Mirjam Grobbel

Emergence of human pandemic O25:H4-ST131 CTX-M-15 extended-spectrum-β-lactamase-producing Escherichia coli among companion animals

OBJECTIVES In view of the intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15 extended-spectrum beta-lactamase (ESBL) in human clinical settings it would be of great interest to explore its existence in animals to unravel a possible reservoir function and the origin and transmission of this group of multiresistant strains. METHODS A total of 177 clinical phenotypically ESBL-producing E. coli isolates, mainly obtained from companion animals with urinary tract infections, wound infections and diarrhoea, were collected in a veterinary diagnostic laboratory covering a European-wide service area. They were screened for molecular subtype O25b and multilocus sequence type 131. O25b-ST131 isolates were subsequently tested for ESBL types, and phenotypic and genotypic resistance determinants. Further characterization of the strains was performed by PFGE and virulence gene typing. RESULTS Ten (5.6%) of 177 phenotypically ESBL-producing E. coli isolates, nine strains from dogs and one strain from a horse, were allocated to the B2-O25b-ST131 lineage. Nine of these isolates harboured a CTX-M-15-type beta-lactamase enzyme while one strain possessed an SHV-12-type ESBL. Macrorestriction analysis revealed a cluster formation of six of the animal CTX-M-15-type ESBL-producing strains from five different European countries together with a human control strain constituting a group of clonally related strains at a similarity value of 87.0%. CONCLUSIONS Our findings demonstrate that the group of clonally related human B2-O25:H4-ST131 CTX-M-15-type ESBL-producing E. coli strains is present in companion animals from various European countries. This highlights the possibility of inter-species transmission of these multiresistant strains from human to animal and vice versa.

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 β-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-β-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.

Antimicrobial resistance profiles of Escherichia coli from common European wild bird species.

The emergence and spread of multiresistant bacteria in natural environments constitute a serious impact on animal and human health. To gain more insight into the role of wild birds as carriers and reservoir of multiresistant Escherichia coli we tested a broad spectrum of common European bird species for the occurrence of E. coli strains and their antimicrobial resistance by minimal inhibitory concentration testing and PCR analysis of several resistance genes. Nine of the 187 E. coli isolates (4.8%) exhibited multiresistant phenotypes including resistances against beta-lactams, aminoglycosides, fluoroquinolones, tetracyclines and sulfonamides. By comparing avian E. coli resistance frequencies with frequencies known for E. coli isolated from livestock and companion animals analogous profiles were identified. Multiresistant E. coli strains were isolated from synanthropic avian species as well as from birds of prey, waterfowl and passerines. By that, all these avian hosts are suggested to represent a considerable reservoir of resistant E. coli strains. Consequently wild birds might constitute a potential hazard to human and animal health by transmitting multiresistant strains to waterways and other environmental sources via their faecal deposits.

CTX-M-15-type extended-spectrum beta-lactamases-producing Escherichia coli from wild birds in Germany.

The isolation of Escherichia coli from wild birds in Germany revealed the occurrence of four CTX-M-15-producing strains from four different birds (2.3% of 172 isolates). CTX-M producers were recovered from two Eurasian Blackbirds, one Rock Pigeon and a Greater White-fronted Goose. All CTX-M-producing E. coli revealed a clonal relationship as determined by pulsed-field gel electrophoresis (PFGE) and were assigned to multilocus sequence type (ST) 648. Our findings suggest the emergence of a new clone with epidemiological importance and strengthen the role of wild bird species other than waterfowl as possible reservoirs of ESBL-producing Enterobacteriaceae.

Antimicrobial susceptibility of Escherichia coli from swine, horses, dogs and cats as determined in the BfT-GermVet monitoring program 2004-2006.

A total of 417 isolates of Escherichia coli collected from five animal species/organ system combinations from swine [urinary/genital tract (UGT) incl. mastitis metritis agalactia syndrome], horses [genital tract (GT)] and dogs/cats [respiratory tract (RT), UGT and gastrointestinal tract (GIT)] were analysed quantitatively for their susceptibility against different antimicrobial agents by determination of minimum inhibitory concentrations. Regardless of which animal species the strains originated from, resistance appeared most frequently against sulfamethoxazole (18-59%), tetracycline (14-54 %), and ampicillin (14-39%). High percentages of intermediate isolates were observed for cephalothin (39-46 %). In general, low prevalences of resistance were detected for amoxicillin/clavulanic acid (1-4%), gentamicin (1-9%), and cefazolin (0-11%). Generally speaking, the antimicrobial resistance situation among E. coli isolates from horses and small animals is relatively good.

First insights into antimicrobial resistance among faecal Escherichia coli isolates from small wild mammals in rural areas

Wild rodents can be carriers of antimicrobial resistant Escherichia coli. As rodents are known to be involved in the transmission of bacteria of human and animal health concern, they could likewise contribute to the dissemination of antimicrobial resistant bacteria in the environment. The aim of this study was therefore to get first insights into the antimicrobial resistance status among E. coli isolated from wild small mammals in rural areas. We tested 188 faecal isolates from eight rodent and one shrew species originating from Germany. Preselected resistant isolates were screened by minimal inhibitory concentration (MIC) testing or agar diffusion test and subsequent PCR analysis of resistance genes. The prevalence of antimicrobial resistant isolates was low with only 5.5% of the isolates exhibiting resistant phenotypes against at least one antimicrobial compound including beta-lactams, tetracyclines, aminoglycosides and sulfonamides. These results suggest a minor role of wild rodents from rural areas in the cycle of transmission and spread of antimicrobial resistant E. coli into the environment. Nevertheless E. coli with multiple antimicrobial resistances were significantly more often detected in wildlife rodents originating from areas with high livestock density suggesting a possible transmission from livestock to wild rodents.

Antimicrobial susceptibility of coagulase-positive and coagulase-variable Staphylococci from various indications of swine, dogs and cats as determined in the BfT-GermVet monitoring program 2004-2006.

A total of 248 coagulase-positive and coagulase-variable staphylococci from two indications of swine (infections of the urinary/genital tract including strains from the mastitis metritis agalactia syndrome as well as infections of the skin) as well as two indications of dogs/cats (respiratory tract infections and infections of skin/ear/mouth) were investigated for their susceptibility to numerous antimicrobial agents. Regardless of the animal origin and indication, the most frequently detected resistance properties were resistances against penicillin G (53-77%) and ampicillin (42-75%), tetracycline (33-52%) as well as erythromycin (13-27%). Oxacillin-resistant staphylococci were rarely detected.

Companion animals: a relevant source of extended-spectrum β-lactamase-producing fluoroquinolone-resistant Citrobacter freundii.

Citrobacter spp. are usually considered to be of low athogenicity, commonly present in water, soil and food, whilst hey occasionally colonise the gastrointestinal tract of animals nd humans. However, in immunocompromised hosts a range of nfections such as urinary tract infections, pneumonia, skin and oft-tissue infections, sepsis and meningitis are likely to occur [1,2]. n recent years, reports of high mortality rates associated with nvasive Citrobacter infections in human clinical settings appeared, ocumenting the pathogenic potential of this bacterial species that s additionally impaired by high rates of antimicrobial resistance, ncluding production of extended-spectrum -lactamases (ESBLs). s similar reports are exceptionally rare in the veterinary medcal literature [3], we explored the presence of ESBL-producing trains amongst 77 clinical Citrobacter spp. isolates (31 Citrobacter reundii, 32 Citrobacter koseri, 5 Citrobacter braakii, 3 Citrobacter malonaticus, 4 Citrobacter youngae and 2 Citrobacter spp.) from varous animal species and European countries collected consecutively y a veterinary diagnostic service laboratory from April 2008 to anuary 2010. A positive confirmatory test for the production of ESBLs [4] as only observed in C. freundii strains (9/31; 29%) This high roportion of ESBL-producing C. freundii, which is currently the ost commonly encountered Citrobacter spp. amongst hospitalssociated infections, reflected the upper rates that have been eported for human clinical strains worldwide (2.5–41.2%) [5]. ll ESBL-producing isolates were further characterised using reviously summarised protocols [6]. As determined by sequence analysis, five ESBL-producing isoates harboured the blaCTX-M-1 gene, whereas four clinical strains s well as a wild bird strain that was adopted from a previous tudy [7] for comparative purposes possessed the blaSHV-12 gene Fig. 1). Thus, animal strains appear to produce ESBL types that re frequently observed amongst multiresistant Citrobacter spp. n human clinical settings, where CTX-M (blaCTX-M-1, -3, -15) and HV-12 appear to be the most often encountered ESBL types orldwide [8,9]. Southern blotting of the resistance plasmids of ransconjugants showed that the bla genes and other resistance enes detected amongst the ESBL-producers were located on arge plasmids (>100 kb) with single replicon types IncHI1, IncHI2,

Real-time PCR assay for the detection of species of the genus Mannheimia

Infections caused by species of the genus Mannheimia cause diverse disease complexes in many wild and domestic animals worldwide. Fast and accurate detection of single species within the genus remains an unsolved problem till today. To resolve this diagnostic challenge, we developed a real-time PCR assay for the rapid and specific identification of five species of the genus Mannheimia (M. haemolytica, M. varigena, M. ruminalis, M. granulomatis and M. glucosida) from bacterial cultures and tissue samples. The assay was validated with reference strains, field isolates and bacteria spiked tissue samples. The sodA gene was used as target region for species-specific primer pairs. The real-time PCR assay demonstrated species specificity for all five examined Mannheimia spp. and a rapid test completion time of less than 5 h. This is a considerable advantage compared to the traditional phenotyping methods currently used to distinguish between the species of the genus. The assay was able to detect approximately 10(3) bacterial cells per gram lung tissue sample, as determined with spiked tissue samples. We assume that the assay could become useful for fast laboratory diagnostic assessment particularly of respiratory infections caused by Mannheimia in animals.

Antimicrobial susceptibility of Pasteurella multocida and Bordetella bronchiseptica from dogs and cats as determined in the BfT-GermVet monitoring program 2004-2006.

A total of 92 canine/feline Pasteurella multocida strains form respiratory tract infections or infections of skin/ear/mouth as well as 42 canine/feline Bordetella bronchiseptica strains from respiratory tract infections were investigated for their susceptibility to antimicrobial agents. While the P. multocida strains were susceptible to all antimicrobial agents tested - except sulfonamides -, a considerable number of the B. bronchiseptica strains was resistant or exhibited high MIC values against a number of antimicrobial agents including penicillin G, oxacillin, cefazolin, ceftiofur, cefquinome, sulfamethoxazole, and trimethoprim/sulfamethoxazole.