IncC plasmid genome rearrangements influence the vertical and horizontal transmission tradeoff in Escherichia coli.

It has been shown that an evolutionary tradeoff between vertical (host growth rate) and horizontal (plasmid conjugation) transmissions contributes to global plasmid fitness. As conjugative IncC plasmids are important for the spread of multidrug resistance (MDR), in a broad range of bacterial hosts, we investigated vertical and horizontal transmissions of two multidrug-resistant IncC plasmids according to their backbones and MDR-region rearrangements, upon plasmid entry into a new host. We observed plasmid genome deletions after conjugation in three diverse natural Escherichia coli clinical strains, varying from null to high number depending on the plasmid, all occurring in the MDR region. The plasmid burden on bacterial fitness depended more on the strain background than on the structure of the MDR region, with deletions appearing to have no impact. Besides, we observed an increase in plasmid transfer rate, from ancestral host to new clinical recipient strains, when the IncC plasmid was rearranged. Finally, using a second set of conjugation experiments, we investigated the evolutionary tradeoff of the IncC plasmid during the critical period of plasmid establishment in E. coli K-12, by correlating the transfer rates of deleted or non-deleted IncC plasmids and their costs on the recipient strain. Plasmid deletions strongly improved conjugation efficiency with no negative growth effect. Our findings indicate that the flexibility of the MDR-region of the IncC plasmids can promote their dissemination, and provide diverse opportunities to capture new resistance genes. In a broader view, they suggest that the vertical-horizontal transmission tradeoff can be manipulated by the plasmid to improve its fitness.

Emergence of a clinical Salmonella enterica serovar 1,4,[5], 12: i:-isolate, ST3606, in China with susceptibility decrease to ceftazidime-avibactam carrying a novel blaCTX-M-261 variant and a blaNDM-5.

PURPOSE: The detection rate of Salmonella enterica serovar 1,4,[5], 12: i: - (S. 1,4,[5], 12: i: -) has increased as the most common serotype globally. A S. 1,4,[5], 12: i: - strain named ST3606 (sequence type 34), isolated from a fecal specimen of a child with acute diarrhea hospitalized in a tertiary hospital in China, was firstly reported to be resistant to carbapenem and ceftazidime-avibactam. The aim of this study was to characterize the whole-genome sequence of S. 1,4,[5], 12: i: - isolate, ST3606, and explore its antibiotic resistance genes and their genetic environments. METHODS: The genomic DNA of S. 1,4,[5], 12: i: - ST3606 was extracted and performed with single-molecule real-time sequencing. Resistance genes, plasmid replicon type, mobile elements, and multilocus sequence types (STs) of ST3606 were identified by ResFinder 3.2, PlasmidFinder, OriTfinder database, ISfinder database, and MLST 2.0, respectively. The conjugation experiment was utilized to evaluate the conjugation frequency of pST3606-2. Protein expression and enzyme kinetics experiments of CTX-M were performed to analyze hydrolytic activity of a novel CTX-M-261 enzyme toward several antibiotics. RESULTS: Single-molecule real-time sequencing revealed the coexistence of a 109-kb IncI1-Iα plasmid pST3606-1 and a 70.5-kb IncFII plasmid pST3606-2. The isolate carried resistance genes, including blaNDM-5, sul1, qacE, aadA2, and dfrA12 in pST3606-1, blaTEM-1B, aac(3)-lld, and blaCTX-M-261, a novel blaCTX-M-1 family member, in pST3606-2, and aac(6')-Iaa in chromosome. The blaCTX-M-261 was derived from blaCTX-M-55 by a single-nucleotide mutation 751G>A leading to amino acid substitution of Val for Met at position 251 (Val251Met), which conferred CTX-M increasing resistance to ceftazidime verified by antibiotics susceptibility testing of transconjugants carrying pST3606-2 and steady-state kinetic parameters of CTX-M-261. pST3606-1 is an IncI1-α incompatibility type that shares homology with plasmids of pC-F-164_A-OXA140, pE-T654-NDM-5, p_dm760b_NDM-5, and p_dmcr749c_NDM-5. The conjugation experiment demonstrated that pST3606-2 was successfully transferred to the Escherichia coli recipient C600 with four modules of OriTfinder. CONCLUSION: Plasmid-mediated horizontal transfer plays an important role in blaNDM-5 and blaCTX-M-261 dissemination, which increases the threat to public health due to the resistance to most ß-lactam antibiotics. This is the first report of blaCTX-M-261 and blaNDM-5 in S. 1,4,[5], 12: i: -. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of CTX-M enzymes and confirms urgency to control resistance of S. 1,4,[5], 12: i: -.

Prevalence of streptomycin and tetracycline resistance and increased transmissible third-generation cephalosporin resistance in Salmonella enterica isolates derived from food handlers in Japan from 2006 to 2021.

The increasing prevalence of AmpC- and extended-spectrum ß-lactamase (ESBL)- producing food pathogens is a serious public health concern. AmpC- and ESBL-producing Salmonella species pose a high risk of food contamination. AIMS: This study aimed to investigate changes in the prevalence of Salmonella among food handlers in Japan from 2006 to 2021 using 100 randomly selected isolates from 2006, 2012, 2018, and 2021 with different serotypes and antimicrobial resistance patterns. METHODS AND RESULTS: The average Salmonella isolation rate was 0.070% (19,602/27,848,713). Serotyping revealed that the most common serotypes were Enteritidis in 2006, Infantis in 2012, Agoueve/Cubana in 2018, and Schwarzengrund in 2021. Antimicrobial susceptibility testing showed that Salmonella isolates exhibited the highest resistance to streptomycin (< 40%), followed by tetracycline (< 20%-40%). Moreover, 6% of the Salmonella isolates produced cephalosporinases with the blaCMY-2, blaCTX-M-14, and blaTEM genes. The annual incidence of cephalosporin resistance has increased. Plasmid conjugation assays revealed that cephalosporin-resistant Salmonella spp. transmitted their resistance to Escherichia coli. Additionally, plasmid genome analysis showed that the insertion sequence IS26 was encoded in the upstream and downstream regions of blaCTX-M-14 and qnrS1 in the IncHI1 plasmid, which could be transmitted to other bacteria. CONCLUSIONS: The tested Salmonella isolates showed high resistance to specific antibiotics, with differences in resistance depending on the serotype. Further increase and spread of transmissible cephalosporin-resistant strains should be noted.

Prevalence and characteristics of ESBL-producing Escherichia coli in clinically healthy pigs: implications for antibiotic resistance spread in livestock.

AIM: This study aimed to compare and characterize the resistance profile and the presence of extended-spectrum beta-lactamase (ESBL) related genes in Escherichia coli isolated from healthy finishing pigs fed with or without antibiotics in their diets. METHODS AND RESULTS: A total of 27 ceftiofur-resistant E. coli isolates were obtained from 96 healthy pigs. The antibiotic resistance profile was tested, and all 27 isolates were classified as multidrug-resistant (MDR). A high proportion of isolates were resistant to cephalosporins, ampicillin, ciprofloxacin, and tetracyclines. The ESBL production was observed in 85% of isolates by double-disc synergy test. The MDR-E. coli isolates harbored ESBL genes, such as blaTEM, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-8,25. In addition, other antibiotics resistance genes (ARGs) were also detected, such as sul2, ant(3″)-I, tetA, and mcr-1. The mobilization of the blaCTX-M gene was confirmed for nine E. coli isolates by conjugation assays. The presence of blaCTX-M on mobile genetic elements in these isolates was demonstrated by Southern blot hybridization, and the resistance to cephalosporins was confirmed in the transconjugants. Our results indicate the prevalence of CTX-M-producing E. coli strains harboring mobile genetic elements in the normal microbiota of healthy pigs. CONCLUSIONS: These findings highlight the significance of ESBL genes as a global health concern in livestock and the potential spread of antimicrobial resistance to other members of the gastrointestinal tract microbiota.

Extended-spectrum ß-lactamase-producing Plesiomonas shigelloides isolated from the stool of a Japanese traveler returning from Rwanda: A case report.

A 21-year-old previously healthy Japanese woman visited an outpatient clinic because of abdominal pain, watery diarrhea, vomiting, and mild fever that had started on the previous day. She traveled to rural and urban areas of Rwanda and returned to Japan 3 days before. Stool culture yielded the Plesiomonas shigelloides strain TMCH301018, against which minimum inhibitory concentrations of cefotaxime and cefotaxime-clavulanate were 128 and ≤0.12/4 µg/mL, respectively. The strain had the blaCTX-M-27 gene and an IncA/C replicon-type plasmid. Moreover, a transformant produced by introduction of an IncA/C plasmid extracted from TMCH301018 into Escherichia coli DH5α was positive for the blaCTX-M-27 gene and fulfilled the criteria of extended-spectrum ß-lactamase (ESBL) production described by the Clinical and Laboratory Standards Institute, indicating that TMCH301018 produced ESBL of CTX-M-27 and the ESBL-encoding gene was located on an IncA/C plasmid. Pathogenicity of TMCH301018 for the patient's complaints was uncertain because a molecular assay detected other enteropathogens in the stool specimen and the symptoms improved within 2 days with administration of oral ciprofloxacin, to which TMCH301018 was not susceptible. To our knowledge, this is the first report describing the isolation of ESBL-producing P. shigelloides.

Sensitive and Specific Loop-Mediated Isothermal Amplification Assays for Detection of Salmonella, CTX-M-1 Group Genes, mph(A), and ermB in Stool and Blood Samples Based on Orange to Green Visible Dye.

Salmonella is a globally prevalent foodborne bacterium, and ceftriaxone and azithromycin have been regarded as drugs of choice for treating Salmonella infections, particularly in children. With the growing incidence of ceftriaxone and azithromycin resistance in Salmonella, there is an urgent requirement for a rapid and dependable gene testing approach to enhance the efficacy of treating Salmonella infections. Utilizing the orange to green visible dye approach, this study developed loop-mediated isothermal amplification (LAMP) assays for the sensitive and specific detection of Salmonella, ceftriaxone and azithromycin resistance genes (including CTX-M-1 group, mph(A), and ermB genes) in stool and blood samples. The specificity and sensitivity of primers during the LAMP assays for detection of Salmonella, CTX-M-1 group, mph(A), and ermB genes were determined in this study. The detection threshold for Salmonella was found to be 1.5 × 103 colony-forming units (CFU)/mL, while it was 1.5 × 102 CFU/mL for CTX-M-1 group genes (including blaCTX-M-3, blaCTX-M-15, and blaCTX-M-55), 1.5 × 102 CFU/mL for mph(A), and 1.5 × 102 CFU/mL for ermB, showing 10-103-fold, 103-fold, and 105-fold increased sensitivity compared with the polymerase chain reaction assay, respectively. Results indicated that the LAMP primers designed for Salmonella, CTX-M-1 group, mph(A), and ermB genes possess high specificity (100%) and sensitivity (over 94%). This novel approach advocates its application in detecting Salmonella, CTX-M-1 group, mph(A), and ermB genes.

Is Carbapenem Therapy Necessary for the Treatment of Non-CTX-M ESBL-Producing Enterobacterales Bloodstream Infections?

BACKGROUND: Investigations into antibiotics for extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) bloodstream infections (BSIs) have focused on blaCTX-M genes. Outcomes of patients with non-CTX-M-producing ESBL-E BSIs and optimal treatment are unknown. METHODS: A multicenter observational study investigating 500 consecutive patients with ceftriaxone-resistant Enterobacterales BSIs during 2018-2022 was conducted. Broth microdilution and whole genome sequencing confirmed antibiotic susceptibilities and ESBL gene presence, respectively. Inverse probability weighting (IPW) using propensity scores was employed to ensure patients infected with non-CTX-M and CTX-M ESBL-E BSIs were similar prior to evaluation of outcomes. RESULTS: 396 patients (79.2%) were confirmed to have an ESBL-E BSI. ESBL gene family prevalence was as follows: blaCTX-M (n=370), blaSHV (n=16), blaOXY (n=12), and blaVEB (n=5). ESBL gene identification was not limited to Escherichia coli and Klebsiella species. In the IPW cohort, there was no difference in 30-day mortality or ESBL-E infection recurrence between the non-CTX-M and CTX-M groups (OR=.99, 95% CI 0.87-1.11; p=0.83) and (OR=1.10, 95% CI 0.85--1.42; p=0.47), respectively. In an exploratory analysis limited to the non-CTX-M group, 86% of the 21 patients receiving meropenem were alive on day 30; none of the 5 patients receiving piperacillin-tazobactam were alive on day 30. CONCLUSIONS: Our findings suggest that non-CTX-M and CTX-M ESBL-producing Enterobacterales BSIs are equally concerning and associated with similar clinical outcomes. Meropenem may be associated with improved survival in patients with non-CTX-M ESBL-E BSIs, underscoring the potential benefit of comprehensive molecular diagnostics to enable early antibiotic optimization for patients with ESBL-E BSI, beyond just blaCTX-M genes.

Chromosome-Borne CTX-M-65 Extended-Spectrum ß-Lactamase-Producing Salmonella enterica Serovar Infantis, Taiwan.

A CTX-M-65‒producing Salmonella enterica serovar Infantis clone, probably originating in Latin America and initially reported in the United States, has emerged in Taiwan. Chicken meat is the most likely primary carrier. Four of the 9 drug resistance genes have integrated into the chromosome: blaCTX-M-65, tet(A), sul1, and aadA1.

Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M ß-Lactamases: Biochemical and Structural Analyses.

Design of novel ß-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two α-amido-ß-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum ß-lactamase [ESBL]) ß-lactamases were evaluated. The 50% inhibitory concentrations (IC50s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k2/K for CTX-M-96 (24,000 M-1 s-1) was twice the reported value for KPC-2 (12,000 M-1 s-1); for MB_076, the k2/K values ranged from 1,200 M-1 s-1 (KPC-2) to 3,900 M-1 s-1 (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-Å resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to ß-lactamase inhibitor design.

Dissemination of IncI plasmid encoding bla CTX-M-1 is not hampered by its fitness cost in the pig's gut.

Multiresistance plasmids belonging to the IncI incompatibility group have become one of the most pervasive plasmid types in extended-spectrum beta-lactamase-producing Escherichia coli of animal origin. The extent of the burden imposed on the bacterial cell by these plasmids seems to modulate the emergence of "epidemic" plasmids. However, in vivo data in the natural environment of the strains are scarce. Here, we investigated the cost of a bla CTX-M-1-IncI1 epidemic plasmid in a commensal E. coli animal strain, UB12-RC, before and after oral inoculation of 15 6- to 8-week- old specific-pathogen-free pigs. Growth rate in rich medium was determined on (i) UB12-RC and derivatives, with or without plasmid, in vivo and/or in vitro evolved, and (ii) strains that acquired the plasmid in the gut during the experiment. Although bla CTX-M-1-IncI1 plasmid imposed no measurable burden on the recipient strain after conjugation and during the longitudinal carriage in the pig's gut, we observed a significant difference in the bacterial growth rate between IncI1 plasmid-carrying and plasmid-free isolates collected during in vivo carriage. Only a few mutations on the chromosome of the UB12-RC derivatives were detected by whole-genome sequencing. RNA-Seq analysis of a selected set of these strains showed that transcriptional responses to the bla CTX-M-1-IncI1 acquisition were limited, affecting metabolism, stress response, and motility functions. Our data suggest that the effect of IncI plasmid on host cells is limited, fitness cost being insufficient to act as a barrier to IncI plasmid spread among natural population of E. coli in the gut niche.

Evaluation of Phenotypic Tests to Detect Extended-Spectrum ß-Lactamase (ESBL)-Producing Klebsiella oxytoca Complex Strains.

Klebsiella oxytoca complex (KoC) species may overproduce their chromosomal class A OXY ß-lactamases, conferring reduced susceptibility to piperacillin-tazobactam, expanded-spectrum cephalosporins and aztreonam. Moreover, since clavulanate maintains its ability to inhibit these enzymes, the resulting resistance phenotype may falsely resemble the production of acquired extended-spectrum ß-lactamases (ESBLs). In this work, a collection of 44 KoC strains of human and animal origin was characterized with whole-genome sequencing (WGS) and broth microdilution (BMD) susceptibility testing. Comparison of ESBL producers (n = 11; including CTX-M-15 [n = 6] and CTX-M-1 [n = 5] producers) and hyperproducers of OXYs (n = 21) showed certain phenotypic differences: piperacillin-tazobactam (MIC90s: 16 versus >64 µg/mL), cefotaxime (MIC90s: 64 versus 4 µg/mL), ceftazidime (MIC90s: 32 versus 4 µg/mL), cefepime (MIC90s: 8 versus 4 µg/mL) and associated resistance to non-ß-lactams (e.g., trimethoprim-sulfamethoxazole: 90.9% versus 14.3%, respectively). However, a clear phenotype-based distinction between the two groups was difficult. Therefore, we evaluated 10 different inhibitor-based confirmatory tests to allow such categorization. All tests showed a sensitivity of 100%. However, only combination disk tests (CDTs) with cefepime/cefepime-clavulanate and ceftazidime/ceftazidime-clavulanate or the double-disk synergy test (DDST) showed high specificity (100%, 95.5%, and 100%, respectively). All confirmatory tests in BMD or using the MIC gradient strip did not perform well (specificity, ≤87.5%). Of note, ceftazidime/ceftazidime-avibactam tests also exhibited low specificity (CDT, 87.5%; MIC gradient strip, 77.8%). Our results indicate that standard antimicrobial susceptibility profiles can raise some suspicion, but only the use of cefepime/cefepime-clavulanate CDT or DDST can guarantee distinction between ESBL-producing KoC strains and those hyperproducing OXY enzymes.

Polluted wetlands contain multidrug-resistance plasmids encoding CTX-M-type extended-spectrum ß-lactamases.

While most detailed analyses of antibiotic resistance plasmids focus on those found in clinical isolates, less is known about the vast environmental reservoir of mobile genetic elements and the resistance and virulence factors they encode. We selectively isolated three strains of cefotaxime-resistant Escherichia coli from a wastewater-impacted coastal wetland. The cefotaxime-resistant phenotype was transmissible to a lab strain of E. coli after one hour, with frequencies as high as 10-3 transconjugants per recipient. Two of the plasmids also transferred cefotaxime resistance to Pseudomonas putida, but these were unable to back-transfer this resistance from P. putida to E. coli. In addition to the cephalosporins, E. coli transconjugants inherited resistance to at least seven distinct classes of antibiotics. Complete nucleotide sequences revealed large IncF-type plasmids with globally distributed replicon sequence types F31:A4:B1 and F18:B1:C4 carrying diverse antibiotic resistance and virulence genes. The plasmids encoded extended-spectrum ß-lactamases blaCTX-M-15 or blaCTX-M-55, each associated with the insertion sequence ISEc9, although in different local arrangements. Despite similar resistance profiles, the plasmids shared only one resistance gene in common, the aminoglycoside acetyltransferase aac(3)-IIe. Plasmid accessory cargo also included virulence factors involved in iron acquisition and defense against host immunity. Despite their sequence similarities, several large-scale recombination events were detected, including rearrangements and inversions. In conclusion, selection with a single antibiotic, cefotaxime, yielded conjugative plasmids conferring multiple resistance and virulence factors. Clearly, efforts to limit the spread of antibiotic resistance and virulence among bacteria must include a greater understanding of mobile elements in the natural and human-impacted environments.

ESBL-producing Vibrio vulnificus and V. alginolyticus harbour a plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

In recent years, trade liberalisation has led to the spread of antibiotic-resistant bacteria (ARB) in food products. Because ARB has reportedly been found in imported foods, the spread of plasmid-mediated ARB through food products is a concern. Here, we report the complete genome sequences of ESBL-producing Vibrio vulnificus and V. alginolyticus strains harbouring a plasmid isolated from imported seafood. First, V. vulnificus and V. alginolyticus were isolated from purchased frozen and thawed Litopenaeus vannamei shrimp, and genome extraction and sequencing were performed. Hybrid genome assemblies were performed using Unicycler and annotated using DFAST. Then genome analysis was performed using BRIG. Plasmid comparisons showed that the plasmids carried by both Vibrios are remarkably similar and encode the same antibiotic-resistance genes. The 270-310 kb region specific to both Vibrios were isolated in this study and encodes the antibiotic-resistance genes blaCTX-M and qnr. Furthermore, the mobile genetic factors ISEc9, ISVch4, and ISVpa4 are located upstream and downstream of these genes. This is the first report of ESBL-producing V. vulnificus and V. alginolyticus harbouring a common plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

Traditional marketed meats as a reservoir of multidrug-resistant Escherichia coli.

This study aimed to analyze Escherichia coli from marketed meat samples in Peru. Sixty-six E. coli isolates were recovered from 21 meat samples (14 chicken, 7 beef), and antimicrobial resistance levels and the presence of mechanisms of antibiotic resistance, as well as clonal relationships and phylogeny of colistin-resistant isolates, were established. High levels of antimicrobial resistance were detected, with 93.9% of isolates being multi-drug resistant (MDR) and 76.2% of samples possessing colistin-resistant E. coli; of these, 6 samples from 6 chicken samples presenting mcr-1-producer E. coli. Colistin-resistant isolates were classified into 22 clonal groups, while phylogroup A (15 isolates) was the most common. Extended-spectrum ß-lactamase- and pAmpC-producing E. coli were found in 18 and 8 samples respectively, with blaCTX-M-55 (28 isolates; 16 samples) and blaCIT (8 isolates; 7 samples) being the most common of each type. Additionally, blaCTX-M-15, blaCTX-M-65, blaSHV-27, blaOXA-5/10-like, blaDHA, blaEBC and narrow-spectrum blaTEM were detected. In addition, 5 blaCTX-M remained unidentified, and no sought ESBL-encoding gene was detected in other 6 ESBL-producer isolates. The tetA, tetE and tetX genes were found in tigecycline-resistant isolates. This study highlights the presence of MDR E. coli in Peruvian food-chain. The high relevance of CTX-M-55, the dissemination through the food-chain of pAmpC, as well as the high frequency of unrelated colistin-resistant isolates is reported.

Longitudinal study of ESBL/AmpC-producing Enterobacterales strains sharing between cohabiting healthy companion animals and humans in Portugal and in the United Kingdom.

Extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales (ESBL/AmpC-E) are an increasing healthcare problem in both human and veterinary medicine. The aim of this study was to investigate the possible sharing of ESBL/AmpC-E strains between healthy companion animals and humans of the same household in Portugal (PT) and the United Kingdom (UK). In a prospective longitudinal study, between 2018 and 2020, faecal samples were collected from healthy dogs (n=90), cats (n=20) and their cohabiting humans (n=119) belonging to 41 PT and 44 UK households. Samples were screened for the presence of ESBL/AmpC-E and carbapenemase-producing bacteria. Clonal relatedness between animal and human strains was established by using REP-PCR fingerprinting method, followed by whole-genome sequencing (WGS) of selected strains. ESBL/AmpC-E strains were detected in companion animals (PT=12.7%, n=8/63; UK=8.5%, n=4/47) and humans (PT=20.7%, n=12/58; UK=6.6%, n=4/61) in at least one timepoint. REP-PCR identified paired multidrug-resistant ESBL/AmpC-producing Escherichia coli strains from companion animals and owners in two Portuguese households (4.8%) and one UK household (2.3%). WGS analysis of nine E. coli strains from these three households confirmed that interhost sharing occurred only between the two animal-human pairs from Portugal. Three shared strains were identified: one CTX-M-15-producing E. coli strain in a cat-human pair (O15-H33-ST93) and two CTX-M-15- and CTX-M-55/CMY-2-producing E. coli strains, in a dog-human pair (O8:H9-ST410 and O11:H25-ST457, respectively) at different timepoints. These E. coli clonal lineages are human pandemic, highlighting the role of companion animals living in close contact with humans in the dissemination and persistence of antimicrobial resistance in the household environment.

Phenotypic and genotypic characterization of multidrug resistant and extended-spectrum ß-lactamase-producing Enterobacterales isolated from clinical samples in the western region in Cameroon.

BACKGROUND: The 2017 World Health Organization (WHO) report has listed extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) as critical pathogens for public health and requiring urgently new antibiotics. The aim of this study was to characterize phenotypically and genotypically ESBL-E isolated among clinical samples in Dschang, Cameroon. METHODS: A cross-sectional study was conducted during a four-month periods from February to May 2022 in the two biggest hospitals of Dschang. Clinical samples were collected and cultured on Eosin Methylene Blue agar. Suspected growing colonies were biochemically identified using the Enterosystem Kit 18R. Antimicrobial susceptibility testing (AST) was done using the Kirby Bauer disc diffusion method and interpretated according to the CA-SFM recommendations. ESBL phenotypes were double screened using CHROMagar™ ESBL and double disk synergy test (DDST). The detection of resistance genes was performed using conventional and multiplex PCR methods. Results were analyzed with SPSS (version 21) and a p-value < 0.05 was considered statistically significant. RESULTS: A total of 152 Enterobacterales were isolated among 597 clinical samples including urine, blood, cervico-vaginal, urethral swabs and wound samples. The overall prevalence of ESBL-Enterobacterales was 29.61% (45/152). The most represented ESBL species were Escherichia coli (n = 23; 51.11%), Klebsiella pneumoniae (n = 8; 17.78%) and Citrobacter freundii (n = 6; 13.33%). CONCLUSION: This study reveals the high burden of ESBL-E among clinical samples in the regional hospital in Dschang with the most common species being E. coli and K. pneumoniae. It confirmed the high occurrence of blaCTX-M and blaTEM among ESBL-E. The study suggests that implementing antimicrobial stewardship program and real-time surveillance of antimicrobial resistance are needed in the Western region of Cameroon. Moreover, the implementation of infection prevention and control measures (IPC) is essential to curb the dissemination of these bacteria from community to hospital settings. Implementation of national action plan to fight against antimicrobial resistance at the local levels is urgently needed.

Prevalence of extended spectrum beta lactamase and molecular detection of blaTEM, blaSHV and blaCTX-M genotypes among Gram negative bacilli isolates from pediatric patient population in Gaza strip.

BACKGROUND: Extended-spectrum ß lactamases (ESBLs), have the ability to hydrolyze and cause resistance to various types of the ß-lactam antibiotics, including the extended-spectrum (or third-generation) cephalosporins (e.g., cefotaxime, ceftriaxone, ceftazidime) and monobactams (e.g., aztreonam). ESBL-producing Gram negative bacteria is still posing significant therapeutic challenges. OBJECTIVES: To assess the prevalence and molecular characteristics of ESBL producing Gram negative bacilli, isolated from a cohort of pediatric patients in Gaza hospitals. METHODS: A total of 322 isolates of Gram-negative bacilli were collected from four referral pediatric hospitals in Gaza, namely: Al-Nasr, Al-Rantisi, Al-Durra and Beit Hanoun hospitals. These isolates were tested for ESBL production using the double disk synergy and CHROMagar phenotypic methods. Molecular characterization of the ESBL producing strains was performed using PCR targeting the CTX-M, TEM and SHV genes. Antibiotic profile was done using Kirby Bauer method according to Clinical and Laboratory Standard Institute. RESULTS: Out of 322 isolates tested by phenotypic methods, 166 (51.6%) were ESBL positive. The prevalence of ESBL production in Al-Nasr, Al-Rantisi, Al-Durra and Beit Hanoun hospitals was 54%, 52.5%, 45.5% and 52.8% respectively. The prevalence of ESBL production among Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp., Proteus mirabilis, Enterobacter spp., Citrobacter spp., and Serratia marcescens is 55.3%, 63.4%, 17.8%, 57.1%, 33.3%, 28.5%, 38.4%, and 4% respectively. ESBL production among urine, pus, blood, CSF and sputum was 53.3%, 55.2%, 47.4%, 33.3%, and 25% respectively. Out of the 322 isolates, 144 were screened for CTX-M, TEM and SHV production. Using PCR, 85 (59%) had at least one gene. The prevalence rate of CTX-M, TEM and SHV genes was 60%, 57.6%, and 38.3% respectively. Meropenem and amikacin were highest rates of susceptibility antibiotics against ESBLs producers (83.1% and 82.5% respectively), while the least effective antibiotics were amoxicillin (3.1%) and cephalexin (13.9%). Moreover, ESBLs producers showed high resistance rate to cefotaxime, ceftriaxone and ceftazidime (79.5%, 78.9% and 79.5% respectively). CONCLUSION: Our results show high prevalence of ESBL production among Gram negative bacilli isolated from children in different pediatric hospitals in Gaza strip. A substantial level of resistance to first and second generation cephalosporins was also observed. This ascertains the need for a rational antibiotic prescription and consumption policy.

Ceftriaxone resistant Salmonella enterica serovar Paratyphi A identified in a case of enteric fever: first case report from Pakistan.

BACKGROUND: Enteric fever is an acute systemic infectious disease associated with substantial morbidity and mortality in low- and middle-income countries (LMIC), with a global burden of 14.3 million cases. Cases of enteric fever or paratyphoid fever, caused by Salmonella enterica serovar Paratyphi A (S. Para A) have been found to rise in many endemic and non-endemic countries. Drug resistance is relatively uncommon in S. Para A. Here we report a case of paratyphoid fever caused by ceftriaxone resistant S. Para A from Pakistan. CASE PRESENTATION: A 29-year-old female presented with a history of fever, headache, and shivering. Her blood culture revealed a S. Para A isolate (S7), which was resistant to ceftriaxone, cefixime, ampicillin and ciprofloxacin. She was prescribed oral Azithromycin for 10 days, which resulted in resolution of her symptoms. Two other isolates of S. Para A (S1 and S4), resistant to fluoroquinolone were also selected for comparison. DST and whole genome sequencing was performed for all three isolates. Sequence analysis was performed for identification of drug resistance and phylogeny. Whole Genome Sequencing (WGS) of S7 revealed the presence of plasmids, IncX4 and IncFIB(K). blaCTX-M-15 and qnrS1 genes were found on IncFIB(K). The gyrA S83F mutation conferring fluoroquinolone resistance was also found present. Multi-locus sequence typing (MLST) showed the S7 isolate to belong to ST129. S1 and S4 had the gyrA S83Y and S83F mutations respectively. CONCLUSIONS: We highlight the occurrence of plasmid-mediated ceftriaxone resistant strain of S. Para A. This is of significance as ceftriaxone is commonly used to treat paratyphoid fever and resistance in S. Para A is not known. Continuous epidemiological surveillance is required to monitor the transmission and spread of antimicrobial resistance (AMR) among Typhoidal Salmonellae. This will guide treatment options and preventive measures including the need for vaccination against S. Para A in the region.

Tn3-like structures co-harboring of blaCTX-M-65, blaTEM-1 and blaOXA-10 in the plasmids of two Escherichia coli ST1508 strains originating from dairy cattle in China.

The purpose of this study was to determine the level of horizontal transmission of the blaCTX-M-65 gene and the role of its associated mobile genetic elements (MGEs) in the bovine-derived Escherichia coli. After PCR identification, two plasmids carrying blaCTX-M-65 were successfully transferred to the recipient E. coli J53 Azr through conjugation assays and subsequently selected for Whole-Genome sequencing (WGS) analysis. The resistance profiles of these two positive strains and their transconjugants were also determined through antimicrobial susceptibility tests. Whole genome data were acquired using both the PacBio sequencing platform and the Illumina data platform. The annotated results were then submitted to the Genbank database for accession number recording. For comparison, the genetic environment of plasmids carrying the resistance gene blaCTX-M-65 was mapped using the Easyfig software. WGS analysis revealed Tn3-like composite transposons bearing blaCTX-M-65, blaTEM-1, and blaOXA-10 in the IncHI2-type plasmids of these two E. coli ST1508 strains. A phylogenetic tree was generated from all 48 assembled E. coli isolates blaCTX-M-65, blaTEM-1, and blaOXA-10 from the NCBI Pathogen Detection database with our two isolates, showing the relationships and the contribution of SNPs to the diversity between genetic samples. This study suggests that the transmissibility of blaCTX-M-65 on Tn3-like composite transposons contributes to an increased risk of its transmission in E. coli derived from dairy cattle.

CRISPR/cas9 cassette targeting Escherichia coli blaCTX-M specific gene of mastitis cow milk origin can alter the antibiotic resistant phenotype for cefotaxime.

CTX-M beta-lactamases are one of the most important extended spectrum beta-lactamase (ESBL) resistance enzymes found in E. coli. In the present study, 59% of E. coli isolates from mastitis cow milk were reported to be positive for ESBL types. The prevalence of beta-lactam (ß-lactam) antibiotic resistance was reported to be 84%, 72.7%, 52.27%, 50%, and 45.4% for cefotaxime, cefepime, cefuroxime, oxacillin, and cephalexine, respectively. The blaCTX-M gene was found in 65% (n = 17) of the E. coli isolates when they were genotyped. Further, the use of a CRISPR/cas9 cassette to target the E. coli blaCTX-M gene revealed changes in antibiotic phenotypes for cefotaxime.