Search Results (90)

Background: There are many methods of identifying microbial resistance to therapeutic agents; however, they can generally be classified into two main categories: phenotypic and genotypic. The study aims to determine drug sensitivity and to analyze the correlation between the results obtained from cultures on commercial chromogenic media Brilliance^TM CRE (OXOID) and Brilliance^TM ESBL (OXOID) and the occurrence of specific resistance genes carbapenemase (IMP, NDM, VIM, KPC, OXA), ESBL β-lactamase (TEM, SHV, CTX-M), and AmpC (CMY, DHA), which will be used in drug sensitivity tests. Methods: The present study used bacteria, including Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli, obtained from patients hospitalized in military hospitals in Poland. All strains were plated on the commercial chromogenic media and subjected to antimicrobial susceptibility testing. Additionally, molecular assays detecting three main classes according to the mechanism of action, enzyme type carbapenemase (IMP, NDM, VIM, KPC, OXA), ESBL β-lactamase (TEM, SHV, CTX-M), and AmpC (CMY, DHA) were performed using the real-time PCR method. Results: The results of the studies indicate the presence of carbapenemases and ESBL genes. Among K. pneumoniae strains, the dominant gene was CTX-M-15 (88.89%), followed by the SHV (84.12%), NDM (46.03%), TEM (41.26%), KPC (34.92%), and OXA-48 (19.04%). In contrast, A. baumanii was dominated by carbapenemases from the OXA family (OXA-51 in 96.00% and OXA-24/40 in 84.00%). E. coli exhibits a high prevalence of CTX-M-15 (53.85%), TEM (46.15%), NDM (38.46%), and CMY-2 (30.77%). It was observed that the CTX-M-15 gene was commonly co-identified with SHV (n = 43). All tested strains grew on chromogenic Brilliance^TM CRE medium. In the case of Brilliance^TM ESBL medium, the genes determining the resistance mechanism were detected in 41.7% for A. baumannii, 53.8% for E. coli, and 100% for K. pneumoniae. Chromogenic media perfectly differentiate strains to species. A moderate positive correlation of the occurrence of the antibiotic resistance genes was observed for OXA-51 and OXA-24/40 genes, which were resistant to meropenem (rho = 0.45, p < 0.001). K-means cluster analysis performed on integrated genotype–phenotype data allowed for the identification of three distinct clusters characterized by distinct resistance gene profiles. These results demonstrate that selective agar media enable faster identification compared to other conventional techniques; however, the obtained results should be confirmed by other validated phenotypic methods, and, if possible, by a molecular assay. Full article

Background/Objectives: Antimicrobial resistance (AMR) threatens global public health. This systematic review and meta-analysis, as part of the “ENVIRE” project (interventions to control the dynamics of antimicrobial resistance from chickens through the environment), assesses the prevalence of phenotypic and genotypic resistance, including extended-spectrum beta-lactamases (ESBLs), AmpC beta-lactamases, carbapenemases, colistin, and fluoroquinolone resistance, in broiler chickens and their environment. Methods: The analysis covers the years 2002–2022, focusing on Escherichia (E.) coli, Klebsiella spp., Enterobacter spp., and Citrobacter spp. in fecal, meat, environmental, and other-than-feces samples from observational studies published in PubMed and Web of Science. Quality assessment was performed using the Alberta Heritage Foundation criteria. Results: Data from 170 studies, conducted in Europe, North Africa, and North America, were included. The most frequently studied resistance was to beta-lactam, with focus on ESBL-producing and AmpC beta-lactamase isolates. The pooled prevalence of ESBL-resistant E. coli observed in meat samples at 41% and in fecal samples at 38% demonstrated significant heterogeneity between the studies. The negative binomial regression analysis of prevalence data revealed significantly higher ESBL-producing E. coli rates in European meat samples compared to North African samples. Conclusions: This systematic review revealed substantial variation in prevalence and emphasizes the need for standardized surveillance systems and robust study designs. Full article

Antimicrobial resistance (AMR) among commensal Escherichia coli from poultry is a growing concern for food safety and public health. This study investigated AMR patterns in E. coli isolated from broiler neck skin at slaughter, comparing organic, antibiotic-free (ATB-free), and conventional production systems. A total of 375 samples were collected from two Italian slaughterhouses and tested by broth microdilution following EU protocols. E. coli was recovered from 358 samples, and 37.9% were presumptively positive for ESBL/AmpC-producing strains. Conventional broilers showed the highest resistance to ampicillin (73.8%), sulfonamides (72.5%), and fluoroquinolones (nalidixic acid, 62.5%; ciprofloxacin, 67.5%), while organic and ATB-free systems showed significantly lower levels. Intermediate resistance occurred for trimethoprim (21.4–47.9%) and tetracycline (36–54%), and low prevalence (<10%) was found for gentamicin, tigecycline, and third-generation cephalosporins. No relevant resistance was detected to colistin or carbapenems (≤1.2%). Total E. coli counts did not differ among systems, suggesting differences in resistant strain proportions rather than bacterial load. ATB-free flocks processed after conventional batches displayed higher resistance, indicating possible cross-contamination during slaughter. These results highlight the influence of farming practices and slaughterhouse hygiene on AMR dissemination, underscoring the need for integrated farm-to-slaughter control strategies. Full article

Background: Fluoroquinolone (FQ) resistance in Escherichia coli (E. coli) undermines empiric therapy and often coincides with multidrug resistance (MDR). Because sequencing is not routinely available in many laboratories, we evaluated a phenotype-first, sequencing-independent diagnostic framework deployable on standard platforms. Methods: We profiled 45 archived E. coli isolates for susceptibility (Clinical and Laboratory Standards Institute [CLSI]-guided), extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC) phenotypes, MDR, and multiple-antibiotic resistance (MAR) indices. Ten founders (five FQ-susceptible [FQ-S], five low-level resistant [LLR]) seeded 20 parallel lineages exposed to stepwise ciprofloxacin. We tracked minimum inhibitory concentrations (MICs), collateral resistance, growth kinetics, and biofilm biomass using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification, automated and reference antimicrobial susceptibility testing (AST), growth-curve analysis, and crystal violet microtiter assays. The intended use is a sequencing-independent workflow for routine laboratories—especially where whole-genome sequencing is not readily available—working with archived or prospective clinical E. coli. This workflow is best applied when local FQ nonsusceptibility threatens empiric reliability; inputs include standard ID/AST with simple growth and biofilm assays. Primary outputs include: (i) MIC trajectories with time to high-level resistance (HLR), (ii) ΔMAR-summarized collateral resistance with class-level susceptible-to-resistant conversions, and (iii) concise fitness/biofilm summaries to guide empiric-policy refresh and early de-escalation. Results: At baseline, ciprofloxacin nonsusceptibility was 40.0%; ESBL and AmpC phenotypes were confirmed in 28.9% and 15.6%, respectively; 46.7% met the MDR definition; and the median MAR index was 0.29. During evolution, 70% of lineages reached HLR (MIC ≥ 4 μg/mL), with earlier conversion from LLR versus FQ-S founders (median 7 vs. 11 passages). Collateral resistance emerged most often to third-generation cephalosporins (3GCs), trimethoprim–sulfamethoxazole, and tetracyclines, while carbapenem activity was preserved. MAR increased in parallel with rising MICs. Resistance acquisition imposed modest fitness costs (slightly reduced growth rates and longer lag phases) that were partly offset under subinhibitory ciprofloxacin, whereas biofilm biomass changed little. Conclusions: this phenotype-first, routine-laboratory workflow rapidly maps FQ resistance and clinically relevant co-selection in E. coli. In high-resistance settings, empiric FQ use is difficult to justify, and MAR trends provide practical co-selection signals for stewardship. This reproducible framework complements genomic surveillance and is directly applicable where sequencing is unavailable. Full article

Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC (pAmpC) β-lactamases represent a threat for public health. Their dissemination is often mediated by mobile genetic elements (MGEs), but plasmid identification and characterization could be hindered by sequencing limitations. Hybrid assembly may overcome these barriers. Eight ESBL/pAmpC-producing E. coli isolates from broilers were sequenced using Illumina (short-read) and Oxford Nanopore MinION (long-read). Assemblies were generated individually and using a hybrid approach. Plasmids were typed, annotated, and screened for antimicrobial resistance genes (ARGs), MGEs, and virulence factors. Short-read assemblies were highly fragmented, while long reads improved contiguity but showed typing errors. Hybrid assemblies produced the most accurate and complete plasmids, including more circularized plasmids. Long and hybrid assemblies detected IS26 associated with ESBL genes and additional virulence genes not identified by short reads. ARG profiles were consistent across methods, but structural resolution and contextualization of resistance loci were superior in hybrid assembly. Hybrid assembly integrates the strengths of short- and long-read sequencing, enabling accurate plasmid reconstruction and improved detection of resistance-associated MGEs. This approach may enhance genomic surveillance of ESBL/pAmpC plasmids and support strategies to mitigate antimicrobial resistance. Full article

Antimicrobial resistance (AMR) is a significant public health concern in companion animals, yet systematic surveillance in North Macedonia is lacking. This study investigated the prevalence of resistance in Escherichia coli isolated from 112 fecal samples from dogs in six shelters in North Macedonia and evaluated the associated risk factors, providing the first baseline dataset for this population. High resistance was observed for sulfamethoxazole (68.75%), ampicillin (52.68%), and ciprofloxacin (41.07%). Multidrug resistance was present in 50% of the isolates, with 17 (15.17%) confirmed as ESBL producers. Additionally, 18 isolates (16.1%) were identified as AmpC producers, 16 of which carried the blaCMY-2 gene. Notably, 72.2% of ESBL/AmpC isolates were resistant to ertapenem despite the absence of carbapenemase genes, a finding that warrants further investigation. Risk factors such as shared housing, longer shelter stays, and frequent empirical antimicrobial use were identified as probable contributors to the carriage of ESBL-/AmpC-producing E. coli. None of the shelters had antimicrobial stewardship protocols or routine diagnostic testing, revealing critical gaps in infection control and antimicrobial practices. These findings underscore the urgent need for antimicrobial stewardship and surveillance in North Macedonia’s companion animal populations within the One Health framework. Full article

Background/Objectives: The widespread use of antibiotics in animal husbandry has led to an increase in antimicrobial-resistant Escherichia coli, particularly strains producing extended-spectrum β-lactamases (ESBL) and AmpC β-lactamases. This study aimed to isolate and characterize such strains from fecal samples of broiler chickens (n = 71) and slaughtered turkeys (n = 31) in northwestern Romania. Methods: Antimicrobial susceptibility testing and PCR were used to evaluate phenotypic resistance patterns and detect the presence of resistance genes (AmpC, blaZ, and blaTEM). Results: The results showed that 55% of turkey and 61% of broiler isolates were presumptive ESBL/AmpC producers. Among all isolates, 50% were classified as extensively drug-resistant (XDR), 44% as multidrug-resistant (MDR), and only 6% were fully susceptible. Gene detection revealed an overall prevalence of 44.2% for AmpC, 72.7% for blaZ, and 58.1% for blaTEM, yielding a total penetrance of 51.09%. The diagnostic odds ratio (DOR) values, ranging from 0.67 to 81, suggest the efficacy of the antibiotic susceptibility testing method used in detecting the presence of these resistance genes. Conclusion: Overall, these findings highlight a significant burden of antimicrobial-resistant, poultry-associated E. coli strains, warranting stricter antimicrobial stewardship. Full article

Objective: To evaluate the antimicrobial susceptibility of Enterobacterales isolated from patients with intra-abdominal infections (IAI) in United States (US) medical centres. Methods: A total of 2036 isolates (1/patient) were consecutively collected from patients with IAI in 63 US hospitals in 2019–2023 and susceptibility tested by broth microdilution. Carbapenem-resistant Enterobacterales (CRE) were screened for carbapenemases by whole genome sequencing. Results: The most common Enterobacterales species were E. coli (47.1%), K. pneumoniae (18.7%), and E. cloacae species complex (9.8%). The most active agents were aztreonam-avibactam (MIC_50/90, ≤0.03/0.12 mg/L), ceftazidime-avibactam (MIC_50/90, 0.12/0.25 mg/L), and meropenem-vaborbactam (MIC_50/90, 0.03/0.06 mg/L) with 99.9% susceptibility. A multidrug-resistant (MDR) phenotype (nonsusceptibility to ≥3 classes) was observed in 21.4% of Enterobacterales (n = 436). Piperacillin-tazobactam was active against 87.2% of Enterobacterales overall and 50.2% of MDR isolates, and meropenem was active against 99.2% of Enterobacterales and 96.1% of MDR isolates. Only 51.6% of Enterobacterales were susceptible to ampicillin-sulbactam. An acquired broad-spectrum β-lactamase gene was identified in 207 (10.2%) isolates and included extended-spectrum β-lactamases (ESBL; n = 182), transferable AmpC (n = 24) and carbapenemases (n = 9). Eight isolates produced two β-lactamase classes. Conclusions: Aztreonam-avibactam, ceftazidime-avibactam, and meropenem-vaborbactam exhibited almost complete activity (99.9% susceptibility) against Enterobacterales causing IAI in US hospitals. In contrast, piperacillin-tazobactam exhibited limited activity against these organisms, especially those with a MDR phenotype. Full article

Background: Mapping the local etiology and susceptibility of common pathogens causing complicated urinary tract infection (cUTI) is important for promoting evidence-based antimicrobial prescribing. Evaluating the prevalence of extended-spectrum beta-lactamase (ESBL), AmpC beta-lactamase (AmpC), and carbapenemase-producing Enterobacterales (CPEs) is equally important as it informs treatment guidelines and empiric management. Whole genome sequencing (WGS) enhances antimicrobial resistance (AMR) surveillance by complementing phenotypic antimicrobial susceptibility testing, offering deeper insights into resistance mechanisms, transmissions, and evolutions. Integrating it into routine AMR monitoring can significantly improve global efforts to combat antimicrobial resistance. Methods: Antimicrobial susceptibility profiles of isolates from cUTI were collected from patients presenting with Sultan Qaboos University Hospital, Muscat and Suhar Hospital, Suhar, Oman. Automated systems as well as manual methods were used for detection of ESBL, AmpC, and CPE. ESBLs, AmpC β-lactamases, and CPEs were further detected by manual methods: double-disk synergy test for ESBL; disk approximation assay and D69C AmpC detection set for AmpC, and mCIM and KPC/IMP/NDM/VIM/OXA-48 Combo test kit for CPE. WGS was carried out in 11 FOX-resistant E. coli and (22 carbapenem-resistant K. pneumoniae) isolates with varying susceptibilities to identify circulating clades, AMR genes, and plasmids. Bioinformatic analysis was performed using online tools. Results: The susceptibility patterns of E. coli from cUTI were as follows: nitrofurantoin (96%), fosfomycin (100%), fluoroquinolones (44%), aminoglycosides (93%), piperacillin-tazobactam (95%), and carbapenems (98%). In comparison, susceptibility rates of K. pneumoniae were far lower: nitrofurantoin (38%), fosfomycin (89%), aminoglycosides (82%), piperacillin-tazobactam (72%), and carbapenems (83%). K. pneumoniae, however, was more susceptible to fluoroquinolones at 47% in comparison to E. coli. The prevalence of ESBL among E. coli and K. pneumoniae was 37.2% and CRE was 6.2% while the estimated prevalence of AmpC was 5.4%. It was observed that E. coli was the predominant ESBL and AmpC producer, while K. pneumoniae was the major carbapenem-resistant Enterobacterales (CREs) producer. No predominant multi-locus sequence typing (MLST) lineage was observed in AmpC-producing E. coli with nine E. coli MLST lineages being identified from eleven isolates: ST-10, ST-69, ST-77, ST-131, ST-156, ST-167, ST-361, ST-1125, and ST-2520. On the other hand, a less diverse MLST spectrum (ST-2096, ST-231, ST-147, ST-1770, and ST-111) was observed in the CRE K. pneumoniae. Among the five MLST lineages, ST-2096 (twelve isolates) and ST-147 (seven isolates) predominated. WGS revealed that DHA-1 was the predominant plasmid-mediated AmpC gene in E. coli, while OXA-232 and NDM-5 were the most common carbapenemase genes in K. pneumoniae. All E. coli DHA-1-positive isolates co-harbored the quinolone resistance gene qnrB4 and the sulfonamide resistance gene sul1 while no aminoglycoside resistance genes were detected. The majority of CPE CRE K. pneumoniae carried other β-lactamase genes, such as blaCTX-M-15, blaSHV, and blaTEM; all co-harbored the quinolone resistance gene OqxAB; and 77% carried the aminoglycoside resistance gene armA. Conclusions: Our results suggest that fosfomycin is an excellent empiric choice for treating complicated cystitis caused by both E. coli and K. pneumoniae, while nitrofurantoin is an appropriate choice for E. coli cystitis but not for K. pneumoniae. Aminoglycosides and piperacillin-tazobactam are excellent intravenous alternatives that spare carbapenems. DHA-1 was the predominant AmpC in E. coli, while OXA-232 and NDM-5 were the predominant carbapenemases in K. pneumoniae. In AmpC-producing E. coli, no MLST predominated, suggesting a significant flux in E. coli with lack of stable clades in this region. In contrast, ST-2096 and ST-147 predominated in CRE Klebsiella pneumoniae, suggesting a stable circulation of these in Oman. WGS profiling provides a deeper understanding of the genetic basis of resistance and enhances surveillance and offers comprehensive insights into pathogen evolution and transmission patterns. Full article

Objectives: This study aimed to analyze the antibiotic resistance patterns of microorganisms isolated from intensive care unit (ICU) patients and evaluate their impact on mortality and length of ICU stay. Given the increasing prevalence of multidrug-resistant (MDR) pathogens in critically ill patients, understanding their resistance profiles is crucial for optimizing empirical antibiotic therapy and improving patient outcomes. Methods: This retrospective study included 237 ICU patients admitted between 1 July 2022, and 1 January 2024. The initial culture growth results from blood and urine samples were analyzed. Microorganism identification was performed using VITEK 2 Compact and conventional bacteriological methods, while antibiotic susceptibility testing followed CLSI 2022 and EUCAST 2022 guidelines. Results: A total of 237 ICU patients were included in this study. The most frequently isolated microorganisms were Escherichia coli (E. coli) (44.3%), Klebsiella pneumoniae (K. pneumoniae) (35.0%), and Pseudomonas aeruginosa (P. aeruginosa) (25.3%), with Acinetobacter baumannii (A. baumannii) (31.2%) being the most resistant pathogen. Among Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) (12.2%) and vancomycin-resistant enterococci (VRE) (21.5%) were the most frequently identified multidrug-resistant (MDR) pathogens. Regarding antimicrobial resistance, carbapenem resistance was highest in A. baumannii (55%), followed by P. aeruginosa (40%) and K. pneumoniae (30%). Additionally, ESBL-producing E. coli (43.2%) and K. pneumoniae (38.5%), as well as carbapenemase-producing K. pneumoniae (18.6%) and E. coli (9.2%), were identified as key resistance mechanisms impacting clinical outcomes. Patients with MDR infections had significantly longer ICU stays (p < 0.05) and higher mortality rates. The Kaplan–Meier survival analysis revealed that A. baumannii infections were associated with the highest mortality risk (HR: 4.6, p < 0.001), followed by MRSA (HR: 3.5, p = 0.005) and P. aeruginosa (HR: 2.8, p = 0.01). Among laboratory biomarkers, elevated procalcitonin (≥2 ng/mL, OR: 2.8, p = 0.008) and CRP (≥100 mg/L, OR: 2.2, p = 0.01) were significantly associated with ICU mortality. Additionally, patients who remained in the ICU for more than seven days had a 1.4-fold increased risk of mortality (p = 0.02), further emphasizing the impact of prolonged hospitalization on adverse outcomes. Conclusions: MDR pathogens, particularly A. baumannii, MRSA, P. aeruginosa, and K. pneumoniae, are associated with longer ICU stays and higher mortality rates. Carbapenem, cephalosporin, fluoroquinolone, and aminoglycoside resistance significantly impact clinical outcomes, emphasizing the urgent need for antimicrobial stewardship programs. ESBL, p-AmpC, and carbapenemase-producing Enterobacterales further worsen patient outcomes, highlighting the need for early infection control strategies and optimized empirical antibiotic selection. Biomarkers such as procalcitonin and CRP, alongside clinical severity scores, serve as valuable prognostic tools for ICU mortality. Full article

Extended-spectrum-β-lactamase (ESBL) and carbapenemase-producing Escherichia coli, Klebsiella pneumoniae and Acinetobacter spp. are associated with hospital-acquired infections and are commonly isolated across the poultry food production chain. Comprehensive data regarding the prevalence, spatiotemporal variations, and characterization of β-lactam-resistant bacteria in poultry farms and slaughterhouses is scarce. This study examines the prevalence and characteristics of β-lactam-resistant E. coli, K. pneumoniae, and Acinetobacter spp. isolated from poultry farms, slaughterhouses, and associated personnel in Greece. Strains were selectively isolated and identified via MALDI-TOF MS, which was also employed to identify possible relatedness. E. coli isolates were further classified into phylogenetic groups. The prevalence of β-lactam-resistant strains in farm and slaughterhouse environments was 15.0% (n = 15 strains)/57.3% (n = 71 strains) for E. coli, 11.0% (n = 11 strains)/1.6% (n = 2 strains) for K. pneumoniae, and 1.0% (n = 1 strain)/25.8% (n = 38 strains) for Acinetobacter spp., respectively. The prevalence of Acinetobacter spp. and E. coli on farmers’ skin was 16.7% (n = 2 strains) and 8.3% (n = 1 strain), correspondingly. Significantly higher E. coli isolation rates were observed in warmer seasons. All strains were multidrug-resistant and most carried ESBL/AmpC genes. Most E. coli isolates belonged to phylogroups A (41.4%, n = 36) and B1 (24.1%, n = 21). Proteomic analysis indicated relatedness among strains from different regions and seasons. Thus, poultry farms and slaughterhouses may serve as significant reservoirs of β-lactam-resistant strains of E. coli, K. pneumoniae, and Acinetobacter spp. Full article

Background/Objectives: Reptiles are known reservoirs for members of the Enterobacterales. We investigated antimicrobial resistance (AMR) patterns, the diversity of extended-spectrum-/AmpC-β-lactamases (ESBL/AmpC) genes and the genomic organization of the ESBL/AmpC producers. Methods: A total of 92 shipments with 184 feces, skin, and urinate samples of live healthy reptiles were obtained during border inspections at Europe’s most important airport for animal trade and screened for AMR bacteria by culture, antimicrobial susceptibility testing, and whole genome sequencing (WGS) of selected isolates. Results: In total, 668 Enterobacterales isolates with phenotypic evidence for extended-spectrum-/AmpC-β-lactamases (ESBL/AmpC) were obtained, from which Klebsiella (n = 181), Citrobacter (n = 131), Escherichia coli (n = 116), Salmonella (n = 69), and Enterobacter (n = 52) represented the most common groups (other genera (n = 119)). Seventy-nine isolates grew also on cefotaxime agar and were confirmed as ESBL (n = 39) or AmpC (n = 39) producers based on WGS data with respective genes localized on chromosomes or plasmids. Isolates of E. coli contained the most diverse set of ESBL genes (n = 29), followed by Klebsiella (n = 9), Citrobacter, and Enterobacter (each n = 1). Contrarily, AmpC genes were detected in E. coli and Citrobacter (n = 13 each), followed by Enterobacter (n = 12) and Klebsiella (n = 4). Isolates of Salmonella with ESBL/AmpC genes were not found, but all genera contained a variety of additional AMR phenotypes and/or genotypes. MLST revealed 36, 13, 10, and nine different STs in E. coli, Klebsiella, Citrobacter, and Enterobacter, respectively. Conclusions: A significant fraction of the studied Enterobacterales isolates possessed acquired AMR genes, including some high-risk clones. All isolates were obtained from selective media and also wild-caught animals carried many AMR genes. Assignment of AMR to harvesting modes was not possible. Full article

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 bla_CTX family were detected with other bla genes or alone; the most frequently observed β-lactamase gene was bla_CTX-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. Full article

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 bla_CTX-M and/or bla_TEM in ESBL-producing E. coli; bla_CMY-2 and the presence of putative low-level AmpC production in AmpC-producing E. coli; and bla_OXA-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. Full article

Background: Wastewater is a major source of Antibiotic-Resistant Bacteria (ARB) and a hotspot for the exchange of Antibiotic-Resistant Genes (ARGs). The occurrence of Carbapenem-Resistant Bacteria (CRB) in wastewater samples is a major public health concern. Objectives: This study aimed to analyze Antibiotic resistance in bacteria from wastewater sources in Pakistan. Methods: We analyzed 32 bacterial isolates, including 18 Escherichia coli, 4 Klebsiella pneumoniae, and 10 other bacterial isolates using phenotypic antibiotic susceptibility assay and whole-genome sequencing. This study identified the ARGs, plasmid replicons, and integron genes cassettes in the sequenced isolates. One representative isolate was further sequenced using Illumina and Oxford nanopore sequencing technologies. Results: Our findings revealed high resistance to clinically important antibiotics: 91% of isolates were resistant to cefotaxime, 75% to ciprofloxacin, and 62.5% to imipenem, while 31% showed non-susceptibility to gentamicin. All E. coli isolates were resistant to cephalosporins, with 72% also resistant to carbapenems. Sequence analysis showed a diverse resistome, including carbapenamases (blaNDM-5, blaOXA-181), ESBLs (blaCTX-M-15, blaTEM), and AmpC-type β-lactamases (blaCMY). Key point mutations noticed in the isolates were pmrB_Y358N (colistin) and ftsI_N337NYRIN, ftsI_I336IKYRI (carbapenem). The E. coli isolates had 11 different STs, with ST410 predominating (28%). Notably, the E. coli phylogroup A isolate 45EC1, (ST10886) is reported for the first time from wastewater, carrying blaNDM-5, blaCMY-16, and pmrB_Y358N with class 1 integron gene cassette of dfrA12-aadA2-qacEΔ1 on a plasmid-borne contig. Other carbapenamase, blaNDM-1 and blaOXA-72, were detected in K. pneumoniae 22EB1 and Acinetobacter baumannii 51AC1, respectively. The integrons with the gene cassettes encoding antibiotic resistance, and the transport and bacterial mobilization protein, were identified in the sequenced isolates. Ten plasmid replicons were identified, with IncFIB prevalent in 53% of isolates. Combined Illumina and Oxford nanopore sequencing revealed blaNDM-5 on an IncFIA/IncFIC plasmid and is identical to those reported in the USA, Myanmar, and Tanzania. Conclusions: These findings highlight the environmental prevalence of high-risk and WHO-priority pathogens with clinically important ARGs, underscoring the need for a One Health approach to mitigate ARB isolates. Full article