Third-generation cephalosporin resistant Escherichia coli in dogs and cats in Germany in 2019-2021.

OBJECTIVES: Antimicrobial resistance (AMR) poses a worldwide challenge, threatening global health. The objective of this research was to determine the 3rd generation cephalosporin resistance (3GCR) proportion in Escherichia (E.) coli isolated from clinical samples of dogs and cats in Germany. METHODS: The study utilized result data from antimicrobial susceptibility testing (AST) of isolates obtained from diagnostic samples collected from dogs and cats send in for bacterial examination. Data includes AST results from 3,491 veterinary practices in Germany spanning the years 2019 to 2021, representing 33.1% of practices and clinics nationwide. Out of 175,171 clinical samples, a total of 25,491 E. coli strains (14,6%) were evaluated for their susceptibility to antimicrobials, in particular the 3rd generation cephalosporin cefovecin, but also aminoglycosides (gentamicin, GEN), fluoroquinolones (enrofloxacin, ENR), tetracyclines (doxycycline), phenicols (chloramphenicol), folate pathway inhibitors (sulfamethoxazole + trimethoprim), and nitrofurans (nitrofurantoin). RESULTS: The cefovecin resistance proportion was 11.6% in the study period. Geographical analysis showed local variations in 3GCR in E. coli of ±3%. Regarding all E. coli isolates investigated, resistance proportions were observed as follows: 12% for sulfamethoxazole-trimethoprim, 7% for enrofloxacin, 8% for chloramphenicol and 4% for gentamicin. Notably, 3GCR E. coli showed significantly higher resistance proportions, specifically 30% for sulfamethoxazole-trimethoprim, 28% for chloramphenicol, 18% for enrofloxacin and 14% for gentamicin. CONCLUSIONS: This study represents the first of its kind to utilize an extensive dataset encompassing dogs and cats across Germany. Companion animals have close contact to their owners and transmission of 3GCR between them is likely as well as acquisition from other environmental sources. Resistance proportions (6.7%) against the antibiotic ceftazidime as reported by the German AMR surveillance for human medicine were lower than in our veterinary data. Our study provides an overview of the current 3GCR resistance proportion in Germany and demonstrates the importance of integrated AMR monitoring.

Panton-Valentine Leukocidin-Positive Staphylococcus aureus in Family and Pet Cat.

Continued detection of Panton-Valentine leukocidin-positive Staphylococcus aureus in samples from a family with severe repeated skin infections and their pet cat suggests transmission between the family and the cat. Decolonizing the pet led to successful elimination of the bacteria from the household. Clinicians should consider pet cats as possible reinfection sources.

Prevalence of MRSA in canine and feline clinical samples from one-third of veterinary practices in Germany from 2019-2021.

BACKGROUND: MRSA is a major contributor to AMR-related deaths. The WHO's global action plan emphasizes a One Health approach, acknowledging the connection between humans and their companion animals. It is agreed on that comprehensive AMR surveillance is needed. OBJECTIVES: This study provides a large-scale overview of MRSA occurrence in cats and dogs in Germany, serving as a foundation for continuous surveillance. METHODS: The study analysed all results of canine and feline bacterial diagnostic samples from a large laboratory, encompassing samples received from veterinary practices between January 2019 and December 2021. MRSA prevalence between host species, sample types and geographical distribution were compared. Additionally, data were contrasted with human MRSA surveillance data from Germany. RESULTS: Samples originated from 3491 German veterinary practices, representing 33.1% of practices and clinics nationally. Bacterial examination results from 175 171 samples were analysed, identifying S. aureus in 5526 of these samples (3.2% isolation rate). S. aureus in clinical samples was more prevalent in cats (5.6%) than dogs (2.0%). Methicillin resistance was found in 17.8% of S. aureus samples and was higher in dogs (20.4%, 95%CI 18.9-22.0) than cats (15.6%, 95%CI 14.3-17.0). The highest MRSA prevalence was found in canine wound samples (32%), compared to skin/soft tissue, respiratory tract and other (<23% respectively). CONCLUSION: The study reveals a 17.8% MRSA prevalence, which is higher than the human outpatient MRSA prevalence (5.4%). Restriction and regulation of veterinary antibiotic use should be validated with AMR surveillance. Our study shows that this is feasible in companion animals with significant coverage.

Occurrence of Antimicrobial Resistance in Canine and Feline Bacterial Pathogens in Germany under the Impact of the TÄHAV Amendment in 2018.

The occurrence of antimicrobial resistance due to the use of antimicrobials is considered to be a main cause for treatment failure of bacterial infections in humans and animals. The right of German veterinarians to use and prescribe medications such as antimicrobials is regulated by the Regulation of Veterinary Pharmacies (TÄHAV). The aim of this study was to investigate the impact of the second amendment to the TÄHAV in 2018 on the occurrence of antimicrobial resistance in selected bacterial pathogens isolated from dogs and cats in Germany. For this purpose, we analyzed antimicrobial susceptibility data from 38 German small animal practices gathered between 2015 and 2021 in cooperation with Laboklin (Labor für klinische Diagnostik GmbH & Co.KG, Bad Kissingen, Germany). Annual cumulative susceptibility data of eight bacterial species were analyzed and compared. The mean value of resistant isolates was determined for each year and supplemented by 95% confidence intervals. Encouraged by the amendment, an increase in sample submissions was observed in Germany. The highest resistance rates to the analyzed substances penicillin G, ampicillin, amoxicillin-clavulanic acid, cefovecin, and enrofloxacin were found for Staphylococcus pseudintermedius (S. pseudintermedius), S. aureus, and Escherichia coli (E. coli). In contrast, resistance rates were low for Pasteurella multocida (P. multocida) and ß-hemolytic streptococci. Significant resistance trends (p < 0.05) assumed as influenced by the TÄHAV amendment could be the significant decreases in resistance rates of S. pseudintermedius against penicillin G to 67% (n = 322/479), and ampicillin to 63% (n = 286/453), as well as S. felis against amoxicillin-clavulanic acid and cefovecin to 2% (n = 2/109), furthermore, the reduction in the occurrence of resistance of S. aureus against enrofloxacin to 4% (n = 3/76) in 2021. Moreover, for all species, the efficacy against the analyzed substances was maintained over the study period.

Biocide Susceptibility and Antimicrobial Resistance of Escherichia coli Isolated from Swine Feces, Pork Meat and Humans in Germany.

Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum ß-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials.

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.

How to survive pig farming: Mechanism of SCCmec element deletion and metabolic stress adaptation in livestock-associated MRSA.

Previous research on methicillin susceptible Staphylococcus aureus (MSSA) belonging to livestock-associated (LA-) sequence type (ST) 398, isolated from pigs and their local surroundings, indicated that differences between these MSSA and their methicillin resistant predecessors (MRSA) are often limited to the absence of the staphylococcal cassette chromosome mec (SCCmec) and few single nucleotide polymorphisms. So far, our understanding on how LA-MRSA endure the environmental conditions associated with pig-farming as well as the putative impact of this particular environment on the mobilisation of SCCmec elements is limited. Thus, we performed in-depth genomic and transcriptomic analyses using the LA-MRSA ST398 strain IMT38951 and its methicillin susceptible descendant. We identified a mosaic-structured SCCmec region including a putative replicative SCCmecVc which is absent from the MSSA chromosome through homologous recombination. Based on our data, such events occur between short repetitive sequences identified within and adjacent to two distinct alleles of the large cassette recombinase genes C (ccrC). We further evaluated the global transcriptomic response of MRSA ST398 to particular pig-farm associated conditions, i.e., contact with host proteins (porcine serum) and a high ammonia concentration. Differential expression of global regulators involved in stress response control were identified, i.e., ammonia-induced alternative sigma factor B-depending activation of genes for the alkaline shock protein 23, the heat shock response and the accessory gene regulator (agr)-controlled transcription of virulence factors. Exposure to serum transiently induced the transcription of distinct virulence factor encoding genes. Transcription of genes reported for mediating the loss of methicillin resistance, especially ccrC, was not significantly different compared to the unchallenged controls. We concluded that, from an evolutionary perspective, bacteria may save energy by incidentally dismissing a fully replicative SCCmec element in contrast to the induction of ccr genes on a population scale. Since the genomic SCCmec integration site is a hot-spot of recombination, occasional losses of elements of 16 kb size may restore capacities for the uptake of foreign genetic material. Subsequent spread of resistance, on the other hand, might depend on the autonomous replication machinery of the deleted SCCmec elements that probably enhance chances for reintegration of SCCmec into susceptible genomes by mere multiplication.

A role for ColV plasmids in the evolution of pathogenic Escherichia coli ST58.

Escherichia coli ST58 has recently emerged as a globally disseminated uropathogen that often progresses to sepsis. Unlike most pandemic extra-intestinal pathogenic E. coli (ExPEC), which belong to pathogenic phylogroup B2, ST58 belongs to the environmental/commensal phylogroup B1. Here, we present a pan-genomic analysis of a global collection of 752 ST58 isolates from diverse sources. We identify a large ST58 sub-lineage characterized by near ubiquitous carriage of ColV plasmids, which carry genes encoding virulence factors, and by a distinct accessory genome including genes typical of the Yersiniabactin High Pathogenicity Island. This sub-lineage includes three-quarters of all ExPEC sequences in our study and has a broad host range, although poultry and porcine sources predominate. By contrast, strains isolated from cattle often lack ColV plasmids. Our data indicate that ColV plasmid acquisition contributed to the divergence of the major ST58 sub-lineage, and different sub-lineages inhabit poultry, swine and cattle.

Antimicrobial and Biocide Resistance among Feline and Canine Staphylococcus aureus and Staphylococcus pseudintermedius Isolates from Diagnostic Submissions.

A total of 114 Staphylococcus isolates from various infections of companion animals, including 43 feline Staphylococcus aureus, 19 canine S. aureus, 11 feline Staphylococcus pseudintermedius and 41 canine S. pseudintermedius were investigated for (i) their susceptibility to 24 antimicrobial agents and three combinations of antimicrobial agents by broth microdilution following CLSI recommendations and (ii) the corresponding resistance genes. In addition, the isolates were tested for their susceptibility to the four biocides benzalkonium chloride, chlorhexidine, polyhexanide and octenidine by a recently developed biocide susceptibility testing protocol. Penicillin resistance via blaZ was the dominant resistance property in all four groups of isolates ranging between 76.7 and 90.9%. About one quarter of the isolates (25.4%) proved to be methicillin-resistant and carried the genes mecA or mecC. Macrolide resistance was the second most prevalent resistance property (27.2%) and all isolates harbored the resistance genes erm(A), erm(B), erm(C), erm(T) or msr(A), alone or in combinations. Fluoroquinolone resistance was detected in 21.1% of all isolates tested, whereas tetracycline resistance via tet(K) and/or tet(M) occurred in 19.3% of the isolates. Resistance to last resort antimicrobial agents in human medicine was seen only in single isolates, if at all. The minimal inhibitory concentrations (MICs) of the four biocides showed unimodal distributions and were very similar for the four groups of staphylococci. Because of the large number of (multi)resistant isolates, antimicrobial susceptibility testing of feline and canine S. aureus and S. pseudintermedius isolates is highly recommended before the start of an antimicrobial chemotherapy. Moreover, no hints towards the development of biocide resistance were detected.

Antimicrobial and Biocide Resistance among Canine and Feline Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii Isolates from Diagnostic Submissions.

A total of 215 isolates from infections of dogs and cats, including 49 Enterococcus faecalis, 37 Enterococcus faecium, 59 Escherichia coli, 56 Pseudomonas aeruginosa, and 14 Acinetobacter baumannii, were investigated for their susceptibility to 27 (Gram-positive bacteria) or 20 (Gram-negative bacteria) antimicrobial agents/combinations of antimicrobial agents by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute. Moreover, all isolates were analysed for their susceptibility to the biocides benzalkonium chloride, chlorhexidine, polyhexanide, and octenidine by a recently published broth microdilution biocide susceptibility testing method. While the E. faecalis isolates did not show expanded resistances, considerable numbers of the E. faecium isolates were resistant to penicillins, macrolides, tetracyclines, and fluoroquinolones. Even a single vancomycin-resistant isolate that carried the vanA gene cluster was detected. Expanded multiresistance phenotypes were also detected among the E. coli isolates, including a single carbapenem-resistant, blaOXA-48-positive isolate. In addition, multiresistant A. baumannii isolates were detected. The minimal inhibitory concentrations of the biocides showed unimodal distributions but differed with respect to the biocide and the bacterial species investigated. Although there were no indications of a development of biocide resistance, some P. aeruginosa isolates exhibited benzalkonium MICs higher than the highest test concentration.

A Real-Time Thermal Sensor System for Quantifying the Inhibitory Effect of Antimicrobial Peptides on Bacterial Adhesion and Biofilm Formation.

The increasing rate of antimicrobial resistance (AMR) in pathogenic bacteria is a global threat to human and veterinary medicine. Beyond antibiotics, antimicrobial peptides (AMPs) might be an alternative to inhibit the growth of bacteria, including AMR pathogens, on different surfaces. Biofilm formation, which starts out as bacterial adhesion, poses additional challenges for antibiotics targeting bacterial cells. The objective of this study was to establish a real-time method for the monitoring of the inhibition of (a) bacterial adhesion to a defined substrate and (b) biofilm formation by AMPs using an innovative thermal sensor. We provide evidence that the thermal sensor enables continuous monitoring of the effect of two potent AMPs, protamine and OH-CATH-30, on surface colonization of bovine mastitis-associated Escherichia (E.) coli and Staphylococcus (S.) aureus. The bacteria were grown under static conditions on the surface of the sensor membrane, on which temperature oscillations generated by a heater structure were detected by an amorphous germanium thermistor. Bacterial adhesion, which was confirmed by white light interferometry, caused a detectable amplitude change and phase shift. To our knowledge, the thermal measurement system has never been used to assess the effect of AMPs on bacterial adhesion in real time before. The system could be used to screen and evaluate bacterial adhesion inhibition of both known and novel AMPs.

High-Zinc Supplementation of Weaned Piglets Affects Frequencies of Virulence and Bacteriocin Associated Genes Among Intestinal Escherichia coli Populations.

To prevent economic losses due to post-weaning diarrhea (PWD) in industrial pig production, zinc (Zn) feed additives have been widely used, especially since awareness has risen that the regular application of antibiotics promotes buildup of antimicrobial resistance in both commensal and pathogenic bacteria. In a previous study on 179 Escherichia coli collected from piglets sacrificed at the end of a Zn feeding trial, including isolates obtained from animals of a high-zinc fed group (HZG) and a corresponding control group (CG), we found that the isolate collection exhibited three different levels of tolerance toward zinc, i.e., the minimal inhibitory concentration (MIC) detected was 128, followed by 256 and 512 µg/ml ZnCl2. We further provided evidence that enhanced zinc tolerance in porcine intestinal E. coli populations is clearly linked to excessive zinc feeding. Here we provide insights about the genomic make-up and phylogenetic background of these 179 E. coli genomes. Bayesian analysis of the population structure (BAPS) revealed a lack of association between the actual zinc tolerance level and a particular phylogenetic E. coli cluster or even branch for both, isolates belonging to the HZG and CG. In addition, detection rates for genes and operons associated with virulence (VAG) and bacteriocins (BAG) were lower in isolates originating from the HZG (41 vs. 65% and 22 vs. 35%, p < 0.001 and p = 0.002, resp.). Strikingly, E. coli harboring genes defining distinct pathotypes associated with intestinal disease, i.e., enterotoxigenic, enteropathogenic, and Shiga toxin-producing E. coli (ETEC, EPEC, and STEC) constituted 1% of the isolates belonging to the HZG but 14% of those from the CG. Notably, these pathotypes were positively associated with enhanced zinc tolerance (512 µg/ml ZnCl2 MIC, p < 0.001). Taken together, zinc excess seems to influence carriage rates of VAGs and BAGs in porcine intestinal E. coli populations, and high-zinc feeding is negatively correlated with enteral pathotype occurrences, which might explain earlier observations concerning the relative increase of Enterobacterales considering the overall intestinal microbiota of piglets during zinc feeding trials while PWD rates have decreased.

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs.

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from "pigs fed with high zinc concentrations" (high zinc group, [HZG]: n = 99) and a corresponding "control group" ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 µg/ml (HZG, 2%; CG, 6%), 256 µg/ml (HZG, 64%; CG, 91%) and 512 µg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between "higher" ZnCl2 MICs and isolates representing the HZG as well as "lower ZnCl2 MICs" with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 µg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and - detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes - a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems.

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

Lysogenic conversion of atypical enteropathogenic Escherichia coli (aEPEC) from human, murine, and bovine origin with bacteriophage Φ3538 Δstx2::cat proves their enterohemorrhagic E. coli (EHEC) progeny.

Bacteriophages play an important role in the evolution of bacterial pathogens. A phage-mediated transfer of stx-genes to atypical enteropathogenic E. coli (aEPEC) which are prevalent in different hosts, would convert them to enterohemorrhagic E. coli (EHEC). We decided to confirm this hypothesis experimentally to provide conclusive evidence that aEPEC isolated from different mammalian hosts are indeed progenitors of typical EHEC which gain the ability to produce Shiga-Toxin by lysogeny with stx-converting bacteriophages, utilizing the model phage Φ3538 Δstx2::cat. We applied a modified in vitro plaque-assay, using a high titer of a bacteriophage carrying a deletion in the stx2 gene (Φ3538 Δstx2::cat) to increase the detection of lysogenic conversion events. Three wild-type aEPEC strains were chosen as acceptor strains: the murine aEPEC-strain IMT14505 (sequence type (ST)28, serotype Ont:H6), isolated from a striped field mouse (Apodemus agrarius) in the surrounding of a cattle shed, and the human aEPEC-strain 910#00 (ST28, Ont:H6). The close genomic relationship of both strains implies a high zoonotic potential. A third strain, the bovine aEPEC IMT19981, was of serotype O26:H11 and ST21 (STC29). All three aEPEC were successfully lysogenized with phage Φ3538 Δstx2::cat. Integration of the bacteriophage DNA into the aEPEC host genomes was confirmed by amplification of chloramphenicol transferase (cat) marker gene and by Southern-Blot hybridization. Analysis of the whole genome sequence of each of the three lysogens showed that the bacteriophage was integrated into the known tRNA integration site argW, which is highly variable among E. coli. In conclusion, the successful lysogenic conversion of aEPEC with a stx-phage in vitro underlines the important role of aEPEC as progenitors of EHEC. Given the high prevalence and the wide host range of aEPEC acceptors, their high risk of zoonotic transmission should be recognized in infection control measures.

Molecular study on Pasteurella multocida and Mannheimia granulomatis from Kenyan Camels (Camelus dromedarius).

BACKGROUND: Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890. Yet the aetiology of this condition remains elusive. This study is the first to apply state of the art molecular methods to shed light on the nasopharyngeal carrier state of Pasteurellaceae in camels. The study focused on HS causing Pasteurella multocida capsular types B and E. Other Pasteurellaceae, implicated in common respiratory infections of animals, were also investigated. METHODS: In 2007 and 2008, 388 nasopharyngeal swabs were collected at 12 locations in North Kenya from 246 clinically healthy camels in 81 herds that had been affected by HS-like disease. Swabs were used to cultivate bacteria on blood agar and to extract DNA for subsequent PCR analysis targeting P. multocida and Mannheimia-specific gene sequences. RESULTS: Forty-five samples were positive for P. multocida genes kmt and psl and for the P. multocida Haemorrhagic Septicaemia (HS) specific sequences KTSP61/KTT72 but lacked HS-associated capsular type B and E genes capB and capE. This indicates circulation of HS strains in camels that lack established capsular types. Sequence analysis of the partial 16S rRNA gene identified 17 nasal swab isolates as 99% identical with Mannheimia granulomatis, demonstrating a hitherto unrecognised active carrier state for M. granulomatis or a closely related Mannheimia sp. in camels. CONCLUSIONS: The findings of this study provide evidence for the presence of acapsular P. multocida or of hitherto unknown capsular types of P. multocida in camels, closely related to P. multocida strains causing HS in bovines. Further isolations and molecular studies of camelid P. multocida from healthy carriers and from HS-like disease in camels are necessary to provide conclusive answers. This paper is the first report on the isolation of M. granulomatis or a closely related new Mannheimia species from camelids.

Effects of Ex Vivo Infection with ETEC on Jejunal Barrier Properties and Cytokine Expression in Probiotic-Supplemented Pigs.

BACKGROUND AND AIM: Enterotoxigenic Escherichia coli (ETEC) strains are involved in piglet post-weaning diarrhea. Prophylactic measures including probiotics have been examined in infection experiments with live piglets. In the present study, we have tested whether the early effects of ETEC infection can also be evoked and studied in a model in which ETEC is added to whole mucosal tissues ex vivo, and whether this response can be modulated by prior supplementation of the piglets with probiotics. METHODS: Jejunal barrier and transport properties of Enterococcus faecium-supplemented or control piglets were assessed in Ussing chambers. Part of the epithelia was challenged with an ETEC strain at the mucosal side. Fluxes of fluorescein as a marker of paracellular permeability, and the expression of selected tight junction (TJ) proteins and of proinflammatory cytokines were measured. RESULTS: The addition of ETEC ex vivo induced an increase in transepithelial resistance peaking in the first 2 h with a concomitant reduction in fluorescein fluxes, indicating tightening effects on barrier function. The response of short-circuit current after stimulation with PGE2 or glucose was reduced in epithelia treated with ETEC. ETEC induced a decrease in the TJ protein claudin-4 in the control diet group after 280 min and an increase in the mRNA expression of the proinflammatory cytokines interleukin-8 and TNF-α in both groups after 180 min. CONCLUSIONS: The addition of ETEC ex vivo affected barrier function and transport properties of the jejunal tissues and enhanced cytokine expression. The differences in claudin-4 expression in the jejunum might indicate a beneficial effect of E. faecium prefeeding.

Putative connection between zoonotic multiresistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in dog feces from a veterinary campus and clinical isolates from dogs.

INTRODUCTION: To contribute to the understanding of multiresistant bacteria, a 'One Health' approach in estimating the rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and getting insights into the transmission from clinical settings to the surrounding environment was employed by collecting fecal samples of dogs in a public area. Isolates were compared to those from samples of diseased dogs from a nearby small-animal clinic. MATERIALS AND METHODS: One hundred fecal samples of dogs were collected on a single day in the public area of a veterinary faculty with a small-animal clinic and adjacent residential neighborhoods. All identified ESBL-producing strains were isolated by selective plating, genotypically analyzed by DNA microarray, polymerase chain reaction, sequence analysis, and pulsed-field gel electrophoresis and compared to 11 clinical ESBL/AmpC-producing E. coli isolated from diseased dogs treated in the small-animal clinic 2 months before and 2 months following the environmental sampling collection. RESULTS AND DISCUSSION: Fourteen percent (14/100) of the extra-clinical samples harbored phenotypic ESBL/putative AmpC-producing E. coli with additional resistances against other antimicrobials. One ESBL-strain displayed an identical macrorestriction pattern to one clinical, another one to three clinical clonal ESBL-producing strains. The genotypic ESBL-determinants (blaCTX-M-1 and blaCTX-M-15) and detection rates (10%) in dog feces collected outside of the small-animal clinic are comparable to the rates and ESBL-types in the healthy human population in Germany and to clinical and non-clinical samples of humans and companion animals in Europe. The occurrence of identical strains detected both outside and inside the clinical setting suggests a connection between the small-animal clinic and the surrounding environment. In conclusion, dog feces collected in proximity to veterinary facilities should be considered as a non-point infection source of zoonotic ESBL-producing E. coli for both animals and humans. The common sniffing behavior of dogs further urges hygienic measures on the part of dog-patient owners, who should be educated to remove their pet's feces immediately and effectively.

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.