become a serious pathogen in both hospital and community settings. The β-lactam class of antibiotics is a primary treatment option for A. baumannii infections, and expression of β-lactamases is the most frequent mechanism of resistance in this bacterium. New approaches to treating multidrug-resistant A. baumannii strains are needed. In this study, we demonstrate that overexpressing the OXA-23 β-lactamase leads to significant collateral changes, where peptidoglycan structure is altered. We have identified genes that become selectively essential in OXA-23-expressing strains and confirmed the relationship between altered peptidoglycan and OXA-23 expression by demonstrating that OXA-23 overexpression sensitizes cells to genetic and chemical inhibition of peptidoglycan synthesis. This work paves the way for the identification of new antimicrobial targets, where inhibitors would selectively kill β-lactamase-expressing strains.

Introduction: Extended spectrum beta-lactamases (ESBL) produced by gram-negative bacteria have been associated with increased hospital morbidity and mortality, longer hospital stays, use of carbapenems, and higher health care costs. Few studies have assessed the risk factors for infection by ESBL-producing germs in intensive care units (ICU). Objective: To determine the risk factors for healthcare-associated infections (HAIs) by ESBL-producing Klebsiella pneumoniae and Escherichia coli in an ICU of a public tertiary care hospital and to assess the impact of this type of infection on mortality. Materials and methods: A case-control study with a 1:2 ratio (97 cases and 194 controls) was conducted in critically ill patients admitted to the ICU of a public tertiary care hospital in Bogotá D.C. (Colombia) between January 2016 and December 2019, and in which a HAI by ESBL-producing K. pneumoniae or E. coli (cases: n=97) or K. pneumoniae or E. coli with a normal antibiotic resistance pattern (controls: n=194) was documented. Bivariate analyses were performed using the chi-squared and the Mann-Whitney U tests. A logistic regression model was used in the multivariate analysis, and a two-tailed test was performed with the p-values obtained in the bivariate analyses. Results: ESBL-producing K. pneumoniae and E. coli isolates were identified in 57 (58.76%) and 40 (41.24%) patients, respectively. These isolates were obtained mainly from urine samples (30,92%), followed by peritoneal fluid (27.80%). In the multivariate analysis, the presence of urinary tract infection on admission to the ICU was identified as a risk factor (OR=5.63, 95%CI: 1.918-16.53;; p=0.002). The mortality rate was 28.17% (26.29% in the controls and 31.95% in the cases), but no significant difference was observed between groups (p=0.311). Conclusion: Urinary tract infection on admission to the ICU was a risk factor for HAIs by ESBL-producing K. pneumoniae or E. coli. Furthermore, no significant difference was observed between cases and controls in terms of mortality.

The diversity of beta-lactam antibiotic resistance genes, with particular emphasis on carbapenemase genes, during the treatment process at two wastewater treatment plants (WWTPs) with different levels of hospital wastewater inflow was investigated using high-throughput sequencing. An additional aspect of the study was to determine the taxonomic diversity of microorganisms in the studied samples. The obtained results suggest that bacteria of the Fusobacteriaceae family, not associated to date with this phenomenon, may be involved in the spread of antibiotic resistance in the environment. In samples from both wastewater treatment plants, the dominant beta-lactamase genes included blaOXA, blaGES, blaBEL, blaCfxA, and blaTEM. It is worth noting that the blaKPC and blaNDM genes were only found in untreated municipal wastewater with a higher hospital wastewater content. Moreover, an increase in the abundance of the blaIMP gene after the biological treatment stage in the studied treatment plants was found. In wastewater characterized by a higher proportion of hospital wastewater, 94 correlations were observed, while in wastewater with its lower proportion, 41 correlations were noted. Considering the above, the current research indicates that the inflow of hospital wastewater contributes to the spread of antibiotic resistance in the aquatic environment.

The growth rate of the population aging is increasing worldwide. To assess antimicrobial susceptibility of extended-spectrum β-lactamase- (ESBL-) producing Klebsiella pneumoniae Isolated from Patients aging in Rasool Akram, Hospital, as well as to identify AmpC genes. 100 K. pneumoniae strain isolated from different clinical samples. Isolates resistant to oxyimino-cephalosporins and to cefoxitin evaluated to phenotypic ESBL production and to phenotypic AmpC production, respectively. Detection of resistance genes was then performed using primers specific for AmpC genes. Piperacillin/tazobactam and carbapenems remained the active β-lactam antibiotic against K. pneumoniae. ESBLs were detected among 40 (40%) of K. pneumoniae isolates. CMY-1 gene was detected in 34.3% of all AmpC-positive isolates, whereas CMY-2 and FOX genes were 14.2% and 28.6%, respectively. The consumption of Carbapenem family drugs is high in Iranian hospitals which are used as a first line of treatment without antibiotic susceptibility testing. Therefore, increase in antibiotic resistance to this family drugs is unavoidable in the near future. Therefore, it is necessary to take the necessary measures to modify the administration and use of antibiotics.

Hospital-built environment colonization by healthcare-associated infections-related bacteria (HAIrB) and the interaction with their occupants have been studied to support more effective tools for HAI control. To investigate HAIrB dynamics and antimicrobial resistance (AMR) profile we carried out a 6-month surveillance program in a developing country public hospital, targeting patients, hospital environment, and healthcare workers, using culture-dependent and culture-independent 16S rRNA gene sequencing methods. The bacterial abundance in both approaches shows that the HAIrB group has important representativeness, with the taxa Enterobacteriaceae, Pseudomonas, Staphylococcus, E. coli, and A. baumannii widely dispersed and abundant over the time at the five different hospital units included in the survey. We observed a high abundance of HAIrB in the patient rectum, hands, and nasal sites. In the healthcare workers, the HAIrB distribution was similar for the hands, protective clothing, and mobile phones. In the hospital environment, the healthcare workers resting areas, bathrooms, and bed equipment presented a wide distribution of HAIrB and AMR, being classified as contamination hotspots. AMR is highest in patients, followed by the environment and healthcare workers. The most frequently detected beta-lactamases genes were, blaSHV–like, blaOXA–23–like, blaOXA–51–like, blaKPC–like, blaCTX–M–1, blaCTX–M–8, and blaCTX–M–9 groups. Our results demonstrate that there is a wide spread of antimicrobial resistance due to HAIrB in the hospital environment, circulating among patients and healthcare workers. The contamination hotspots identified proved to be constant over time. In the fight for patient safety, these findings can reorient practices and help to set up new guidelines for HAI control.

Zeqiang Zhan,1 Xuebin Xu,2 Zhen Gu,3 Jianghong Meng,4 Xiayidan Wufuer,5 Mingliu Wang,6 Meilian Huang,7 Jianhui Chen,8 Chunmei Jing,9 Zhiying Xiong,1 Mei Zeng,10 Ming Liao,1 Jianmin Zhang1 1National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Key Laboratory of Zoonoses, Ministry of Agriculture, Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Animal Infectious Diseases Laboratory, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; 2Shanghai Municipal Center for Disease Control and Prevention, Shanghai 200336, China; 3Emerging Infections Program China Office, Beijing, 102206, China; 4Department of Food Science and Nutrition, University of Maryland, College Park, Maryland 20742, USA; 5Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention, Urumchi 830001, China; 6Guangxi Zhuang Autonomous Region Disease Prevention and Control Center, Nanning 530028, China; 7Xiamen City Children’s Hospital, Xiamen 361006, China; 8Fujian Provincial Center for Disease Control and Prevention, Fuzhou 350001, China; 9Chongqing City Children’s Hospital, Chongqing 400014, China; 10Department of Infectious Diseases, Children’s Hospital of Fudan University, Shanghai 200336, ChinaCorrespondence: Mei ZengDepartment of Infectious Diseases, Children’s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102, ChinaEmail zengmeigao@aliyun.comJianmin ZhangCollege of Veterinary Medicine, South China Agricultural University, No. 483 Wushan Road, Tianhe District, Guangzhou 510642, ChinaTel +86 20 8528 0240Fax +86 20 8528 5282Email junfeng-v@163.comPurpose: Human infections caused by invasive non-typhoidal Salmonella (iNTS) are highly prevalent worldwide. However, data for such infections in China are scarce. This study reports the epidemiology of iNTS in China.Methods: INTS isolates were recovered from blood and other clinical specimens collected during 2007–2016 across five provinces (Shanghai, Xinjiang, Fujian, Guangxi, and Chongqing) in China. Antimicrobial susceptibility was performed using the agar dilution method and molecular epidemiology was performed using standard microbiological techniques.Results: A total of 178 iNTS isolates were recovered from approximately 9700 patient specimens during 2007–2016. The predominant serovars were Salmonella Enteritidis (57/178, 32%), Salmonella Choleraesuis (47/178, 26.4%), and Salmonella Typhimurium (24/178, 13.5%). Up to 50 isolates (28.1%) were from patients who were ≤1 year of age, while 28 (15.7%) were from patients who were ≥60 years. Among these isolates, high rates of resistance to nalidixic acid (114/178, 64%), sulfisoxazole (59%), ciprofloxacin (15.2%), and cefotaxime (8.4%) were found. Moreover, 53.4% (95/178) exhibited multidrug resistance, and 3.9% (7/178) showed co-resistance to third-generation cephalosporins and ciprofloxacin. Steadily increasing numbers of nalidixic acid, cefotaxime, and ciprofloxacin-resistant isolates, but decreasing numbers of multidrug resistance isolates were detected during the study period. Detection of quinolone genes in 114 nalidixic acid-resistant isolates showed that 58.3% (67/114) harbored plasmid-mediated quinolone resistance (PMQR) genes [aac(6´)-Ib-cr, qnrA, qnrB, oqxAB, qepA, qnrS, and qnrD] and 98.2% (112/114) exhibited mutations in quinolone resistance determining regions [gyrA, parC, and parE]. Furthermore, we detected beta-lactamases genes in the ceftriaxone-resistant isolates. The most common were blaTEM-1 (93.3%), followed by blaCTX-M-55 (40%), blaCMY-2 (33.3%), and blaOXA-1 (33.3%). Finally, a range of pulsed-field gel electrophoresis patterns were detected among the Salmonella Enteritidis and Salmonella Typhimurium isolates.Conclusion: High rates of multidrug resistance and steadily increasing cefotaxime and ciprofloxacin-resistant iNTS could pose a significant challenge for the effective treatment of salmonellosis in China.Keywords: invasive non-typhoidal Salmonella, fluoroquinolones, multidrug resistant, beta-lactamases, pulsed field gel electrophoresis, China

Objective(s): Pseudomonas aeruginosa is one of the most important nosocomial pathogens causing a high rate of mortality among hospitalized patients. Herein, we report the prevalence of antibiotic resistance genes, class 1 integrons, major virulence genes and clonal relationship among multidrug- resistant (MDR) P. aeruginosa, isolated from four referral hospitals in the southeast of Iran.Materials and Methods: In this study, 208 isolates of P. aeruginosa were collected from four referral hospitals in southeast of Iran. Disk diffusion method was used to determine susceptibility to 13 antibacterial agents. AmpC was detected by phenotypic method and β-lactamase genes, virulence genes and class 1 integrons were detected by PCR. Clonal relationship of the isolates was determined by RAPD-PCR.Results: All the isolates were susceptible to polymyxin-B and colistin. Overall, 40.4% of the isolates were MDR, among which resistance to third generation cephalosporins, aminoglycosides, and carbapenems was 47.5%, 32.3% and 40%, respectively. None of the isolates was positive for blaNDM-1 genes, while 84.5% and 4.8% were positive for the blaIMP-1 and blaVIM, metallo-β-lactamase genes, respectively. Incidence of class 1 integrons was 95% and AmpC was detected in 33% of the isolates. Prevalence of exoA, exoS, exoU, pilB and nan1 were 98.8%, 44%, 26%, 8.3% and 33.3%, respectively. RAPD profiles identified four large clusters consisting of 77 isolates, and two small clusters and three singletons.Conclusion: The rate of MDR P. aeruginosa isolates was high in different hospitals in this region. High genetic similarity among MDR isolates suggests cross-acquisition of infection in the region.

Introduction: The treatment of urinary tract infections has become more challenging due to the increasing frequency of multidrug-resistant Escherichia coli in human populations. Objective: To characterize multidrug-resistant E. coli isolates causing community-acquired urinary tract infections in Cumaná, Venezuela, and associate possible risk factors for infection by extended-spectrum beta-lactamases (ESBL)-producing isolates. Materials and methods: We included all the patients with urinary tract infections attending the urology outpatient consultation and emergency unit in the Hospital de Cumaná, Estado Sucre, Venezuela, from January through June, 2014. blaTEM, blaSHV and blaCTX-M genes detection was carried out by PCR. Results: We found a high prevalence of multidrug-resistant E. coli (25.2%) with 20.4% of the isolates producing ESBL. The ESBL-producing isolates showed a high frequency (66.7%) of simultaneous resistance to trimethoprim-sulphamethoxazole, fluoroquinolones and aminoglycosides compared to non-producing isolates (2.4%). Of the resistant isolates, 65.4% carried the blaTEM gene, 34.6% the blaCTX-M and 23.1% the blaSHV. The blaCTX-M genes detected belonged to the CTX-M-1 and CTX-M-2 groups. Plasmid transfer was demonstrated by in vitro conjugation in 17 of the 26 ESBL-producing isolates. All three genes detected were transferred to the transconjugants. Age over 60 years, complicated urinary tract infections and previous use of a catheter predisposed patients to infection by ESBL-producing E. coli. Conclusions: The high frequency of multidrug-resistant ESBL-producing isolates should alert the regional health authorities to take measures to reduce the risk of outbreaks caused by these types of bacteria in the community.

Abstract Background Environmental and commensal bacteria maintain a diverse and largely unknown collection of antibiotic resistance genes (ARGs) that, over time, may be mobilized and transferred to pathogens. Metagenomics enables cultivation-independent characterization of bacterial communities but the resulting data is noisy and highly fragmented, severely hampering the identification of previously undescribed ARGs. We have therefore developed fARGene, a method for identification and reconstruction of ARGs directly from shotgun metagenomic data. Results fARGene uses optimized gene models and can therefore with high accuracy identify previously uncharacterized resistance genes, even if their sequence similarity to known ARGs is low. By performing the analysis directly on the metagenomic fragments, fARGene also circumvents the need for a high-quality assembly. To demonstrate the applicability of fARGene, we reconstructed β-lactamases from five billion metagenomic reads, resulting in 221 ARGs, of which 58 were previously not reported. Based on 38 ARGs reconstructed by fARGene, experimental verification showed that 81% provided a resistance phenotype in Escherichia coli. Compared to other methods for detecting ARGs in metagenomic data, fARGene has superior sensitivity and the ability to reconstruct previously unknown genes directly from the sequence reads. Conclusions We conclude that fARGene provides an efficient and reliable way to explore the unknown resistome in bacterial communities. The method is applicable to any type of ARGs and is freely available via GitHub under the MIT license.