Predominance of enterotoxigenic Escherichia coli among ESBL/plasmid-mediated AmpC-producing strains isolated from diarrheic foals: a public health concern.

BACKGROUND: The upsurge of diarrheagenic E. coli pathotypes carrying extended-spectrum beta-lactamases (ESBLs)/plasmid-mediated AmpC ß-lactamase (pAmpC) among animals constitutes an emerging threat for humans and animals. This study investigated the burden of ESBL-/pAmpC-producing diarrheagenic E. coli among diarrheic foals and its potential public health implications. Rectal swabs were collected from 80 diarrheic foals. These swabs were processed to isolate and identify ESBL/pAmpC-producing E. coli using a selective culture medium, biochemical tests, phenotypic identification, and molecular identification of ESBL- and pAmpC-encoding genes. Moreover, all ESBL-/pAmpC-producing E. coli isolates were examined for different virulence genes related to diarrheagenic E. coli pathotypes. RESULTS: Out of 80 examined foals, 26 (32.5%) were confirmed as ESBL-/pAmpC-producing E. coli, of which 14 (17.5%) animals carried only ESBL-producing E. coli, whereas 12 (15%) animals possessed ESBL-pAmpC-producing E. coli. The only detected diarrheagenic pathotype was enterotoxigenic, encoded by the heat-stable enterotoxin gene (ST) with a prevalence rate of 80.8% (21/26). The ST gene was further characterized where STa, STb, and STa + STb were found in one, four, and 16 strains, respectively. Moreover, all enterotoxigenic E. coli (ETEC) isolates exhibited a multidrug-resistance pattern. The phylogenetic analysis of 3 obtained partial STb sequences revealed high genetic relatedness to ETEC isolates retrieved from humans, conferring such sequences' public health significance. CONCLUSIONS: These findings highlight that diarrheic foals could serve as a potential reservoir for multidrug-resistant ESBL-/pAmpC-producing enterotoxigenic E. coli.

Hazard characterization of antibiotic-resistant Aeromonas spp. isolated from mussel and oyster shellstock available for retail purchase in Canada.

Surveillance and monitoring of foods for the presence of antimicrobial-resistant (AMR) bacteria is required to assess the risks these bacteria pose to human health. Frequently consumed raw or lightly cooked, live bivalve shellfish such as mussels and oysters can be a source of exposure to AMR bacteria. This study sought to determine the prevalence of third generation cephalosporin (3GC) and carbapenem resistant bacteria in live mussel and oyster shellstock available for retail purchase through the course of one calendar year. Just over half of the 180 samples (52%) tested positive for the presence 3GC-resistant bacteria belonging to thirty distinct bacterial species. Speciation of the isolates was carried out using the Bruker MALDI Biotyper. Serratia spp., Aeromonas spp., and Rahnella spp. were the most frequently isolated groups of bacteria. Antibiotic resistance testing confirmed reduced susceptibility for 3GCs and/or carbapenems in 15 of the 29 Aeromonas isolates. Based on AMR patterns, and species identity, a sub-set of ten Aeromonas strains was chosen for further characterization by whole genome sequence analysis. Genomic analysis revealed the presence of multiple antibiotic resistance and virulence genes. A number of mobile genetic elements were also identified indicating the potential for horizontal gene transfer. Differences in gene detection by the bioinformatic tools and databases used (ResFinder. CARD RGI, PlasmidFinder, and MobSuite) are discussed. This study highlights the strengths and limitations of using genomics tools to perform hazard characterization of diverse foodborne bacterial species.

Association between the presence of CRISPR-Cas system genes and antibiotic resistance in Klebsiella pneumoniae isolated from patients admitted in Ahvaz teaching hospitals.

BACKGROUND: This study aims to investigate the frequency of cas1 and cas3 and CRISPR1,2,3 genes in Klebsiella pneumoniae isolates, as well as their connection with antibiotic resistance. MATERIALS AND METHODS: 106 K. pneumoniae isolates were identified by biochemical assays and PCR. The susceptibility to antibiotics was determined by Kirby-Bauer disk diffusion method. Screening of ESBLs was undertaken by using double disk diffusion and standard disk diffusion methods. The E-test and mCIM techniques was used to confirm the disc diffusion-based carbapenem resistance profiles. CRISPR-Cas system genes were identified using PCR. RESULTS: ESBL production was found in 19% of isolates. Carbapenemase production was found in 46% of the isolates. Furthermore, the bacteria were classified as multidrug (76%), extensively drug-resistant (4%), or pan-drug-resistant (2%). When CRISPR/Cas systems were present, antibiotic resistance was lower; conversely, when they were absent, resistance was higher. CONCLUSIONS: If the CRISPR/Cas modules aren't present, the bacteria can still acquire foreign DNA, including antibiotic resistance genes. K. pneumoniae isolates with a CRISPR-Cas system were less likely to carry antibiotic-resistance genes than those lacking this defense system.

Antibiotic Susceptibility of Aerobic and Facultative Anaerobic Gram-Negative Rods in Hong Kong and Implications on Usefulness of Ceftazidime-Avibactam and Ceftolozane-Tazobactam.

Due to the increasing resistance of aerobic and facultative anaerobic Gram-negative rods, ceftazidime-avibactam and ceftolozane-tazobactam have been launched in the market in the last few years. In this study, we analyzed the susceptibility pattern of the major aerobic and facultative anaerobic Gram-negative rods in Hong Kong for ceftazidime-avibactam, ceftolozane-tazobactam, four other broad-spectrum antibiotics commonly used in Hong Kong and colistin. For 300 isolates collected from January to December 2021, non-ESBL-producing Enterobacterales, ESBL-producing Enterobacterales and Pseudomonas aeruginosa were highly susceptible to ceftazidime-avibactam (all 100%) and ceftolozane-tazobactam (98.7%, 99.7% and 94.3%). For 32 archived ESBL-producing Klebsiella pneumoniae isolates collected between January 2014 and March 2023, all were susceptible to ceftazidime-avibactam and ceftolozane-tazobactam. For 101 archived carbapenemase-producing Enterobacterales, their susceptibilities to ceftazidime-avibactam and ceftolozane-tazobactam varied depending on the type of carbapenemase produced. Both had high activities against OXA-producing strains (97.1% and 76.5%, respectively) but were 100% resistant for NDM-producing and NDM+OXA-producing strains. All KPC-producing strains were susceptible to ceftazidime-avibactam but resistant to ceftolozane-tazobactam. Ceftazidime-avibactam and ceftolozane-tazobactam are good alternatives for the management of infections caused by ESBL-producing Enterobacterales and selective strains of carbapenemase-producing Enterobacterales in Hong Kong.

Avian Pathogenic Escherichia coli: An Overview of Infection Biology, Antimicrobial Resistance and Vaccination.

Avian Pathogenic Escherichia coli (APEC) is an extraintestinal pathotype of E. coli that leads to a range of clinical manifestations, including respiratory, systemic and reproductive infections of chickens in both egg and meat production. Unlike most E. coli pathotypes, APEC is not defined by specific virulence genes but rather is a collection of several distinct genotypes that can act as both primary and secondary pathogens leading to colibacillosis. Recent measures to reduce antimicrobials both as growth promoters and as flock-level therapeutics are considered to have led to increased numbers of animals affected. Nevertheless, antimicrobial resistance is a considerable problem in APEC, with resistance to third and fourth-generation cephalosporins via extended-spectrum beta-lactamases (ESBLs), fluoroquinolones and colistin seen as a particular concern. The need to control APEC without antimicrobial use at the flock level has seen an increased focus on vaccination. Currently, a few commercial vaccines are already available, and a range of approaches are being applied to develop new vaccines, and other controls, such as bacteriophage or probiotics, are attracting interest. The lack of a single defined APEC genotype presents challenges to these approaches.

Genetic Characterization of Extended-Spectrum Beta-Lactamase (ESBL) and Metallo-Beta-Lactamase (MBL) Producing Klebsiella pneumoniae from Diabetic Foot Ulcer (DFU).

Background: Antibiotic resistance in common pathogenic bacteria is linked with the genetic makeup. The genetic basis of antibiotic resistance may vary in different species or pathophysiological conditions. Objectives: We studied the antibiotic resistance in Klebsiella pneumonia isolates from DFU in the western Indian population. We also studied the presence of ESBL and MBL mechanisms of antibiotic resistance along with the prevalence of the genes involved in ESBL (TEM ESBL , SHV ESBL , and CTX-M ESBL ) and MBL (NDM-1 bla , KPC bla , OXA-48 bla , and VIM bla ) production. Results: A total of 161 K. pneumoniae isolates were analyzed; among which 50.93% were positive for ESBL and 45.96% were positive for MBL production. Most of the isolates were resistant to antibiotics used in the present study and partially resistant to Imipenem and Amikacin. There was no relation between the antibiotic resistance of the isolates and the production of ESBL or MBL mechanism of antibiotic resistance. Further, TEM ESBL was the most prevalent gene in K. pneumoniae isolates followed by CTX-M ESBL , NDM-1 bla , SHV ESBL , and KPC bla . VIM bla was the least prevalent gene found in K. pneumoniae isolates. There was no difference in the prevalence of the genes with respect to the presence or absence of ESBL and MBL mechanism of resistance. Further, there was no relation between the prevalence of the genes and antibiotic resistance in K. pneumoniae isolates. Conclusion: These results along with the literature review suggest that the prevalence of the genes involved in antibiotic resistance mechanisms are widespread in India and their distribution varies in different studies.

Molecular insights into the evolutionary trajectory of a Klebsiella aerogenes clinical isolate with a complex trade-off between resistance and virulence.

The fitness cost associated with antimicrobial resistance has an important influence on evolutionary dynamics. We compared the genomes of three Klebsiella aerogenes isolates recovered from blood samples or deep abscess cultures from the same patient: the wild-type strain (CT_WT), a piperacillin-tazobactam-resistant strain (CT_PENI), and an extended-spectrum-cephalosporin (ESC)-resistant strain (CT_R). Whole-genome sequencing revealed that CT_PENI had acquired a TEM-1 ß-lactamase with a mutated promoter, accounting for overproduction. CT_PENI then acquired an E240G substitution in the TEM-1 ß-lactamase (resulting in TEM-207) and lost the porin-encoding ompK36 gene to give CT_R. All three strains showed the same virulence in a mouse model of intraperitoneal infection. The results of recombination and transformation assays indicated that when present separately, the TEM-207 overproduction and the ompK36 gene deletion had only small effects on susceptibility to ESCs. However, the combination of the two changes led to a much lower susceptibility to ESCs. Moreover, the levels of fitness in vitro and in vivo in a murine model of gut colonization were significantly lower after TEM-1 ß-lactamase overproduction and lower still after E240G substitution and OmpK36 loss. We hypothesize that the chosen courses of antibiotics led to the stepwise emergence of a clone with resistance to penicillins and ESCs and no loss of virulence. However, acquired resistance may have a fitness cost that limits evolutionary success. Our results might explain why the overproduction of extended-spectrum ß-lactamases (which should confer a high level of piperacillin-tazobactam resistance) is not observed in clinical practice and why TEM-207 has rarely been detected in clinical isolates.

Microbiological Quality of Coconut Water Sold in the Grande Vitória Region, Brazil, and Phenogenotypic Antimicrobial Resistance of Associated Enterobacteria.

This study aimed to evaluate the microbiological quality of coconut water sold from street carts equipped with cooling coils or refrigerated at bakeries in the Grande Vitória Region, Brazil. Additionally, it assessed the phenotypic and genotypic antimicrobial resistance profiles of isolated enterobacteria. The results indicated that coconut water sold at street carts had lower microbiological quality compared to refrigerated samples, as evidenced by significantly higher counts of mesophilic microorganisms. Using MALDI-TOF, the following opportunistic pathogens were identified: Citrobacter freundii, Enterobacter bugandensis, E. kobei, E. roggenkampii, Klebsiella pneumoniae, and Kluyvera ascorbata. Three isolates-E. bugandensis, K. pneumoniae, and K. ascorbata-were classified as multidrug-resistant (MDR). Widespread resistance to ß-lactams and cephalosporins was detected, and some isolates were resistant to quinolones, nitrofurans, and phosphonic acids. The gene blaCTX-M-2 was detected in C. freundii, E. bugandensis, E. kobei, and K. ascorbata. However, genes blaNDM, blaKPC, blaCMY-1, and blaCMY-2 were not detected in any isolate. The findings underscore the need to enhance good manufacturing practices in this sector to control the spread of antimicrobial resistance (AMR). To our knowledge, this is the first study documenting the presence of potentially pathogenic enterobacteria in coconut water samples and their associated phenotypic and genotypic AMR profiles.

Detection of Salmonella Mbandaka Carrying the blaCTX-M-8 Gene Located on IncI1 Plasmid Isolated from a Broiler Flock Environment.

Salmonella Mbandaka is one of the most globally widespread serovars, occurring in many sources and included among twenty serovars that contribute to human salmonellosis in Europe. In Poland, it has been noted in non-human sources since 1996, being found firstly in feeds and later in waterfowl and chicken. Over the years, it gained epidemiological importance, being isolated from a wide range of animal species, including livestock. Generally, it is characterized by sensitivity to most antimicrobials and the ability to form biofilms. The occurrence of cephalosporin-resistant Salmonella in non-human sources is an extremely rare phenomenon in Poland. In this report, we characterized the full genome of the ESBL-producing S. Mbandaka strain isolated from a broiler farm environment (boot swab sample) in Poland in 2022. The isolate was serotyped as S. Mbandaka according to the White-Kaufmann-Le Minor scheme. Antimicrobial susceptibility testing performed with the microbroth dilution method showed its resistance to ampicillin, cefotaxime, ceftazidime, ciprofloxacin, and nalidixic acid. The whole-genome sequence was reconstructed using short and long reads and assembled into the complete chromosome and three plasmids: IncI1 pST113 (89,439 bp), Col(pHAD28) (2699 bp), and Col440 (2495 bp). The strain belonged to sequence type ST413. Plasmid analysis showed blaCTX-M-8 mobilization on IncI1(alpha) surrounded with insertion sequences. The analyzed genome content draws attention to the possibility of the horizontal spread of the resistance genes. To the best of our knowledge, this is the first report of blaCTX-M-8-positive Salmonella in Poland.

Convergence of mcr-1 and broad-spectrum ß-lactamase genes in Escherichia coli strains from the environmental sector.

The mcr-type gene encodes the main plasmid-mediated mechanism of colistin resistance and has been reported in several bacterial species obtained from different sources. Anthropogenic activities in the environment favor the evolution of antimicrobial resistance. Indeed, mcr-1-positive Escherichia coli strains were susceptible to non-polymyxins antimicrobials, but now emerging as multidrug-resistant (MDR) lineages. In this regard, hundreds of surface water and agricultural soil samples were screened for the presence of E. coli carrying the mcr-type genes and mcr-1-positive strains were subjected to in-depth genomic analysis. Almost all colistin-resistant strains were classified as MDR, highlighting those obtained from soils that showed resistance to extended-spectrum cephalosporins and carbapenems. International and high-risk clones of E. coli were identified, with ST10 and ST1720 shared between water and soil samples. Resistome analysis showed a broad resistome (AMR, metal tolerance, and biocide resistance). The mcr-1.1 and mcr-1.26 allelic variants were detected on IncX4 and IncI2 plasmids. Curiously, mcr-1-positive E. coli strains from agricultural soils harbored plasmid-mediated blaCTX-M-1, blaCTX-M-8, or blaKPC-2 genes. Virulome analysis demonstrated traits of a high putative virulence potential, with the presence of extraintestinal pathogenic E. coli. Comparative analysis revealed the persistence and dissemination of plasmid-mediated antimicrobial resistance genes in genetically diversity E. coli strains at the human-animal-environmental interface. These findings demonstrate a possible emerging AMR trend with the convergence of resistance to colistin and broad-spectrum ß-lactams in environmental-derived E. coli strains.

Transmission dynamics of ESBL/AmpC and carbapenemase-producing Enterobacterales between companion animals and humans.

Antimicrobial resistance mediated by extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales, as well as carbapenemase-producing Enterobacterales have globally increased among companion animals, posing a potential health risk to humans in contact with them. This prospective longitudinal study investigates the transfer of ESBL/AmpC- and carbapenemase-producing Enterobacterales between companion animals and their cohabitant humans in Portugal (PT) and the United Kingdom (UK) during animal infection. Fecal samples and nasal swabs collected from dogs and cats with urinary tract infection (UTI) or skin and soft tissue infection (SSTI), and their cohabitant humans were screened for resistant strains. Relatedness between animal and human strains was established by whole-genome sequencing (WGS). ESBL/AmpC-producing Enterobacterales were detected in companion animals (PT = 55.8%; UK = 36.4%) and humans (PT = 35.9%; UK = 12.5%). Carbapenemase-producing Enterobacterales carriage was observed in one dog from Portugal (2.6%) and another dog from the UK (4.5%). Transmission of index clinical ESBL-producing Escherichia coli and Klebsiella pneumoniae strains to cohabitant humans was observed in three Portuguese households (6.9%, n = 43), with repeated isolation of the index strains on fecal samples from the animals and their cohabiting humans. In addition, longitudinal sharing of E. coli strains carried by companion animals and their owners was observed in other two Portuguese households and two households from the UK. Furthermore, a multidrug-resistant ACT-24-producing Enterobacter hormaechei subsp. hoffmannii strains were also shared within another Portuguese household. These results highlight the importance of the household as an epidemiological unit in the efforts to mitigate the spread of antimicrobial resistance, further emphasizing the need for antimicrobial surveillance in this context, capable of producing data that can inform and evaluate public health actions.

New Insights Into The Antibacterial Activity Of Hydroxytyrosol Extracted From Olive Leaves: Molecular Docking Simulations Of Its Antibacterial Mechanisms.

This study investigated the biological activities of a hydroxytyrosol-rich extract from Oleaeuropaea leaves, particularly its ability to eradicate severe pathogenic bacteria producing Extended-Spectrum Beta-Lactamases (ESBLs). The latter bacteria are emerging microorganisms that pose significant challenges due to their resistance to a broad range of potent therapeutic drugs. The extract was prepared through an accessible acid hydrolysis method. In vitro and In silico analyses through MIC, MBC analysis and molecular docking were conducted to evaluate the antibacterial properties. The extract showed remarkable antioxidant activity and significant antibacterial potential against reference species and ESBL bacteria. MIC and MBC calculations confirmed the extract's capacity to kill bacteria rather than just inhibit their growth. Further in silico analyzes demonstrated the high binding affinity of HT to the active sites of the gyrase B subunit and the peptidoglycan DD-transpeptidase domain from proteins located in the cytoplasm and the cell wall of the bacteria, respectively. Results confirmed the structure-activity relationship and the ability of HT to disrupt essential bacterial functions. This study validates the debated antimicrobial potential of HT and highlights its importance as a potential therapeutic agent against resistant bacteria, which is a critical area of research given the global challenge of antibiotic resistance.

Antibiotic resistant Escherichia coli strains in healthy pets from Tamaulipas, Mexico.

Antibiotic resistance constitutes a significant public health challenge, with diverse reservoirs of resistant bacteria playing pivotal roles in their dissemination. Among these reservoirs, pets are carrying antibiotic-resistant strains. The objective of this study was to assess the resistance profiles of Escherichia coli, and the prevalence of extended-spectrum ß-lactamase (ESBL) producing E. coli strains in dogs and cats from Tamaulipas, Mexico. A total of 300 stool samples (150 dogs and 150 cats) from healthy pets were subjected to analysis. Antibiotic susceptibility testing and the identification of ESBLs were carried out by disc diffusion method. The presence of resistance genes, class 1, 2, and 3 integrons (intI1, intI2, and intI3) and phylogroups was determined by PCR analysis. The findings reveal that 42.6% (128/300) of the strains exhibited resistance to at least one of the eight antibiotics assessed, and 18.6% (56/300) demonstrated multidrug resistance (MDR), that distributed across 69 distinct resistance patterns. Altogether 2.6% of E. coli strains (8/300) were confirmed as TEM and CTX-M type ESBL producers. These outcomes underscore the roles of dogs and cats in Tamaulipas as reservoirs for the dissemination of MDR and/or ESBL strains. The results underscore the necessity for conducting prevalence studies on ESBL-producing E. coli, forming a foundation for comprehending the present scenario and formulating strategies for the control and mitigation of this issue.

The role of colonization with resistant Gram negative bacteria in the treatment of febrile neutropenia after stem cell transplantation.

INTRODUCTION: Febrile neutropenia (FN) is one of the most common complications of stem cell transplantation. The aim of this analysis was to evaluate the frequency of sepsis in patients with FN colonized with resistant Gram negative bacteria (Extended spectrum ß-lactamase positive, multidrug resistant (MDR) P. aeruginosa) and the choice of primary antibiotic in colonized patients. PATIENTS AND METHODS: This was a retrospective study analyzed data from patients who underwent hematopoietic stem cell transplantation from 01/2018 to 09/2022. Data were extracted from the hospital information system. RESULTS: Carbapenem as the primary antibiotic of choice was chosen in 10.9% of non-colonized +/-AmpC patients, 31.5% of ESBL+ patients, and 0% of MDR P. aeruginosa patients. Patients with FN and MDR P. aeruginosa colonization had a high prevalence of sepsis (namely 100%, p = 0.0197). The spectrum of sepsis appeared to be different, with Gram negative bacilli predominating in the ESBL+ group (p = 0.0123, OR 5.39 [95% CI 1.55-18.76]). Colonizer sepsis was present in 100% of sepsis with MDR P. aeruginosa colonization (p=0.002), all in allogeneic transplantation (p=0.0003), with a mortality rate of 33.3% (p=0.0384). The incidence of sepsis in patients with ESBL+ colonization was 25.9% (p=0.0197), with colonizer sepsis in 50% of sepsis cases (p=0.0002), most in allogeneic transplantation (p=0.0003). CONCLUSION: The results show a significant risk of sepsis in FN with MDR P. aeruginosa colonization, this state is almost exclusively caused by the colonizer. At the same time, a higher risk of Gram negative sepsis has been demonstrated in patients colonized with ESBL+ bacteria.

Development of a Multiplex PCR Assay for the Detection of Extended-Spectrum Beta-Lactamase Genes in Acinetobacter Baumannii Isolates in Tehran City, Iran.

Extended­spectrum ß­lactamase (ESBL) genes are responsible for creating Multidrug­resistant and Extensive drug resistance (XDR) patterns in Acinetobacter baumanii isolates, so limit treatment options and increase mortality and morbidity. This study aimed to development of a multiplex PCR assay for the detection of extended-spectrum beta-lactamase genes including bla CTX-M, bla SHV and bla TEM among clinical samples of Acinetobacter baumanii isolates in Tehran, Iran. In present study, 100 clinical Acinetobacter baumannii strains have been gathered from patients in Motahhari hospital in Tehran city, Iran. Antibiotic susceptibility test was conducted by Kirby-Bauer disc diffusion method. To identify ESBL-producing strains, used combined disk test and Multiplex PCR method was used for Simultaneous diagnosis of bla CTX-M, bla SHV, and bla TEM genes. Out of 100 isolates, 93% were ESBL-positive according to the phenotypic test. Most of the isolates were XDR and the highest sensitivity was for colistin. The frequency of bla CTX-M, bla SHV and bla TEM genes was 95, 1, and 2% respectively. The high percentage of antibiotic resistance and high prevalence of the bla CTX-M gene in A. baumannii isolates is a serious threat to the effectiveness of available antibiotics. This study showed Multiplex PCR can be a reliable and sensitive technique for the fast detection of ESBL genes in Acinetobacter baumannii isolates.

First detection of VEB-1 extended-spectrum ß-lactamase-producing Escherichia coli clinical isolate in Japan.

The extended-spectrum ß-lactamase (ESBL) gene, blaVEB-1, was identified for the first time in an Escherichia coli clinical isolate, JARB-RN-0061, from blood cultures in a Japanese general hospital in 2021. The isolate exhibited high resistance to broad-spectrum cephalosporins, including ceftazidime (MIC >128 mg/L) and cefepime (MIC = 16 mg/L). blaVEB-1 was identified during whole-genome sequencing and characterization of the isolate. JARB-RN-0061 belonged to the B2-O2:K1:H7-ST95-fimH41 lineage and was classified as presumptive extraintestinal pathogenic E. coli (ExPEC) and uropathogenic E. coli (UPEC). Moreover, the strain harbored multiple virulence genes on the chromosome. The Col156/IncFIB(AP001918)/IncFII(29)-type plasmid (114,216 bp), with clbB and tcpC genes involved in bacteremia, was unique to the fimH41 subclone. The blaVEB-1 gene was located on a non-typeable and non-conjugative plasmid, pJARB-RN-0061_VEB-1 (17,093 bp). It was embedded in the class 1 integron In1883-like, with multidrug resistance gene cassettes for aacA4, aadB, cmlA5, qnrVC4, and dfrA14. Notably, comparative analysis of the complete sequence of plasmid pJARB-RN-0061_VEB-1 revealed that it was highly homologous to the blaVEB-1-harboring plasmid, pMS2H7VEB-1 (100% coverage and 99.99% identity), except for the Tn3 family transposon (4,931 bp) and the plasmid pRHBSTW-00138_5 (97% coverage and 100% identity) harbored by Klebsiella quasipneumoniae subsp. similipneumoniae strains from hospital sewage in Japan and wastewater influent in the United Kingdom, respectively. The emergence of a human pathogenic E. coli clinical isolate with the blaVEB-1-carrying plasmid in the B2-ST95 worldwide pandemic lineage, characterized by the virulence potential of ExPEC or UPEC but a low prevalence of antimicrobial resistance, would raise public health concerns. IMPORTANCE: ESBLs are plasmid-mediated enzymes that confer resistance to clinically significant antimicrobial agents, such as broad-spectrum cephalosporins. Recently, the rapid spread of CTX-M-type ESBL-producing E. coli has become a global issue, including in Japan, where ESBL production in human pathogenic E. coli, such as the ExPEC and UPEC lineages, which typically harbor several virulence genes, is a severe public health concern. To date, VEB (Vietnamese extended-spectrum ß-lactamase) producers have been found only in hospital wastewater and rivers in Japan. Thus, we describe the first detection of a very rare human-derived blaVEB-1 gene in the E. coli B2-ST95 pandemic clonal lineage that is highly associated with ExPEC and UPEC in a Japanese clinical setting. Furthermore, we characterized the genomic features of plasmids harboring the class 1 integron-borne blaVEB-1. Our findings highlight the significance of closely monitoring ESBL-producing E. coli isolates to prevent the potential dissemination of this resistance determinant in Japan.

ESBL Producing E. coli in Chickens and Poultry Farms Environment in Selangor, Malaysia: A Cross-Sectional Study on Their Occurrence and Associated Risk Factors With Environment and Public Health Importance.

INTRODUCTION: Extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) are widespread multidrug-resistant zoonotic bacteria that threaten animal production, food safety and antimicrobial therapy worldwide including Malaysia. Poultry has been reported as one of the pathways for human exposure to ESBL-EC. There has been little research on the occurrence of ESBL-EC within the Malaysian poultry food chain. Hence, the objectives of the study were to determine the occurrence of ESBL-EC in chickens and to identify the potential risk factors associated with their occurrence in poultry farms. METHODS: A cross-sectional study was conducted on 400 samples, consisting of 240 cloacal swabs from chickens and 160 from poultry farms environments in eight districts in Selangor, Malaysia using culture and disk combination methods and multiple polymerase chain reaction assays. In the determination of possible factors associated with the presence of ESBL-EC at poultry farms, a questionnaire was used to obtain the information and data. RESULTS: The findings demonstrated the wide distribution of ESBL-EC in all the farms with an overall occurrence of 37.2%. Farms in Gombak, Klang and Hulu Selangor had the highest occurrence rates at 62%, 50% and 50%, respectively, followed by farms in Petaling 38%, Sepang at 34%, Kuala Langat at 26% and Kuala Selangor at 24%, and the lowest was in Hulu Langat 14%. Among the study samples, chickens had the highest occurrence rate at 45.4%, followed by chicken house floors at 40% and flies at 30%, while feed and water samples at 17.5% and 12.5%, respectively. The present study indicated the high occurrence and wide dissemination of ESBL-EC in chickens and poultry farms environment. The ESBL-EC occurrence was associated with several factors including imprudent use of antibiotics, poor husbandry, management and biosecurity practices at the farms. CONCLUSIONS: Our study showed the presence and spread of ESBL EC among chickens in the farms and their environment; this may lead to being spread to outside of farm environment by flies, vermins, flying birds, litter and farm wastes and possibly to humans upon contact with the contaminated environment and by poultry meat. Thus, the findings of the study can assist to serve as a piece of useful information to veterinary authority in designing evidence-based mitigation strategies for the control of ESBL-EC in poultry farms.

Sensitivity and specificity of Nanopore sequencing for detecting carbapenem and 3rd-generation cephalosporin-resistant Enterobacteriaceae in urine samples: Real-time simulation with public antimicrobial resistance gene database.

Objectives: To evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing. Methods: DNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST). Results: We processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (<1 ng/µl) for a final case of 53 in total. The sensitivity of antimicrobial resistance gene detection within 6 h, the optimal duration from real-time simulation, of Nanopore sequencing for the diagnosis of carbapenem-resistant and ceftriaxone-resistant phenotypes was 73.9 % (95%CI 56.0-91.9 %) and 81.1 % (95%CI 68.5-93.7 %), while the specificity was 96.7 % (95%CI 90.2-100.0 %) and 56.3 % (95%CI 31.9-80.6 %), respectively. The median times for MinION to generate DNA reads containing carbapenemase and ESBL/AmpC genes were 93 min (IQR 17-245.5) and 99 min (IQR 31.25-269.75) after sequencing commencement, respectively. Conclusions: Nanopore sequencing can identify bacterial genotypic resistance in urine and may enable clinicians to adjust antimicrobial therapy earlier than routine AST.

Evaluation of virulence determinants and cell surface properties associated with biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) Escherichia coli from livestock and poultry origin.

Antibiotic resistance poses a persistent threat to modern medicine due to the emergence of novel antibiotic-resistant strains. Therefore, a timely understanding of antibiotic resistance and the virulence biology of pathogenic bacteria, particularly those of public health significance, is crucial for implementing effective mitigation strategies. This study aimed to investigate the virulence profiles of ten S. aureus isolates (NDa to NDj) and ten E. coli isolates (ND1 to ND10) originating from livestock and poultry, and to assess how various cell surface properties and biofilm formation abilities influence antibiotic resistance phenotypes. Antibiotic resistance profiling through phenotypic (AST) and genotypic methods (PCR) confirmed that NDa to NDe were methicillin-resistant S. aureus (MRSA) and ND1 to ND5 were extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Virulence properties such as hemolytic activity, coagulase activity, and nuclease activity were found to be independent of the antibiotic resistance phenotype in S. aureus. In contrast, biofilm formation phenotype was observed to influence antibiotic resistance phenotypes, with MRSA and ESBL E. coli isolates demonstrating higher biofilm formation potency. Chemical and enzymatic analysis of S. aureus and E. coli biofilms revealed proteins and polysaccharides as major components, followed by nucleic acids. Furthermore, cell surface properties such as auto-aggregation and hydrophobicity were notably higher in isolates with strong to medium biofilm-forming capabilities (ESBL and MRSA isolates), corroborated by genomic confirmation of various genes associated with biofilm, adhesion, and colonization. In conclusion, this study highlights that surface hydrophobicity and biofilm formation ability of MRSA (NDa to NDe) and ESBL E. coli (ND1 to ND5) isolates may influence antibiotic resistance phenotypes.

Enteropathogenic and Multidrug-Resistant blaCTX-M-Carrying E. coli Isolates from Dogs and Cats.

Enteropathogenic Escherichia coli (EPEC) are pathogens associated with gastrointestinal illnesses. Dogs and cats can harbor EPEC, and antimicrobial resistance may impair necessary treatments. This study characterized E. coli strains from dogs and cats, focusing on phylogroup classification, virulence factors, and antimicrobial resistance profiles. Ninety-seven E. coli isolates from fecal samples of 31 dogs and 3 cats were obtained from a private diagnostic laboratory in Botucatu, Brazil, from March to October 2021. The antimicrobial susceptibility was assessed using the disk diffusion method. Polymerase chain reaction (PCR) was employed to screen for blaCTX-M and genes encoding virulence factors, as well as to classify the isolates into phylogroups. Twenty isolates were positive for intimin encoding gene eae and, consequently, these isolates were classified as EPEC (20.62%). Notably, 5.1% (5/97) of the isolates exhibited extended-spectrum ß-lactamase (ESBL) production and 13.4% (13/97) were identified as multidrug-resistant bacteria. Phylogroups A and B2 were the most prevalent, comprising 29.9% (29/97) and 26.8% (26/97) of the bacterial isolates, respectively. This characterization highlights the prevalence of EPEC in domestic animals, emphasizing the potential risk they pose to public health and highlighting the urgency of responsible antimicrobial use in veterinary practices and the important role of laboratories in the surveillance of pathogenic multidrug-resistant bacteria.