Comparative phylogenomics of ESBL-, AmpC- and carbapenemase-producing Klebsiella pneumoniae originating from companion animals and humans.

BACKGROUND: WHO considers ESBL- and carbapenemase-producing Klebsiella pneumoniae a major global concern. In animals, ESBL- and carbapenemase-producing K. pneumoniae of human-related ST11, ST15 and ST307 have been reported, but not in the context of large WGS-based One Health investigations. OBJECTIVES: To perform comparative phylogenomics on a large collection of multidrug-resistant (MDR) K. pneumoniae recovered from diseased companion animals and humans. METHODS: MDR K. pneumoniae (n = 105) recovered from companion animals in France during 2010-18 were phenotypically characterized. All isolates were whole-genome sequenced using the NovaSeq technology and phylogenomic analysis across animal and human K. pneumoniae was performed using appropriate pipelines. RESULTS: bla CTX-M-15, blaDHA-1 and blaOXA-48 were strongly associated with IncFIIk, IncR and IncL plasmids, respectively. When compared with human K. pneumoniae genomes, four groups of closely related French human and animal isolates belonging to ST11, ST15 and ST307 were detected, suggesting the circulation of clones between the human and animal sectors at country level. A large cluster of 31 ST11-KL105 animal isolates from France and Switzerland suggested it corresponds to a sub-lineage that is particularly well-adapted to the animal host. CONCLUSIONS: This study demonstrates the spread of blaCTX-M-15-carrying ST15 and ST307, and blaDHA-1-carrying ST11 K. pneumoniae clones in animal populations. ST11 was the main vector of blaOXA-48/IncL, despite the absence of carbapenem use in French animals. Comparative phylogenomics suggests cross-transmission of K. pneumoniae sub-lineages more prone than others to colonize/infect the animal host. Our data also evidenced the emergence of convergent hypervirulent and MDR K. pneumoniae in animals.

Spread of blaCTX-M-15-Producing Enterobacteriaceae and OXA-23-Producing Acinetobacter baumannii Sequence Type 2 in Tunisian Seafood.

Bivalves are filter-feeding animals and markers of bacterial pollution. We report a massive spread of blaCTX-M-15 through dominant Escherichia coli and Klebsiella pneumoniae lineages and/or plasmid subtypes (F31:A4:B1) as well as the presence of OXA-23-producing Acinetobacter baumannii sequence type 2 (ST2) in seafood, highlighting a direct risk for the consumer. These findings should urge authorities to consider hospital effluents, and also farm and urban effluents, as important sources of extended-spectrum-beta-lactamase (ESBL)/carbapenemase producers that filter-feeding animals can concentrate and further spread to humans.

Emergence of blaCTX-M-55 associated with fosA, rmtB and mcr gene variants in Escherichia coli from various animal species in France.

Objectives: In Asian countries, blaCTX-M-55 is the second most common ESBL-encoding gene. blaCTX-M-55 frequently co-localizes with fosA and rmtB genes on epidemic plasmids, which remain sporadic outside Asia. During 2010-13, we investigated CTX-M-55-producing Escherichia coli isolates and their co-resistance to fosfomycin, aminoglycosides, fluoroquinolones and colistin as part of a global survey of ESBLs in animals in France. Methods: blaCTX-M-55, fosA, rmtB and plasmidic quinolone and colistin resistance genes were characterized by PCR, sequencing and hybridization experiments. Plasmids were classified according to their incompatibility groups and subtypes. Genotyping was performed by MLST and repetitive extragenic palindromic sequence-based PCR. Results: Twenty-one E. coli isolates from bovines (n = 16), dogs (n = 2), horses (n = 2) and a monkey harboured blaCTX-M-55, were MDR and belonged to ST744 (n = 9) and 10 other clones. blaCTX-M-55 was mostly located on IncF (n = 19), but also on IncI1 (n = 2) plasmids. On IncF33:A1:B1 plasmids, blaCTX-M-55 co-localized with the rmtB and aac(6')-Ib genes and in one isolate with the fosA3 allele. Ten IncF46:A-:B20 plasmids, which were found in different clones from unrelated animals, also carried the mcr-3 gene. blaCTX-M-55-carrying IncF18:A-:B1 plasmids were found in different animal species from distinct locations and periods, and one additionally carried the fosA4 gene. One isolate harboured the mcr-1 gene, which did not co-localize with blaCTX-M-55. Conclusions: A large diversity of E. coli clones and plasmid types supported the spread of blaCTX-M-55, together with atypical resistance genes, in various animal species in France. fosA and rmtB genes are emerging among animals in Europe and this issue is of concern for public health.

High prevalence of international ESBL CTX-M-15-producing Enterobacter cloacae ST114 clone in animals.

OBJECTIVES: The objective of this study was to characterize ESBL-producing Enterobacter cloacae isolated from animals and to compare their clonal distribution with that of human-related isolates. METHODS: Among 635 clinical E. cloacae from horses, dogs and cats collected in France between 2010 and 2013, 36 were resistant to ceftiofur as determined by disc diffusion. ESBL genes were identified by sequencing. Plasmids carrying ESBL-encoding genes were characterized by PCR-based replicon typing, S1-PFGE and Southern blotting. IncHI2 plasmids were subtyped using the plasmid double-locus sequence typing scheme and multiplex amplification of the hipA, smr0092 and smr0183 genes. All E. cloacae were typed by PFGE and MLST. ST clustering was analysed by eBURST. RESULTS: All 36 ceftiofur-resistant E. cloacae produced an ESBL. Their PFGE patterns formed 23 clusters of high similarity and 13 STs and were isolated from epidemiologically unrelated animals (14 horses, 11 dogs and 11 cats) distributed throughout France. ST114, the most prevalent clone in humans, was over-represented in animals (16/36) compared with other human-related clones detected here. The blaCTX-M-15 gene was dominant (66.7%) and mostly carried on IncHI2 plasmids (ST1 subtype). ST114 isolates always produced CTX-M-15. CONCLUSIONS: Most ESBL-producing E. cloacae from animals studied here (69.4%) belonged to potentially high-risk clones in humans, in particular ST114 (44.4%). These data raise questions and potential concerns about the transfer of E. cloacae between animals and humans.

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.

Multiple host colonization and differential expansion of multidrug-resistant ST25-Acinetobacter baumannii clades.

The Acinetobacter baumannii clonal lineage ST25 has been identified in humans and animals and found associated with outbreaks globally. To highlight possible similarities among ST25 A. baumannii of animal and human origins and to gather clues on the dissemination and evolution of the ST25 lineage, we conducted a phylogenetic analysis on n = 106 human and n = 35 animal A. baumannii ST25 genomes, including 44 sequenced for this study. Resistance genes and their genetic background were analyzed, as well. ST25 genomes are clustered into four clades: two are widespread in South America, while the other two are largely distributed in Europe, Asia and America. One particular clade was found to include the most recent strains and the highest number of acquired antibiotic resistance genes. OXA-23-type carbapenemase was the most common. Other resistance genes such as blaNDM-1, blaPER-7, and armA were found embedded in complex chromosomal regions present in human isolates. Genomic similarity among multidrug resistant ST25 isolates of either animal or human origin was revealed, suggesting cross-contaminations between the two sectors. Tracking the clonal complex ST25 between humans and animals should provide new insights into the mode of dissemination of these bacteria, and should help defining strategies for preserving global health.

Healthcare Equipment and Personnel Reservoirs of Carbapenem-Resistant Acinetobacter baumannii Epidemic Clones in Intensive Care Units in a Tunisian Hospital.

Carbapenem-resistant Acinetobacter baumannii (CRAB) strains can cause severe and difficult-to-treat infections in patients with compromised general health. CRAB strains disseminate rapidly in nosocomial settings by patient-to-patient contact, through medical devices and inanimate reservoirs. The occurrence of CRAB in patients residing in the intensive care units (ICUs) of the Sahloul University hospital in Sousse, Tunisia is high. The objective of the current study was to determine whether the surfaces of items present in five ICU wards and the medical personnel there operating could serve as reservoirs for CRAB strains. Furthermore, CRAB isolates from patients residing in the ICUs during the sampling campaign were analyzed for genome comparison with isolates from the ICUs environment. Overall, 206 items were screened for CRAB presence and 27 (14%) were contaminated with a CRAB isolate. The items were located in several areas of three ICUs. Eight of the 54 (15%) screened people working in the wards were colonized by CRAB on the hands. Patients residing in the ICUs were infected with CRAB strains sharing extensive genomic similarity with strains recovered in the nosocomial environment. The strains belonged to three sub-clades of the internationally disseminated clone (ST2). A clone emerging in the Mediterranean basin (ST85) was detected as well. The strains were OXA-23 or NDM-1 producers and were also pan-aminoglycoside resistant due to the presence of the armA gene. Hygiene measures are urgent to be implemented in the Sahloul hospital to avoid further spread of difficult-to-treat CRAB strains and preserve health of patients and personnel operating in the ICU wards.

Non-ST131 Escherichia coli from cattle harbouring human-like bla(CTX-M-15)-carrying plasmids.

OBJECTIVES: To characterize bla(CTX-M-15)-carrying plasmids and lineages of nine strains of Escherichia coli from cattle. METHODS: Plasmid DNA was analysed using PCR-based replicon typing and plasmid sub-typing schemes, restriction fragment length polymorphism, S1 nuclease-PFGE and Southern hybridization. Strains were characterized by PFGE, multilocus sequence typing, phylogenetic grouping and B2-O25b:H4-ST131 (where ST stands for sequence type) clone screening. Susceptibilities to antimicrobials were determined by agar diffusion and resistance genes were characterized by PCR and sequencing. RESULTS: The bla(CTX-M-15) gene was found on F31:A4:B1/IncFII and F2:A-:B-/IncFII plasmids, which have been reported abundantly in humans. On F31:A4:B1/IncFII plasmids, the bla(CTX-M-15) gene was associated with the bla(TEM-1), bla(OXA-1) and aac(6')-Ib-cr resistance genes. The bla(CTX-M-15) gene was also found on IncI1 plasmids of the CC31 clonal complex, recently identified in the human epidemic and virulent E. coli clone O104:H4. None of the cattle isolates belonged to the human and widespread clone B2-O25b:H4/ST131, but were mostly of new STs and of the phylogenetic groups A (n=4), B1 (n=3) or D (n=2). The E. coli isolates harbouring the bla(CTX-M-15)-carrying plasmids were genetically diverse, and were recovered from different geographical locations and farms and at different times. CONCLUSIONS: This study demonstrates that bla(CTX-M-15)-carrying plasmids from cattle-derived non-ST131 E. coli isolates were highly similar to those found in ST131 E. coli isolates commonly reported in humans. It also exemplifies the key role of plasmids versus clonal dissemination in the spread of the bla(CTX-M-15) gene among cattle, and possibly between E. coli isolates detected in humans and cattle.

A USA300 variant and other human-related methicillin-resistant Staphylococcus aureus strains infecting cats and dogs in France.

OBJECTIVES: To characterize methicillin-resistant Staphylococcus aureus (MRSA) clinical strains from cats and dogs in France, and to compare the clones identified with the distribution of French human MRSA. METHODS: Susceptibilities to antimicrobials were assessed by disc diffusion. Resistance and virulence genes were screened using a microarray-based assay. Isolates were additionally characterized by SmaI macrorestriction analysis and spa typing. RESULTS: From 2006 to 2010, the proportion of MRSA infections in pets in France was low (1.8%), but most isolates (87.0%, 20/23) belonged to human clones. The most common clones were the Lyon clone (69.6%, 16/23), the livestock-associated CC398 (13.0%, 3/23) and the Geraldine clone (8.7%, 2/23). Interestingly, we report the first USA300 clone infecting a European dog, which was probably imported by a US patient. CONCLUSIONS: Over a 5 year period, the proportion of MRSA infections in pets appears low (<2%) in France, but the distribution of the clones mostly mirrors the epidemiology of human invasive clones. These data highlight the role of pets as both victims and reservoirs of endemic, epidemic and/or invasive MRSA.

CTX-M-15 extended-spectrum ß-lactamase in a shiga toxin-producing Escherichia coli isolate of serotype O111:H8.

We report the discovery of a CTX-M-15-producing Escherichia coli (STEC) of serogroup O111:H8, a major serotype responsible for human enterohemorrhagic Escherichia coli (EHEC) infections. In line with the recent CTX-M-15/O104:H4 E. coli outbreak, these data may reflect an accelerating spread of resistance to expanded-spectrum cephalosporins within the E. coli population, including STEC isolates.

Prevalence and Characterization of Extended-Spectrum ß-Lactamase- and Carbapenemase-Producing Enterobacterales from Tunisian Seafood.

Aquaculture is a rapidly expanding sector in which it is important to monitor the occurrence of multi-drug resistant (MDR) bacteria. The presence of extended-spectrum ß-lactamase (ESBL-) or carbapenemase-producing Enterobacterales is a commonly used indicator of the resistance burden in a given sector. In this study, 641 pieces of farmed fish (sea bream and sea bass), as well as 1075 Mediterranean clams, were analyzed. All ESBL- and carbapenemase-producing Enterobacterales collected were whole-genome sequenced. The proportion of ESBL-producing Enterobacterales was 1.4% in fish and 1.6% in clams, carried by Escherichia coli (n = 23) and Klebsiella pneumoniae (n = 4). The ESBL phenotype was exclusively due to the presence of blaCTX-M genes, the most frequent one being blaCTX-M-15. The blaCTX-M-1 gene was also identified in six E. coli, among which four were carried by IncI1/pST3 plasmids, possibly betraying an animal origin. Carbapenemases were absent in fish but identified in two K. pneumoniae isolates from clams (blaNDM-1 and blaOXA-48). Several sequence types (STs) identified were associated with human MDR clones such as E. coli ST131 and ST617, or K. pneumoniae ST307 and ST147. Our results might indicate that bacteria from hospital or farm effluents can reach the open sea and contaminate seafood and fish that are living or raised nearby. Therefore, monitoring the quality of water discharged to the sea and the presence of MDR bacteria in seafood is mandatory to ensure the quality of fishery products.

Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis.

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.

Dynamics and molecular features of OXA-48-like-producing Klebsiella pneumoniae lineages in a Tunisian hospital.

OBJECTIVES: The aim of this study was to elucidate the molecular features of genes, plasmids and clones of OXA-48-like producingKlebsiella pneumoniae isolates recovered in Sahloul Hospital (Sousse, Tunisia) in the period 2012-2014. METHODS: In vitro antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting and PCR-based replicon typing (PBRT) were performed. Extended-spectrum ß-lactamase (ESBL) and carbapenemases genes were detected by PCR and sequencing. The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). RESULTS: Klebsiella pneumoniae accounted for 26.8% (1095/4083) of clinical Enterobacterales isolates identified during 2012-2014, of which 21.9% (240/1095) were resistant to carbapenems, mostly harbouring blaOXA-48-like genes (196/240; 81.7%). Plasmid analysis showed that blaOXA-204 and blaOXA-48 were mostly carried by IncA/C and IncL plasmids, respectively. The current data highlight the dominance of two ST101 and ST147 lineages spreading OXA-48 and OXA-204, respectively, through successive clonal spreads at this hospital. In addition, a large diversity of other K. pneumoniae lineages was also identified, such as ST15, ST36 and ST525 spreading OXA-48 as well as ST340, ST2032, ST301, ST199 and ST1561 spreading OXA-48 or OXA-204, constituting a reservoir of possible dominant clones in the future. CONCLUSION: This study reports the full molecular characterisation of carbapenem resistance in K. pneumoniae and the predominance of a few clones responsible for the dissemination of OXA-48 and OXA-204 enzymes in a Tunisian hospital.