Clonal, Plasmidic and Genetic Diversity of Multi-Drug-Resistant Enterobacterales from Hospitalized Patients in Tripoli, Libya.

Resistance to extended-spectrum cephalosporins (ESC) and carbapenems in Enterobacterales is a major issue in public health. Carbapenem resistance in particular is associated with increased morbidity and mortality. Moreover, such resistance is often co-harbored with resistance to non-beta-lactam antibiotics, and pathogens quickly become multi-drug-resistant (MDR). Only a few studies have been published on AMR in Libyan hospitals, but all reported worrisome results. Here, we studied 54 MDR isolates that were collected from 49 patients at the Tripoli University Hospital between 2019 and 2021. They were characterized using phenotypic methods, PCR and PFGE, and a sub-set of isolates were short- and long-read whole-genome sequenced. The results showed the frequent occurrence of Klebsiella pneumoniae (49/54), among which several high-risk clones were responsible for the spread of resistance, namely, ST11, ST17, ST101 and ST147. ESC and carbapenem resistance was due to a wide variety of enzymes (CTX-M, OXA-48, NDM, KPC), with their corresponding genes carried by different plasmids, including IncF-IncHI2 and IncF-IncR hybrids. This study highlights that implementation of infection prevention, control and surveillance measures are needed in Libya to fight against AMR.

Escherichia coli ST224 and IncF/blaCTX-M-55 plasmids drive resistance to extended-spectrum cephalosporins in poultry flocks in Parana, Brazil.

Resistant Enterobacterales of avian intestinal origin can contaminate carcasses during broiler processing and thereby spread through the human food chain. This study aimed at assessing the prevalence, diversity and genomic characteristics of ESBL/AmpC Enterobacterales in poultry flocks from different farms and cities in the state of Paraná, Brazil. Enterobacterales isolated from cloacal samples were subjected to antimicrobial susceptibility testing (AST). ESBL/AmpC isolates were whole-genome sequenced and subjected to S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blotting to determine the location of resistant genes on plasmids. A surprisingly high proportion of E. coli (40.6 %) collected on non-selective plates presented an ESBL/AmpC phenotype. Multidrug resistance was statistically not higher in ESBL/AmpC E. coli having the potential to be Avian Pathogenic (APEC-like) compared to non-APEC-like ESBL/AmpC E. coli isolates. Resistance to antibiotics not authorized for use in poultry in the State of Paraná was observed, suggesting that antimicrobial resistance (AMR) is co-selected by the use of veterinary-licensed antibiotics. Phylogenetic analyzes revealed the presence of identical or highly similar ESBL/AmpC E. coli clones on farms distant up to 100 km of each other; this strongly suggests that the centralization and verticalization of the poultry industry can facilitate the spread of resistant bacteria among different farms, companies, and cities. The molecular characterization of clones and plasmids proved the dominance of the ST224 E. coli lineage and the IncF/blaCTX-M-55 plasmid, possibly indicating the emergence of successful clones and plasmids adapted to the chicken host. Our data contribute to the epidemiological tracking of resistance mechanisms in Enterobacterales from poultry and to knowledge for further One Health studies to control the spread of resistant bacteria from food animals to humans.

Spread of the blaOXA-48/IncL Plasmid within and between Dogs in City Parks, France.

The blaOXA-48/IncL plasmid is increasingly reported in dogs, even in the absence of carbapenem use in animals. In this study, we witnessed the spread of this plasmid within and between dogs sharing the same relaxing area. This indicates a very dynamic situation where carbapenem resistance can be transmitted between dogs and expanded in the dogs' gut. As a consequence, picking up dog feces may lower both this dynamic and the global antimicrobial resistance burden. IMPORTANCE The use of carbapenems in animals is forbidden in France due to their critical importance to treat human diseases. Nevertheless, blaOXA-48-producing Enterobacterales were sporadically recovered in cats and dogs, most likely as a spill over from the human reservoir. This study highlights the rapid spread of blaOXA-48 once transmitted to dogs, suggesting that companion animals can play a role in the transmission routes of carbapenemase genes.

Interplay between Bacterial Clones and Plasmids in the Spread of Antibiotic Resistance Genes in the Gut: Lessons from a Temporal Study in Veal Calves.

Intestinal carriage of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing, and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over 6 months. We characterized the bacterial clones and plasmids carrying blaESBL genes with a combination of genotyping methods, whole genome sequencing, and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [blaCTX-M-1 (64.7%), blaCTX-M-14 (33.5%), or blaCTX-M-15 (1.8%)] were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and blaCTX-M-carrying plasmids, few blaCTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario "clone versus plasmid spread" depended on the farm. Thus, epistatic interactions between resistance genes, plasmids, and bacterial clones contribute to optimize fitness in specific environments. IMPORTANCE The gut microbiota is the epicenter of the emergence of resistance. Considerable amount of knowledge on the molecular mechanisms of resistance has been accumulated, but the ecological and evolutionary forces at play in nature are less studied. In this context, we performed a field work on temporal intestinal carriage of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli in veal farms. Veal calves are animals with one of the highest levels of ESBL producing E. coli fecal carriage, due to early high antibiotic exposure. We were able to show that calves were colonized successively by different ESBL-producing E. coli clones, and that two main scenarios were at play in the spread of blaCTX-M genes among calves: efficient colonization of several calves by a few bacterial clones and successful plasmid spread in various bacterial clones. Such knowledge should help develop new strategies to fight the emergence of antibiotic-resistance.

Selection of ESBL-producing Escherichia coli in the gut of calves experimentally fed with milk containing antibiotic residues.

In the bovine sector, the spread of Enterobacterales producing extended-spectrum and AmpC ß-lactamases (ESBL/AmpC) mostly concerns veal calves, and the use of waste milk containing antibiotic residues has been recurrently incriminated. In this study, calves were experimentally fed with milk containing either 2,000 µg/L or 20,000 µg/L of the critically important antibiotic cefquinome. The total counts of enterobacterales and ESBL-producing E. coli were monitored using non-selective and selective media. Our data highlighted the important combination of two main factors (cefquinome exposure and initial ESBL colonization level) in the ESBL selection and amplification process in the gut of calves. Results also proved the dose-independent effect of cefquinome administration on the selection and amplification of ESBL-producing E. coli. Finally, the blaCTX-M-1/IncI1 ST3 plasmid was systematically recovered after cefquinome exposure, highlighting its epidemic success. Altogether, this work is one of the rare experimental studies providing quantitative information on the impact of waste milk containing antimicrobials on the ESBL load in calves' microbiota, and the first one using cefquinome. These data emphasise the need for global guidelines on the use of waste milk on dairy farms in order to decrease the antimicrobial resistance burden in this sector.

An inventory of 44 qPCR assays using hydrolysis probes operating with a unique amplification condition for the detection and quantification of antibiotic resistance genes.

Early antibiotic resistance determinants (ARDs) detection in humans or animals is crucial to counteract their propagation. The ARDs quantification is fundamental to understand the perturbation caused by disruptors, such as antibiotics, during therapies. Forty-three qPCRs on the most diffused ARDs and integrons among human and animal Enterobacterales, and one on the 16S rDNA for bacteria quantification, were developed. The qPCRs, using hydrolysis probes, operated with a unique amplification condition and were tested analytically and diagnostically performing 435 reactions on five positive and negative controls for each qPCR. Diagnostic sensitivity and specificity were confirmed by PCR and genome sequencing of control isolates, demonstrating 100% performance for all qPCRs. An easy and rapid discrimination method for the epidemiologically relevant blaCTX-Ms is provided. This large, noncommercial qPCRs inventory could serve for precise quantification of ARDs, but also as a rapid screening tool for surveillance purposes, providing the basis for further high-throughput developments.

Characterization of cultivable airborne bacteria and their antimicrobial resistance pattern in French milking parlour.

The main goal of this preliminary study was to quantify airborne particles and characterize the dominant cultivable bacterial species as well as some Gram-positive species, and their antibiotic resistance pattern, from environmental samples taken inside and outside of a dairy milking parlour. Sampling was performed over 2 days, in different seasons. The small viable particulate matter < 10 µm (bioaerosols) and cultivable bacteria reached their highest concentrations in the milking parlour. The majority of airborne bacteria in the milking parlour belonged to the genera Staphylococcus (41.9%) and Bacillus (20.9%). A total of 32 different bacterial species of Staphylococcus, Aerococcus, Bacillus, Pseudomonas, Serratia and Acinetobacter were identified. Many of these bacteria may be opportunistic pathogens, causing disease in humans or animals. We found low levels of acquired resistance to the antibiotics commonly used in human or animal infections caused by these opportunistic bacteria. More specifically, resistance to tetracyclines (13.4%), penicillin G (13.4%) and macrolides (7.5%) was identified in Staphylococcus sp. as was a methicillin-resistant S. hominis and resistance to spiramycin (n = 1), lincomycin (n = 1) and streptomycin (n = 2) in Aerococcus sp. An assessment of the occupational risk run by dairy farmers for contracting infections after long- or short-term exposure to micro-organisms requires further studies on the concentration of opportunistic pathogenic bacteria in dairy farm environments.

Wide Spread of bla CTX-M-9/mcr-9 IncHI2/ST1 Plasmids and CTX-M-9-Producing Escherichia coli and Enterobacter cloacae in Rescued Wild Animals.

Wildlife has recently been pinpointed as one of the drivers of dissemination of genes conferring resistances to clinically important antimicrobials. The presence of both extended-spectrum beta-lactamase- (ESBL) and carbapenemase-encoding genes has notably been reported in wild birds, that can act as sentinels of antimicrobial resistance (AMR) contamination but also as long-distance spreaders in case of migratory birds. Here, 424 wild birds brought to a rescue center in France were sampled over a 6-month period. These birds encompassed 62 different sedentary or migratory species. A further 16 wild mammals present in the center were also investigated. No carbapenemase-producer was found, but we identified a surprisingly high proportion (24.1%) of ESBL-positive isolates. A total of 144 non-duplicate isolates were collected, including Escherichia coli (n = 88), Enterobacter cloacae (n = 51), and Citrobacter freundii (n = 5), of which 123 carried the bla CTX-M- 9 gene. PFGE, phylogroup, and MLST revealed the presence of a limited number of ESBL-positive clones circulating in these animals, all presenting multiple associated resistances. Next-generation sequencing on a subset of isolates, followed by Southern blot hybridization, showed the wide dissemination of an IncHI2/ST1 plasmid carrying the bla CTX-M- 9, bla SHV- 12 and mcr-9 genes. In all, our results undoubtedly reflect cross transmissions of ESC-resistance (ESC-R) Enterobacteriaceae within the rescue center - similarly to nosocomial spreads observed at hospital, rather than the true bacterial flora of birds. We also showed that the spread of ESC-R in this rescue center did not only rely on clonal but also on a highly successful plasmidic transmission. Since most animals are intended to get back to nature after a few days or weeks, this is obviously an issue with regard to ESBL dissemination in natural environments.

Epidemic spread of IncI1/pST113 plasmid carrying the Extended-Spectrum Beta-Lactamase (ESBL) blaCTX-M-8 gene in Escherichia coli of Brazilian cattle.

INTRODUCTION: The prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide and the Agri-Food sector acts as a reservoir of clinically relevant ESBL genes. Our study aimed at detecting and characterizing ESBL-producing Enterobacteriaceae responsible for intestinal colonization of Brazilian bovines. MATERIAL AND METHODS: ESBL-producing E. coli isolates were recovered from fecal samples of healthy cattle in Northwest Brazil. Antimicrobial susceptibility was determined by disk diffusion. Resistance and virulence genes were identified by PCR and amplicons were sequenced, clonality was assessed by PFGE and MLST, and plasmids were characterized by replicon typing, S1-PFGE and Southern blot hybridizations. Transferability of ESBL genes was assessed by conjugation assay. RESULTS: A total of 40 ESBL-producing E. coli were characterized, which originated from 34/191 animals (17.8 %) and 15/22 farms (68.2 %). The blaCTX-M-8 gene was the most frequent ESBL gene (62.5 %), followed by blaSHV-2a (20.0 %), blaCTX-M-2 (10.0 %), and blaCTX-M-15 (7.5 %). The blaCTX-M-8 gene was localized on the IncI1/pST113 plasmid in multiple E. coli sequence types across unrelated animals and farms. DISCUSSION: We report the first characterization and a high prevalence of ESBL-producing E. coli in the beef cattle sector in Brazil, which is mainly supported by the spread of an epidemic IncI1/pST113/blaCTX-M-8 plasmid. Since Brazil is one of the biggest beef meat exporters worldwide, the spread of this ESBL plasmid across other sectors, countries and continents should be considered with attention.

Spread of ESBL/AmpC-producing Escherichia coli and Klebsiella pneumoniae in the community through ready-to-eat sandwiches in Algeria.

The spread of Extended-Spectrum ß-Lactamases (ESBLs) or AmpC ß-Lactamases (AmpC) encoding genes in healthy human populations is of major concern. The role of the food chain has been questioned since numerous studies reported surface contamination of retail meat or crude vegetables with ESBL/AmpC-producing Enterobacteriaceae (ESBL/AmpC-E). Nonetheless, these food products are intended to be cooked or washed before consumption so that the risk of human transfer might be low. Here, the presence of ESBL/AmpC-E was investigated in ready-to-eat (RTE) sandwiches purchased in the street in the city of Bejaia, Algeria. Thirty ESBL/AmpC-producing E. coli (n=18), K. pneumoniae (n=11) and K. oxytoca (n=1) were recovered from 21 sandwiches purchased in 14 of the 100 shops that were visited (14%). Twenty-four isolates (13 E. coli, 10 K. pneumoniae, 1 K. oxytoca) produced one or two ESBLs, while 5 E. coli and 1 K. pneumoniae isolates produced an AmpC. Among those, 12 E. coli harbored blaCTX-M-1 (n=7), blaCTX-M-15 (n=3), blaCTX-M-14 (n=1) or blaCTX-M-2 (n=1) and one E. coli co-harbored the blaCTX-M-15 and blaSHV-2 genes. The 10 K. pneumoniae displayed blaCTX-M-15 (n=7), blaSHV-2 (n=3), blaSHV-12 (n=1) or blaCTX-M-1 (n=1), including two isolates presenting a blaCTX-M-15/blaSHV-2 or blaCTX-M-1/blaSHV-2 combination. The K. oxytoca harbored the blaSHV-2 gene, and one K. pneumoniae and four E. coli displayed blaDHA and blaCMY-2, respectively. Most isolates (26/30, n=87%) also possessed the aac(6')-Ib-cr gene. Identical ESBL/AmpC-producing E. coli or K. pneumoniae clones were detected at different places across the city. This may reflect cross-contamination through poor handling practices, contaminated equipment, common ingredients or environmental factors. Of note, the emergent ST405 K. pneumoniae human clone was identified as a CTX-M-15 producer. This study highlights the presence of ESBL/AmpC-E in RTE sandwiches, which are a source of direct transfer to the human gut. These data indicate that fast food shops should be regarded as ESBL/AmpC reservoirs, and a risk for humans. Major efforts should be made in Algeria through guidelines on good practices in the food chain, and more globally in all countries sharing similar poor levels of food hygiene worldwide.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Horses, Cats, and Dogs Over a 5-Year Period in France.

Methicillin-resistant Staphylococcus aureus (MRSA) has been reported as a worldwide pathogen in humans and animals including companion animals, i.e., cats, dogs, and horses. France lacked a comprehensive nationwide study describing the molecular features of MRSA circulating among companion animals over a large period of time. Here is reported the characterization of 130 non-duplicate clinical MRSA isolates collected from those three animal species from 2010 to 2015 through the French national Resapath network. Characterization of isolates was performed using phenotypic (antimicrobial susceptibility tests) and molecular (DNA arrays, spa-typing) methods. A horse-specific epidemiology was observed in France with the large dissemination of a unique clone, the CC398 clone harboring a Staphylococcal chromosomal cassette mec (SCCmec) type IV and spa-type t011. It was even the unique clone collected in 2015 whereas the clone CC8 USA500 (SCCmec type IV), classically described in horses, was present until 2014. Contrarily, cats and dogs were mainly infected by human-related MRSA isolates, i.e., clones usually reported in human infections, thus mirroring the human epidemiology in hospitals in France. Isolates belonging to the CC398 clone (SCCmec type IV or V) were also identified in 21.4% of dogs' and 26.5% of cats' MRSA isolates. In order to differentiate human-related from CC398 MRSA, tetracycline-resistance [or tet(M) detection] could be useful since this resistance is scarce in human-related strains but constant in CC398 MRSA isolates. In all, our data give a nationwide epidemiological picture of MRSA in companion animals over a 5-year period in France, adding further epidemiological information on the contribution of those animal species to a major public health issue. Considering the wide dissemination of CC398 MRSA isolates and the fact that 11/64 (17.2%) of them presented the Immune Evasion Cluster which enhances CC398 capacities to colonize humans, a specific attention should be paid in the coming years to determine the risk associated to the transmission in people in frequent contacts with companion animals. Our data also show that the prevalence of MRSA has likely decreased in cats, dogs, and horses between 2012 and 2015 in France. This trend should be monitored in the years to come.

Assessment of Adhesins as an Indicator of Pathovar-Associated Virulence Factors in Bovine Escherichia coli.

The CS31A, F17, and F5 adhesins are usually targeted by serology-based methods to detect pathogenic Escherichia coli associated with calf enteritis. However, the virulence traits of the selected isolates are still poorly described. Here, from a set of 349 diarrheagenic E. coli isolates from cattle, we demonstrated a 70.8% concordance rate (Cohen's kappa, 0.599) between serology- and PCR-based approaches for the detection of adhesins under field conditions. A 79% to 82.4% correspondence between the two methods was found for fimbrial adhesins, whereas major discrepancies (33%) were observed for CS31A-type antigens. Various F17A variants were found, such as F17Ac (20K) (50%), F17Aa (FY) (18.9%), F17Ab (8.1%), and F17Ad (111K) (5.4%), including a high proportion (17.6%) of new F17A internal combinations (F17Aab, F17Aac, and F17Abc) or untypeable variants. In addition, the highest proportion of pathovar-associated virulence factor (VF) genes was observed among E. coli isolates that produced F5/F41 adhesins. A specific link between the heat-stable toxins related to the enterotoxigenic E. coli (ETEC) pathovar and adhesins was identified. STa was significantly linked to F5/F41 and EAST1 to CS31A adhesins (P < 0.001), respectively, whereas NTEC was associated with F17 adhesin (P = 0.001). Clustering between phylogroups according to the adhesin types was also observed. Also, few Shiga toxin-producing E. coli (STEC) or enteropathogenic E. coli (EPEC) pathovars were identified. Finally, no statistically significant difference was observed in the occurrence of extended-spectrum beta lactamase (ESBL) production according to the adhesins expressed by the isolates (P = 0.09). Altogether, this study gives new insights into the relationship between adhesins, VF, and antimicrobial resistance in calf enteritis and supports the need for further standardization of methodologies for such approaches.

High prevalence of blaCTX-M-1/IncI1/ST3 and blaCMY-2/IncI1/ST2 plasmids in healthy urban dogs in France.

In the community, close contacts between humans and dogs may promote the transfer of extended-spectrum beta-lactamase/plasmidic AmpC cephalosporinase (ESBL/pAmpC) genes. Large-scale prevalence studies on ESBL/pAmpC carriage in dogs are rare, and data on ESBL/pAmpC plasmids are even more limited. Here, a considerable rate of 18.5% ESBL/pAmpC carriers was found among 368 unrelated healthy dogs in Paris, France. This prevalence is much higher than the one found in healthy humans in the same city (6%) but close to that recently reported in dogs in China (24.5%). All isolates were identified as Escherichia coli, except one Salmonella enterica and one Klebsiella pneumoniae isolate. The sequence type 131 (ST131) clone was rare (2/73 isolates). Interestingly, two plasmids (blaCTX-M-1/IncI1/ST3 and blaCMY-2/IncI1/ST2) were unexpectedly highly predominant, raising the question of their successful spread. Considering that CTX-M-1 was recently found to be equally as abundant as CTX-M-15 in healthy Parisian subjects, the question of dogs being a CTX-M-1 reservoir for humans is open. Such a high prevalence of the blaCMY-2/IncI1/ST2 plasmid may result from the use of cephalexin in veterinary medicine, as previously demonstrated experimentally. In all, our study points out healthy urban dogs as a potential source of ESBL/pAmpC genes that can further disseminate to the human community.

Comparative prevalence and characterization of ESBL-producing Enterobacteriaceae in dominant versus subdominant enteric flora in veal calves at slaughterhouse, France.

Food-producing animals have become a growing reservoir of Extended-Spectrum Beta-Lactamase (ESBL)-producing bacteria. In cattle, veal calves are exposed to high amounts of antibiotics but ESBL prevalence data are still limited compared to other food sectors such as poultry production. Based on the investigation of 491 veal calves from different slaughtering batches at 12 abattoirs, this study shows a prevalence of 29.4% of ESBL producers in the faecal flora of veal calves in France in 2012. A variety of blaCTX-M genes was found, reflecting possible diverse pathways of dissemination in cattle. Another major conclusion is the comparison of the ESBL prevalence in the dominant versus sub-dominant Escherichia coli population of the same calves (1% and 29.4%, respectively). Also, the ESBL E. coli clones in the sub-dominant flora mostly differed from the non-ESBL dominant E. coli clones of the same calves. Of note, the distribution of blaCTX-M genes and E. coli phylogroups were similar to the ones previously found in ESBL E. coli clones from diseased calves. The hypothesis that ESBL genes may distribute more abundantly in certain backgrounds of E. coli was also discussed. In all, as recently reported in the Netherlands, these results strongly suggest a recent increase in the prevalence of ESBL carriage in French veal calves, which should be considered one of the major ESBL reservoirs in food animals.

Characterization of extended-spectrum beta-lactamase (ESBL)-carrying plasmids and clones of Enterobacteriaceae causing cattle mastitis in France.

Extended-spectrum beta-lactamases (ESBLs) have become widespread enzymes in food-producing and companion animals worldwide. However, in cattle mastitis, a major cause of economic loss in the dairy industry, ESBL-producers were rarely described. In this study, from a collection of 1427 Escherichia coli and Klebsiella pneumoniae isolates causing clinical mastitis in France, we report 0.4% (6/1427) of the isolates carrying an ESBL gene. These six isolates were genetically unrelated and recovered over a 3-year period of time. The bla(CTX-M-14) gene was found in 4/6 isolates, and was predominantly located on F2:A-:B- IncFII plasmids. The bla(CTX-M-1) IncI1/ST3, which is widespread in various animal species in France, was found as well. Interestingly, among the five E. coli isolates, the ST23 and ST58 clones were found twice, together with the ST10 clone, all of which were previously found as ESBL-carriers in humans. Despite the very limited number of ESBL-producers recovered, this study shows a surprisingly low molecular diversity of the strains causing mastitis in France with respect to ESBL genes, plasmids and clones. Further work is needed to understand the major driving forces of the ESBL epidemiology in animals, including for different infections within the same animal species.