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.

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.

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.

Investigation of an XDR-Acinetobacter baumannii ST2 outbreak in an intensive care unit of a Lebanese tertiary care hospital.

Aim: We sought to investigate the genetic epidemiological relatedness of carbapenem-resistant Acinetobacter baumannii (CRAB) strains of a suspected outbreak in a Lebanese tertiary care hospital to implement necessary infection prevention and control measures. Methods: Twenty-eight nonduplicate CRAB isolates detected among hospitalized patients between January 2016 and July 2017 were studied by real-time polymerase chain reaction (PCR), pulsed-field gel electrophoresis and multilocus sequence typing analyses. Results: Twenty-seven isolates harbored blaOXA-23, of which one also carried blaNDM-1. The isolates distributed temporally in two presumably episodes were stratified by pulsed-field gel electrophoresis into many clusters. Although several clones have become endemic in the hospital, we have rapidly implemented appropriate infection prevention and control measures, achieving full eradication from August 2017 to November 2019. Conclusion: We have successfully investigated and controlled a polyclonal outbreak of OXA-23 producing ST2 CRAB.

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.

High Genetic Diversity of Enterobacteriaceae Clones and Plasmids Disseminating Resistance to Extended-Spectrum Cephalosporins and Colistin in Healthy Chicken in Tunisia.

Enterobacteriaceae resistant to extended-spectrum cephalosporins (ESC-R) are listed as "priority pathogens" by the World Health Organization, and the Agri-food sector has regularly been pointed out as a potential source of ESC-R for humans through food consumption and animal handling. Chicken industry and chicken meat have recurrently been under specific scrutiny due to the high proportions of ESC-R reported worldwide in this sector. In Tunisia, recent studies suggested that the plasmidic AmpC blaCMY-2 gene may have emerged in chicken. We thus collected 258 cloacal swabs from five different farms and selected ESC-R isolates to determine the current ESC-R prevalence and epidemiology. All five farms were ESC-R positive with proportions ranging from 4% to 67.3%. blaCTX-M-1/IncI1/ST3 was the dominant gene/plasmid association in chicken, but several other CTX-M genes and plasmid backgrounds were shown to spread ESC-R. Surprisingly, the CMY-2 enzyme was only identified in one isolate. In addition, we also reported the sporadic presence of the mcr-1 gene carried by an IncHI2 plasmid. Our data suggest that the high diversity of Enterobacteriaceae clones and plasmids circulating in healthy chicken in Tunisia maintains a high ESC-R proportion in flocks and constitutes a major source of ESC-R determinants further disseminating in the food chain.

Genomic characterisation of a multidrug-resistant TEM-52b extended-spectrum ß-lactamase-positive Escherichia coli ST219 isolated from a cat in France.

OBJECTIVES: TEM-52 extended-spectrum ß-lactamases (ESBLs) have been detected in members of the Enterobacteriaceae isolated from human and non-human reservoirs, mainly in European countries. Here we report the first draft genome of a multidrug-resistant TEM-52b-positive Escherichia coli isolated from a companion animal in France. METHODS: Whole genomic DNA from E. coli 39590 was extracted and was sequenced using an Illumina NextSeq platform. De novo genome assembly was performed using Velvet v.1.2.10 and the draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline v.3.2. Genomic analyses were performed through bioinformatics tools from the Center for Genomic Epidemiology. RESULTS: The genome size was calculated as 5362108bp, with 5268 protein-coding sequences and a GC content of 50.5%. E. coli strain 39590 belonged to ST219, serotype O4:H34 and phylogroup E. The antimicrobial resistome consisted of genes encoding resistance to ß-lactams (blaTEM-52b), aminoglycosides [aph(3″)-Ib, aph(6)-Id, aadA2, aadA24], phenicols (catA1), sulfonamides (sul1, sul2), trimethoprim (dfrA1, dfrA14), lincosamides (lnuG) and tetracycline (tetA) as well as mutations in gyrA (Ser83Leu, Asp87Asn) and parC (Ser80Ile) conferring resistance to quinolones. Virulome analysis revealed iss, astA and eilA genes, and IncQ1, IncX4, IncX1, IncFIB and IncFIC plasmid incompatibility groups were identified. CONCLUSION: This draft genome can be used as a reference sequence for comparative studies using human and non-human E. coli isolates to identify genetic events that have contributed to pathogenicity and adaptation of TEM-52-producing E. coli clones at the human-animal interface as well as to elucidate dynamics of the spread of blaTEM-52 ESBL genes.

Prevalence and molecular features of ESBL/pAmpC-producing Enterobacteriaceae in healthy and diseased companion animals in Brazil.

Extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated AmpC (pAmpC)-carrying Enterobacteriaceae have widely disseminated in human, animal and environmental reservoirs. Pets have been recognized as a source of ESBL/pAmpC worldwide, and are possibly also a source of human contamination. The aim of this study was to document to what extent cats and dogs may act as a driving force in the spread of ESBLs and pAmpCs in Brazil. A total of 113 healthy stray cats and dogs and 74 sick pets were sampled, and extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESC-R) were detected in 28/113 (24.8%) and 8/74 (10.8%) tested animals, respectively. Different Enterobacteriaceae isolates (mostly E. coli), a large number of E. coli clones (with ST90, ST457, ST973 and ST2541 being predominant), and several ESBL/pAmpC genes and plasmids were characterized, highlighting the ability of stray and pet cats and dogs to further spread a wide range of ESC-resistance determinants. The ESBL phenotype was due to the blaCTX-M-2 and blaCTX-M-8 genes, as found in human epidemiology in Brazil, but blaCTX-M-9 and blaCTX-M-15 were also identified. The pAmpC phenotype was systematically due to the presence of the blaCMY-2 gene, mostly carried by IncI1 ST12 plasmids. Our results showed that pets can be considered a significant reservoir of multidrug-resistant bacteria in Brazil. This is especially true for healthy stray dogs that displayed the highest prevalence (24.8%) of ESBLs/pAmpC resistance determinants, which can then be further spread both to the environment and to other animals or humans by contact.

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.

Is blaCTX-M-1 Riding the Same Plasmid Among Horses in Sweden and France?

A predominance of the blaCTX-M-1/IncHI1 plasmid combination in horses has been reported in Czech-Republic, Denmark, and The Netherlands. To clarify a possible specific plasmid epidemiology of blaCTX-M-1 in horses in a European perspective, a collection of 74 extended-spectrum beta-lactamase-producing Escherichia coli recovered from diseased horses in France and Sweden during the period 2009-2014 was investigated in respect of their genetic relatedness, plasmid content, and molecular features. Overall, 80% of E. coli isolates from diseased horses harbored blaCTX-M-1 on large IncHI1 plasmids with plasmid sequence type (pST) 2 and pST9 more prevalent in Sweden and France, respectively. In French isolates, IncI1/pST3 plasmids were also identified. The CTX-M-1-producing E. coli belonged principally to the clonal complex 10. ST641, together with its single locus variant, and ST1730 constituted two other major groups. The rep-PCR clustering highlighted a clonal dissemination of E. coli CTX-M-1 producers in different regions of the same country and during several years. The STs found in our isolates were also reported in The Netherlands, suggesting a common source of contamination in Europe, although only further cooperative investigation will clarify this issue. On the other hand, the spread of IncI1/pST3 plasmids among horses constitutes another worrisome issue considering their successful spread in other animal hosts such as chicken or bovines. Monitoring evolution and propagation of antimicrobial resistance in equine environment is a priority to avoid further propagation of antimicrobial resistance mechanisms threatening human and animal health.

Extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae in water sources in Lebanon.

Extended-spectrum beta-lactamases (ESBLs) have been recurrently reported in both human and veterinary medicine, and carbapenemases have also emerged in these two sectors. Such resistance phenotypes were increasingly reported in the environment, which both receives and further disseminates multidrug-resistant (MDR) bacteria. Here, we report the high contamination of water samples (68.2%; 15/22) collected in estuaries in Lebanon. From these 15 contaminated sites, a total of 21 ESBL-producing (mostly harbouring the blaCTX-M-15 gene) and four carbapenemase-producing (two blaOXA-48 and two blaOXA-244) Enterobacteriaceae were recovered. ESBL contamination was also identified in water samples collected from rural wells and spring water, although at a lower frequency. Indeed, 1.9% (3/155) and 6.1% (7/115) of the wells and springs were contaminated, respectively, and all identified isolates were CTX-M-15-producing E. coli. Interestingly, sequence types (STs) previously associated both with animal and human reservoirs were detected (ST38, ST10 and ST131), suggesting a complex source of contamination. This situation is alarming since water drawn from wells or springs is directly intended for human consumption in Lebanon without any further treatment. Moreover, even though water from estuaries is not intended for human consumption, it is used to water animals and irrigate crops. Consequently, water contamination by ESBLs and carbapenemases in Lebanon is potentially a major risk to public health. Part of this work was presented at the 7th Symposium on Antimicrobial Resistance in Animals and the Environment (ARAE).

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.

OXA-48 and CTX-M-15 extended-spectrum beta-lactamases in raw milk in Lebanon: epidemic spread of dominant Klebsiella pneumoniae clones.

Raw milk has recently been reported as a source of extended-spectrum beta-lactamase (ESBL) and carbapenemase genes. We thus investigated the prevalence of ESBL- and carbapenemase-producing Enterobacteriaceae in raw milk in Lebanon in order to assess the risk of transfer of these bacteria to humans. A high prevalence (30.2 %) of CTX-M-15-producing K. pneumoniae was detected in raw bovine milk. Three main K. pneumoniae clones were identified by PFGE and MLST typing. Southern blot experiments revealed that one of these clones carried the blaCTX-M-15 gene chromosomally. Moreover, one OXA-48-producing K. pneumoniae ST530 and seven CTX-M-15-producing Escherichia coli sharing the same ST were also detected. These findings highlight the spread of dominant CTX-M-15-producing K. pneumoniae clones and OXA-48-producing isolates in the food chain. Milk, which is mostly consumed raw in Lebanon, may be a source of human exposure to ESBLs and carbapenemases.

Outbreak of colistin-resistant carbapenemase-producing Klebsiella pneumoniae in Tunisia.

OBJECTIVES: Mechanisms of colistin and carbapenem resistance among a collection of Klebsiella pneumoniae isolates recovered in a university hospital in Tunisia were studied. 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-lactamases (ESBLs), carbapenemases, AmpC-type enzymes and mgrB genes were detected by PCR and sequencing. Clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). RESULTS: Of 940 Enterobacteriaceae isolates recovered from June 2015 to March 2016 in Tahar Sfar Hospital (Mahdia, Tunisia), 220 were identified as K. pneumoniae, among which 29 were carbapenem-resistant. Carbapenem resistance was mostly due to expression of blaOXA-48 or blaOXA-204 in combination with blaCMY-4. Seven isolates carried blaNDM-1, of which two also harboured blaOXA-48, together with blaCMY-16 in one of them. All but two isolates also harboured blaCTX-M-15. All 20 blaOXA-48 genes were part of transposon Tn1999 on an IncL plasmid, whereas blaOXA-204 was found on transposon Tn2016 on an IncA/C plasmid. Finally, all blaNDM-1 genes were located within a Tn125 transposon on an IncFIIk plasmid. Interestingly, 7 (24.1%) of 29 carbapenem-resistant isolates were resistant to colistin, of which 6 were assigned to ST101, had similar PFGE profiles and presented the same 2-kb insertion in the mgrB gene. CONCLUSIONS: This study reports, for the first time in Tunisia, the full molecular characterisation of colistin resistance in K. pneumoniae. There is an urgent need for control measures and prudent use of colistin in treatment of infections with carbapenemase-producing K. pneumoniae.

High prevalence of ESBLs in retail chicken meat despite reduced use of antimicrobials in chicken production, France.

Extended-spectrum cephalosporins (ESCs) are critically important antibiotics for humans and their use in animals poses a potential threat for public health. Chicken represents an increasing part of the human diet and has also been regarded as a source of ESC-resistant Enterobacteriaceae because of the worldwide off-label use of ceftiofur, a broad-spectrum cephalosporin. Thus, numerous studies pointed out chicken as a reservoir of ESBL/pAmpC genes, plasmids and/or clones at risk for humans. In France, late 2011, strong political pressure led to a drastic reduction of ceftiofur use and all other antibiotics in chicken production. Here, we ascertained the potential impact of those efforts on the prevalence of ESC-resistant E. coli in retail chicken. From October 2015 to January 2016, of 48 unrelated pieces of meat (chicken legs) belonging to four different brands, 44 (91.7%) were positive for ESC-resistant E. coli. The blaCTX-M-1 gene was highly prevalent (68/74, 91.9%), mostly located on IncI1/ST3 plasmids (65/68, 95.6%). Other ESBL/pAmpC genes (blaTEM-52, blaSHV-12, blaCMY-2) were carried by IncX1, IncI1/ST36, IncI1/ST95, IncA/C or IncK plasmids. The positive isolates were non-clonal, suggesting a horizontal spread of the ESBL/pAmpC genes. Obviously, the strong decrease of antimicrobial use in chicken farms had no impact yet on the ESBL/pAmpC prevalence in retail chicken meat in France. A human source of these ESBL/pAmpC genes is unlikely as blaCTX-M-1 IncI1/ST3 plasmids are dominant in animals and rare in humans. Our data question the real impact of the decrease of antimicrobial use in chicken production on ESBL contamination of chicken meat and point out the risk of ESBL/AmpCs human transfer through the food chain.