Andrej Kirbiš

Implementation of the Bacillus cereus microbiological plate used for the screening of tetracyclines in raw milk samples with STAR protocol – the problem with false-negative results solved

In antibiotic residue analyses the first step of screening is just as important as the following steps. Screening methods need to be quick and inexpensive, but above all sensitive enough to detect the antibiotic residue at or below the maximum residue limit (MRL). In the case of a positive result, the next step is conducted and further methods are used for confirmation. MRLs stated in European Union Regulation 37/2010 for tetracyclines in raw milk are: 100 µg kg–1 for tetracycline, 100 µg kg–1 for oxytetracycline, 100 µg kg–1 for chlortetracycline and no limit for doxycycline because it is prohibited for use in animals from which milk is produced for human consumption. The current five-plate microbiological screening method for the detection of antibiotic residues in raw milk was found to be simple and inexpensive, but not specific, sensitive and reliable enough to detect tetracycline at MRL in routine raw milk screening procedures. Spiking samples with tetracycline at the MRL level and applying them on Bacillus cereus ATCC 11778 microbiological plates often gave false-negative results, indicating that tetracyclines may have to be inactivated or masked. Tetracyclines seem to bind to a certain component in milk. Consequently, when applying samples to the B. cereus microbiological plate the antibiotic cannot inhibit the growth of B. cereus which disables the formation of inhibition zones on the test plate. After adding the appropriate amount of citric acid into the milk samples, we solved the problem of false-negative results. During the validation 79 samples of milk were spiked with tetracyclines at different concentrations: 100 µg kg–1 for tetracycline, 100 µg kg–1 for oxytetracycline, 80 µg kg–1 for chlortetracycline and 30 µg kg–1 for doxycycline. Concentrations used in the validation matched the requirements for MRLs (they were either at or below the MRLs) stated in European Union Regulation 37/2010. The sensitivity of the validation was 100%.

Residues of certain veterinary drugs in raw milk in Slovenia in the 2000?2002 period

In order to ensure the hygienic suitability of foodstuffs of animal origin, and from the aspect of human health protection and improvement, we studied the contamination of raw milk with microbial inhibitors, chloramphenicol, sulphonamides and ivermectin in one part of Slovenia - the central Stajerska region. Our own sampling in 2002 included industrial areas along with areas of intensive and extensive milk production. Thus, in the summer, autumn and winter periods of that year, 27 samples of raw milk were taken and tested for residues of microbial inhibitors, chloramphenicol, sulphonamides and ivermectin. Microbial inhibitors, sulphonamides and ivermectin were, in fact, not found in any of the tested samples. Yet in two samples of raw milk taken in the autumn of 2002, the presence of chloramphenicol was confirmed. These results of testing milk from the central Stajerska region were compared with the results of systematic veterinary-sanitary control in Slovenia during 2000 and 2001 (the residual monitoring programme). No residues of certain veterinary drugs tested for were found in any of the samples that were examined.

The first detection and whole genome characterization of the G6P[15] group A rotavirus strain from roe deer.

Although rotaviruses have been detected in a variety of host species, there are only limited records of their occurrence in deer, where their role is unknown. In this study, group A rotavirus was identified in roe deer during a study of enteric viruses in game animals. 102 samples of intestinal content were collected from roe deer (56), wild boars (29), chamois (10), red deer (6) and mouflon (1), but only one sample from roe deer was positive. Following whole genome sequence analysis, the rotavirus strain D38/14 was characterized by next generation sequencing. The genotype constellation, comprising 11 genome segments, was G6-P[15]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Phylogenetic analysis of the VP7 genome segment showed that the D38/14 rotavirus strain is closely related to the various G6 zoonotic rotavirus strains of bovine-like origin frequently detected in humans. In the VP4 segment, this strain showed high variation compared to that in the P[15] strain found in sheep and in a goat. This finding suggests that rotaviruses from deer are similar to those in other DS-1 rotavirus groups and could constitute a source of zoonotically transmitted rotaviruses. The epidemiological status of group A rotaviruses in deer should be further investigated.

DETECTION OF VIBRIO PARAHAEMOLYTICUS IN MEDITERRANEAN MUSSELS (MYTILUS GALLOPROVINCIALIS) IN SLOVENIA

The aim of this study was to determine the prevalence of Vibrio parahaemolyticus in shellfish samples harvested along the Slovenian coast. Shellfish samples of Mediterranean mussels (Mytilus galloprovincialis) were collected along the Slovenian coast at four locations (Seča, Piran, Strunjan and Debeli Rtič) between 2006 and 2008. Samples were examined and analysed for the presence of V. parahaemolyticus by conventional and molecular methods. The presence of Vibrio in the samples was examined by conventional methods on plate grown bacterial cells before and after enrichment in alkaline saline peptone water (ASPW). PCR methods were used for the detection of V. parahaemolyticus-specific toxR and tlh genes and of the virulence-associated tdh and trh genes. Out of 168 samples examined, 24 were positive for toxR and tlh genes by PCR from enrichment broth. Five out of 62 (8.1%), 4 out of 32 (12.5%) and 15 out of 74 (20.2%) samples were positive in 2006, 2007 and 2008, respectively. Colonies of V. parahaemolyticus were isolated from only one sample positive for V. parahaemolyticus by PCR.

Antimicrobial Resistance and Molecular Characterization of Extended-Spectrum β-Lactamases and Other Escherichia coli Isolated from Food of Animal Origin and Human Intestinal Isolates

Antibiotics have always appeared miraculous, saving innumerable lives. However, the unwise use of antimicrobial drugs has led to the appearance of resistant bacteria. The purpose of this study was to evaluate antimicrobial resistance in Escherichia coli (n =160) isolated from food of animal origin. The focus was on E. coli -producing extended-spectrum β-lactamases. E. coli was chosen because it is a part of the normal microbiota in mammals and can enter the food chain during slaughtering and food manipulation. Subsequently, its resistance genes can be transferred to pathogenic bacteria and human microbiota. Phenotypic and genotypic analyses of selected antimicrobial resistances were carried out together with a molecular analysis of virulence genes. E. coli isolates from food of animal origin were compared with clinical E. coli strains isolated from the human intestinal tract. Extended-spectrum β-lactamase-producing E. coli isolates were found in 9.4% of food isolates and in 1.8% of intestinal isolates. Phylogenetically, the majority of food (86.3%) and intestinal E. coli (58.1%) isolates were found to belong to the commensal phylogenetic groups A and B1. The distribution of 4 of 14 analyzed virulence factors was similar in the food and intestinal isolates. Strains isolated from food in Slovenia harbored resistance genes and virulence factors, which can constitute a problem for food safety if not handled properly.

A comprehensive study of hepatitis E virus infection in pigs entering a slaughterhouse in Slovenia

Hepatitis E is a zoonotic viral disease of pigs with increasing public health concern in industrialized countries. Presented broad study of hepatitis E virus (HEV) presence in pigs in Slovenia is the first attempt to overview the HEV situation in pigs entering a slaughterhouse and, further, to analyse the possibility of HEV entering into the food supply chain. 2433 samples from 811 clinically healthy pigs were collected at four slaughterhouses in Slovenia. Sampling covered three different age groups of pigs and three different types of samples (faeces, bile and liver) important for tracing HEV in a pig population. In addition, 63 swab samples were collected systematically from three different sites on the slaughter line, as well as 22 samples of minced meat and 30 bratwurst samples. All the samples were screened for the presence of HEV nucleic acids by specific real-time RT-PCR assay. In the group of three month old pigs 13.7% of faeces, 13.0% of bile and 2.1% of liver samples were HEV positive. In the group of six months old pigs only 0.25% of liver and 0.25% of bile samples were positive. In the category of sows, no positive samples were found. Two out of 63 swab samples collected on the slaughter line were HEV positive. All tested samples of minced meat and bratwurst were negative. The phylogenetic analysis of 50 HEV positive samples, with comparison of 366 nucleotides in ORF1 region, revealed high diversity of identified strains of HEV in pigs, belonging into subtypes 3a, 3b, 3c and 3e.

Development and Validation of a New Protocol for Detecting and Recovering Clostridium difficile from Meat Samples

There is no recommended protocol for detecting and isolating Clostridium difficile present in food samples. Here, we have evaluated the recovery of C. difficile in meat samples after incubating them in various enrichment broths. The media were as follows: cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme; cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme; and cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, C. difficile moxalactam norfloxacin selective supplement, and lysozyme. Samples were inoculated with various strains and quantities of C. difficile and then enriched in the different broths for 1, 4, and 7 days. C. difficile was isolated on agar plates and detected with quantitative real-time PCR (qPCR). The procedure using enrichment in cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme and incubation for 4 days for qPCR detection and 7 days for isolation (plating on C. difficile agar base with added C. difficile selective supplement and 7% [v/v] defibrinated horse blood after alcoholic shock and centrifugation) was validated. Samples of different kinds of meat and meat preparation were contaminated and used for validation of the chosen protocol. The sensitivity of detection with qPCR was 100%, and the sensitivity of the isolation method was 96%.

Hepatitis E – a “new” foodborne disease

Hepatitis E (HE) is a zoonosis caused by hepatitis E virus (HEV). The disease that used to be problematic only in developing regions with inadequate water supplies and poor sanitary conditions is now considered one of the foodborne diseases in industrialized countries as well. According to current knowledge, the main reservoir of the virus is linked to domestic swine and wild boar. Consumption of raw or undercooked pork meat and liver is considered as a risk factor for HE human infection, together with some other sources of infection like blood transfusion or organ transplantation. Although the number of cases has been rising in the last decade, HEV is still a generally unknown virus among the general public. Consumers need to be warned and educated about HEV and its potential sources of contamination within the food supply chain.

The microbiological quality of Slovenian raw milk from vending machines and their hygienic-technical conditions

Purpose The purpose of this paper is to study the microbiological quality of raw milk delivered by 17 vending machines (VM) owned by different Slovenian milk producers. Design/methodology/approach For the determination of hygiene-technical conditions of VM, an observation list that included criteria for estimation of hygiene-technical suitability was made. A total of 51 milk samples were collected in three different seasons. The swabs and the cleaning liquid (eluates) of dispensing nozzles and chambers were also sampled. The main groups of microorganisms were determined by colony count technique according to international standards in all collected samples. Findings The aerobic colony count was higher than 100,000 CFU/mL in 20 (39.2 per cent) of milk samples. Its mean value was 4.8 log10 CFU/mL. The mean values of Enterobacteriaceae, psychrotrophic microorganisms, lipolytes, proteolytes, yeasts and moulds together, coagulase-positive staphylococci and somatic cell count were 3.3 log10 CFU/mL, 4.1 log10 CFU/mL, 3.2 log10 CFU/mL, 3.9 log10 CFU/mL, 2.2 log10 CFU/mL, 2.8 log10 CFU/mL and 5.3 log10 cells/mL, respectively. E. coli was found in 33.3 per cent of milk samples, while Listeria monocytogenes and antibiotics were not detected. The inner surface contamination of the dispensing nozzles and chambers was estimated in the range from 1.8 log10 CFU to 6.0 log10 CFU/cm2. The presence of detergents and disinfectants in supply valve eluates was determined in more than one-third of the samples. The hygienic-technical conditions of observed VM show some deviations from specified hygienic-technical requirements which could influence the safety of raw milk. Research limitations/implications The data about construction and the cleaning practice of VM, included in the experiment, were not available during the inspection facility. Originality/value In the paper the pathogenic and also the spoilage microorganisms in milk in the combination with hygienic conditions of inside surfaces of VM were studied.

Whole genome sequence and a phylogenetic analysis of the G8P[14] group A rotavirus strain from roe deer

BackgroundGroup A rotaviruses (RVA) are associated with acute gastroenteritis in children and in young domestic and wild animals. A RVA strain was detected from a roe deer for the first time during a survey of game animals in Slovenia in 2014. A further RVA strain (SLO/D110–15) was detected from a roe deer during 2015. The aim of this study was to provide a full genetic profile of the detected RVA strain from roe deer and to obtain additional information about zoonotic transmitted strains and potential reassortments between human rotavirus strains and zoonotic transmitted rotavirus strains. The next generation sequencing (NGS) analysis on Ion Torrent was performed and the whole genome sequence has been determined together with a phylogenetic analysis.ResultsThe whole genome sequence of SLO/D110–15 was obtained by NGS analyses on an IonTorrent platform. According to the genetic profile, the strain SLO/D110–15 clusters with the DS-1-like group and expresses the G8-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genome constellation. Phylogenetic analysis shows that this roe deer G8P[14] strain is most closely related to RVA strains found in sheep, cattle and humans. A human RVA strain with the same genotype profile was detected in 2009 in Slovenia.ConclusionsThe G8P[14] genotype has been found, for the first time, in deer, a newly described host from the order Artiodactyla for this RVA genotype. The finding of a rotavirus with the same genome segment constellation in humans indicates the possible zoonotic potential of this virus strain.