Matjaž Ocepek

Antimicrobial susceptibility of animal and human isolates of Clostridium difficile by broth microdilution.

A total of 188 human (n = 92) and animal (n = 96) isolates of Clostridium difficile of different PCR ribotypes were screened for susceptibility to 30 antimicrobials using broth microdilution. When comparing the prevalence of antimicrobial resistance, the isolates of animal origin were significantly more often resistant to oxacillin, gentamicin and trimethoprim/sulfamethoxazole (P<0.01). The most significant difference between the animal and human populations (P = 0.0006) was found in the level of imipenem resistance, with a prevalence of 53.3 % in isolates of human origin and 28.1 % in isolates of animal origin. Overall, the results show similar MICs for the majority of tested antimicrobials for isolates from human and animal sources, which were collected from the same geographical region and in the same time interval. This supports the hypothesis that C. difficile could be transmissible between human and animal hosts. Resistant isolates have been found in all animal species tested, including food and companion animals, and also among non-toxigenic isolates. The isolates of the most prevalent PCR ribotype 014/020 had low resistance rates for moxifloxacin, erythromycin, rifampicin and daptomycin, but a high resistance rate for imipenem. Multiresistant strains were found in animals and humans, belonging to PCR ribotypes 012, 017, 027, 045, 046, 078 and 150, and also to non-toxigenic strains of PCR ribotypes 010 and SLO 080.

MIRU–VNTR typing of Mycobacterium avium in animals and humans: Heterogeneity of Mycobacterium avium subsp. hominissuis versus homogeneity of Mycobacterium avium subsp. avium strains

Epidemiological studies on Mycobacterium avium are requisite for revealing infection sources and disease transmission. They are based upon genotyping methods like RFLP and MIRU-VNTR. In our study, MIRU-VNTR typing was applied to 121 previously RFLP typed M. avium field isolates to compare the discriminatory power of both methods. The applicability of MIRU-VNTR typing was studied for isolates from a limited geographic area, namely 41 M. avium subsp. avium and 80 M. avium subsp. hominissuis isolates. Among the former, exhibiting 12 IS901 RFLP types, five MIRU-VNTR types were found with discriminatory index (DI) of 0.716. Among the latter, exhibiting 56 IS1245 RFLP types, 18 MIRU-VNTR types were found with DI of 0.866. Concomitant use of both methods increased DI to 0.981 and 0.995, respectively. MIRU-VNTR typing employing the selected markers provided discernible discrimination among M. avium subsp. hominissuis isolates, but more discriminative markers are needed for M. avium subsp. avium isolates.

Aphanomyces astaci in wild crayfish populations in Slovenia: first report of persistent infection in a stone crayfish Austropotamobius torrentium population

All 5 crayfish species inhabiting Slovenian freshwaters, of which 3 are indigenous crayfish species (ICS: Astacus astacus, Austropotamobius pallipes, and A. torrentium) and 2 are non-indigenous (NICS: Pacifastacus leniusculus and Cherax quadricarinatus), were inspected for the presence of Aphanomyces astaci, the causative agent of crayfish plague. Wild crayfish populations showing no clinical signs of infection were inspected using A. astaci-specific real-time PCR. In addition, a conventional PCR assay was employed and confirmative sequencing was performed. Out of 88 analyzed crayfish, 15/27 (55.6%) specimens of A. torrentium from Borovnišcˇ%%KERN_ERR%%ica Brook and 4/35 (11.4%) of P. leniusculus from the Mura River tested positive, showing low to moderate levels of infection (agent levels A1-A4 and A1-A3, respectively). Results revealed the presence of A. astaci not only in the resistant NICS but also in ICS, since the infected population of A. torrentium presumably had no contact with the NICS carrier and appeared to sustain A. astaci infection in the 2 sampling years. Although the A. astaci genotype has not yet been identified, a connection between the latent infection in ICS and a Group A strain of A. astaci, co-evolving with A. torrentium since its first introduction to Slovenia, is suggested as the most plausible conclusion. This is the first reported population of the genus Austropotamobius with persistent infection, in addition to the already known populations of the genus Astacus. Findings of the presumed co-evolution of A. astaci and ICS hosts open new perspectives, necessitating additional studies on the presence of A. astaci genotypes in the persistently infected ICS populations.

IS1245 RFLP-based genotyping study of Mycobacterium avium subsp. hominissuis isolates from pigs and humans

Mycobacterium avium subsp. hominissuis, ubiquitous environmental mycobacterium, is an opportunistic pathogen that is regularly isolated from pigs and humans in Slovenia. Genetic diversity of 114 isolates from pigs (n = 57) and humans (n = 57), identified by means of bacteriology, DNA-RNA hybridization techniques, IS901 PCR and IS1245 PCR, was investigated in this study, using IS1245 restriction fragment length polymorphism (RFLP) analysis. Identical IS1245 RFLP profiles were found in isolates from pigs, isolates from humans and isolates from both origins. The proportion of clustered isolates varied as it depended on the similarity level (100% and 75%) chosen for the cluster limits. Using IS1245 RFLP, it was possible to detect monoclonal, polyclonal and recent infections and to monitor the genetic variability of the strains from individual patients. Our findings indicate the environment as the source of infection for both pigs and humans. The questions about the possibility of intra- and inter-species transmission of infection remain to be answered.

Evidence of Anaplasma phagocytophilum in game animals from Slovenia

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen responsible for granulocytic anaplasmosis in mammalian hosts including humans. Wild animals may play an important role in the epidemiology of this disease. The aim of this study was to estimate the prevalence of infection with A. phagocytophilum among wildlife in Slovenia. Serum samples (n = 376) from the most important game species [red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa), chamois (Rupicapra rupicapra) and brown bear (Ursus arctos)] were examined by A. phagocytophilum-specific indirect fluorescent-antibody assay (IFA) and wild boar spleen samples (n = 160) were tested by polymerase chain reaction (PCR). A. phagocytophilum-specific antibodies were found in 72% of sera and A. phagocytophilum DNA was present in 6.2% of spleens. The data indicate that A. phagocytophilum is present and widespread in Slovenian game animals and that game species are involved in the natural life cycle of A. phagocytophilum.

High genetic similarity of ciprofloxacin-resistant Campylobacter jejuni in central Europe.

Campylobacteriosis is the leading zoonosis in the European Union with the majority of cases attributed to Campylobacter jejuni. Although the disease is usually self-limiting, some severe cases need to be treated with antibiotics, primarily macrolides and quinolones. However, the resistance to the latter is reaching alarming levels in most of the EU countries. To shed light on the expansion of antibiotic resistance in central Europe, we have investigated genetic similarity across 178 ciprofloxacin-resistant C. jejuni mostly isolated in Slovenia, Austria and Germany. We performed comparative genetic similarity analyses using allelic types of seven multilocus sequence typing housekeeping genes, and single nucleotide polymorphisms of a quinolone resistance determining region located within the DNA gyrase subunit A gene. This analysis revealed high genetic similarity of isolates from clonal complex ST-21 that carry gyrA allelic type 1 in all three of these central-European countries, suggesting these ciprofloxacin resistant isolates arose from a recent common ancestor and are spread clonally.

Cutaneous listeriosis in a veterinarian with the evidence of zoonotic transmission - a case report.

A case of Listeria monocytogenes skin infection in a man is presented. A 54‐year‐old male veterinary practitioner developed pustular changes on the skin of arms and hands after assisting with the delivery of a stillborn calf. Listeria monocytogenes was isolated from the skin lesions on the arms and from the bovine placenta. Listeria monocytogenes isolates were serotyped and genotyped with pulsed‐field gel electrophoresis (PFGE) to confirm the suspected transmission of the pathogen from animal to human. All isolates were of serotype 4b with identical pulsotype. To the best of our knowledge, this is the first case of cutaneous listeriosis in which the evidence for zoonotic transmission of L. monocytogenes is supported by genotyping methods.

New Approaches on Quantification of Campylobacter jejuni in Poultry Samples: The Use of Digital PCR and Real-time PCR against the ISO Standard Plate Count Method

Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

The First Report of Mycobacterium celatum Isolation from Domestic Pig (Sus scrofa domestica) and Roe Deer (Capreolus capreolus) and an Overview of Human Infections in Slovenia

Mycobacterium celatum, a slowly growing potentially pathogenic mycobacterium first described in humans, is regarded as an uncommon cause of human infection, though capable of inducing invasive disease in immunocompromised hosts. According to some reports, a serious disease due to M. celatum may also occur in individuals with no apparent immunodeficiency. In animals, an M. celatum-related disease has been described in three cases only: twice in a domestic ferret (Mustela putorius furo) and once in a white-tailed trogon (Trogon viridis). In this paper, we report the first detection of M. celatum in a domestic pig (Sus scrofa domestica) and roe deer (Capreolus capreolus). A nation-wide overview of human M. celatum infections recorded in Slovenia between 2000 and 2010 is also given. Pulmonary disease due to M. celatum was recognized in one patient with a history of a preexisting lung disease.

A pulsed-field gel electrophoresis study of the genetic diversity of Campylobacter jejuni and Campylobacter coli in poultry flocks in Slovenia.

Campylobacter jejuni and C. coli have recently become the most frequent cause of bacterial foodborne enteric infection in most industrialised countries. Consumption and handling of undercooked contaminated poultry meat was identified as an important risk factor for human campylobacteriosis. The aim of this study was to ascertain the genetic diversity of C. jejuni and C. coli strains isolated from poultry in Slovenia. A total of 68 isolates (42 C. jejuni , 26 C. coli ) from faeces (n = 48), meat (n = 15) and skin/carcasses (n = 5) of chicken (n = 60) and turkey samples (n = 5) were analysed by pulsed-field gel electrophoresis. Sma I macrorestriction discriminated between C. jejuni and C. coli isolates. C. jejuni isolates exhibited a higher degree of diversity compared to C. coli isolates. In the C. jejuni group, a number of small clusters were apparent, while C. coli strains formed less but larger clusters. Additional Kpn I digestion of selected isolates resulted in poor subtyping. Strains with identical or very similar profiles were found on different farms, either in the same or different regions and time periods. Some of the clones indicated possible cross-contamination at slaughterhouses.