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AimsQuantile regression is an alternate type of regression analysis that has been shown to have numerous advantages over standard linear regression. Unlike linear regression, which uses the mean to fit a linear model, quantile regression uses a data set's quantiles (or percentiles), which leads to a more comprehensive analysis of the data. However, while relatively common in other scientific fields such as economic and environmental modeling, it is infrequently used to understand biological and microbiological systems.Methods and resultsWe analyzed a set of bacterial growth rates using quantile regression analysis to better understand the effects of antibiotics on bacterial fitness. Using a bacterial model system containing 16 variant genotypes of the TEM β-lactamase enzyme, we compared our quantile regression analysis to a previously published study that uses the Tukey's range test, or Tukey honestly significantly difference (HSD) test. We find that trends in the distribution of bacterial growth rate data, as viewed through the lens of quantile regression, can distinguish between novel genotypes and ones that have been clinically isolated from patients. Quantile regression also identified certain combinations of genotypes and antibiotics that resulted in bacterial populations growing faster as the antibiotic concentration increased-the opposite of what was expected. These analyses can provide new insights into the relationships between enzymatic efficacy and antibiotic concentration.ConclusionsQuantile regression analysis enhances our understanding of the impacts of sublethal antibiotic concentrations on enzymatic (TEM β-lactamase) efficacy and bacterial fitness. We illustrate that quantile regression analysis can link patterns in growth rates with clinically relevant mutations and provides an understanding of how increasing sub-lethal antibiotic concentrations, like those found in our modern environment, can affect bacterial growth rates, and provide insight into the genetic basis for varied resistance.

Abstract Background Klebsiella pneumoniae is a clinically relevant pathogen that has raised considerable public health concerns. This study aims to determine the presence of beta-lactamase genes and perform molecular genotyping of multidrug-resistant (MDR) K. pneumoniae clinical isolates. Methods Clinical isolates of MDR K. pneumoniae were collected from educational hospitals affiliated with Babol University of Medical Sciences. The isolates of K. pneumoniae were identified through standard microbial and biochemical tests. Antibiotic resistance was assessed using disk diffusion, modified Hodge test (MHT), combined disk, and polymerase chain reaction (PCR) methods. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was performed for molecular typing. Results A total of 42 MDR K. pneumoniae isolates were obtained from various clinical specimens. The highest antibiotic resistance was observed for ampicillin (100%), while the lowest resistance was noted for amikacin (19.04%). The MHT indicated that 38.09% of K. pneumoniae isolates produced carbapenemase enzymes. Metallo-beta-lactamase (MBL) production was found in 54.76% of isolates. Molecular detection of beta-lactamase genes revealed the presence of bla NDM (21.42%), bla KPC (42.85%), bla TEM (76.19%), bla SHV (47.16%), and bla CTX−M (80.95%) genes. ERIC-PCR molecular typing identified seven distinct genetic patterns among the isolates. Conclusions This investigation demonstrates the high resistance levels of K. pneumoniae strains. The beta-lactamase genes with the highest and lowest frequencies correspond to bla CTX−M and bla NDM genes, respectively. ERIC-PCR dendrograms suggest a common origin for K. pneumoniae clinical isolates and the propagation of similar clones within hospital wards. These findings indicate that K. pneumoniae isolates are highly virulent, necessitating the development of more effective resistance-fighting techniques and gene transfer research. Clinical trial number Not applicable.

We describe a case of New Delhi metallo-β-lactamase 1-producing carbapenem-resistant Pseudomonas aeruginosa (CRPA) in a transplant patient with multiple hospitalizations in California, USA. Whole-genome sequencing revealed the isolate was genetically distinctive, despite ≈95% similarity to other global strains. The patient's lack of international travel suggests this CRPA was acquired domestically.

Recurrent urinary tract infections (rUTI) are common in kidney transplant recipients (KTR) and are associated with multidrug resistance and increased morbidity/mortality. Novel antibiotic alternatives to reduce UTI recurrence are critically needed. We describe a case of rUTI due to extended spectrum beta lactamase (ESBL) Klebsiella pneumoniae in a KTR that was treated successfully with 4 weeks of adjunctive intravenous bacteriophage therapy alone, without concomitant antibiotics, and with no recurrence in a year of follow-up.

BackgroundThe role of piperacillin/tazobactam for treatment of serious infections due to AmpC-producing organisms remains debatable, particularly in immunocompromised patients.MethodsThis was a retrospective cohort study in immunocompromised patients that investigated the effect of definitive treatment with either piperacillin/tazobactam versus cefepime or carbapenems for bacteraemia caused by cefoxitin-non-susceptible Enterobacterales. The primary endpoint was a composite of clinical and microbiological failure. A logistic regression model was constructed to assess the impact of definitive treatment choice on the primary endpoint.ResultsA total of 81 immunocompromised patients with blood cultures positive for cefoxitin-non-susceptible Enterobacterales were included for analysis. There was more microbiological failure in the piperacillin/tazobactam arm compared with the cefepime/carbapenem arm (11.4% versus 0.0%, P = 0.019). Definitive treatment with cefepime or a carbapenem was associated with a decreased odds of clinical or microbiological failure (OR 0.303, 95% CI 0.093-0.991, P = 0.048) when controlling for baseline characteristics.ConclusionsIn immunocompromised patients with bacteraemia due to cefoxitin-non-susceptible Enterobacterales, definitive treatment with piperacillin/tazobactam was associated with an increased risk of microbiological failure and higher odds of clinical or microbiological failure compared with cefepime or carbapenems.

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.

BackgroundUrinary tract infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL-E. coli) may occur as outbreaks due to common-source exposures. Yet, it is currently unknown if they cluster geographically as would be expected as part of an outbreak.MethodsWe collected electronic health record data on all patients living in San Francisco with culture-documented community-onset E. coli bacteriuria in a safety-net public healthcare system from January 2014 to March 2020 (diagnosed

BackgroundAntimicrobial resistant infections continue to be a leading global public health crisis. Mobile genetic elements, such as plasmids, have been shown to play a major role in the dissemination of antimicrobial resistance (AMR) genes. Despite its ongoing threat to human health, surveillance of AMR in the United States is often limited to phenotypic resistance. Genomic analyses are important to better understand the underlying resistance mechanisms, assess risk, and implement appropriate prevention strategies. This study aimed to investigate the extent of plasmid mediated antimicrobial resistance that can be inferred from short read sequences of carbapenem resistant E. coli (CR-Ec) in Alameda County, California. E. coli isolates from healthcare locations in Alameda County were sequenced using an Illumina MiSeq and assembled with Unicycler. Genomes were categorized according to predefined multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) schemes. Resistance genes were identified and corresponding contigs were predicted to be plasmid-borne or chromosome-borne using two bioinformatic tools (MOB-suite and mlplasmids).ResultsAmong 82 of CR-Ec identified between 2017 and 2019, twenty-five sequence types (STs) were detected. ST131 was the most prominent (n = 17) followed closely by ST405 (n = 12). blaCTX-M were the most common ESBL genes and just over half (18/30) of these genes were predicted to be plasmid-borne by both MOB-suite and mlplasmids. Three genetically related groups of E. coli isolates were identified with cgMLST. One of the groups contained an isolate with a chromosome-borne blaCTX-M-15 gene and an isolate with a plasmid-borne blaCTX-M-15 gene.ConclusionsThis study provides insights into the dominant clonal groups driving carbapenem resistant E. coli infections in Alameda County, CA, USA clinical sites and highlights the relevance of whole-genome sequencing in routine local genomic surveillance. The finding of multi-drug resistant plasmids harboring high-risk resistance genes is of concern as it indicates a risk of dissemination to previously susceptible clonal groups, potentially complicating clinical and public health intervention.

Antimicrobial resistance in urinary tract infections (UTIs) is a major public health concern. This study aims to characterize the phenotypic and genetic basis of multidrug resistance (MDR) among expanded-spectrum cephalosporin-resistant (ESCR) uropathogenic Escherichia coli (UPEC) causing UTIs in California patient populations. Between February and October 2019, 577 ESCR UPEC isolates were collected from patients at 6 clinical laboratory sites across California. Lineage and antibiotic resistance genes were determined by analysis of whole-genome sequence data. The lineages ST131, ST1193, ST648, and ST69 were predominant, representing 46%, 5.5%, 4.5%, and 4.5% of the collection, respectively. Overall, 527 (91%) isolates had an expanded-spectrum β-lactamase (ESBL) phenotype, with blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaCTX-M-14 being the most prevalent ESBL genes. In the 50 non-ESBL phenotype isolates, 40 (62%) contained blaCMY-2, which was the predominant plasmid-mediated AmpC (pAmpC) gene. Narrow-spectrum β-lactamases, blaTEM-1B and blaOXA-1, were also found in 44.9% and 32.1% of isolates, respectively. Among ESCR UPEC isolates, isolates with an ESBL phenotype had a 1.7-times-greater likelihood of being MDR than non-ESBL phenotype isolates (P < 0.001). The cooccurrence of blaCTX-M-15, blaOXA-1, and aac(6')-Ib-cr within ESCR UPEC isolates was strongly correlated. Cooccurrence of blaCTX-M-15, blaOXA-1, and aac(6')-Ib-cr was associated with an increased risk of nonsusceptibility to piperacillin-tazobactam, cefepime, fluoroquinolones, and amikacin as well as MDR. Multivariate regression revealed the presence of blaCTX-M-55, blaTEM-1B, and the ST131 genotype as predictors of MDR. IMPORTANCE The rising incidence of resistance to expanded-spectrum cephalosporins among Escherichia coli strains, the most common cause of UTIs, is threatening our ability to successfully empirically treat these infections. ESCR E. coli strains are often MDR; therefore, UTI caused by these organisms often leads to treatment failure, increased length of hospital stay, and severe complications (D. G. Mark, Y.-Y. Hung, Z. Salim, N. J. Tarlton, et al., Ann Emerg Med 78:357-369, 2021, https://doi.org/10.1016/j.annemergmed.2021.01.003). Here, we performed an in-depth analysis of genetic factors of ESCR E. coli associated with coresistance and MDR. Such knowledge is critical to advance UTI diagnosis, treatment, and antibiotic stewardship.

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.

BackgroundThe rapid spread of extended-spectrum beta-lactamase-producing E. coli (ESBL-EC) is an urgent global health threat. We examined child caretaker knowledge, attitudes, and practices (KAP) towards proper antimicrobial agent use and whether certain KAP were associated with ESBL-EC colonization of their children.MethodsChild caretakers living in semi-rural neighborhoods in peri-urban Quito, Ecuador were visited and surveyed about their KAP towards antibiotics. Fecal samples from one child (less than 5 years of age) per household were collected at two time points between July 2018 and May 2019 and screened for ESBL-EC. A repeated measures analysis with logistic regression was used to assess the relationship between KAP levels and child colonization with ESBL-EC.ResultsWe analyzed 740 stool samples from 444 children living in households representing a range of environmental conditions. Of 374 children who provided fecal samples at the first household visit, 44 children were colonized with ESBL-EC (11.8%) and 161 were colonized with multidrug-resistant E. coli (43%). The prevalences of ESBL-EC and multidrug-resistant E. coli were similar at the second visit (11.2% and 41.3%, respectively; N = 366). Only 8% of caretakers knew that antibiotics killed bacteria but not viruses, and over a third reported that they "always" give their children antibiotics when the child's throat hurts (35%). Few associations were observed between KAP variables and ESBL-EC carriage among children. The odds of ESBL-EC carriage were 2.17 times greater (95% CI: 1.18-3.99) among children whose caregivers incorrectly stated that antibiotics do not kill bacteria compared to children whose caregivers correctly stated that antibiotics kill bacteria. Children from households where the caretaker answered the question "When your child's throat hurts, do you give them antibiotics?" with "sometimes" had lower odds of ESBL-EC carriage than those with a caretaker response of "never" (OR 0.48, 95% CI 0.27-0.87).ConclusionCaregivers in our study population generally demonstrated low knowledge regarding appropriate use of antibiotics. Our findings suggest that misinformation about the types of infections (i.e. bacterial or viral) antibiotics should be used for may be associated with elevated odds of carriage of ESBL-EC. Understanding that using antibiotics is appropriate to treat infections some of the time may reduce the odds of ESBL-EC carriage. Overall, however, KAP measures of appropriate use of antibiotics were not strongly associated with ESBL-EC carriage. Other individual- and community-level environmental factors may overshadow the effect of KAP on ESBL-EC colonization. Intervention studies are needed to assess the true effect of improving KAP on laboratory-confirmed carriage of antimicrobial resistant bacteria, and should consider community-level studies for more effective management.

BackgroundThe prevalence of infections caused by extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli) is increasing worldwide, but the setting in which this increase is occurring is not well defined. We compared trends and risk factors for ESBL-E. coli bacteriuria in community vs healthcare settings.MethodsWe collected electronic health record data on all patients with E. coli isolated from urine cultures in a safety-net public healthcare system from January 2014 to March 2020. All analyses were stratified by healthcare-onset/associated (bacteriuria diagnosed > 48 h after hospital admission or in an individual hospitalized in the past 90 days or in a skilled nursing facility resident, N = 1277) or community-onset bacteriuria (bacteriuria diagnosed  65 (RR 1.88, CI 1.17, 3.05), male gender (RR 2.12, CI 1.65, 2.73), and Latinx race/ethnicity (RR 1.52, CI 0.99, 2.33) were associated with community-onset ESBL-E. coli. Only male gender (RR 1.53, CI 1.03, 2.26) was associated with healthcare-onset/associated ESBL-E. coli.ConclusionsESBL-E. coli bacteriuria frequency increased at a faster rate in healthcare-associated settings than in the community between 2014 and 2020. Male gender was associated with ESBL-E. coli bacteriuria in both settings, but additional risks-age > 65 and Latinx race/ethnicity-were observed only in the community.

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.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) frequently causes hospital-acquired infections and is associated with high morbidity and mortality. CRKP can have multiple resistance mechanisms and only a few can be routinely detected by commercial molecular or phenotypic assays making surveillance for CRKP particularly challenging. In this report, we identified and characterized an unusual non-carbapenemase-producing CRKP carrying a rare plasmid-borne inducible AmpC gene, blaDHA-1 . The isolate was recovered from blood culture of a 67-year-old female presenting with sepsis post bladder surgery and ureteral stent removal. The primary isolate displayed an indeterminate susceptibility pattern for ceftriaxone by broth microdilution, but was susceptible by disk diffusion with one colony growing within the zone of inhibition. The ceftriaxone resistant colony was sub-cultured and had a minimum inhibitory concentration (MIC) of 2 ug/ml for imipenem (intermediate) and a zone size of 18 mm for ertapenem (resistant), but remained susceptible to cefepime and meropenem. Further phenotypic characterization of this sub-cultured isolate showed carbapenemase activity. Whole genome sequencing (WGS) revealed the presence of two subpopulations of a K. pneumoniae (MLST sequence type 11) from the primary blood culture isolate: one pan-susceptible to beta-lactams tested and the other resistant to the 3rd generation cephalosporins and ertapenem. WGS analysis identified the resistant K. pneumoniae harboring IncFIB(K) and IncR plasmids and the presence of plasmid-borne beta-lactam resistance genes bla OXA-1 and bla DHA-1, an inducible AmpC gene. Additional resistance genes against quinolones (aac(6')-Ib-cr, oqxA, oqB), aminoglycoside (aph(3')-Ia), sulfonamide (sul1), and tetracycline (tet(A)) were also identified. DHA-1 positive K. pneumoniae have been previously identified outside the US, particularly in Asia and Europe, but limited cases have been reported in the United States and may be underrecognized. Our study highlights the importance of using both extended phenotypic testing and WGS to identify emerging resistance mechanisms in clinical Enterobacterales isolates with unusual antimicrobial resistance patterns.

Community-acquired urinary tract infections (UTIs) are one of the most common bacterial infections worldwide. Escherichia coli is the most common cause of community-acquired UTI. In general, UTI results from E. coli in the intestine that enters the bladder via the urethra. However, whether these E. coli strains that cause UTI represent members of the intestinal commensal E. coli or a distinct subgroup of pathogenic E. coli remains unestablished. Here, we analyzed E. coli isolates from fecal samples of healthy volunteers and urine samples of UTI patients obtained from a university-affiliated health center. The E. coli isolates were genotyped by multilocus sequence typing (MLST). From May to October 2018, we analyzed 89 E. coli isolates from 76 (75%) rectal swabs from 113 unique healthy volunteers. We also analyzed 106 (27%) E. coli isolates from 398 unique urine samples collected between August and October 2018. Fecal and urine E. coli isolates each contained 31 distinct sequence types (STs). Nine STs were shared by fecal and urine E. coli isolates, which accounted for approximately 50% of urine isolates typed by MLST. Among the shared genotypes, ST10 and ST131 were significantly more frequently found in fecal samples, whereas ST95 and ST127 were significantly more frequently recovered from UTI samples. ST73 was found only among urine samples. These E. coli genotypes clustered and fluctuated over time. These observations suggest that E. coli genotypes found to cause UTI transiently colonize the intestine and that their primary reservoir may reside outside of the human intestine.

We report here a fatal case of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in a renal transplant patient without a travel history in the prior year, from whom 2 genetically different CRKP (sequence type 14 [ST14] and ST2497) strains carrying the same plasmids and antimicrobial resistance genes, including blaNDM-1, blaOXA-232, blaCTX-M-15, armA, and tet(D), were isolated from blood and the abdominal cavity. The isolates were susceptible to colistin, tigecycline, eravacycline, and cefiderocol, which was used to treat the CRKP in combination with ceftazidime-avibactam and polymyxin B and resulted in bacterial clearance. Despite the aggressive treatment, the patient died of ischemic colitis and multiorgan failure.

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.

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.

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.

Aeromonas hydrophila resides in a variety of aquatic environments. Infections with A. hydrophila mainly occur after contact with fresh or brackish water. Nosocomial infections with A. hydrophila can also occur. A. hydrophila infections can be difficult to treat due to both intrinsic and acquired antimicrobial resistance (AMR) mechanisms. In 2018-19, we isolated multi-drug resistant (MDR) A. hyrodphila from two solid organ transplant patients with intra-abdominal infections. We aimed to characterize their AMR mechanisms and to determine their genetic relatedness to aid epidemiological investigation. We performed whole genome sequencing (WGS) using Illumina MiSeq and Nanopore MinIon on 3 A. hydrophila isolates, with one isolate from Patient A (blood) and two isolates from Patient B (abdominal and T-tube fluid, isolated 2 weeks apart). Phenotypic assays included: Broth Microdilution (BMD), Modified Hodge Test (MHT), Modified Carbapenem Inactivation Method (mCIM), and EDTA Carbapenem Inactivation Method (eCIM). Data analyses were performed using CLCbio and Geneious. AMR genomic analysis revealed that all three isolates possess chromosomally encoded genes including bla OXA-12(oxacillinase), bla cepS (AmpC), and bla cphA7(metallo-beta-lactamase). All isolates tested strongly positive by MHT and mCIM, but only Patient B's second isolate (after 2 weeks of meropenem treatment) tested positive by eCIM. More intriguingly, Patient B's first isolate (before meropenem treatment) tested falsely susceptible to carbapenems by BMD, suggesting bla cphA7 gene was not expressed constitutively. Phylogenetic analysis showed the two isolates from Patient B were highly similar with only 1 SNP difference. The isolate from Patient A only differed from Patient B's isolates by 35 and 36 SNPs, respectively, suggesting close genetic relatedness. Further epidemiological investigation is undergoing. We report the first cases of CphA-mediated carbapenem resistant A. hydrophila in the U.S. It is concerning that 1 out of 3 isolates tested falsely susceptible to carbapenems by BMD despite clear carbapenemase production shown by strongly positive MHT and mCIM. In both cases, meropenem was initially used to treat the patients. Clinicians and microbiologists in the US should be aware of the emerging MDR Aeromonas nosocomial infections and the potential false carbapenem susceptible results due to CphA-type carbapenemase, which may be induced during treatment.

NON-TECHNICAL SUMMARY:Antibiotic resistance is a global human health problem. We partnered with Dignity Health Mercy Medical Center to study antibiotic resistance in clinical isolates. We tested whether growth rates, a sensitive assay used to measure the fitness of bacterial samples, correlate with a clinical test to measure antibiotic resistance. We found a strong correlation between these two methods suggesting that growth rates could be reliably applied to evolutionary studies of clinically relevant problems. Moreover, the sensitivity of the growth rates assay enabled us to identify fitness effects of specific antibiotic resistance genes.

BackgroundAntibiotic-resistant (AR) bacteria are a global threat. AR bacteria can be acquired in early life and have long-term sequelae. Limiting the spread of antibiotic resistance without triggering the development of additional resistance mechanisms is of immense clinical value. Here, we show how the infant gut microbiome can be modified, resulting in a significant reduction of AR genes (ARGs) and the potentially pathogenic bacteria that harbor them.MethodsThe gut microbiome was characterized using shotgun metagenomics of fecal samples from two groups of healthy, term breastfed infants. One group was fed B. infantis EVC001 in addition to receiving lactation support (n = 29, EVC001-fed), while the other received lactation support alone (n = 31, controls). Coliforms were isolated from fecal samples and genome sequenced, as well as tested for minimal inhibitory concentrations against clinically relevant antibiotics.ResultsInfants fed B. infantis EVC001 exhibited a change to the gut microbiome, resulting in a 90% lower level of ARGs compared to controls. ARGs that differed significantly between groups were predicted to confer resistance to beta lactams, fluoroquinolones, or multiple drug classes, the majority of which belonged to Escherichia, Clostridium, and Staphylococcus. Minimal inhibitory concentration assays confirmed the resistance phenotypes among isolates with these genes. Notably, we found extended-spectrum beta lactamases among healthy, vaginally delivered breastfed infants who had never been exposed to antibiotics.ConclusionsColonization of the gut of breastfed infants by a single strain of B. longum subsp. infantis had a profound impact on the fecal metagenome, including a reduction in ARGs. This highlights the importance of developing novel approaches to limit the spread of these genes among clinically relevant bacteria. Future studies are needed to determine whether colonization with B. infantis EVC001 decreases the incidence of AR infections in breastfed infants.Trial registrationThis clinical trial was registered at ClinicalTrials.gov, NCT02457338.

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.

CTX-M is the most prevalent family of extended-spectrum β-lactamases. We recently developed a tetrazole-derived noncovalent inhibitor of CTX-M-9. Here, we present the biochemical and microbiological activity of this inhibitor across a representative panel of serine β-lactamases and Gram-negative bacteria. The compound displayed significant activity against all major subgroups of CTX-M, including CTX-M-15, while it exhibited some low-level inhibition of other serine β-lactamases. Complex crystal structures with the CTX-M-14 S237A mutant and CTX-M-27 illustrate the binding contribution of specific active-site residues on the β3 strand. In vitro pharmacokinetic studies revealed drug-like properties and positive prospects for further optimization. These studies suggest that tetrazole-based compounds can provide novel chemotypes for future serine β-lactamase inhibitor discovery.

Objectives:Antibiotic selective pressure may result in changes to antimicrobial susceptibility throughout the course of infection, especially for organisms that harbour chromosomally encoded AmpC β-lactamases, notably Enterobacter spp., in which hyperexpression of ampC may be induced following treatment with cephalosporins. In this study, we document a case of bacteraemia caused by a blaSME-1-harbouring Serratia marcescens that subsequently developed resistance to expanded-spectrum cephalosporins, piperacillin/tazobactam and fluoroquinolones, over the course of several months of treatment with piperacillin/tazobactam and ciprofloxacin. Methods:Susceptibility testing and WGS were performed on three S. marcescens isolates from the patient. β-Lactamase activity in the presence or absence of induction by imipenem was measured by nitrocefin hydrolysis assays. Expression of ampC and blaSME-1 under the same conditions was determined by real-time PCR. Results:WGS demonstrated accumulation of missense and nonsense mutations in ampD associated with stable derepression of AmpC. Gene expression and β-lactamase activity of both AmpC and SME-1 were inducible in the initial susceptible isolate, but were constitutively high in the resistant isolate, in which total β-lactamase activity was increased by 128-fold. Conclusions:Although development of such in vitro resistance due to selective pressure imposed by antibiotics is reportedly low in S. marcescens, our findings highlight the need to evaluate isolates on a regular basis during long-term antibiotic therapy.

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.

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 μg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.

BACKGROUND:Antimicrobial resistance is an emerging global health issue. Data on the epidemiology of multidrug-resistant organisms are scarce for Africa, especially in HIV-infected individuals who often have frequent contact with healthcare. We investigated the prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in stool among HIV-infected children attending an HIV outpatient department in Harare, Zimbabwe. METHODS:We recruited children who were stable on antiretroviral therapy (ART) attending a HIV clinic from August 2014 to June 2015. Information was collected on antibiotic use and hospitalization. Stool was tested for ESBL-E through combination disc diffusion. API20E identification and antimicrobial susceptibility was performed on the positive samples followed by whole genome sequencing. RESULTS:Stool was collected from 175/202 (86.6 %) children. Median age was 11 [inter-quartile range (IQR) 9-12] years. Median time on ART was 4.6 years (IQR 2.4-6.4). ESBL-Es were found in 24/175 samples (13.7 %); 50 % of all ESBL-Es were resistant to amoxicillin-clavulanate, 100 % to co-trimoxazole, 45.8 % to chloramphenicol, 91.6 % to ceftriaxone, 20.8 % to gentamicin and 62.5 % to ciprofloxacin. ESBL-Es variously encoded CTX-M, OXA, TEM and SHV enzymes. The odds of ESBL-E carriage were 8.5 times (95 % CI 2.2-32.3) higher in those on ART for less than one year (versus longer) and 8.5 times (95 % CI 1.1-32.3) higher in those recently hospitalized for a chest infection. CONCLUSION:We found a 13.7 % prevalence of ESBL-E carriage in a population where ESBL-E carriage has not been described previously. Antimicrobial resistance (AMR) in Africa merits further study, particularly given the high HIV prevalence and limited diagnostic and therapeutic options available.

Based upon knowledge of the hydrolytic profile of major β-lactamases found in Gram-negative bacteria, we tested the efficacy of the combination of ceftazidime-avibactam (CAZ-AVI) with aztreonam (ATM) against carbapenem-resistant enteric bacteria possessing metallo-β-lactamases (MBLs). Disk diffusion and agar-based antimicrobial susceptibility testing were initially performed to determine the in vitro efficacy of a unique combination of CAZ-AVI and ATM against 21 representative Enterobacteriaceae isolates with a complex molecular background that included blaIMP, blaNDM, blaOXA-48, blaCTX-M, blaAmpC, and combinations thereof. Time-kill assays were conducted, and the in vivo efficacy of this combination was assessed in a murine neutropenic thigh infection model. By disk diffusion assay, all 21 isolates were resistant to CAZ-AVI alone, and 19/21 were resistant to ATM. The in vitro activity of CAZ-AVI in combination with ATM against diverse Enterobacteriaceae possessing MBLs was demonstrated in 17/21 isolates, where the zone of inhibition was ≥21 mm. All isolates demonstrated a reduction in CAZ-AVI agar dilution MICs with the addition of ATM. At 2 h, time-kill assays demonstrated a ≥4-log10-CFU decrease for all groups that had CAZ-AVI with ATM (8 μg/ml) added, compared to the group treated with CAZ-AVI alone. In the murine neutropenic thigh infection model, an almost 4-log10-CFU reduction was noted at 24 h for CAZ-AVI (32 mg/kg every 8 h [q8h]) plus ATM (32 mg/kg q8h) versus CAZ-AVI (32 mg/kg q8h) alone. The data presented herein require us to carefully consider this new therapeutic combination to treat infections caused by MBL-producing Enterobacteriaceae.

BackgroundWhole-genome sequencing (WGS) is an emerging and powerful technique by which to perform epidemiological studies in outbreak situations.MethodsWGS was used to identify and evaluate an outbreak of OXA-232-expressing carbapenem-resistant Klebsiella pneumoniae (CRKP) transmitted to 16 patients over the course of 40 weeks via endoscopic retrograde cholangiopancreatography procedures at a single institution. WGS was performed on 32 OXA-232 CRKP isolates (1-7 per patient) and single-nucleotide variants (SNVs) were analyzed, with reference to the index patient's isolate.ResultsInterhost genetic diversity of isolates was between 0 and 15 SNVs during the outbreak; molecular clock calculations estimated 12.31 substitutions per genome per year (95% credibility interval, 7.81-17.05). Both intra- and interpatient diversification at the plasmid and transposon level was observed, significantly impacting the antibiogram of outbreak isolates. The majority of isolates evaluated (n = 27) harbored a blaCTX-M-15 gene, but some (n = 5) lacked the transposon carrying this gene, which resulted in susceptibility to aztreonam and third- and fourth-generation cephalosporins. Similarly, an isolate from a colonized patient lacked the transposon carrying rmtF and aac(6')lb genes, resulting in susceptibility to aminoglycosides.ConclusionsThis study broadens the understanding of how bacteria diversify at the genomic level over the course of a defined outbreak and provides reference for future outbreak investigations.

AimExtended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs.Materials & methodsSamples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced.ResultsThe isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1, bla TEM-1B, bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage.ConclusionThis is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

AimExtended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs.Materials & methodsSamples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced.ResultsThe isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1, bla TEM-1B, bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage.ConclusionThis is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

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.

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.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., blaOXA-23, blaOXA-24/40, and blaOXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen.

BackgroundAntimicrobial resistance (AMR) is a growing and significant threat to public health on a global scale. Escherichia coli comprises Gram-negative, fecal-borne pathogenic and commensal bacteria that are frequently associated with antibiotic resistance. AMR E. coli can be ingested via food, water and direct contact with fecal contamination.MethodsWe estimated the prevalence of AMR Escherichia coli from select drinking water sources in northern Tanzania. Water samples (n = 155) were collected and plated onto Hi-Crome E. coli and MacConkey agar. Presumptive E. coli were confirmed by using a uidA PCR assay. Antibiotic susceptibility breakpoint assays were used to determine the resistance patterns of each isolate for 10 antibiotics. Isolates were also characterized by select PCR genotyping and macro-restriction digest assays.ResultsE. coli was isolated from 71 % of the water samples, and of the 1819 E. coli tested, 46.9 % were resistant to one or more antibiotics. Resistance to ampicillin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim was significantly higher (15-30 %) compared to other tested antibiotics (0-6 %; P

For 2013-2014, we prospectively identified US adults with flank pain, temperature >38.0°C, and a diagnosis of acute pyelonephritis, confirmed by culture. Cultures from 453 (86.9%) of 521 patients grew Escherichia coli. Among E. coli isolates from 272 patients with uncomplicated pyelonephritis and 181 with complicated pyelonephritis, prevalence of fluoroquinolone resistance across study sites was 6.3% (range by site 0.0%-23.1%) and 19.9% (0.0%-50.0%), respectively; prevalence of extended-spectrum β-lactamase (ESBL) production was 2.6% (0.0%-8.3%) and 12.2% (0.0%-17.2%), respectively. Ten (34.5%) of 29 patients with ESBL infection reported no exposure to antimicrobial drugs, healthcare, or travel. Of the 29 patients with ESBL infection and 53 with fluoroquinolone-resistant infection, 22 (75.9%) and 24 (45.3%), respectively, were initially treated with in vitro inactive antimicrobial drugs. Prevalence of fluoroquinolone resistance exceeds treatment guideline thresholds for alternative antimicrobial drug strategies, and community-acquired ESBL-producing E. coli infection has emerged in some US communities.

Whole genome sequencing (WGS) was compared to pulse-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA, as methods by which to evaluate a potential transmission of carbapenem-resistant Klebsiella pneumoniae between 2 hospital inpatients. PFGE result demonstrated only 1-band difference between the isolates, suggesting probable relatedness. In contrast, while WGS data demonstrated the same sequence type and very similar chromosomal sequences, over 20 single nucleotide variants were identified between the isolates, bringing into question whether there was a transmission event. WGS also identified an additional plasmid, with an XbaI restriction site in the isolates of the second patient that was not identified by PFGE. While WGS provided additional information that was not available by PFGE, in this study, neither method could definitively conclude the relatedness between the isolates.

Ceftazidime-avibactam is the first antimicrobial approved by the U.S. FDA for the treatment of carbapenem-resistant Enterobacteriaceae. Avibactam, a non-β-lactam β-lactamase inhibitor, inactivates class A serine carbapenemases, including Klebsiella pneumoniae carbapenemase (KPC). We report a KPC-producing K. pneumoniae isolate resistant to ceftazidime-avibactam (MIC, 32/4 μg/ml) from a patient with no prior treatment with ceftazidime-avibactam.

The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using bla OXA-48-like-specific primers coupled with an unlabeled 3'-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including bla KPC, bla SME, bla IMP, bla NDM-1, bla VIM, bla OXA-48, bla OXA-162, bla OXA-181, bla OXA-204, bla OXA-244, bla OXA-245, and bla OXA-232. Our assay identified the presence of bla OXA-48-like β-lactamase genes and clearly distinguished between bla OXA-48 and its variants in control strains, including between bla OXA-181 and bla OXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.

Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis--a complete reversion of mutational effects, particularly in protein evolution--yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in TEM-1 β-lactamase--an enzyme that endows antibiotics resistance. Separated by 10 Å, these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that R164S causes local disorder whereas G238S induces discrete conformations. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures.

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.

ObjectiveThe emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria is now a critical concern. The ESBL-producing Klebsiella pneumoniae constitutes one of the most common multidrug-resistant (MDR) groups of gram-negative bacteria involved in nosocomial infections worldwide. In this study we report on the molecular characterization through whole genome sequencing of an ESBL-producing K. pneumoniae strain, LAU-KP1, isolated from a stool sample from a patient admitted for a gastrointestinal procedure/surgery at the Lebanese Amrican University Medical Center-Rizk Hospital (LAUMCRH) in Lebanon.MethodsIllumina paired-end libraries were prepared and sequenced, which resulted in 4,220,969 high-quality reads. All sequence processing and assembly were performed using the A5 assembly pipeline.ResultsThe initial assembly produced 86 contigs, for which no scaffolding was obtained. The final collection of contigs was submitted to GenBank. The final draft genome sequence consists of a combined 5,632,663 bases with 57% G+C content. Automated annotation was performed using the RAST annotation server. Sequencing analysis revealed that the isolate harbored different β-lactamase genes, including bla oxa-1, bla CTX-M-15, bla SHV-11, and bla TEM-1b. The isolate was also characterized by the concomitant presence of other resistance determinants most notably acc(6')-lb-cr and qnrb1. The entire plasmid content was also investigated and revealed homology with four major plasmids pKPN-IT, pBS512_2, pRSF1010_SL1344, and pKPN3.ConclusionsThe potential role of K. pneumonia as a reservoir for ESBL genes and other resistance determinants is along with the presence of key factors that favor the spread of antimicrobial resistance a clear cause of concern and the problem that Carbapenem-non-susceptible ESBL isolates are posing in hospitals should be reconsidered through systematic exploration and molecular characterization.

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing.

Acinetobacter baumannii contains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system of Escherichia coli. We expressed adeABC and adeIJK of A. baumannii, as well as E. coli acrAB, in an E. coli host from which acrAB was deleted. The A. baumannii pumps were functional in E. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in the E. coli host.