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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.

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.

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.

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.

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.

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

A total of 199 Gram-negative bacterial isolates from urinary tract infections and 162 from bloodstream infections were collected from 12 healthcare systems throughout the United States between May 2021 and August 2022. The isolates, phenotypically non-susceptible to 2nd or 3rd generation cephalosporins or carbapenems, were characterized through antimicrobial susceptibility testing and whole genome sequence analysis to obtain a broad snapshot of beta-lactamase-mediated resistance among these two sample types. Overall, 23 different carbapenemase genes were detected among 13 species (20.5% of isolates). The blaKPC-3 and blaKPC-2 subtypes were the most common carbapenemase genes identified, followed by blaNDM and the co-carriage of two different blaOXA carbapenemases by Acinetobacter baumannii isolates. All carbapenemase-producing A. baumannii isolates were mCIM negative. Extended-spectrum beta-lactamase genes were identified in 66.2% of isolates; blaCTX-M-15 was the most common. AmpC genes, both plasmid and chromosomal, were detected in 33.2% of isolates. Importantly, 2.8%, 8.3%, and 22.2% of blaKPC-positive organisms were susceptible to ertapenem, imipenem, and meropenem, respectively. The correlation between broth microdilution and disk diffusion results was high for most drugs except cefepime, where the detection of resistance was statistically lower by disk diffusion. Thus, there were gaps in the accuracy of susceptibility testing for some mechanisms of resistance.

Antimicrobial resistance is considered a topic of utmost interest under the concept of “One Health”, having severe implications in both human and veterinary medicine. Among the antibiotic-resistant bacteria, gram-negative bacteria, especially those belonging to the order of Enterobacterales (such as Escherichia coli), hold a prominent position in terms of both virulence and possessing/disseminating antimicrobial resistance (AMR) traits. The aim of this study was to examine the presence of extended-spectrum β-lactamase producing E. coli isolates in raw poultry carcasses collected from a university club. Five hundred raw poultry skin samples were collected from the Aristotle University of Thessaloniki (AUTh) club in Thessaloniki, Greece. A total of 64% of the samples were positive for the presence of extended-spectrum β-lactamase (ESBL)-producing E. coli. The isolates were further examined for their susceptibility to selected antibiotics by the disc diffusion method and were characterized as true ESBL, as producing class C cephalosporinases (AmpC) or “of unknown etiology” by the combination disc test. The 86 of the 120 isolates (71.67%) were classified as true ESBL, 24 (20.00%) as AmpC, and 10 (8.33%) as “of unknown etiology”. The isolates were screened for the occurrence of β-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaOXA). Thirty-six isolates (32 ESBL- and 4 AmpC-phenotype) harbored both blaTEM and blaCTX-M genes, twenty-two isolates (among which 19 ESBL-phenotype and 2 AmpC-phenotype) harbored blaCTX-M only, whereas twenty-six (14 ESBL- and 12 AmpC-phenotype) isolates harbored blaTEM alone. No isolate harboring blaSHV or blaOXA was detected. The results demonstrate the existence of E. coli isolates producing extended-spectrum β-lactamases in poultry carcasses from Greece, pausing a risk for antibiotic resistance transfer to humans.

Dogs have been reported as potential carriers of antimicrobial-resistant bacteria, but the role of cats has been poorly studied. The aim of this study was to investigate the presence and the risk factors associated with the fecal carriage of extended-spectrum β-lactamase and AmpC (ESBL/AmpC)-producing Escherichia coli (E. coli) in pet and stray cats. Fecal samples were collected between 2020 and 2022 from healthy and unhealthy cats and screened for ESBL/AmpC-producing E. coli using selective media. The presence of ESBL/AmpC-producing E. coli was confirmed by phenotypic and molecular methods. The evaluation of minimum inhibitory concentrations (MICs) was performed on positive isolates. Host and hospitalization data were analyzed to identify risk factors. A total of 97 cats’ samples were collected, and ESBL/AmpC-producing E. coli were detected in 6/97 (6.2%), supported by the detection of blaCTX-M (100%), blaTEM (83.3%), and blaSHV (16.7%) genes and the overexpression of chromosomal ampC (1%). All E. coli isolates were categorized as multidrug-resistant. Unhealthy status and previous antibiotic therapy were significantly associated with ESBL/AmpC-producing E. coli fecal carriage. Our results suggest that cats may be carriers of ESBL/AmpC-producing E. coli, highlighting the need for antimicrobial stewardship in veterinary medicine and an antimicrobial-resistance surveillance program focusing on companion animals, including stray cats.

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

Antibiotic resistance (AR) remains one of the greatest threats to global health, and Aeromonas species have the potential to spread AR in the aquatic environment. The spread of resistance to antibiotics important to human health, such as third-generation cephalosporins (3GCs) and carbapenems, is of great concern. We isolated and identified 15 cefotaxime (3GC)- and 51 carbapenem-resistant Aeromonas spp. from untreated hospital and treated municipal wastewater in January 2020. The most common species were Aeromonas caviae (58%), A. hydrophila (17%), A. media (11%), and A. veronii (11%). Almost all isolates exhibited a multidrug-resistant phenotype and harboured a diverse plasmidome, with the plasmid replicons ColE, IncU, and IncR being the most frequently detected. The most prevalent carbapenemase gene was the plasmid-associated blaKPC-2 and, for the first time, the blaVIM-2, blaOXA-48, and blaIMP-13 genes were identified in Aeromonas spp. Among the 3GC-resistant isolates, the blaGES-5 and blaMOX genes were the most prevalent. Of the 10 isolates examined, three were capable of transferring carbapenem resistance to susceptible recipient E. coli. Our results suggest that conventionally treated municipal and untreated hospital wastewater is a reservoir for 3GC- and carbapenem-resistant, potentially harmful Aeromonas spp. that can be introduced into aquatic systems and pose a threat to both the environment and public health.

The genus Cedecea (family Enterobacteriaceae) causes a wide spectrum of acute infections in immunocompromised hosts, from pneumonia and bacteremia to oral ulcers and dialysis-related peritonitis. While Cedecea infections are reported infrequently in the literature, documented clinical cases of this emerging opportunistic human pathogen have occurred worldwide. Cedecea neteri has clinical significance and exhibits antimicrobial drug resistance. However, little is known about the molecular basis underlying the resistance phenotypes in C. neteri. We previously hypothesized that the open-reading frame cnt10470 in the C. neteri SSMD04 genome encodes a chromosomal Ambler class C (AmpC) β-lactamase based on sequence homology. In this study, recombinant polyhistidine-tagged proteins were created by cloning the putative ampC genes from SSMD04 and C. neteri ATCC 33855 (a clinical isolate) into the pET-6xHN expression vector, overexpressing the proteins, and then purifying the recombinant AmpCs (rAmpCs) using immobilized metal affinity chromatography (Ni-NTA). The in vitro enzymatic analysis of the purified rAmpCs was performed to determine the Km and kcat for various β-lactam substrates. The rAmpCs are functional class C β-lactamases when assayed using the chromogenic β-lactamase substrate, nitrocefin. The presence of functional AmpCs in both C. neteri strains underscores the necessity of performing antibiotic susceptibility testing in the management of C. neteri infections.

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, aac(6′)-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was blaRANH-2, a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant β-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC β-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.

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 bla CTX -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.

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.

Pseudomonas aeruginosa is an important nosocomial pathogen which frequently becomes resistant to most antibiotics used in chemotherapy, resulting in treatment failure among infected individuals. Although the evolutionary trajectory and molecular mechanisms for becoming β-lactam resistant have been well established for P. aeruginosa, the molecular basis of reversion from β-lactam resistant to susceptible is largely unexplored. In this study, we investigated the molecular mechanisms by which a ceftazidime-resistant clinical strain is converted to a ceftazidime-susceptible isolate under the clinical setting. RNA sequencing and genomic DNA reference mapping were conducted to compare the transcriptional profiles and chromosomal mutations between these two isolates. Our results demonstrate that a gain-of-function mutation in ampD, via deletion of a 53 bp duplicated nucleotide sequence, is the contributory factor for the conversion. Furthermore, we show for the first time that AmpD is involved in intraspecies competitiveness in P. aeruginosa. We also found that AmpD is not responsible for phenotypic changes between R1 and S2, including growth rate, motilities, pyocyanin, rhamnolipid, and biofilm production. This finding provides novel insights into the alteration of β-lactam sensitivity in P. aeruginosa under the clinical setting.

Extended-spectrum-β-lactamase (ESBL) and AmpC-lactamase-producing Enterobacteriaceae are serious public health threats. Due to an increasing number of reports of ESBL and AmpC producing Escherichia coli in agricultural settings, it is critical to understand the relationship between the use of two of the highest priority critically important human antibiotics (e.g., third generation cephalosporins [3GC] and macrolides) in food animals and their potential contribution to the selection of ESBL/AmpC E. coli. The objective of our randomized controlled feedlot trial was to measure the effects of ceftiofur crystalline-free acid and tulathromycin on 3GC resistant fecal E. coli populations in cattle before and at various time points after treatment up to and including at slaughter. Multi-level mixed-effects linear regression showed no effect of ceftiofur and tulathromycin on 3GC E. coli CFU counts at slaughter (Day 99); however, a significant (p < 0.05) population shift was observed from susceptible to 3GC resistant E. coli immediately after ceftiofur administration (Day 7). Among 799 fecal samples screened using selective media, 17.7% were ESBL/AmpC E. coli positive, which were further tested for phenotypic antibiotic susceptibility. The majority of the isolates from these plates were multidrug-resistant (94.3%) and expressed either AmpC (78.1%) or ESBL (28.1%) phenotype. A subset of isolates was whole-genome sequenced (n = 20) and identified to harbor chromosomal and/or plasmidal bla genes such as CMY-2, CTX-M, and TEM. Our findings show a time-dependent selection of antibiotics on 3GC-resistant E. coli. High prevalence of multidrug-resistant ESBL/AmpC E. coli found in cattle feces highlights the importance of prudent use of antibiotics in livestock.

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are a universal public health alarm frequently identified among humans, animals, and poultry. Livestock and poultry production are a possible source of multidrug-resistant microorganisms, including ESBL-producing Enterobacteriaceae, which confer antimicrobial resistance to different β-lactam antimicrobial agents. From January to May 2020, a cross-sectional study was carried out in three dairy cattle farms and four poultry farms in different districts of northern Egypt to assess the prevalence of ESBLs, AmpC beta-lactamase-producing E. coli and Klebsiella in livestock, poultry, and human contacts, and to investigate the genetic relatedness of the recovered isolates. In total, 140 samples were collected, including human fecal samples (n = 20) of workers with intimate livestock contact, cattle rectal swabs (n = 34), milk (n = 14), milking machine swabs (n = 8), rations (n = 2), and water (n = 2) from different cattle farms, as well as cloacal swabs (n = 45), rations (n = 5), water (n = 5) and litter (n = 5) from poultry farms. The specimens were investigated for ESBL-producing E. coli and Klebsiella using HiCrome ESBL media agar. The agar disk diffusion method characterized the isolated strains for their phenotypic antimicrobial susceptibility. The prevalence of ESBL-producing Enterobacteriaceae was 30.0%, 20.0%, and 25.0% in humans, cattle, and poultry, respectively. Further genotypic characterization was performed using conventional and multiplex PCR assays for the molecular identification of ESBL and AmpC genes. The majority of the ESBL-producing Enterobacteriaceae showed a multi-drug resistant phenotype. Additionally, blaSHV was the predominant ESBL genotype (n = 31; 93.94%), and was mainly identified in humans (n = 6), cattle (n = 11), and poultry (14); its existence in various reservoirs is a concern, and highlights the necessity of the development of definite control strategies to limit the abuse of antimicrobial agents.

Extended-spectrum beta-lactamases (ESBLs) and AmpC producing Enterobacteriaceae are public health threats. This study aims to characterize ESBL and AmpC producing Enterobacteriaceae isolated from sepsis patients. A multicenter study was conducted at four hospitals located in central (Tikur Anbessa and Yekatit 12), southern (Hawassa) and northern (Dessie) parts of Ethiopia. Blood culture was performed among 1416 sepsis patients. Enterobacteriaceae (n = 301) were confirmed using MALDI-TOF and subjected for whole genome sequencing using the Illumina (HiSeq 2500) system. The overall genotypic frequencies of ESBL and AmpC producing Enterobacteriaceae were 75.5% and 14%, respectively. The detection of ESBL producing Enterobacteriaceae at Hawassa, Yekatit 12, Tikur Anbessa and Dessie was 95%, 90%, 82% and 55.8%, respectively. The detection frequency of blaCTX-M, blaTEM and blaSHV genes was 73%, 63% and 33%, respectively. The most frequently detected ESBL gene was blaCTX-M-15 (70.4%). The common AmpC genes were blaACT (n = 22) and blaCMY (n = 13). Of Enterobacteriaceae that harbored AmpC (n = 42), 71% were ESBL co-producers. Both blaTEM-1B (61.5%) and blaSHV-187 (27.6%) were the most frequently detected variants of blaTEM and blaSHV, respectively. The molecular epidemiology of ESBL producing Enterobacteriaceae showed high frequencies and several variants of ESBL and AmpC genes. Good antimicrobial stewardship and standard bacteriological laboratory services are necessary for the effective treatment of ESBL producing Enterobacteriaceae.

The emergence and spread of multidrug-resistant Salmonella enterica (S. enterica) to humans through food of animal origin are considered a major global public health concern. Currently, little is known about the prevalence of important antimicrobial resistance genes in S. enterica from retail food in Africa. Therefore, the screening and characterization of the extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes in S. enterica isolated from retail meats and slaughterhouses in Egypt were done by using PCR and DNA sequencing techniques. Twenty-eight out of thirty-four (82.4%) non-duplicate S. enterica isolates showed multidrug-resistance phenotypes to at least three classes of antimicrobials, and fourteen (41.2%) exhibited an ESBL-resistance phenotype and harbored at least one ESBL-encoding gene. The identified β-lactamase-encoding genes included blaCTX-M-1, blaCTX-M-3, blaCTX-M-13, blaCTX-M-14, blaCTX-M-15, and blaSHV-12 (ESBL types); blaCMY-2 (AmpC type); and blaTEM-1 and blaOXA-1 (narrow-spectrum types). PMQR genes (included qnrA, qnrB, qnrS, and aac(6′)-Ib-cr) were identified in 23 (67.6%) isolates. The presence of ESBL- and PMQR-producing S. enterica with a high prevalence rate in retail meats and slaughterhouses is considered a major threat to public health as these strains with resistance genes could be transmitted to humans through the food chain.

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.

In the last decade, detection of antibiotic resistant bacteria from wildlife has received increasing interest, due to the potential risk posed by those bacteria to wild animals, livestock or humans at the interface with wildlife, and due to the ensuing contamination of the environment. According to World Health Organization, cephalosporins are critically important antibiotics to human health. However, acquired resistance to β-lactams is widely distributed and is mainly mediated by extended-spectrum beta-lactamase and AmpC beta-lactamases, such as cephalosporinases. This work thus aimed to compile and analyse the information available on the emergence and dissemination of cephalosporinases in wildlife worldwide. Results suggest a serious scenario, with reporting of cephalosporinases in 46 countries from all continents (52% in Europe), across 188 host species, mainly birds and mammals, especially gulls and ungulates. The most widely reported cephalosporinases, CTX-M-1, CTX-M-14, CTX-M-15 and CMY-2, were also the most common in wild animals, in agreement with their ubiquity in human settings, including their association to high-risk clones of Escherichia coli (E. coli), such as the worldwide distributed CTX-M-15/ST131 E. coli. Altogether, our findings show that anthropogenic activities affect the whole ecosystem and that public policies promoting animal and environmental surveillance, as well as mitigation measures to avoid antimicrobial misuse and AMR spread, are urgently needed to be out in practise.

Disease transmission among wild boars, domestic animals and humans is a public health concern, especially in areas with high wild boar densities. In this study, fecal samples of wild boars (n = 200) from different locations of the Metropolitan Area of Barcelona were analyzed by PCR to explore the frequency of β-lactamases and extended cephalosporin and carbapenem resistance genes (ESBLs) in Escherichia coli strains and the presence of toxigenic Clostridioides difficile. The prevalence of genes conferring resistance to β-lactam antimicrobials was 8.0% (16/200): blaCMY-2 (3.0%), blaTEM-1b (2.5%), blaCTX-M-14 (1.0%), blaSHV-28 (1.0%), blaCTX-M-15 (0.5%) and blaCMY-1 (0.5%). Clostridioides difficile TcdA+ was detected in two wild boars (1.0%), which is the first report of this pathogen in wild boars in Spain. Moreover, the wild boars foraging in urban and peri-urban locations were more exposed to AMRB sources than the wild boars dwelling in natural environments. In conclusion, the detection of E. coli carrying ESBL/AmpC genes and toxigenic C. difficile in wild boars foraging in urban areas reinforces the value of this game species as a sentinel of environmental AMRB sources. In addition, these wild boars can be a public and environmental health concern by disseminating AMRB and other zoonotic agents. Although this study provides the first hints of the potential anthropogenic sources of AMR, further efforts should be conducted to identify and control them.

Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC) producing Enterobacteriaceae occur frequently in livestock animals and the subsequent stages of the meat production chain and are therefore considered a risk for human health. Strict biosecurity measures and optimal farm management should reduce or even prevent poultry flock colonization at farm level. This review summarizes and evaluates published information on the effectiveness of specific intervention measures and farm management factors aiming to reduce the occurrence and spread of ESBL/AmpC producing or commensal or pathogenic E. coli in broiler chicken farms. In this systematic literature review, a total of 643 publications were analyzed, and 14 studies with significant outcome about the effectiveness of specific measures against E. coli and ESBL/AmpC producing E. coli in broiler chicken farms were found. Different feed additives seem to have an impact on the occurrence of those microorganisms. The measures ‘cleaning and disinfection’ and ‘competitive exclusion’ showed strong effects in prevention in some studies. In summary, some intervention measures showed potential to protect against or eliminate ESBL/AmpC-producing, commensal or pathogenic E. coli at farm level. Due to the high variability in the outcome of the studies, more specific, detailed investigations are needed to assess the potential of the individual intervention measures.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) represent major healthcare concerns. The role of wildlife in the epidemiology of these bacteria is unclear. The purpose of this study was to determine their prevalence in wild boars in Germany and to characterize individual isolates. A total of 375 fecal samples and 439 nasal swabs were screened for the presence of ESBL-/AmpC-E. coli and MRSA, respectively. The associations of seven demographic and anthropogenic variables with the occurrence of ESBL-/AmpC-E. coli were statistically evaluated. Collected isolates were subjected to antimicrobial susceptibility testing, molecular typing methods, and gene detection by PCR and genome sequencing. ESBL-/AmpC-E. coli were detected in 22 fecal samples (5.9%) whereas no MRSA were detected. The occurrence of ESBL-/AmpC-E. coli in wild boars was significantly and positively associated with human population density. Of the 22 E. coli, 19 were confirmed as ESBL-producers and carried genes belonging to blaCTX-M group 1 or blaSHV-12. The remaining three isolates carried the AmpC-β-lactamase gene blaCMY-2. Several isolates showed additional antimicrobial resistances. All four major phylogenetic groups were represented with group B1 being the most common. This study demonstrates that wild boars can serve as a reservoir for ESBL-/AmpC-producing and multidrug-resistant E. coli.

(1) Background: Resistance plasmids are under selective conditions beneficial for the bacterial host, but in the absence of selective pressure, this carriage may cause fitness costs. Compensation of this fitness burden is important to obtain competitive ability under antibiotic-free conditions. In this study, we investigated fitness effects after a conjugative transfer of plasmids containing various beta-lactamase genes transferred into Escherichia coli. (2) Methods: Fourteen beta-lactamase-encoding plasmids were transferred from clinical donor strains to E. coli J53. Growth rates were compared for all transconjugants and the recipient. Selected transconjugants were challenged in long-term growth experiments. Growth rates were assessed at different time points during growth for 500 generations. Whole-genome sequencing (WGS) of initial and evolved transconjugants was determined. Results: Most plasmid acquisitions resulted in growth differences, ranging from −4.5% to 7.2%. Transfer of a single blaCMY-16-carrying plasmid resulted in a growth burden and a growth benefit in independent mating. Long-term growth led to a compensation of fitness burdens and benefits. Analyzing WGS revealed genomic changes caused by Single Nucleotide Polymorphisms (SNPs) and insertion sequences over time. Conclusions: Fitness effects associated with plasmid acquisitions were variable. Potential compensatory mutations identified in transconjugants’ genomes after 500 generations give interesting insights into aspects of plasmid–host adaptations.

A global increase in Escherichia coli (E. coli) resistant to cephalosporins (extended-spectrum β-lactamases (ESβLs) and AmpC β-lactamases) has been recorded in the last 20 years. Similarly, several studies have reported the spread of colistin resistance in Enterobacteriaceae isolated from food and the environment. The aim of the present study was to evaluate the prevalence of ESβL, AmpC and colistin-resistant E. coli isolated from pork and wild boar meat products in the Emilia Romagna region (North Italy). The isolates were analysed phenotypically (considering both resistant and intermediate profiles) and genotypically. The prevalence of genotypically confirmed ESβL and AmpC E. coli was higher in pork meat products (ESβL = 11.1% vs. AmpC = 0.3%) compared to wild boar meat (ESβL = 6.5% vs. AmpC = 0%). Intermediate profiles for cefotaxime (CTX) and ceftazidime (CAZ) were genotypically confirmed as ESβL in pork meat isolates but not for wild boar. Four E. coli from wild boar meat were resistant to colistin but did not harbour the mcr-1 gene. E. coli isolated from wild boar meat seem to show aspecific antimicrobial resistance mechanisms for cephalosporins and colistin. The prevalence of resistant isolates found in wild boar is less alarming than in pork from farmed domestic pigs. However, the potential risk to consumers of these meat products will require further investigations.

The aim of this work was to assess the prevalence of extended spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae in fecal samples recovered from rural and urban healthy dogs in Northwest Spain (Galicia) to identify potential high-risk clones and to molecularly characterize positive isolates regarding the genes coding for ESBL/pAmpC resistance and virulence. Thirty-five (19.6%) out of 179 dogs were positive for cephalosporin-resistant Enterobacteriaceae, including Escherichiacoli and Klebsiella pneumoniae (39 and three isolates, respectively). All the isolates were multidrug resistant, with high rates of resistance to different drugs, including ciprofloxacin (71.4%). A wide diversity of ESBL/pAmpC enzymes, as well as E. coli phylogroups (A, B1, C, D, E, F and clade I) were found. The eight isolates (20.5%) found to conform to the ExPEC status, belonged to clones O1:H45-clade I-ST770 (CH11-552), O18:H11-A-ST93-CC168 (CH11-neg), O23:H16-B1-ST453-CC86 (CH6-31), and O83:H42-F-ST1485-CC648 (CH231-58), with the latter also complying the uropathogenic (UPEC) status. The three K. pneumoniae recovered produced CTX-M-15 and belonged to the ST307, a clone previously reported in human clinical isolates. Our study highlights the potential role of both rural and urban dogs as a reservoir of high-risk Enterobacteriaceae clones, such as the CC648 of E. coli and antimicrobial resistance traits. Within a One-Health approach, their surveillance should be a priority in the fight against antimicrobial resistance.

Objectives: Chromosomally mediated AmpC-producing Enterobacteriaceae (CAE) display high susceptibility to fluoroquinolones; minimal clinical data exist supporting comparative clinical outcomes. The objective of this study was to compare treatment outcomes between fluoroquinolone and nonfluoroquinolone definitive therapy of bloodstream infections caused by CAE. Methods: This retrospective cohort assessed adult patients with positive blood cultures for CAE that received inpatient treatment for ≥48 h. The primary outcome was difference in clinical failure between patients who received fluoroquinolone (FQ) versus non-FQ treatment. Secondary endpoints included microbiological cure, infection-related length of stay, 90-day readmission, and all-cause inpatient mortality. Results: 56 patients were included in the study (31 (55%) received a FQ as definitive therapy; 25 (45%) received non-FQ). All non-FQ patients received a beta-lactam (BL). Clinical failure occurred in 10 (18%) patients, with 4 (13%) in the FQ group and 6 (24%) in the BL group (p = 0.315). Microbiological cure occurred in 55 (98%) patients. Median infection-related length of stay was 10 (6–20) days, with a significantly longer stay occurring in the BL group (p = 0.002). There was no statistical difference in 90-day readmissions between groups (7% FQ vs. 17% BL; p = 0.387); one patient expired. Conclusion: These results suggest that fluoroquinolones do not adversely impact clinical outcomes in patients with CAE. When alternatives to beta-lactam therapy are needed, fluoroquinolones may provide an effective option.

Objectives: There is debate on whether the use of third-generation cephalosporins (3GC) increases the risk of clinical failure in bloodstream infections (BSIs) caused by chromosomally-mediated AmpC-producing Enterobacterales (CAE). This study evaluates the impact of definitive 3GC therapy versus other antibiotics on clinical outcomes in BSIs due to Enterobacter, Serratia, or Citrobacter species. Methods: This multicenter, retrospective cohort study evaluated adult hospitalized patients with BSIs secondary to Enterobacter, Serratia, or Citrobacter species from 1 January 2006 to 1 September 2014. Definitive 3GC therapy was compared to definitive therapy with other non-3GC antibiotics. Multivariable Cox proportional hazards regression evaluated the impact of definitive 3GC on overall treatment failure (OTF) as a composite of in-hospital mortality, 30-day hospital readmission, or 90-day reinfection. Results: A total of 381 patients from 18 institutions in the southeastern United States were enrolled. Common sources of BSIs were the urinary tract and central venous catheters (78 (20.5%) patients each). Definitive 3GC therapy was utilized in 65 (17.1%) patients. OTF occurred in 22/65 patients (33.9%) in the definitive 3GC group vs. 94/316 (29.8%) in the non-3GC group (p = 0.51). Individual components of OTF were comparable between groups. Risk of OTF was comparable with definitive 3GC therapy vs. definitive non-3GC therapy (aHR 0.93, 95% CI 0.51–1.72) in multivariable Cox proportional hazards regression analysis. Conclusions: These outcomes suggest definitive 3GC therapy does not significantly alter the risk of poor clinical outcomes in the treatment of BSIs secondary to Enterobacter, Serratia, or Citrobacter species compared to other antimicrobial agents.

The aim of the present study was to investigate the diversity of broad-spectrum cephalosporin-resistant Klebsiella spp. isolated from horses in Austria that originated from diseased horses. A total of seven non-repetitive cefotaxime-resistant Klebsiella sp. isolates were obtained during diagnostic activities from autumn 2012 to October 2019. Antimicrobial susceptibility testing was performed. The isolates were genotyped by whole-genome sequencing (WGS). Four out of seven Klebsiella isolates were identified as K. pneumoniae, two as K. michiganensis and one as K. oxytoca. All isolates displayed a multi-drug resistant phenotype. The detection of resistance genes reflected well the phenotypic resistance profiles of the respective isolates. All but one isolate displayed the extended-spectrum β-lactamases (ESBL) phenotype and carried CTX-M cefotaximases, whereas one isolate displayed an ESBL and AmpC phenotype and carried cephamycinase (CMY)-2 and sulfhydryl variable (SHV)-type b and Temoniera (TEM) β-lactamases. Among Klebsiella pneumoniae isolates, for different sequence types (ST) could be detected (ST147, ST307, ST1228, and a new ST4848). Besides resistance genes, a variety of virulence genes, including genes coding for yersiniabactin were detected. Considering the high proximity between horses and humans, our results undoubtedly identified a public health issue. This deserves to be also monitored in the years to come.

β-Lactam-resistant Klebsiella isolates continue to cause multidrug resistance infections worldwide. This study aimed to describe the geographical distribution of extended spectrum β-lactamase (ESBL), AmpC β-lactamase (AmpC), and carbapenemase production among 139 Klebsiella isolates recovered from patients at major referral health facilities in Ghana. The phenotypic methods of combined disc diffusion test, modified three-dimensional test, modified Hodge test (MHT), and combined disc test were performed for each isolate to detect ESBL, AmpC, carbapenemase, and metallo-β-lactamase (MBL) producers, respectively. Except for MBL, all other β-lactam resistance mechanisms were highest in the healthcare facilities situated in the northern belt of Ghana. Significant regional difference of ESBL producers was observed between the northern and middle belts as well as the northern and southern belts. Genotypic detection with polymerase chain reaction (PCR) revealed the presence of bla TEM 36/139 (25.9%), bla SHV 40/139 (28.8%), bla CTX-M 37/139 (26.6%), bla OXA-48 3/139 (2.16%), and bla NDM 1/139 (0.72%) genotypes. In conclusion, there were variations in β-lactam resistance among Klebsiella spp. from health facilities situated in the northern, middle, and southern belts of Ghana. The study provides preliminary evidence that emphasizes the need to direct more attention to antimicrobial resistance control, especially in the northern belt of Ghana. Findings from this study may be critical for creating and fine-tuning effective antimicrobial resistance control strategies and for informing accurate antibiotic prescription by practitioners.

Background and Objectives: Given the considerable spatial, temporal, and ecological factors, heterogeneity, which affects emergency response, persistence, and dissemination of genetic determinants that confer microorganisms their resistance to antibiotics, several authors claim that antibiotics’ resistance must be perceived as an ecological problem. The aim of this study was to determine the prevalence of broad-spectrum bla genes, not only Extended-spectrum β-lactamases (ESBL) but also AmpC-types, in clinical strains of Escherichia coli isolated from Portugal (in the highest region of the country, Serra da Estrela) to disclose susceptibility profiles among different genotypes, and to compare the distribution of bla genes expressing broad-spectrum enzymes. Materials and Methods: Clinical strains of Escherichia coli presenting resistance to third generation (3G) cephalosporins and susceptibility to inhibition by clavulanic acid were studied by means of phenotypic and molecular profiling techniques for encoding β-lactamases genes. Results: Strains were mainly isolated from hospital populations (97%). Molecular analysis enabled the detection of 49 bla genes, in which 55% (27/49) were identified as blaOXA-1-like, 33% (16/49) as blaCTX-M-group-1, 10% (5/49) as blaTEM, and 2% (1/49) were identified as genes blaCIT (AmpC). Among all blaOXA-1-like detected, about 59% of strains expressed at least another bla gene. Co-production of β-lactamases was observed in 40% of strains, with the co-production of CTX-M group 1 and OXA-1-like occurring as the most frequent. Conclusions: This is the first study using microorganisms isolated from native people from the highest Portuguese mountain regions, showing an unprecedent high prevalence of genes blaOXA-1-like in this country.

This study examined the utility of combination therapy for bloodstream isolates of Enterobacteriaceae and non-fermenting Gram-negative bacilli (NFGN) from adults at two community hospitals from January 2010 through to June 2015. Changes to in vitro antimicrobial susceptibilities by adding ciprofloxacin or gentamicin to third-generation cephalosporins (3GC) were examined overall and in patients with risk factors for 3GC resistance. Overall ceftriaxone susceptibility among Enterobacteriaceae was 996/1063 (94%) and 247/295 (84%) in patients with 3GC resistance risk factors. Susceptibilities increased marginally by adding ciprofloxacin or gentamicin (mean difference 2.4% (95% CI 1.5, 3.4) and 3.0% (95% CI 2.0, 4.0), respectively, overall and 5.4% (95% CI 2.8, 8.0) and 7.1% (95% CI 4.2, 10.1), respectively, in patients with risk factors). Eighty-three of 105 (79%) NFGN were susceptible to ceftazidime overall and 20/29 (69%) in patients with prior beta-lactam use. Overall mean increase in susceptibilities was 15.2% (95% CI: 8.3, 22.2) and 17.1% (95% CI: 9.8, 24.5) for ciprofloxacin and gentamicin combinations, respectively; and 27.6% (95% CI: 10.3, 44.9) for either one with recent beta-lactam use. In this setting, empirical combination therapy had limited utility for Enterobacteriaceae bloodstream isolates but provided significant additional antimicrobial coverage to ceftazidime for NFGN, particularly in patients with prior beta-lactam use.

The consumption of milk contaminated with antibiotic-resistant bacteria poses a significant health threat to humans. This study aimed to investigate the prevalence of Enterobacteriaceae producing β-lactamases (ESBL, MBL, and AmpC) in cow and buffalo milk samples from two Indian states, Haryana and Assam. A total of 401 milk samples were collected from dairy farmers and vendors in the specified districts. Microbiological assays, antibiotic susceptibility testing, and PCR-based genotyping were employed to analyze 421 Gram-negative bacterial isolates. The overall prevalence of β-lactamase genes was 10% (confidence interval (CI) (7-13)), with higher rates in Haryana (13%, CI (9-19)) compared to Assam (7%, CI (4-11)). The identified β-lactamase genes in isolates were bla CMY , bla MOX , bla FOX , bla EBC , and bla DHA , associated with AmpC production. Additionally, bla CTX-M1 , bla SHV , and bla TEM were detected as ESBL producers, while bla VIM , bla IMP , bla SPM , bla SIM , and bla GIM were identified as MBL producers. Notably, Shigella spp. were the dominant β-lactamase producers among identified Enterobacteriaceae. This study highlights the presence of various prevalent β-lactamase genes in milk isolates, indicating the potential risk of antimicrobial-resistant bacteria in dairy products. The presence of β-lactam resistance raises concern as this could restrict antibiotic options for treatment. The discordance between genotypic and phenotypic methods emphasizes the necessity for comprehensive approaches that integrate both techniques to accurately assess antibiotic resistance. Urgent collaborative action incorporating rational and regulated use of antibiotics across the dairy value chain is required to address the global challenge of β-lactam resistance.

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

A total of 199 Gram-negative bacterial isolates from urinary tract infections and 162 from bloodstream infections were collected from 12 healthcare systems throughout the United States between May 2021 and August 2022. The isolates, phenotypically non-susceptible to 2nd or 3rd generation cephalosporins or carbapenems, were characterized through antimicrobial susceptibility testing and whole genome sequence analysis to obtain a broad snapshot of beta-lactamase-mediated resistance among these two sample types. Overall, 23 different carbapenemase genes were detected among 13 species (20.5% of isolates). The bla KPC-3 and bla KPC-2 subtypes were the most common carbapenemase genes identified, followed by bla NDM and the co-carriage of two different bla was the most common. AmpC genes, both plasmid and chromosomal, were detected in 33.2% of isolates. Importantly, 2.8%, 8.3%, and 22.2% of OXA carbapenemases by Acinetobacter baumannii isolates. All carbapenemase-producing A. baumannii isolates were mCIM negative. Extended-spectrum beta-lactamase genes were identified in 66.2% of isolates; bla CTX-M-15 was the most common. AmpC genes, both plasmid and chromosomal, were detected in 33.2% of isolates. Importantly, 2.8%, 8.3%, and 22.2% of bla KPC -positive organisms were susceptible to ertapenem, imipenem, and meropenem, respectively. The correlation between broth microdilution and disk diffusion results was high for most drugs except cefepime, where the detection of resistance was statistically lower by disk diffusion. Thus, there were gaps in the accuracy of susceptibility testing for some mechanisms of resistance.

In this study, we sought to profile the abundances and drivers of antibiotic resistance genes in an urban river impacted by combined sewage overflow (CSO) events. Water samples were collected weekly during the summer for two years; then, quantitative PCR was applied to determine the abundance of resistance genes associated with tetracycline, quinolones, and β-lactam antibiotics. In addition to sampling a CSO-impacted site near the city center, we also sampled a less urban site ~12 km upstream with no proximal sewage inputs. The tetracycline genes tetO and tetW were rarely found upstream, but were common at the CSO-impacted site, suggesting that the primary source was untreated sewage. In contrast, ampC was detected in all samples indicating a more consistent and diffuse source. The two other genes, qnrA and bla TEM , were present in only 40-50% of samples and showed more nuanced spatiotemporal patterns consistent with upstream agricultural inputs. The results of this study highlight the complex sources of ARGs in urban riverine ecosystems, and that interdisciplinary collaborations across diverse groups of stakeholders are necessary to combat the emerging threat of antibiotic resistance through anthropogenic pollution.

Dogs have been reported as potential carriers of antimicrobial-resistant bacteria, but the role of cats has been poorly studied. The aim of this study was to investigate the presence and the risk factors associated with the fecal carriage of extended-spectrum β-lactamase and AmpC (ESBL/AmpC)-producing Escherichia coli ( E. coli ) in pet and stray cats. Fecal samples were collected between 2020 and 2022 from healthy and unhealthy cats and screened for ESBL/AmpC-producing E. coli using selective media. The presence of ESBL/AmpC-producing E. coli was confirmed by phenotypic and molecular methods. The evaluation of minimum inhibitory concentrations (MICs) was performed on positive isolates. Host and hospitalization data were analyzed to identify risk factors. A total of 97 cats' samples were collected, and ESBL/AmpC-producing E. coli were detected in 6/97 (6.2%), supported by the detection of bla CTX-M (100%), bla TEM (83.3%), and bla isolates were categorized as multidrug-resistant. Unhealthy status and previous antibiotic therapy were significantly associated with ESBL/AmpC-producing SHV fecal carriage. Our results suggest that cats may be carriers of ESBL/AmpC-producing ampC (1%). All E. coli isolates were categorized as multidrug-resistant. Unhealthy status and previous antibiotic therapy were significantly associated with ESBL/AmpC-producing E. coli fecal carriage. Our results suggest that cats may be carriers of ESBL/AmpC-producing E. coli , highlighting the need for antimicrobial stewardship in veterinary medicine and an antimicrobial-resistance surveillance program focusing on companion animals, including stray cats.

There has been a progressive rise in the incidence of blood stream infections (BSI) caused by multidrug-resistant Gram-negative organisms (MDR GN), which cause increased morbidity and mortality. For this reason, recent studies have focused on risk factors of acquisition of carbapenemase-producing Enterobacteriaceae and extended-spectrum beta-lactamase producers. However, there is limited data on risk factors for BSI caused by AmpC-producing Enterobacteriaceae (AmpC EC), especially in low prevalence settings such as Australia. This study was performed to identify risk factors for acquisition of AmpC E. coli, using a retrospective matched case control design over a 3-year period. Patients with BSI caused by AmpC E. coli were matched with controls (third generation cephalosporin susceptible E. coli) by age and site of infection (n = 21). There was no significant difference in age, sex, clinical outcome, time to onset of BSI, recent antibiotic use (last 3 months), comorbidities (type 2 diabetes mellitus, renal failure) intensive care unit admission, underlying hematological condition, immunosuppressant use, APACHE II score, or any recent urological procedures (within last 3 months) between the two groups. On univariate analysis, the AmpC E. coli group were more likely to have had a surgical procedure in hospital and lived in a residential aged care facility. On multivariate logistic regression analysis, a recent surgical procedure was associated with the onset of AmpC E. coli BSI (Odd’s Ratio (OR) 4.78, p = 0.034). We concluded that in a relatively low prevalence setting such as Australia, AmpC E. coli BSI is potentially associated with surgery performed in hospital due to previous antibiotic exposure and longer hospitalization.

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

Antibiotic resistance (AR) remains one of the greatest threats to global health, and Aeromonas species have the potential to spread AR in the aquatic environment. The spread of resistance to antibiotics important to human health, such as third-generation cephalosporins (3GCs) and carbapenems, is of great concern. We isolated and identified 15 cefotaxime (3GC)- and 51 carbapenem-resistant Aeromonas spp. from untreated hospital and treated municipal wastewater in January 2020. The most common species were Aeromonas caviae (58%), A. hydrophila (17%), A. media (11%), and A. veronii (11%). Almost all isolates exhibited a multidrug-resistant phenotype and harboured a diverse plasmidome, with the plasmid replicons ColE, IncU, and IncR being the most frequently detected. The most prevalent carbapenemase gene was the plasmid-associated bla KPC-2 and, for the first time, the bla VIM-2 , bla OXA-48, and bla IMP-13 genes were identified in Aeromonas spp. Among the 3GC-resistant isolates, the bla GES-5 and bla MOX genes were the most prevalent. Of the 10 isolates examined, three were capable of transferring carbapenem resistance to susceptible recipient E. coli . Our results suggest that conventionally treated municipal and untreated hospital wastewater is a reservoir for 3GC- and carbapenem-resistant, potentially harmful Aeromonas spp. that can be introduced into aquatic systems and pose a threat to both the environment and public health.

The genus Cedecea (family Enterobacteriaceae ) causes a wide spectrum of acute infections in immunocompromised hosts, from pneumonia and bacteremia to oral ulcers and dialysis-related peritonitis. While Cedecea infections are reported infrequently in the literature, documented clinical cases of this emerging opportunistic human pathogen have occurred worldwide. Cedecea neteri has clinical significance and exhibits antimicrobial drug resistance. However, little is known about the molecular basis underlying the resistance phenotypes in C. neteri . We previously hypothesized that the open-reading frame cnt10470 in the C. neteri SSMD04 genome encodes a chromosomal Ambler class C (AmpC) β-lactamase based on sequence homology. In this study, recombinant polyhistidine-tagged proteins were created by cloning the putative ampC genes from SSMD04 and C. neteri ATCC 33855 (a clinical isolate) into the pET-6xHN expression vector, overexpressing the proteins, and then purifying the recombinant AmpCs (rAmpCs) using immobilized metal affinity chromatography (Ni-NTA). The in vitro enzymatic analysis of the purified rAmpCs was performed to determine the K m and k cat for various β-lactam substrates. The rAmpCs are functional class C β-lactamases when assayed using the chromogenic β-lactamase substrate, nitrocefin. The presence of functional AmpCs in both C. neteri strains underscores the necessity of performing antibiotic susceptibility testing in the management of C. neteri infections.

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, aac(6')-Ib gene, and integrase genes ( intI1 , intI2 , and intI3 ) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was bla RANH-2 spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Rahnella -related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one Pseudomonas positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant β-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC β-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant β-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC β-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.

Pseudomonas aeruginosa is an important nosocomial pathogen which frequently becomes resistant to most antibiotics used in chemotherapy, resulting in treatment failure among infected individuals. Although the evolutionary trajectory and molecular mechanisms for becoming β-lactam resistant have been well established for P. aeruginosa , the molecular basis of reversion from β-lactam resistant to susceptible is largely unexplored. In this study, we investigated the molecular mechanisms by which a ceftazidime-resistant clinical strain is converted to a ceftazidime-susceptible isolate under the clinical setting. RNA sequencing and genomic DNA reference mapping were conducted to compare the transcriptional profiles and chromosomal mutations between these two isolates. Our results demonstrate that a gain-of-function mutation in ampD , via deletion of a 53 bp duplicated nucleotide sequence, is the contributory factor for the conversion. Furthermore, we show for the first time that AmpD is involved in intraspecies competitiveness in P. aeruginosa . We also found that AmpD is not responsible for phenotypic changes between R1 and S2, including growth rate, motilities, pyocyanin, rhamnolipid, and biofilm production. This finding provides novel insights into the alteration of β-lactam sensitivity in P. aeruginosa under the clinical setting.

Extended-spectrum-β-lactamase (ESBL) and AmpC-lactamase-producing Enterobacteriaceae are serious public health threats. Due to an increasing number of reports of ESBL and AmpC producing Escherichia coli in agricultural settings, it is critical to understand the relationship between the use of two of the highest priority critically important human antibiotics (e.g., third generation cephalosporins [3GC] and macrolides) in food animals and their potential contribution to the selection of ESBL/AmpC E. coli. The objective of our randomized controlled feedlot trial was to measure the effects of ceftiofur crystalline-free acid and tulathromycin on 3GC resistant fecal E. coli populations in cattle before and at various time points after treatment up to and including at slaughter. Multi-level mixed-effects linear regression showed no effect of ceftiofur and tulathromycin on 3GC E. coli CFU counts at slaughter (Day 99); however, a significant (p < 0.05) population shift was observed from susceptible to 3GC resistant E. coli immediately after ceftiofur administration (Day 7). Among 799 fecal samples screened using selective media, 17.7% were ESBL/AmpC E. coli positive, which were further tested for phenotypic antibiotic susceptibility. The majority of the isolates from these plates were multidrug-resistant (94.3%) and expressed either AmpC (78.1%) or ESBL (28.1%) phenotype. A subset of isolates was whole-genome sequenced (n = 20) and identified to harbor chromosomal and/or plasmidal bla genes such as CMY-2, CTX-M, and TEM. Our findings show a time-dependent selection of antibiotics on 3GC-resistant E. coli. High prevalence of multidrug-resistant ESBL/AmpC E. coli found in cattle feces highlights the importance of prudent use of antibiotics in livestock.

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are a universal public health alarm frequently identified among humans, animals, and poultry. Livestock and poultry production are a possible source of multidrug-resistant microorganisms, including ESBL-producing Enterobacteriaceae , which confer antimicrobial resistance to different β-lactam antimicrobial agents. From January to May 2020, a cross-sectional study was carried out in three dairy cattle farms and four poultry farms in different districts of northern Egypt to assess the prevalence of ESBLs, AmpC beta-lactamase-producing E. coli and Klebsiella in livestock, poultry, and human contacts, and to investigate the genetic relatedness of the recovered isolates. In total, 140 samples were collected, including human fecal samples (n = 20) of workers with intimate livestock contact, cattle rectal swabs (n = 34), milk (n = 14), milking machine swabs (n = 8), rations (n = 2), and water (n = 2) from different cattle farms, as well as cloacal swabs (n = 45), rations (n = 5), water (n = 5) and litter (n = 5) from poultry farms. The specimens were investigated for ESBL-producing E. coli and Klebsiella using HiCrome ESBL media agar. The agar disk diffusion method characterized the isolated strains for their phenotypic antimicrobial susceptibility. The prevalence of ESBL-producing Enterobacteriaceae was 30.0%, 20.0%, and 25.0% in humans, cattle, and poultry, respectively. Further genotypic characterization was performed using conventional and multiplex PCR assays for the molecular identification of ESBL and AmpC genes. The majority of the ESBL-producing Enterobacteriaceae showed a multi-drug resistant phenotype. Additionally, bla SHV was the predominant ESBL genotype (n = 31; 93.94%), and was mainly identified in humans (n = 6), cattle (n = 11), and poultry (14); its existence in various reservoirs is a concern, and highlights the necessity of the development of definite control strategies to limit the abuse of antimicrobial agents.