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Abstract In this study delafloxacin resistance mechanisms in Escherichia coli strains were analyzed. Delafloxacin is a new fluoroquinolone, that is approved for clinical application however, resistance against this agent is scarcely reported. In our study 37 E. coli strains were included and antimicrobial susceptibility testing was performed for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, imipenem. Six delafloxacin resistant E. coli strains were selected for whole-genome sequencing and all of them exhibited resistance to other fluoroquinonlones and showed an extended-spectrum beta-lactamase phenotype. The six delafloxacin resistant E. coli strains belonged to different sequence types (STs) namely, ST131 (2 strains), ST57 (2 strains), ST162 and ST15840. Each delafloxacin resistant strain possessed multiple mutations in quinolone resistance-determining regions (QRDRs). Notably, three mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were in strains of ST162, ST57 and ST15840. However, the two strains of ST131 carried five combined mutations namely, gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, parE Ile549Leu. Association of delafloxacin resistance and production of CTX-M-15 in ST131, CMY-2 in ST162 and ST15840 was detected. In this study a new ST, ST15840 of clonal complex 69 was identified. Our results demonstrate, that at least three mutations in QRDRs are required for delafloxacin resistance in E. coli.

The in vitro susceptibility testing interpretive criteria (STIC) for piperacillin/tazobactam (TZP) against Enterobacterales were recently updated by the US Food and Drug Administration, Clinical and Laboratory Standards Institute, and European Committee on Antimicrobial Susceptibility Testing. The United States Committee on Antimicrobial Susceptibility Testing (USCAST) also recently reviewed TZP STIC for Enterobacterales and arrived at different STIC for Enterobacterales. Here, we explain our recommendations and rationale behind them. Based on our review of the available data, USCAST does not recommend TZP STIC for certain Enterobacterales species that have a moderate to high likelihood of clinically significant AmpC production (Enterobacter cloacae , Citrobacter freundii , and Klebsiella aerogenes only) or for third-generation cephalosporin-nonsusceptible Enterobacterales. USCAST recommends a TZP susceptibility breakpoint of ≤ 16/4 mg/L for third-generation cephalosporin-susceptible Enterobacterales and only endorses the use of extended infusion TZP regimens for patients with infections due to these pathogens. [ABSTRACT FROM AUTHOR]

Simple Summary: The role of methicillin-resistant Staphylococcus (S.) aureus (MRSA) and broad-spectrum cephalosporin-resistant Escherichia (E.) coli in cattle has not yet been widely investigated in Austria. This study aimed to understand the presence of these bacteria in certain regions of Lower Austria. A total of 190 milk samples from cows and 123 nasal swabs from cattle were examined for the presence of MRSA, as well as 99 bovine fecal swabs for E. coli. The samples were taken from 66 participating farms between May 2021 and September 2022 as part of the veterinary herd monitoring program of a veterinary practice in Lower Austria. MRSA was detected in a single nasal swab, with no MRSA found in the milk samples. A total of 22 E. coli isolates (22.2%) were detected and displayed an extended-spectrum β-lactamase (ESBL) phenotype. One E. coli isolate also harbored the AmpC gene. Finally, the isolates were analyzed for the following microbiological tests: DNA microarray, PCRs, and spa typing. The results conclusively showed that antibiotic resistance does play a role in cattle in (Lower) Austria. In the field of cattle medicine in Austria, to date, few studies have investigated the presence of methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing Escherichia coli in Austria. For this reason, milk and nasal samples were examined for the presence of methicillin-resistant Staphylococcus aureus as well as fecal samples for extended-spectrum cephalosporin-resistant Escherichia coli. The nasal and fecal swabs were collected during the veterinary treatment of calf pneumonia and calf diarrhea. For the milk samples, the first milk jets were milked into a pre-milking cup and then the teats were cleaned and disinfected before the samples were taken. The cows were selected during the veterinary visits to the farms when treatment was necessary due to mastitis. Depending on the severity of the mastitis (acute mastitis or subclinical mastitis), antibiotics and non-steroidal anti-inflammatory drugs were given immediately (acute disease) or after completion of the antibiogram (subclinical disease). Isolates were characterized by a polyphasic approach including susceptibility pheno- and genotyping and microarray-based assays. No methicillin-resistant Staphylococcus aureus was found in the milk samples, but one nasal swab was positive for methicillin-resistant Staphylococcus aureus. Twenty-two Escherichia coli isolates were detected among the fecal samples. All the Escherichia coli isolates were resistant to ceftazidime. In all the Escherichia coli isolates, genes from the blaCTX family were detected with other bla genes or alone; the most frequently observed β-lactamase gene was blaCTX-M-1/15 (n = 20). In total, 63.6% (n = 14) of the isolates exhibited a multidrug-resistant phenotype and one E. coli isolate (4.5%) harbored the AmpC gene. Precisely because the presence of data regarding extended-spectrum cephalosporin-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus in calves and cows in Austria is rare, this study further expands our understanding of antimicrobial resistance in Austrian cattle, which is highly relevant for successful antibiotic therapy in sick cattle. [ABSTRACT FROM AUTHOR]

Simple Summary: Antimicrobial resistance is a global health threat because microorganisms causing infections no longer respond to antimicrobial medicines, and this is particularly important in the case of resistance to last-resort antibiotics such as carbapenems. The role of dogs in the rise of antimicrobial resistance is under investigation. This study investigated the presence of Escherichia coli (E. coli) showing specific antimicrobial resistance patterns—defined as extended-spectrum β-lactamase (ESBL), AmpC and carbapenemase (CP) production—in dogs' feces from Northern Italy. Out of 100 samples, 14 (14%) dogs showed the presence of these resistant E. coli, with one (1%) bacterium showing resistance to carbapenems. Different combinations of resistance patterns were observed in these 14 E. coli. The majority (13/14, 92.9%) of the E. coli were also resistant to three or more different classes of antibiotics and were classified as multidrug-resistant. The presence of the resistant bacteria in feces tended to be associated with antibiotic treatment. The identification of bacteria known as ESBL-/AmpC-producing E. coli in domestic dogs, along with the presence of a bacterium resistant to carbapenems, although still limited, emphasizes the need for the adequate control of antimicrobial therapy and administration of antibiotics and surveillance programs for carbapenem-resistant bacteria in companion animals. MDR bacteria are an emerging global threat to public health, and the role of dogs in the rise of antimicrobial resistance is under investigation. This study investigated the fecal shedding of extended-spectrum β-lactamase (ESBL)-, AmpC- and carbapenemase (CP)-producing Escherichia coli and associated risk factors in dogs admitted to the Veterinary Teaching Hospital of Lodi, University of Milan, or other veterinary clinics and kennels in Northen Italy. Feces collected in 2020–2022 were microbiologically and molecularly analyzed. ESBL-/AmpC-/CP-producing E. coli was detected in 14/100 (14%) dogs. Eleven (11%), five (5%) and one (1%) dogs carried ESBL-, AmpC- and CP-producing E. coli phenotypes, respectively, supported by the PCR detection of blaCTX-M and/or blaTEM in ESBL-producing E. coli; blaCMY-2 and the presence of putative low-level AmpC production in AmpC-producing E. coli; and blaOXA-48 in CP-producing E. coli. Different combinations of resistance genes and genetic features were observed. Multidrug resistance was observed in 13/14 (92.9%) E. coli isolates. Binary logistic regression analysis showed that ESBL-/AmpC-/CP-producing E. coli fecal shedding tended to be associated with antibiotic treatment (p = 0.058; OR = 3.87). The detection of ESBL-/AmpC-producing E. coli, along with the presence of a carbapenemase-resistant E. coli isolate from domestic dogs, although still limited, emphasizes the need for antimicrobial stewardship and specific surveillance programs, particularly for CP-producing bacteria in companion animals. [ABSTRACT FROM AUTHOR]

Shigellosis, a diarrheal disease caused by Shigella species, is a significant public health concern, particularly in developing countries with inadequate sanitation systems. This study aimed to investigate the patterns of antibiotic resistance, ESBL and AmpC genes, integrons, and enterotoxin genes in Shigella species isolated from patients with gastroenteritis in Northeast Iran. This cross-sectional study was conducted between January 2017 and December 2019 at a tertiary care hospital in Northeast Iran. A total of 110 Shigella isolates were collected from stool samples of patients with gastroenteritis. The isolates were identified using conventional biochemical tests and confirmed by PCR. The highest resistance rates were detected for ampicillin (88.2%) and cotrimoxazole (84.5%). Altogether 64.5% of isolates exhibited multidrug resistance however, ESBL and AmpC phenotypes were detected in 34.54% and 1.81% of isolates, respectively. Interestingly, bla CTX-M-15 and bla TEM were detected in all ESBL-positive isolates but integron class 1, 2, and 3 were identified in 97.3%, 76.4%, and 59.1% of isolates, respectively. The sen gene was present in 72.7% of the isolates. In this study CTX-M-15 production was detected in 31 strains of Shigella sonnei and in 7 strains of Shigella flexneri. The high prevalence of multidrug-resistant Shigella isolates is concerning and shows the need for continuous monitoring and rational use of antibiotics. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the contamination levels of fresh products by ESBLs-producing Enterobacterales (ESBLs-E) or AmpC-producing Enterobacterales and characterize ESBLs genes. A total of 132 samples (67 vegetables and 65 fruits) were collected from markets in Tebessa, eastern Algeria. Among the samples, 16 third-generation cephalosporin-resistant Enterobacterales isolates were identified with a prevalence of 19.40% in vegetable samples, while there was no positive finding in fruit samples. Isolates showed resistance to most β-lactams, and all of them displayed multidrug resistance. Phenotypic tests for ESBLs detection, using double-disk synergy test and double-disk test were positive for 14 strains, including Klebsiella pneumoniae (n = 5), Klebsiella oxytoca (n = 4), Klebsiella terrigena (n = 2), Kluyvera spp. (n = 2), and Enterobacter cloacae (n = 1). Two AmpC-producing strains (Citrobacter freundii and E. cloacae) were identified through the AmpC disk test. Contamination rates of vegetables by ESBLs-E and AmpC-producing Enterobacterales were 19.40% and 2.98%, respectively. PCR results showed the presence of at least one ESBL gene in seven selected strains, with the dominance of blaCTX-M gene. Notably, K. pneumoniae strains showed the co-occurrence of two or three genes. Sequencing identified uncommon variants of ESBLs genes for the first time in Algeria, including blaCTX-M-79 (2/7), blaCTX-M-107 (2/7), blaCTX-M-117 (2/7), blaTEM-112 (1/7), blaTEM-125 (2/7), blaTEM-194 (1/7), and blaSHV-176 (3/7). [ABSTRACT FROM AUTHOR]

In this study delafloxacin resistance mechanisms in Escherichia coli strains were analyzed. Delafloxacin is a new fluoroquinolone, that is approved for clinical application however, resistance against this agent is scarcely reported. In our study 37 E. coli strains were included and antimicrobial susceptibility testing was performed for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, imipenem. Six delafloxacin resistant E. coli strains were selected for whole-genome sequencing and all of them exhibited resistance to other fluoroquinonlones and showed an extended-spectrum beta-lactamase phenotype. The six delafloxacin resistant E. coli strains belonged to different sequence types (STs) namely, ST131 (2 strains), ST57 (2 strains), ST162 and ST15840. Each delafloxacin resistant strain possessed multiple mutations in quinolone resistance-determining regions (QRDRs). Notably, three mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were in strains of ST162, ST57 and ST15840. However, the two strains of ST131 carried five combined mutations namely, gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, parE Ile549Leu. Association of delafloxacin resistance and production of CTX-M-15 in ST131, CMY-2 in ST162 and ST15840 was detected. In this study a new ST, ST15840 of clonal complex 69 was identified. Our results demonstrate, that at least three mutations in QRDRs are required for delafloxacin resistance in E. coli. [ABSTRACT FROM AUTHOR]

Background Klebsiella pneumoniae is capable of resistance to β-lactam antibiotics through expression of β-lactamases (both chromosomal and plasmid-encoded) and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. The purpose of this study was to determine the extent to which β-lactamases and outer membrane porins affected β-lactam resistance. Methods Minimum inhibitory concentrations (MICs) to β-lactams and inhibitor combinations were determined by agar dilution or Etest. Outer membrane porin production was evaluated by Western blot of outer membrane fractions. β-lactamase carriage was determined by whole genome sequencing and expression evaluated by real-time reverse-transcription polymerase chain reaction. Results Plasmid-encoded β--lactamases were important for cefotaxime and ceftazidime resistance. Elevated expression of chromosomal SHV was important for ceftolozane-tazobactam resistance. Loss of outer membrane porins was predictive of meropenem resistance. Extended-spectrum β-lactamases and plasmid-encoded AmpCs (pAmpCs) in addition to porin loss were sufficient to confer resistance to the third-generation cephalosporins, piperacillin-tazobactam, ceftolozane-tazobactam, and meropenem. pAmpCs (CMY-2 and DHA) alone conferred resistance to piperacillin-tazobactam. Conclusions Detection of a resistance gene by whole genome sequencing was not sufficient to predict resistance to all antibiotics tested. Some β-lactam resistance was dependent on the expression of both plasmid-encoded and chromosomal β-lactamases and loss of porins. [ABSTRACT FROM AUTHOR]

Antimicrobial-resistant Escherichia coli is a global health challenge from a One Health perspective. However, data on its emergence in the Caatinga biome are limited. This biome is exclusive to the Brazilian Northeast and offers unique epidemiological conditions that can influence the occurrence of infectious diseases and antimicrobial resistance. In this study, the carriage proportion, antimicrobial susceptibility, and population structure of cephalosporin-resistant E. coli were assessed in 300 cloacal swab samples of free-range chickens from three Brazilian states covered by the Caatinga biome. The results showed that 44 (14.7%) samples were positive for cephalosporin-resistant E. coli, and Paraíba state had the highest frequency of isolates (68.2%). Genes encoding cephotaximase-Munich or ampicillin class C (AmpC) enzymes were identified in 30 (68.2%) and 8 (18.2%) isolates, respectively, comprising 31 E. coli isolates. Overall, molecular typing by genome restriction using XbaI endonuclease followed by pulsed-field gel electrophoresis revealed four clusters from two properties of Paraíba state composed by extended-spectrum β-lactamase-producing and AmpC-producing E. coli carrying blaCTX−M−1−like and blaMIR−1/ACT−1 genes and belonging to different phylogenetic groups. There is a need to control antimicrobial resistance while taking into account the genetic diversity of the strains and their implications for animal and public health, especially in free-range chickens reared in the Brazilian Caatinga biome. [ABSTRACT FROM AUTHOR]

ABSTRACT Beta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to de novo β-lactam resistance in Escherichia coli. However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression of ampC amplification and ampC promoter mutations during the evolution of resistance induced by stepwise increasing amoxicillin concentrations. AmpC promoter mutations occurred by day 2, while the approximately eight-fold amplification occurred after more than 6 days of amoxicillin exposure. The combination of the amplification and the promoter mutations increased the ampC mRNA level by an average factor of 200 after 22 days. An IS1 insertion is identified in the amplification junction after resistance induction in the wild type (WT) and the ampC gene complementation strain (CompA), but not in ∆ampC, suggesting that the amplification depends on mobile genetic element transposition. In order to elucidate the correlation between gene mutations and ampC amplification, the DNA mutations acquired during resistance evolution by the WT, ∆ampC, and CompA were analyzed. Compared to evolved ∆ampC, several resistance-causing mutations are absent in evolved WT, while more mutations accumulated in stress response. The amoxicillin-resistant ∆ampC did not show amplification of the fragment around the original ampC position but exhibited a large duplication or triplication at another position, suggesting the essential role of the duplicated genes in resistance development.IMPORTANCEAmoxicillin is the most used antimicrobial against bacterial infections. DNA fragments containing ampC are amplified upon prolonged and stepwise increasing exposure to amoxicillin, causing resistance. These ampC-containing fragments have been identified in extended-spectrum beta-lactamase plasmids, which are considered the main cause of beta-lactam resistance. In this study, we document the time course of two important factors for ampC transcription enhancement, ampC amplification and ampC promoter mutations, during de novo amoxicillin resistance evolution. We propose that the transposon IS1 contributes to the amplification ampC region, that the sigma factor 70 regulates ampC overexpression, and that these combined form the backbone of a putative mechanism for ampC amplification.

Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC ß-lactamase. Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-β-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2(N=3), and blaNDM-1(N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones. [ABSTRACT FROM AUTHOR]

The increasing prevalence of antibiotic resistance, driven by the production of extended-spectrum beta-lactamases (ESBLs), presents a critical challenge to current medical treatments, particularly in clinical settings. Understanding the distribution and frequency of ESBL-producing bacteria is essential for developing effective control strategies. This study investigated the antibiotic resistance and extended-spectrum beta-lactamase (ESBL) production in bacterial isolates in clinical and non-clinical (food) specimens in Tabuk, KSA. A total of 57 bacterial isolates were analysed, with E. coli and Pseudomonas sp. being the most prevalent. High resistance rates were observed, particularly against third-generation cephalosporins in clinical isolates. ESBL screening revealed a significant prevalence in clinical samples (58.3%), with E. coli showing the highest positivity. Conversely, only a low percentage of food isolates were ESBL positive. Molecular analysis confirmed the presence of various ESBL genes, with blaCTX-M being the most frequent, predominantly found in clinical isolates. This study highlights the concerning levels of antibiotic resistance and ESBL production in the region, emphasising the need for effective infection control measures and prudent antibiotic use. [ABSTRACT FROM AUTHOR]

Distributed drive electric vehicles improve steering response and enhance overall vehicle stability by independently controlling each motor. This paper introduces a control framework based on Adaptive Model Predictive Control (AMPC) for coordinating handling stability, consisting of three layers: the dynamic supervision layer, online optimization layer, and low-level control layer. The dynamic supervision layer considers the yaw rate and maneuverability limits when establishing the β − β ˙ phase plane stability boundary and designs variable weight factors based on this stability boundary. The online optimization layer constructs the target weight-adaptive AMPC strategy, which can adjust the control weights for maneuverability and lateral stability in real time based on the variable weight factors provided by the dynamic supervision layer. The low-level control layer precisely allocates the driver's requested driving force and additional yaw moment by using torque distribution error and tire utilization as the cost function. Finally, experiments are conducted on a Simulink-CarSim co-simulation platform to assess the performance of AMPC. Simulation results show that, compared to the traditional MPC strategy, this control strategy not only enhances maneuverability under normal conditions but also improves lateral stability control under extreme conditions. [ABSTRACT FROM AUTHOR]

Introduction: Bloodstream infections caused by AmpC-producing Enterobacterales pose treatment challenges due to the risk of AmpC overproduction and treatment failure. Current guidelines recommend carbapenems or cefepime as optimal therapy. We aimed to evaluate empiric and definitive non-carbapenem regimens for these infections. Methods: In a retrospective study from June 2014 to March 2023, adult bacteremic patients with Enterobacter cloacae complex strains and Morganella morganii were evaluated. Demographic, clinical and lab data and outcomes were assessed. Results: The cohort comprised 120 bacteremic patients, 17 receiving empiric carbapenem and 103 non-carbapenem regimens. Both groups had similar Charlson and Norton scores and previous antimicrobial exposure. The most common sources of bacteremia were urinary, abdominal and central-line-associated sources. Empiric non-carbapenem regimens (primarily piperacillin–tazobactam and cephalosporins) were not associated with recurrent bacteremia or 30-day mortality. Definitive regimens included mainly carbapenems (n = 41) and ciprofloxacin (n = 46). Beta-lactams were administered to 25 patients. Recurrent bacteremia and 30-day mortality rates were similar among treatment groups. Ciprofloxacin showed comparable outcomes to carbapenems, however, severity of illness among these patients was lower. Conclusions: Empiric and definitive non-carbapenem regimens for bacteremia with AmpC-producing organisms were not associated with treatment failure or increased 30-day mortality. Ciprofloxacin appears promising for selected, stable patients, potentially enabling early discharge. [ABSTRACT FROM AUTHOR]

Objective: Identification of MBL, AmpC and ESBLs in colistin intrinsic and acquired resistant uropathogenic gram negative bacteria. Method: Urine samples were collected from Hayatabad Medical Complex, Peshawar during 17 January to 30 June 2019. Collected urine samples were aseptically transported microbiology lab of Health Research Institution (HRI), National Institute of Health (NIH), Khyber Medical College, Peshawar and streaked on different media. Positive growth was identified by API-10s. Antibiotic sensitivity profile was done by Modified Kirby Bauer disc diffusion method. Detection of metallo βlactamases (MBL) production by Imipenem EDTA synergy test, Double Disc Synergy Test (DDST) for detection of ESBLs and D-test for the detection of inducible AmpC beta lactamases test was used. Colistin resistance was identified via broth micro dilution according to CLSI manual. Colistin resistant bacteria was divided in two categories; acquired and intrinsic resistant bacteria according to CLSI manual. Results: Out of 2000 urine samples, 281(14%) gram-negative bacteria were isolated. Among positive samples, acquired colistin resistant bacteria were 241 and intrinsic resistant bacteria were 40 isolates. MBL was produce by twenty one (11.7%) E.coli and seventeen (40.5%) Pseudomonas aeruginosa. E.coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae, Serratia Oderifora and Proteus Marblis were ESBLs producing bacteria. AmpC production was prevalent in fourteen (7.8%) E. coli and twelve (28.6%) Pseudomonas aeruginosa. Fifty-five samples showed resistance to colistin out of 241 samples. In colistin resistant bacteria, two E.coli were MBL, ESBLs, while one E.coli was ESBLs, AmpC coproducing bacteria. The most prevalent extended drug resistant bacteria were Pseudomonas aeruginosa (28.6%) and Escherichia coli (6.1%), While 155(86.6%) Escherichia coli, 25 (59.5%) Pseudomonas aeruginosa and 22 (95.7%) Serratia Oderifora was multi drug resistant bacteria. Conclusion: Current study concluded that ESBL, MBL AmpC enzymes and their co-expression was observed with colistin resistance in E.coli and Pseudomonas aeruginosa. [ABSTRACT FROM AUTHOR]

ABSTRACT Plasmid-mediated AmpC β-lactamases are a cause of acquired cephalosporin resistance in Gram-negative bacteria. However, consensus regarding the optimal detection method is yet to be achieved and varies depending on local epidemiology and laboratory capacity. We determined the acquired genotypic resistance mechanisms of 250 Escherichia coli isolates with a positive AmpC screen, defined as cefoxitin MIC ≥ 8 mg/L and a positive AmpC double- disc diffusion test, using in-house designed high-resolution melt PCR, detecting plasmid-acquired genes from the CIT and DHA families. A proportion of these isolates (n = 170, 68%) underwent further genotypic characterization using whole- genome sequencing (WGS). Of 250 isolates with a positive screening test, 72 (28.8%) were determined to carry an acquired AmpC gene. There was 100% concordance between PCR and WGS in the identification of acquired AmpC genes. The phenotypic criteria were then assessed to determine their utility in predicting acquired AmpC gene carriage. Criteria 1 (cefoxitin MIC > 8 mg/L plus ceftriaxone MIC > 1 mg/L and/or ceftazidime MIC > 1 mg/L) yielded a sensitivity of 93.1% and a specificity of 47.8%. Criteria 2 (cefoxitin MIC > 16 mg/L plus ceftriaxone MIC > 4 mg/L) had a sensitivity of 33.3% and a specificity of 98.9%. DHA genes, whose expression may be induced following antibiotic exposure, were present in 19% of isolates testing susceptible to ceftriaxone (MIC ≤ 1 mg/L) and were significantly more likely than CIT genes to be detected in susceptible isolates (P < 0.0001). These findings highlight the importance of using genotypic methods to detect acquired AmpC resistance in E. coli isolates that meet phenotypic screening criteria.IMPORTANCEDetection of transmissible AmpC resistance remains a challenging problem for diagnostic laboratories, especially in Escherichia coli where the expression of its intrinsic AmpC gene can result in phenotypic resistance patterns indistinguishable from plasmid-mediated resistance. In conjunction with whole- genome sequencing (WGS), we describe the development and performance of a novel melt-curve PCR to identify the two most prevalent plasmid-mediated AmpC gene families: CIT and DHA. We then describe phenotypic testing algorithms that incorporate this PCR and can differentiate these from non-acquired resistance in E. coli. It is important to distinguish these, not only to spare patients from unnecessarily being treated with infection control precautions, but also to identify plasmid-mediated genes, especially of the DHA family, that have been associated with inducible drug resistance to third- generation cephalosporins.

ABSTRACT In the United States, carbapenem resistance in Pseudomonas aeruginosa is linked to the regulation of chromosomal resistance determinants, AmpC and OprD. The β-lactamase AmpC requires overexpression and genetic modifications to be capable of inhibiting imipenem activity. The outer membrane porin OprD can be downregulated or undergo genetic modifications that strongly correlate with imipenem non-susceptibility. Co-administration of imipenem and the β-lactamase inhibitor, relebactam, can lower imipenem minimal inhibitory concentrations and restore susceptibility. However, it is not understood how this occurs in P. aeruginosa isolates that do not overproduce AmpC or produce a functional OprD for imipenem entry. Therefore, we investigated whether imipenem could enter P. aeruginosa in the absence of OprD and whether any of the chromosomal β-lactamases (AmpC, OXA-50, and PIB-1) contributed to imipenem and/or imipenem/relebactam non-susceptibility. This investigation evaluated 17 imipenem non-susceptible clinical isolates and three laboratory strains of PAO1, two of which were porin transposon mutants for either oprD or opdP. Expression of OXA-50 and PIB-1 RNA was similar to PAO1. However, all 20 isolates exhibited blaampC induction under sublethal exposure to imipenem. This occurred in the absence of detectable OprD protein in 18 isolates. Collectively, our data identify that (i) OprD was not the only channel required for imipenem entry and (ii) imipenem susceptibility can be restored by imipenem/relebactam due to the interaction between relebactam and blaampC overexpression due to imipenem induction.IMPORTANCEInfections caused by Pseudomonas aeruginosa are associated with high mortality and worsened clinical outcomes. The carbapenem, imipenem, has been combined with the β-lactamase inhibitor relebactam to treat carbapenem-resistant P. aeruginosa. Downregulation of the outer membrane porin, OprD is the major mechanism of imipenem resistance; however, relebactam inhibits the chromosomally encoded AmpC β-lactamase. We investigated how relebactam was able to reduce P. aeruginosa imipenem minimal inhibitory concentrations (MICs) in isolates in which OprD was downregulated. Our data show that imipenem is capable of entering the cell in the absence of OprD and capable of inducing the AmpC β-lactamase. The induction of AmpC provides a substrate for relebactam, impacting the imipenem MIC. The data presented support the use of an alternative porin(s) for entry of imipenem. This study provides the basis for further investigation into modifications of imipenem or similar molecules that would increase the affinity for other porins in isolates resistant to imipenem.

Abstract Background The problem of resistance to beta-lactam antibiotics, which is caused by ESBL and AmpC β-lactamases, is getting worse globally. Infections caused by bacterial isolates harboring these enzymes are difficult to treat with carbapenems being the sole effective treatment option for such infections. The objective of this study was to determine the frequency of ESBLs and AmpC-producing Gram-negative bacilli isolated from clinical specimens and to evaluate the sensitivity of cefepime-tazobactam combination against them. Methods This is an observational cross-sectional study carried out on 100 Gram-negative bacilli at Theodor Bilharz Research Institute Hospital during the period from February 2015 to January 2016. ESBL production was screened by using the disc diffusion test followed by confirmation by the combined disc confirmatory test, the screening for AmpC production was conducted using the cefoxitin disc test, which was subsequently confirmed by the AmpC disc test. Isolates confirmed positive for ESBL and/ or AmpC production were investigated for their susceptibility to antibiotics. Results Among 100 Gram-negative bacilli, 44 isolates were confirmed as ESBL producers by the combined disc confirmatory test out of 56 isolates that tested positive for ESBL production through the disc diffusion test. The presence of AmpC production was assessed using the cefoxitin disc test, 32 isolates were screened to be AmpC producers, and the AmpC disc test confirmed AmpC production in 9 isolates of them. Using the Mast® D68C set, 32 isolates were ESBL producers, 3 were AmpC producers, and 4 isolates were ESBL/AmpC co-producers. The highest sensitivity was to cefepime-tazobactam (91.48%) followed by the carbapenems. Conclusion Cefepime-tazobactam showed remarkable activity against ESBL and/or AmpC-producing Gram-negative bacilli and may be considered as a therapeutic alternative to carbapenems.

Abstract Objectives In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. Results In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to bla CTX−M−15 gene (66.1%) followed by bla CTX−M (45.8%), bla CTX−M−14 (30.5%), bla SHV (28.8%), and bla TEM (13.6%). The frequency of bla DHA, bla EBC, bla MOX and bla CIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Purpose: The MAST® D72C test is a phenotypical test which can detect ESBL and AmpC production in Enterobacterales. It can also identify the suspected presence of carbapenemase. The aim of the present study was to assess the sensitivity and specificity of this test and to discuss its usefulness in laboratories, especially those that use only an automated AST system. Method: The performance of the MAST® D72C test was assessed against a collection of 119 non-redundant Enterobacterales isolates characterized for their content in β-lactamases, and compared with that of the reference double disk synergy test. β-lactamase content was established from phenotypic and genotypic analyses to collect a broad diversity of resistance mechanisms and bacterial strains, including 30 ESBL-producing strains, 32 strains overproducing chromosomal AmpC, 10 strains producing plasmid-encoded AmpC, 12 carbapenemase-producing strains, 13 strains combining the production of several β-lactamases, and 22 strains that produced other β-lactamases. Results: The sensitivity and specificity for ESBL-detection were comparable with those of the synergy test, 75 versus 72.5%, and 94.9 versus 93.7%, respectively. The sensitivity and specificity for AmpC-detection were 71.7% and 100%, respectively, and sensitivity reached 78.7% if we excluded carbapenem-resistant isolates. Carbapenemase-detection sensitivity was 90%. Conclusion: These results show that the MAST® D72C test can be a useful tool for the detection of ESBL- and AmpC-production in clinical laboratories. [ABSTRACT FROM AUTHOR]

Background: Currently, the Enterobacteriaceae species are responsible for a variety of serious infections and are already considered a global public health problem, especially in underdeveloped countries, where surveillance and monitoring programs are still scarce and limited. Analyses were performed on the complete genome of an extensively antibiotic-resistant strain of Enterobater hormaechei, which was isolated from a patient with non-Hodgkin's lymphoma, who had been admitted to a hospital in the city of Manaus, Brazil. Methods: Phenotypical identification and susceptibility tests were performed in automated equipment. Total DNA extraction was performed using the PureLink genomic DNA mini-Kit. The genomic DNA library was prepared with Illumina Microbial Amplicon Prep and sequenced in the MiSeq Illumina Platform. The assembly of the whole-genome and individual analyses of specific resistance genes extracted were carried out using online tools and the Geneious Prime software. Results: The analyses identified an extensively resistant ST90 clone of E. hormaechei carrying different genes, including blaCTX-M-15, blaGES-2, blaTEM-1A, blaACT-15, blaOXA-1 and blaNDM-1, [aac(3)-IIa, aac(6′)-Ian, ant(2″)-Ia], [aac(6′)-Ib-cr, (qnrB1)], dfrA25, sul1 and sul2, catB3, fosA, and qnrB, in addition to resistance to chlorhexidine, which is widely used in patient antisepsis. Conclusions: These findings highlight the need for actions to control and monitor these pathogens in the hospital environment. [ABSTRACT FROM AUTHOR]

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Background: The problem of resistance to beta-lactam antibiotics, which is caused by ESBL and AmpC β-lactamases, is getting worse globally. Infections caused by bacterial isolates harboring these enzymes are difficult to treat with carbapenems being the sole effective treatment option for such infections. The objective of this study was to determine the frequency of ESBLs and AmpC-producing Gram-negative bacilli isolated from clinical specimens and to evaluate the sensitivity of cefepime-tazobactam combination against them. Methods: This is an observational cross-sectional study carried out on 100 Gram-negative bacilli at Theodor Bilharz Research Institute Hospital during the period from February 2015 to January 2016. ESBL production was screened by using the disc diffusion test followed by confirmation by the combined disc confirmatory test, the screening for AmpC production was conducted using the cefoxitin disc test, which was subsequently confirmed by the AmpC disc test. Isolates confirmed positive for ESBL and/ or AmpC production were investigated for their susceptibility to antibiotics. Results: Among 100 Gram-negative bacilli, 44 isolates were confirmed as ESBL producers by the combined disc confirmatory test out of 56 isolates that tested positive for ESBL production through the disc diffusion test. The presence of AmpC production was assessed using the cefoxitin disc test, 32 isolates were screened to be AmpC producers, and the AmpC disc test confirmed AmpC production in 9 isolates of them. Using the Mast® D68C set, 32 isolates were ESBL producers, 3 were AmpC producers, and 4 isolates were ESBL/AmpC co-producers. The highest sensitivity was to cefepime-tazobactam (91.48%) followed by the carbapenems. Conclusion: Cefepime-tazobactam showed remarkable activity against ESBL and/or AmpC-producing Gram-negative bacilli and may be considered as a therapeutic alternative to carbapenems. [ABSTRACT FROM AUTHOR]

Objectives In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. Results In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n=50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX−M−15 gene (66.1%) followed by blaCTX−M (45.8%), blaCTX−M−14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates [ABSTRACT FROM AUTHOR]

Citrobacter koseri (formerly classified as Citrobacter diversus) is a gram-negative bacillus (GNB) that occurs as an opportunistic pathogen in neonates and immunocompromised patients. Citrobacter species have been implicated in nosocomial settings leading to infections involving the urinary tract, respiratory tract, liver, biliary tract, meninges, and even in rarer conditions—blood stream infection and infective endocarditis (IE). Gram-negative bacilli are responsible for 3% to 4% of all IE cases and have been traditionally associated with intravenous drug users. Patients with non-HACEK (species other than Haemophilus species, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, or Kinglella species) GNB IE have poor clinical outcomes with higher rates of in-hospital mortality and complications. The American Heart Association (AHA) and Infectious Diseases Society of America (IDSA) both recommend the use of combination antibiotic therapy with a beta-lactam (penicillins, cephalosporins, or carbapenems) and either an aminoglycoside or fluoroquinolones for 6 weeks (about 1 and a half months) to treat IE due to non-HACEK GNB. Citrobacter koseri is becoming more recognized due to its inherent resistance to ampicillin and emerging drug resistance to beta lactams and aminoglycosides requiring carbapenem therapy. Our case is of a 75-year-old male with no previously reported history of primary or secondary immunodeficiency disorders who developed C koseri blood stream infection. His infectious work-up revealed mitral valve IE and septic cerebral emboli resulting in ischemic infarcts. This case illustrates the importance of recognizing GNB organisms as rising human pathogens in IE cases even without active injection drug use or nosocomial exposure. [ABSTRACT FROM AUTHOR]

ABSTRACT The impact of chromosomally encoded wild-type or extended-spectrum (ESAC) AmpC β-lactamases of Escherichia coli on susceptibility to ceftazidime, cefepime, and cefiderocol was evaluated in different genetic backgrounds, including wild-type, PBP3-modified, and porin-deficient E. coli strains. Recombinant E. coli strains possessing the different backgrounds and producing variable ESACs were evaluated. Although ESAC enzymes conferred resistance to ceftazidime and decreased susceptibility to cefepime as expected, we showed here that cefiderocol was also a substrate of ESAC enzymes.IMPORTANCEWe showed here that chromosomally encoded intrinsic extended-spectrum cephalosporinases of Escherichia coli may impact susceptibility not only to ceftazidime and cefepime but also to cefiderocol.

ABSTRACT: Objectives: OCH class C β-lactamases have been reported in several species belonging to the Brucella genus that were formerly known as Ochrobactrum. Moreover, only one complete genome of Brucella pseudintermedia has been published. In this work, we describe the genome of a B. pseudintermedia strain possessing a new blaOCH gene that was isolated from Zophobas morio larvae. Methods: Hybrid whole-genome sequencing analysis (Illumina and Nanopore) was used to identify and characterise the strain (Ops-OCH-23). Phylogenetic analyses based on the 16S rRNA gene sequence and a core-genome alignment were performed to study the relationships among Ops-OCH-23 and deposited genomes. Moreover, all deposited blaOCH genes were compared to the one found in Ops-OCH-23. Results: Ops-OCH-23 showed a susceptibility profile consistent with the production of AmpC β-lactamase(s). Its genome consisted of two chromosomes, of which one carried the blaOCH gene. Such gene encoded a new class C OCH β-lactamase among the fifteen so far reported. Two plasmids (120-Kb and 59-Kb) without any associated antimicrobial resistance genes were also found. Analysis of 16S rRNA revealed that Ops-OCH-23 shared 100% homology with four deposited B. pseudintermedia strains. Moreover, the core-genome analysis indicated that the closest match (279 ΔSNVs) to Ops-OCH-23 was strain CTOTU49018 isolated from an urban environment in Germany in 2013. Conclusion: We described the second complete genome of a B. pseudintermedia that also encoded a new OCH β-lactamase variant. Overall, this report expands our knowledge regarding this rarely isolated Brucella species that have been reported so far only a few times in human sources.

Objectives: This study aimed to evaluate the clinical and microbiological risk factors associated with mortality in patients treated with ceftazidime–avibactam for carbapenem-resistant Gram-negative bacterial infections. Methods: This multicentric prospective cohort study included hospitalized adult patients with a microbiologically confirmed infection treated with ceftazidime–avibactam for ≥48 hours. The clinical and microbiological risk factors for 30-day mortality were evaluated using a Cox regression model. Results: Of the 193 patients evaluated from the five tertiary hospitals, 127 were included in the study. Thirty-five patients (27.6%) died within 30 days. Infections with AmpC beta-lactamase-carrying bacteria were independently related to 30-day mortality (adjusted hazard ratio [aHR] 2.49, 95% confidence interval [CI] 1.28–4.84, P < 0.01) after adjusting for time from infection to antimicrobial prescription (P = 0.04). Further, these bacterial infections were also related to higher in-hospital mortality (aHR 2.17, 95% CI 1.24–3.78, P < 0.01). Only one patient developed resistance to ceftazidime–avibactam during treatment. Conclusions: Treatment with ceftazidime–avibactam had worse clinical outcomes in patients with infections with bacteria with chromosomally encoded AmpC beta-lactamase. However, these findings should be confirmed in future studies.

Introduction: Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC-BL) in Enterobacteriaceae are a global threat. Coproduction of AmpC-BL and ESBL reduces therapeutic options, with more resistance against cephamycin and beta-lactamase inhibitor combinations. This study determines the proportion, incidence, and distribution of AmpC-BL-resistant genes in clinically isolated ESBL-producing Escherichia coli, completing its scarce data in Indonesia. Methods: The samples in this study were ESBL-producing E. coli from blood and urine specimens, confirmed by BD Phoenix semiautomatic examination combined with cefoxitin disk screening method. Confirmation tests of AmpC-BL used the AmpC-disk method and the polymerase chain reaction method. Results: Thirteen (27.8%) of 108 E. coli isolates were nonsusceptible to cefoxitin, and 3 (11.5%) isolates were confirmed to produce AmpC-BL and contained AmpC-BL CITM gene. Of the 3 AmpC-BL and ESBL coproduction isolates, one isolate was a copresentation of ESBL and AmpC-BL genes, namely blaTEM and CITM. Fisher’s exact test showed that the coproduction of AmpC-BL in ESBL-producing isolates was associated with reduced susceptibility to cefoxitin (P = 0.020) and amoxicillin-clavulanate (P = 0.048) compared to isolates producing ESBL alone. The effect of ESBL and AmpC-BL coproduction on reducing susceptibility to carbapenems needs further investigation. Conclusions: AmpC coproduction was found in 3 (2.8%) of 108 ESBL-producing E. coli isolates, and one isolate copresented AmpC-BL and ESBL coding genes blaTEM and CITM. These three isolates were associated with widened antibiotic resistance to cefoxitin and amoxicillin--clavulanate compared to isolates producing ESBL alone.

Vegetables may serve as a reservoir for antibiotic resistant bacteria and resistance genes. AmpC β-lactamases and extended spectrum beta-lactamases (ESBL) inactivate commonly used β-lactam antibiotics, including penicillins and cephalosporins. In this study, we determined the prevalence of AmpC and ESBL-producing Enterobacterales in retail vegetables in the United States. A total of 88 vegetable samples were collected for the screening of AmpC and ESBL-producing Enterobacterales using CHROMagar ESBL agar. These vegetables included washed ready-to-eat salad (23), microgreens/sprouts (13), lettuce (11), herbs (11), spinach (5), mushrooms (5), brussels sprouts (4), kale (3), and other vegetable samples (13). AmpC and ESBL activity in these isolates were determined using double disk combination tests. Two vegetable samples (2.27%), organic basil and brussels sprouts, were positive for AmpC-producing Enterobacterales and eight samples (9.09%), including bean sprouts, organic parsley, organic baby spinach, and several mixed salads, were positive for ESBL-producing Enterobacterales. Whole genome sequencing was used to identify the bacterial species and resistance genes in these isolates. Genes encoding AmpC β-lactamases were found in Enterobacter hormaechei strains S43-1 and 74-2, which were consistent with AmpC production phenotypes. Multidrug-resistant E. hormaechei strains S11-1, S17-1, and S45-4 possess an ESBL gene, blaSHV66 , whereas five Serratia fonticola isolates contain genes encoding a minor ESBL, FONA-5. In addition, we used shotgun metagenomic sequencing approach to examine the microbiome and resistome profiles of three spinach samples. We found that Pseudomonas was the most prevalent bacteria genus in the spinach samples. Within the Enterobacteriaceae family, Enterobacter was the most abundant genus in the spinach samples. Moreover, antibiotic resistance genes encoding 12 major classes of antibiotics, including β-lactam antibiotics, aminoglycoside, macrolide, fluoroquinolone, and others, were found in these spinach samples. Therefore, vegetables can serve as an important vehicle for transmitting antibiotic resistance. The study highlights the need for antibiotic resistance surveillance in vegetable products.

Abstract Objective To evaluate effect of inoculum size of extended-spectrum β-Lactamase (ESBL)-producing-, AmpC-producing-, and KPC-producing Escherichia coli and Klebsiella pneumoniae on the in vitro antibacterial effects of imipenem/relebactam (IMR) and ceftazidime/avibactam (CZA). Methods We compared the impact of inoculum size on IMR and CZA of sixteen clinical isolates and three standard isolates through antimicrobial susceptibility tests, time-kill assays and in vitro PK/PD studies. Results When inoculum size increased from 105 to 107 CFU/mL, an inoculum effect was observed for 26.3% (5/19) and 52.6% (10/19) of IMR and CZA, respectively; time-kill assays revealed that the concentration of CZA increased from ≥ 4 × MIC to 16 × MIC to reach 99.9% killing rate against K. pneumoniae ATCC-BAA 1705 (KPC-2-, OXA-9- and SHV-182-producing) and 60,700 (SHV-27- and DHA-1-producing). While for IMR, a concentration from 1 × MIC to 4 × MIC killed 99.9% of the four strains. When the inoculum size increased to 109 CFU/mL, neither IMR nor CZA showed a detectable antibacterial effect, even at a high concentration. An in vitro PK/PD study revealed a clear bactericidal effect when IMR administered as 1.25 g q6h when inoculum size increased. Conclusion An inoculum effect on CZA was observed more frequent than that on IMR. Among the β-lactamase-producing strains, the inoculum effect was most common for SHV-producing and KPC-producing strains.

The extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is becoming a global public health concern. More comprehensive surveillance of β-lactam resistance in E. coli would improve monitoring strategies and control resistance transmission in contaminated environments. This study investigated the prevalence of β-lactamase genes in E. coli isolated from the Seven Crater Lakes in San Pablo, Laguna, Philippines. Water samples from lakes were collected for the isolation of E. coli (n = 846) and molecular characterization by detecting the presence of the uidA gene. The isolates were then tested for the presence of β-lactamase genes using PCR. Among the screened genes, blaAmpC was the most dominant (91%). Other β-lactamase genes such as blaTEM, blaSHV, and blaCTXM were also detected with percentage occurrence of 34, 5, and 1%, respectively. Multiple genes within individual isolates were also observed, wherein blaTEM/AmpC was the most prevalent gene combination. Moreover, a significant negative correlation between blaAmpC with blaSHV and blaCTXM was depicted in this study. Overall, these findings demonstrate the presence of β-lactamase genes in E. coli in the Seven Crater Lakes of San Pablo and can be used in developing effective strategies to control antibiotic resistance in environmental waters. HIGHLIGHTS blaAmpC was the predominant gene detected among the five screened β-lactamase genes.; A significant association was detected between blaAmpC with blaSHV and blaCTXM.; blaKPC was not detected in any E. coli isolates from the lakes.; Seasonal variations have an effect on the fecal coliform counts and occurrence of β-lactamase-resistant E. coli isolates.;

Background: Antibiotic resistance in Klebsiella pneumoniae isolates, particularly resistance to colistin, has become a growing concern. This study seeks to investigate the upregulation of specific genes (pmrA, pmrB, pmrC, phoQ, phoP, and arnT) that contribute to colistin resistance in K. pneumoniae isolates collected from human clinical samples in Tehran, Iran. Methods: Thirty eight K. pneumoniae isolates were obtained and subjected to antibiotic susceptibility testing, as well as evaluation for phenotypic AmpC and ESBL production according to CLSI guidelines. The investigation of antibiotic resistance genes was conducted using polymerase chain reaction (PCR), whereas the quantification of colistin resistance related genes expressions was performed via Real-Time PCR. Results: The highest and lowest antibiotics resistance were observed for cefotaxime 33 (86.8%) and minocycline 8 (21.1%), respectively. Twenty-four (63.2%) and 31 (81.6%) isolates carried AmpC and ESBLs, respectively. Also, antibiotic resistance genes containing blaNDM, blaIMP, blaVIM, blaSHV, blaTEM, blaCTXM, qnrA, qnrB, qnrS, and aac(6')-Ib were detected in K. pneumoniae isolates. Only 5 (13.1%) isolates were resistant to colistin and the MIC range of these isolates was between 4 and 64 μg ml−1. Upregulation of the pmrA, pmrB, pmrC, phoQ, phoP, and arnT genes was observed in colistin-resistant isolates. The colistin-resistant isolates were found to possess a simultaneous presence of ESBLs, AmpC, fluoroquinolone, aminoglycoside, and carbapenem resistant genes. Conclusions: This study reveals escalating antibiotic resistance in K. pneumoniae, with notable coexistence of various resistance traits, emphasizing the need for vigilant surveillance and innovative interventions.

Escherichia coli (E. coli) plays an important ecological role, and is a useful bioindicator to recognize the evolution of resistance in human, animal and environment. Recently, extended-spectrum β-lactamases (ESBL) producing E.coli has posed a threat to public health. Generally, captive healthy giant pandas are not exposed to antibiotics; however, they still acquire antimicrobial resistant bacteria. In order to understand whether there is an exchange of resistance genes within the ecosystems of captive giant pandas, this study explored resistance characteristics of 330 commensal E. coli isolates from feces of giant pandas, the surroundings, and breeders. Isolates from different sources showed similar resistance phenotype, and ESBL/AmpC-producing isolates showed more profound resistance to antibiotics than non-ESBL/AmpC-producing isolates (P

ABSTRACTSerratia marcescens is an opportunistic pathogen historically associated with sudden outbreaks in intensive care units (ICUs) and the spread of carbapenem-resistant genes. However, the ecology of S. marcescens populations in the hospital ecosystem remains largely unknown. We combined epidemiological information of 1,432 Serratia spp. isolates collected from sinks of a large ICU that underwent demographic and operational changes (2019–2021) and 99 non-redundant outbreak/non-outbreak isolates from the same hospital (2003–2019) with 165 genomic data. These genomes were grouped into clades (1–4) and subclades (A and B) associated with distinct species: Serratia nematodiphila (1A), S. marcescens (1B), Serratia bockelmannii (2A), Serratia ureilytica (2B), S. marcescens/Serratia nevei (3), and S. nevei (4A and 4B). They may be classified into an S. marcescens complex (SMC) due to the similarity between/within subclades (average nucleotide identity >95%–98%), with clades 3 and 4 predominating in our study and publicly available databases. Chromosomal AmpC β-lactamase with unusual basal-like expression and prodigiosin-lacking species contrasted classical features of Serratia. We found persistent and coexisting clones in sinks of subclades 4A (ST92 and ST490) and 4B (ST424), clonally related to outbreak isolates carrying blaVIM-1 or blaOXA-48 on prevalent IncL/pB77-CPsm plasmids from our hospital since 2017. The distribution of SMC populations in ICU sinks and patients reflects how Serratia species acquire, maintain, and enable plasmid evolution in both “source” (permanent, sinks) and “sink” (transient, patients) hospital patches. The results contribute to understanding how water sinks serve as reservoirs of Enterobacterales clones and plasmids that enable the persistence of carbapenemase genes in healthcare settings, potentially leading to outbreaks and/or hospital-acquired infections.IMPORTANCEThe “hospital environment,” including sinks and surfaces, is increasingly recognized as a reservoir for bacterial species, clones, and plasmids of high epidemiological concern. Available studies on Serratia epidemiology have focused mainly on outbreaks of multidrug-resistant species, overlooking local longitudinal analyses necessary for understanding the dynamics of opportunistic pathogens and antibiotic-resistant genes within the hospital setting. This long-term genomic comparative analysis of Serratia isolated from the ICU environment with isolates causing nosocomial infections and/or outbreaks within the same hospital revealed the coexistence and persistence of Serratia populations in water reservoirs. Moreover, predominant sink strains may acquire highly conserved and widely distributed plasmids carrying carbapenemase genes, such as the prevalent IncL-pB77-CPsm (pOXA48), persisting in ICU sinks for years. The work highlights the relevance of ICU environmental reservoirs in the endemicity of certain opportunistic pathogens and resistance mechanisms mainly confined to hospitals.

This study investigated the antimicrobial activity of the green synthesis copper oxide nanoparticles (CuO-NPs) from Eucalyptus globulus leaf extract against S. sonnei clinical isolates and the reference strain S. sonnei ATCC 9290 alone and in synergy with antibiotics. Biosynthesized NPs analyzed for shape, size, and crystal nature. The antimicrobial genotype and phenotype characteristic for selecting the most resistant isolates from 30 S. sonnei clinical isolates performed through the distribution of ipaH, sigA, and virF, CTX-M-1, CIT group, aac(6')-Ib-cr genes by polymerase chain reaction (PCR) technology and Kirby-Bauer susceptibility test. The minimum inhibitory concentration (MIC) of CuO-NPs against S. sonnei was determined within 0–360 min treatment time. The double-disc method was performed for semi-sensitive and antibiotic-resistant strains to evaluate the probable inhibitory effect in synergy form. XRD, TEM, and FTIR analysis confirmed the successful synthesis of NPs with appropriate purity, shape, and size. Only one of the S. sonnei isolates was positive for all MDR-related genes (blaCTX-M-1 and blaCIT, aac(6')-Ib-cr) and showed the highest susceptibility reaction against CuO-NPs, Z = 312.5 mL.µg−1 in comparison to Z = 256.25 mL.µg−1 for the standard strain, and the process continued by performing the optimal ratio of NPs on semi-sensitive and also resistance antibiotic in synergy with NPs for the bacteria isolated. Furthermore, the synergy effect of CuO-NPs with oxacillin, chloramphenicol, imipenem, and levofloxacin was higher than using antibiotics alone. CuO-NPs, enhance the properties and characteristics of antibacterial potency in synergy or developed synthetic functionalized NPs with antibiotics. [ABSTRACT FROM AUTHOR]

Escherichia coli isolates that are resistant to cefixime and amoxicillin/clavulanic acid, but apparently susceptible to cefuroxime, with no ESBL identified, were initially detected in Madrid from urine samples in 2019. Throughout 2020 and 2021, all cases of community UTI by E. coli from six health areas in Madrid were studied. A representative sample of 23 cases was selected for further studies. The broth microdilution method and the agar diffusion method were performed to determine the antibiotic susceptibility. WGS was carried out for phylogeny, resistome and virulome analysis. Community consumption of third-generation oral cephalosporins in Madrid (2017–2021) was analyzed. A total of 582 (1.3%) E. coli isolates had the mentioned resistance profile. The mutation at position –32 (T > A) of the AmpC promoter was found in 21 isolates. No plasmid AmpC- or ESBL-encoding genes were detected. A cluster of 20 ST12 isolates was detected by cgMLST. A 6.2% increase in the consumption of third-generation oral cephalosporins, especially cefixime, was observed in Madrid. Chromosomal AmpC-hyperproducing ST12 E. coli isolates could be implicated in the increase in community UTI cases by cefixime-resistant isolates, which correlates with an increasing trend of cefixime consumption. [ABSTRACT FROM AUTHOR]

BACKGROUND: Urinary tract infection (UTI) remains the common infection diagnosed in outpatients as well as in hospitalized patients. Current knowledge on antimicrobial susceptibility pattern of uropathogens is mandatory for appropriate therapy. ESBL, Amp-C and MBL were clinically important cephalosporinases encoded on the chromosomes of many Enterobacteriaceae and a few other organisms, which mediates resistance to broad spectrum of antibiotics. Detection of these enzymes can be difficult because their presence does not always produce a resistant phenotype on conventional disc diffusion or automated susceptibility testing methods. These enzymes are often associated with potentially fatal outcome to the individual because of false susceptibility to ß-lactams phenotypically in lab reports. Thus, their accurate detection and characterization is important from epidemiological, clinical, laboratory, and infection control point of view. The present study was designed to detect ESBL, Amp-C and MBL from Gram negative bacilli in the urine samples received in Microbiology Department, Govt. Medical College, Thrissur. AIM: To detect the ESBL, Amp-C and MBL production from Gram Negative Bacilli isolated in the urine samples. METHOD: A total number of 390 non repetitive urinary isolates of Gram Negative bacilli were obtained over a period of one year, Isolates resistant to ceftriaxone were tested for ESBL, AmpC, MBL production by double disc synergy test method, Combined disc diffusion method using Cefoxitin (30 mg) dics and confirmed by inhibitor based test using boronic acid as inhibitor and Combined disc diffusion using Imipenem disc and confirmed using EDTA as inhibitor. RESULTS: Among these 390 samples 290 (61.7%) were multidrug resistant (MDR) urinary isolates, with predominant of E.coli-298(76.3%) & Klebsiella spp-92 (23.7%). 290 (74.3%) were multi drug resistant, among these 290 resistant strains, 240 (61.5%) isolates were positive for ESBL production, remaining 50 isolates (12.8%) were negative for ESBL production. Out of 240 ESBL producers, 194 were E.coli (80.8%) & 46 were Klebsiella spp (19.2%). Among the 240 resistant strains, 50 isolates were resistant to Cefoxitin, remaining 190 isolates were sensitive to Cefoxitin. Out of these 50 cefoxitin resistant strain, only 43 were Amp-C producers. Only 7 (1.8%) isolates were MBL producers. CONCLUSION: In the present study a large number of uropathogens were found to be ESBL producers. Most of the ESBL producing isolates were multidrug resistant. Monitoring of ESBL, AmpC & MBL productions and antimicrobial susceptibility testing are necessary to avoid treatment failure in patients with UTI. If irrational use of antibiotics is not controlled, infection with these resistant strain will increase, resulting increase morbidity and mortality. [ABSTRACT FROM AUTHOR]

ABSTRACT: Introduction: Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC-BL) in Enterobacteriaceae are a global threat. Coproduction of AmpC-BL and ESBL reduces therapeutic options, with more resistance against cephamycin and beta-lactamase inhibitor combinations. This study determines the proportion, incidence, and distribution of AmpC-BL-resistant genes in clinically isolated ESBL-producing Escherichia coli , completing its scarce data in Indonesia. Methods: The samples in this study were ESBL-producing E. coli from blood and urine specimens, confirmed by BD Phoenix semiautomatic examination combined with cefoxitin disk screening method. Confirmation tests of AmpC-BL used the AmpC-disk method and the polymerase chain reaction method. Results: Thirteen (27.8%) of 108 E. coli isolates were nonsusceptible to cefoxitin, and 3 (11.5%) isolates were confirmed to produce AmpC-BL and contained AmpC-BL CITM gene. Of the 3 AmpC-BL and ESBL coproduction isolates, one isolate was a copresentation of ESBL and AmpC-BL genes, namely blaTEM and CITM. Fisher's exact test showed that the coproduction of AmpC-BL in ESBL-producing isolates was associated with reduced susceptibility to cefoxitin (P = 0.020) and amoxicillin-clavulanate (P = 0.048) compared to isolates producing ESBL alone. The effect of ESBL and AmpC-BL coproduction on reducing susceptibility to carbapenems needs further investigation. Conclusions: AmpC coproduction was found in 3 (2.8%) of 108 ESBL-producing E. coli isolates, and one isolate copresented AmpC-BL and ESBL coding genes blaTEM and CITM. These three isolates were associated with widened antibiotic resistance to cefoxitin and amoxicillin--clavulanate compared to isolates producing ESBL alone. [ABSTRACT FROM AUTHOR]