Ellen Prenger-Berninghoff

Emergence of OXA-48 carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in dogs

OBJECTIVES To evaluate the possible occurrence of carbapenemase-producing Escherichia coli and Klebsiella spp. strains in domestic animals. METHODS Veterinary clinical E. coli (n = 1175) and Klebsiella spp. (n = 136) isolates consecutively collected from livestock and companion animals in Germany from June 2012 to October 2012 were screened for their susceptibility to carbapenems using the agar disc diffusion test. Carbapenemase genes were characterized by PCR and sequencing; conjugation assays were performed. Carbapenemase-positive isolates were assigned to phylogenetic lineages by multilocus sequence typing and the clonal relatedness was determined using macrorestriction analysis and subsequent PFGE. RESULTS Carbapenem non-susceptible isolates of Klebsiella pneumoniae (n = 5) and E. coli (n = 3) were obtained from six dogs hospitalized in a single veterinary clinic in Hessia, Germany, partly at the same time and consecutively over the study period. All isolates harboured carbapenemase gene blaOXA-48 located within Tn1999.2 transposons on conjugative ~60 kb plasmids. The K. pneumoniae isolates belonged to sequence type ST15, pulsotype 1, and coexpressed CTX-M-15, SHV-28, OXA-1 and TEM-1. Two E. coli isolates were assigned to ST1196 and pulsotype 2 and coproduced CMY-2, SHV-12 and TEM-1, while the third E. coli isolate was of ST1431 (pulsotype 3), and possessed blaCTX-M-1, blaOXA-2 and blaTEM-1. CONCLUSIONS This is the first known report of OXA-48-producing bacteria from companion animals. The clonal nature of the K. pneumoniae and two E. coli isolates suggests a nosocomial dissemination rather than repeated introduction by individual patients into the clinic.

Multiresistant extended-spectrum β-lactamase- producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany

BackgroundMultiresistant Gram-negative bacteria producing extended-spectrum β-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of β-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses.ResultsIn this study 153 (83.6%) of the human isolates (n = 183) and 163 (91.6%) of the animal isolates (n = 178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac(‘6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates.ConclusionsIsolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations.

Multidrug-resistant Acinetobacter baumannii in veterinary clinics, Germany.

An increase in prevalence of multidrug-resistant Acinetobacter spp. in hospitalized animals was observed at the Justus-Liebig-University (Germany). Genotypic analysis of 56 isolates during 2000–2008 showed 3 clusters that corresponded to European clones I–III. Results indicate spread of genotypically related strains within and among veterinary clinics in Germany.

IDENTIFICATION OF BRUCELLA SPECIES AND BIOTYPES USING POLYMERASE CHAIN REACTION-RESTRICTION FRAGMENT LENGTH POLYMORPHISM (PCR-RFLP)

Brucellosis is a worldwide zoonosis causing reproductive failures in livestock and a severe multi-organ disease in humans. The genus Brucella is divided into seven species and various biotypes differing in pathogenicity and host specificity. Although Brucella spp. represent a highly homogenous group of bacteria, RFLPs of selected genes display sufficient polymorphism to distinguish Brucella species and biovars. PCR-RFLP analysis shows excellent typeability, reproducibility, stability, and epidemiological concordance. Consequently, PCR-RFLP assays of specific gene loci can serve as tools for diagnostic, epidemiological, taxonomic, and evolutionary studies. Various PCR-RFLPs used for the identification of Brucella species and biotypes are reviewed.

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 β-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.

Case-control risk factor study of methicillin-resistant Staphylococcus pseudintermedius (MRSP) infection in dogs and cats in Germany

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged as a highly drug-resistant small animal veterinary pathogen. Although often isolated from outpatients in veterinary clinics, there is concern that MRSP follows a veterinary-hospital-associated epidemiology. This studys objective was to identify risk factors for MRSP infections in dogs and cats in Germany. Clinical isolates of MRSP cases (n=150) and methicillin-susceptible S. pseudintermedius (MSSP) controls (n=133) and their corresponding host signalment and medical data covering the six months prior to staphylococcal isolation were analysed by multivariable logistic regression. The identity of all MRSP isolates was confirmed through demonstration of S. intermedius-group specific nuc and mecA. In the final model, cats (compared to dogs, OR 18.5, 95% CI 1.8-188.0, P=0.01), animals that had been hospitalised (OR 104.4, 95% CI 21.3-511.6, P<0.001), or visited veterinary clinics more frequently (>10 visits OR 7.3, 95% CI 1.0-52.6, P=0.049) and those that had received topical ear medication (OR 5.1, 95% CI 1.8-14.9, P=0.003) or glucocorticoids (OR 22.5, 95% CI 7.0-72.6, P<0.001) were at higher risk of MRSP infection, whereas S. pseudintermedius isolates from ears were more likely to belong to the MSSP-group (OR 0.09, 95% CI 0.03-0.34, P<0.001). These results indicate an association of MRSP infection with veterinary clinic/hospital settings and possibly with chronic skin disease. There was an unexpected lack of association between MRSP and antimicrobial therapy; this requires further investigation but may indicate that MRSP is well adapted to canine skin with little need for selective pressure.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for species identification of bacteria of genera Arcanobacterium and Trueperella

In the present study matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for species identification of 98 bacteria previously classified phenotypically and genotypically to genera Arcanobacterium and Trueperella. Species identification was carried out by comparing the main spectra of each strain with the main spectra of reference strains of both genera and 3740 database entries included in the MALDI Biotyper 2.0 software package (Bruker Daltonik GmbH, Bremen, Germany). MALDI-TOF MS correctly identified (log (score) values ≥ 2.0) all investigated strains of the species A. (T.) bialowiezense (n=3), A. (T.) bonasi (n=7), A. haemolyticum (n=10), A. pluranimalium (n=1) and A. (T.) pyogenes (n=77). According to the present results MALDI-TOF MS had a comparable discriminating power than previously conducted tests on DNA level. Further studies with strains isolated from human infections would show the robustness of MALDI-TOF MS for identification of bacteria of these genera.

Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins

The present study was designed to identify phenotypically and genotypically 61 Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. The A. pyogenes isolates showed the typical cultural and biochemical properties of this species and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the superoxide dismutase A encoding gene sodA of reference strains representing 8 species of genus Arcanobacterium and subsequent design of A. pyogenes sodA gene-specific oligonucleotide primer. The A. pyogenes sodA gene-specific oligonucleotide primer allowed, together with previously described A. pyogenes 16S-23S rDNA intergenic spacer region-specific oligonucleotide primer, a reliable molecular identification of all 61 A. pyogenes of various origins. The additionally performed PCR-mediated amplification of 5 known and putative virulence factor encoding genes revealed that 100, 20, 87, 75, and 98% of the A. pyogenes carried the genes plo, cbpA, nanH, nanP, and fimA, which allowed an individual strain characterization. This might help to elucidate the role the putative virulence factors play in bovine mastitis and in various other infections caused by this bacterial pathogen.

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.

Incidence of Brucella species in marine mammals of the German north sea

In this study, organ samples from 426 common seals Phoca vitulina, 298 harbour porpoises Phocoena phocoena, 34 grey seals Halichoerus grypus and 10 other marine mammals were assessed for the presence of Brucella species. Forty-seven common seals, 2 harbour porpoises and 1 grey seal were found to be positive for these bacteria. A total of 91 Brucella strains were successfully isolated, due to the fact that Brucella spp. were found in more than one organ sample in 15 animals. The primary organ in which the bacteria were present was the lung. In addition, 2 strains were isolated from lungworms (Parafilaroides spp.). Forty-nine of the isolated strains were selected for further analysis using conventional phenotyping methods. Molecular characterisation was carried out by analysing the IS711 and omp2 loci. With respect to the distribution of the IS711 loci in the genome, the 49 field isolates differed strongly from the terrestrial Brucella species and marginally from the marine Brucella reference strain NCTC12890. Based on the results of the PCR restriction fragment length polymorphism (PCR-RFLP) investigation of the omp2 locus, the majority of the Brucella field isolates were classified as B. pinnipediae, recently proposed B. pinnipedialis, possessing 1 omp2a gene and 1 omp2b gene. Two field isolates revealed the presence of 2 omp2a genes, as has been described for Brucella ovis. To our knowledge, these results confirm for the first time the presence of Brucella species in the marine mammal population of the German North Sea. These findings highlight the need for additional research on the relevance of these Brucella species for marine hosts and their environment.