Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

Genomic Analysis of Acinetobacter baumannii Isolates Carrying OXA-23 and OXA-58 Genes from Animals Reveals ST1 and ST25 as Major Clonal Lineages.

Acinetobacter baumannii is increasingly being recognized as a relevant pathogen for animals with a putative zoonotic impact. This study aimed at identifying and characterizing carbapenemase-producing A. baumannii from animals. Among 503 A. baumannii, mainly isolated from dogs/cats (75.7%) between 2013 and 2018, 42 isolates from 22 veterinary clinics (VCs) harboured blaOXA-58 (n = 29) or blaOXA-23 (n = 13). The blaOXA-58 gene was located on plasmids (11.4-21.1 kb) within different genetic surroundings (patterns A-D). BlaOXA-23 was embedded in Tn2006 on the chromosome (n = 4; pattern a) or Tn2008 on plasmids (n = 9; 41.2-71.3 kb; patterns b-e). The predominant IC1-ST1P-OXA-58 (66.7%; 96.4% cgMLST complex type (CT)-1808) was disseminated among 11 VCs in Germany. Resistance islands AbaR3-like (n = 15) and AbaR10 (n = 1) have emerged among ST1-isolates since 2016. IC7-ST25P-OXA-23 isolates (21.4%) occurred in seven VCs in Germany, France and Italy and differed in their resistance gene patterns from those of OXA-58 isolates. They were separated into six CTs, basically according to their regional origin. Other STs observed were ST10, ST578 and ST602. In conclusion, OXA-23 and OXA-58 were linked with ST1 and ST25, two globally distributed lineages in humans. The suggested transmission of certain lineages within and among VCs together with the acquisition of AbaR islands hints at a successful dissemination of multidrug-resistant strains in the VC environment.

Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes.

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I-IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches.

Multispecies and Clonal Dissemination of OXA-48 Carbapenemase in Enterobacteriaceae From Companion Animals in Germany, 2009-2016.

The increasing spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a serious threat to public health. Recent studies suggested animals as a putative source of such bacteria. We investigated 19,025 Escherichia coli, 1607 Klebsiella spp. and 570 Enterobacter spp. isolated from livestock, companion animal, horse, and pet samples between 2009 and 2016 in our routine diagnostic laboratory for reduced susceptibility to carbapenems (CP) by using meropenem-containing media. Actively screened CP non-susceptible strains as well as 367 archived ESBL/AmpC-ß-lactamase-producing Enterobacteriaceae were then tested for the presence of CP genes by PCRs. Among 21,569 isolates, OXA-48 could be identified as the sole carbapenemase type in 137 (0.64%) strains. The blaOXA-48 gene was located on an ∼60-kb IncL plasmid and sequence analysis revealed high similarity to reference plasmid pOXA-48a, which has been involved in the global spread of the blaOXA-48 gene in humans for many years. Klebsiella pneumoniae was the predominant OXA-48 producer (n = 86; 6.6% of all K. pneumoniae isolates), followed by E. cloacae (n = 28; 5.0%), Klebsiella oxytoca (n = 1; 0.3%), and E. coli (n = 22, 0.1%). OXA-48 was not found in livestock, but in dogs (120/3182; 3.8%), cats (13/792; 1.6%), guinea pig (1/43; 2.3%), rat (1/23; 4.3%), mouse (1/180; 0.6%), and one rabbit (1/144; 0.7%). Genotyping identified few major clones among the different enterobacteria species, including sequence types ST11 and ST15 for K. pneumoniae, ST1196 for E. coli, and ST506 and ST78 for E. cloacae, most of which were previously involved in the dissemination of multidrug-resistant strains in humans. The majority of OXA-48 isolates (n = 112) originated from a university veterinary clinic (UVC), while animals from further 16 veterinary institutions were positive. Clonal analyses suggested nosocomial events related to different species and STs in two veterinary clinics and horizontal transfer of the pOXA-48-like plasmid between bacterial species and animals. A systematic monitoring is urgently needed to assess the dissemination of CPE not only in livestock but also in companion animals and veterinary clinics.

Complete Genome Sequence of a Burkholderia pseudomallei Strain Isolated from a Pet Green Iguana in Prague, Czech Republic.

Burkholderia pseudomallei was isolated from pus from an abscess of a pet iguana living in a private household in Prague, Czech Republic. This paper presents the complete genome sequence of B. pseudomallei strain VB976100.

Diagnostic microbiology in veterinary dermatology: present and future.

BACKGROUND: The microbiology laboratory can be perceived as a service provider rather than an integral part of the healthcare team. OBJECTIVES: The aim of this review is to discuss the current challenges of providing a state-of-the-art diagnostic veterinary microbiology service including the identification (ID) and antimicrobial susceptibility testing (AST) of key pathogens in veterinary dermatology. METHODS: The Study Group for Veterinary Microbiology (ESGVM) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) identified scientific, technological, educational and regulatory issues impacting the predictive value of AST and the quality of the service offered by microbiology laboratories. RESULTS: The advent of mass spectrometry has significantly reduced the time required for ID of key pathogens such as Staphylococcus pseudintermedius. However, the turnaround time for validated AST methods has remained unchanged for many years. Beyond scientific and technological constraints, AST methods are not harmonized and clinical breakpoints for some antimicrobial drugs are either missing or inadequate. Small laboratories, including in-clinic laboratories, are usually not adequately equipped to run up-to-date clinical microbiologic diagnostic tests. CONCLUSIONS AND CLINICAL IMPORTANCE: ESGVM recommends the use of laboratories employing mass spectrometry for ID and broth micro-dilution for AST, and offering assistance by expert microbiologists on pre- and post-analytical issues. Setting general standards for veterinary clinical microbiology, promoting antimicrobial stewardship, and the development of new, validated and rapid diagnostic methods, especially for AST, are among the missions of ESGVM.

OXA-23 and ISAba1-OXA-66 class D ß-lactamases in Acinetobacter baumannii isolates from companion animals.

Acinetobacter baumannii is recognised as a major pathogen of nosocomial infections that frequently show resistance to last-resort antimicrobials. To investigate whether A. baumannii from companion animals harbour carbapenem resistance mechanisms, 223 clinical isolates obtained from veterinary clinics between 2000 and 2013 in Germany were screened for carbapenem-non-susceptibility employing meropenem-containing Mueller-Hinton agar plates. Minimum inhibitory concentration (MIC) data were obtained using the VITEK®2 system. Assignment to international clones (ICs) was done by multiplex PCR or repetitive sequence-based PCR employing the DiversiLab system. Clonality was studied using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Genes encoding carbapenemases and aminoglycoside-modifying enzymes were detected by PCR. In three samples from dogs, carbapenem-resistant A. baumannii carrying the blaOXA-23 gene on plasmids and located on transposon Tn2008 were identified. The isolates belonged to sequence type ST1P (clonal complex CC1/IC1/pulsotype II) and ST10P (CC10/IC8/pulsotype IV) according to the Pasteur MLST scheme, and to ST231Ox (CC109) and ST585Ox (CC447) following the Oxford scheme. Insertion sequence ISAba1 was identified upstream of blaOXA-66 in 58 A. baumannii isolates. MLST referred them to ST2P (CC2/IC2/pulsotypes I and III), ST208Ox, ST350Ox and ST556Ox (all CC118), respectively. PFGE suggested nosocomial spread of these highly related strains, which frequently demonstrated a multidrug-resistant phenotype, in one veterinary clinic. These data show that A. baumannii from companion animals reveal resistance determinants and clonal lineages of strains globally emerging in humans. This suggests an interspecies transmission and warrants molecular surveillance of A. baumannii in veterinary clinics to mitigate its further spread.

Urinary shedding of leptospires and presence of Leptospira antibodies in healthy dogs from Upper Bavaria.

Leptospirosis is classified as a re-emerging zoonotic disease with global impor- tance. The aim of this study was to determine urinary shedding of leptospires in healthy dogs and to identify the shedded leptospire species. Furthermore, antibody presence against leptospires was evaluated. In a prospective study urine samples of 200 healthy dogs from Upper Bavaria were randomly collected and evaluated by real-time polymerase chain reaction (PCR) specific for the lipL32 gene of pathogenic Leptospira (L) spp. Positive samples were further character- ized via multilocus sequence typing (MLST) to identify the Leptospira species. Microagglutination test (MAT) was performed to determine serum antibody titers. Three of 200 urine samples were found to be PCR-positive resulting in a urinary shedding prevalence of 1.5% (95% confidence interval 0.3-4.5%). All three dogs had been vaccinated before with a bivalent vaccine, covering the serogroups Canicola and lcterohaemorrhagiae. One dog shed leptospires of the species L. borgpetersenii, and two of the species L. interrogans. Of all dogs, 17.0% had antibody titers ≥ 1:100, and 3.5% titers ≥ 1:400 to serovars of non-vaccinal sero- groups. Healthy dogs that shed leptospires represent a possible risk for humans and other animals. The study emphasizes the importance of general hygiene measures in veterinary practice while handling urine of all dogs, and the use of vaccines that protect against a broader range of serogroups and that prevent urinary shedding.

A Look into the Melting Pot: The mecC-Harboring Region Is a Recombination Hot Spot in Staphylococcus stepanovicii.

INTRODUCTION: Horizontal gene transfer (HGT) is an important driver for resistance- and virulence factor accumulation in pathogenic bacteria such as Staphylococcus aureus. METHODS: Here, we have investigated the downstream region of the bacterial chromosomal attachment site (attB) for the staphylococcal cassette chromosome mec (SCCmec) element of a commensal mecC-positive Staphylococcus stepanovicii strain (IMT28705; ODD4) with respect to genetic composition and indications of HGT. S. stepanovicii IMT28705 was isolated from a fecal sample of a trapped wild bank vole (Myodes glareolus) during a screening study (National Network on "Rodent-Borne Pathogens") in Germany. Whole genome sequencing (WGS) of IMT28705 together with the mecC-negative type strain CM7717 was conducted in order to comparatively investigate the genomic region downstream of attB (GenBank accession no. KR732654 and KR732653). RESULTS: The bank vole isolate (IMT28705) harbors a mecC gene which shares 99.2% nucleotide (and 98.5% amino acid) sequence identity with mecC of MRSA_LGA251. In addition, the mecC-encoding region harbors the typical blaZ-mecC-mecR1-mecI structure, corresponding with the class E mec complex. While the sequences downstream of attB in both S. stepanovicii isolates (IMT28705 and CM7717) are partitioned by 15 bp direct repeats, further comparison revealed a remarkable low concordance of gene content, indicating a chromosomal "hot spot" for foreign DNA integration and exchange. CONCLUSION: Our data highlight the necessity for further research on transmission routes of resistance encoding factors from the environmental and wildlife resistome.

Suitable in vitro culture of Eimeria bovis meront II stages in bovine colonic epithelial cells and parasite-induced upregulation of CXCL10 and GM-CSF gene transcription.

We here established a suitable in vitro cell culture system based on bovine colonic epithelial cells (BCEC) for the development of Eimeria bovis merozoites I and the characterization of early parasite-induced innate epithelial host cell reactions as gene transcription of proinflammatory molecules. Both primary and permanent BCEC (BCEC (rim) and BCEC(perm)) were suitable for E. bovis merozoite I invasion and subsequent development of meronts II leading to the release of viable merozoites II. E. bovis merozoite II failed to develop any further neither into gamont nor oocyst stages in BCEC in vitro. E. bovis merozoite I induced innate epithelial host cell reactions at the level of CXC/CCL chemokines (CXCL1, CXCL8, CXCL10, CCL2), IL-6, and GM-CSF gene transcription. Overall, both BCEC types were activated by merozoite I infections since they showed significantly enhanced gene transcript levels of the immunomodulatory molecules CXCL10 and GM-CSF. However, gene transcription profiles of BCEC(prim) and BCEC(perm) revealed different reaction patterns in response to merozoite I infection with regard to quality and kinetics of chemokine/cytokine gene transcription. Although both BCEC types equally showed most prominent responses for CXCL10 and GM-CSF, the induction of CXCL1, CXCL8, CCL2, and IL-6 gene transcripts varied qualitatively and quantitatively. Our results demonstrate that BCEC seem capable to respond to E. bovis merozoite I infection by the upregulation of CXCL10 and GM-CSF gene transcription and therefore probably contribute to host innate effector mechanisms against E. bovis.

Isolation of the highly pathogenic and zoonotic agent Burkholderia pseudomallei from a pet green Iguana in Prague, Czech Republic.

BACKGROUND: Melioidosis caused by Burkholderia (B.) pseudomallei is an endemic zoonotic disease mainly reported from northern Australia and Southeast Asia. In Europe, cases of human melioidosis have been reported only from patients travelling to endemic regions. Besides humans, B. pseudomallei has a very broad host range in domestic and wild animals. There are some reports about importation of B. pseudomallei-infected animals from endemic areas into Europe. The present report describes the first case of B. pseudomallei infection of a pet iguana in Europe. CASE PRESENTATION: In a 5-year-old pet Iguana iguana living in a private household in Prague, Czech Republic, B. pseudomallei was isolated from pus of an abscess. The isolate VB976100 was identified by Vitek®2, MALDI-TOF mass spectrometry and polymerase chain reaction as B. pseudomallei. The molecular typing resulted in multi-locus sequence type 436 hitherto, which has been found only once worldwide in a B. pseudomallei strain isolated in the USA and originating from Guatemala. The identification as internal transcribed spacer type G indicates a close relatedness to strains mainly isolated in the Western Hemisphere. These findings support the hypothesis that the iguana became infected in this region or in a breeding facility through contact to other infected animals. CONCLUSIONS: The present case highlights the risk of importation of the highly pathogenic and zoonotic B. pseudomallei into non-endemic regions through animal trade. Therefore, veterinarians treating animals from these areas and physicians examining patients owning such animals should include melioidosis in differential diagnosis whenever specific symptoms appear. Furthermore, veterinary authorities responsible for supervision of traders and pet shops should be aware of this risk of zoonotic transmission.

Risk factors for MRSA infection in companion animals: results from a case-control study within Germany.

Increasing numbers of companion animals suffering from infections with methicillin-resistant Staphylococcus aureus (MRSA) have been reported in the recent past. These infections are of particular concern because of the limited treatment options for MRSA and their transferability to humans. Since MRSA lineages isolated from infected companion animals often mirror typical human epidemic strains circulating in the same region, successful strategies to combat MRSA need strong and coordinated efforts from both, the human and the veterinary field according to the "One Health" concept. Hence, to identify potential risk factors related to MRSA infections in dogs, cats and horses, a case-control study was conducted, including data on 106 MRSA-infected animal patients as cases and 102 MSSA-infected animals as controls, originating from 155 different veterinary settings within Germany. Demographic data on animal patients, patient history and administration of antibiotics as well as practice/clinic specific parameters were assessed as putative risk factors. Multivariable logistic regression identified the following variables as risk factors for MRSA infection compared to MSSA infection: number of employees working at the veterinary setting (n>10; p<0.001), antibiotic treatment prior to sampling (systemic: p=0.002; local: p=0.049, both: p=0.011) and surgical site infection (p<0.001). Spa typing revealed predominantly clonal complexes well-known for hospital-associated lineages spreading in human health-care settings in Germany (CC5 and CC22) for isolates of dog and cat origin. CC398-MRSA dominated among equine isolates, a CC that was described as a nosocomial pathogen in equine clinical settings before. The identified risk factors and genotyping results are in accordance with numerous study outcomes from the field of human medicine and point towards reasonable problems with nosocomial spread of MRSA, especially within companion animal veterinary clinics. To define targeted infection control strategies against nosocomial pathogens, it is important to accomplish intervention studies addressing routes of transmission in companion animal veterinary settings.

Clonal spread of highly successful ST15-CTX-M-15 Klebsiella pneumoniae in companion animals and horses.

OBJECTIVES: To investigate the clinical relevance and molecular epidemiology of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella species in animals. METHODS: Antimicrobial susceptibilities and presence of ESBLs were examined among Klebsiella spp. (n = 1519) from clinical samples (>1200 senders from Germany and other European countries) mainly from companion animals and horses from October 2008 to March 2010. Multilocus sequence typing (MLST) and PFGE were performed including human isolates for comparative purposes. RESULTS: The overall ESBL rate was 8% for Klebsiella pneumoniae subsp. pneumoniae. Most K. pneumoniae subsp. pneumoniae ESBL producers were isolated from soft tissue infections (29.3%) and urinary tract infections (14.9%). The major ESBL type was CTX-M-15 (85.4%), located on different plasmid scaffolds (HI2, I1, FIA, FIB, FII, A/C, R and N). Other ESBL genes, such as bla(CTX-M-1) (5.6%), bla(CTX-M-3), bla(CTX-M-9), bla(SHV-2) and bla(SHV-12) (1.1% each), were also detected. Additional resistances, e.g. to fluoroquinolones (89.9%), were frequently present. ST15-CTX-M-15, a clonal group that recently emerged in humans, accounted for 75.8% of the strains analysed by MLST and there was evidence for nosocomial events in five veterinary clinics. Human ST15-CTX-M-15 strains shared PFGE clusters with animal isolates, suggesting the dissemination of this clonal group between both populations. CONCLUSIONS: Our data indicate a wide spread of ST15-CTX-M-15 K. pneumoniae subsp. pneumoniae, which should be considered as a zoonotic agent of high clinical relevance for humans and animals. Further research should be undertaken to unravel both microevolutionary and biological aspects probably contributing to this global success.

Alarming proportions of methicillin-resistant Staphylococcus aureus (MRSA) in wound samples from companion animals, Germany 2010-2012.

Staphylococcus (S.) aureus is an important cause of wound infections in companion animals, and infections with methicillin-resistant S. aureus (MRSA) are of particular concern due to limited treatment options and their zoonotic potential. However, comparable epidemiological data on MRSA infections in dogs, cats and horses is scarce, also limiting the knowledge about possible links to MRSA isolates from human populations. To gain more knowledge about the occurrence and genotypic variation of MRSA among wound swabs of companion animal origin in Germany we performed a survey (2010-2012) including 5,229 samples from 1,170 veterinary practices. S. aureus was identified in 201 (5.8%) canine, 140 (12.2%) feline and 138 (22.8%) equine swabs from a total of 3,479 canine, 1,146 feline and 604 equine wounds, respectively. High MRSA rates were identified with 62.7%, 46.4% and 41.3% in S. aureus of canine, feline and equine origin, respectively. Further genotyping including spa typing and multilocus sequence typing (MLST) revealed a comparable distribution of spa types among canine and feline MRSA with CC22 (47.6%; 49.2%) and CC5 (30.2%; 29.2%) as predominant lineages followed by CC398 (13.5%; 7.7%) and CC8 (4.0%; 9.2%). In contrast, the majority of equine MRSA belonged to CC398 (87.7%). Our data highlight the importance of S. aureus and MRSA as a cause of wound infections, particularly in cats and horses in Germany. While "human-associated" MRSA lineages were most common in dogs and cats, a remarkable number of CC398-MRSA was detected in horses, indicating a replacement of CC8-MRSA as the predominant lineage within horses in Germany. These data enforce further longitudinal epidemiological approaches to examine the diversity and temporal relatedness of MRSA populations in humans and animals to assess probable sources of MRSA infections. This would enable a sound risk assessment and establishment of intervention strategies to limit the additional spread of MRSA.

CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence?

OBJECTIVES: To discern the relevance of ST648 extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli as a putative new group of multiresistant and extraintestinal pathogenic strains in animals, its frequency, ESBL types, antimicrobial resistance patterns and virulence gene (VG) profiles should be determined and compared with ST131 strains from the same collection of strains. METHODS: ESBL-producing E. coli isolates (n = 1152), consecutively sampled from predominantly dogs, cats and horses between 2008 and 2011, were assigned to a phylogenetic group by PCR. Partial multilocus sequence typing was performed for group D and B2 strains and strains presumed to be D-ST648 and B2-ST131 were fully typed. ESBL genes and extraintestinal pathogenic E. coli (ExPEC)-like VGs were characterized by PCR and sequence analysis and antimicrobial resistance was determined by broth dilution. Clonal analysis was done by PFGE. RESULTS: Forty (3.5%) ESBL-producing E. coli were determined as D-ST648, whereas B2-ST131 isolates occurred less frequently (2.8%). Although the predominant ESBL type in both groups was CTX-M-15 (72.5% versus 46.9%), ST648 strains from companion animals and horses displayed a lower variety of ESBL types (CTX-M-1, -3, -14, -15 and -61 versus CTX-M-1,-2,-14,-15,-27 and -55 and SHV-12). In contrast to ST131 strains, a higher proportion of ST648 strains showed resistance to most non-ß-lactam antibiotics. Overall, VGs were less abundant in ST648 strains, although some strains had VG profiles comparable to those of ST131 strains. ExPEC-associated serotype O1:H6 was predominant (46.8%) among the ST648 strains. Some PFGE clusters comprised ST648 isolates from pets, horses and wild birds and humans included from previous studies. CONCLUSIONS: Our findings demonstrate that certain subgroups of E. coli D-ST648-CTX-M may represent a novel genotype that combines multiresistance, extraintestinal virulence and zoonotic potential.