David Šilha

Susceptibility to 18 drugs and multidrug resistance of Arcobacter isolates from different sources within the Czech Republic

OBJECTIVES Arcobacter spp. are considered to be potential foodborne pathogens, and consumption of contaminated food containing these bacteria could endanger human and animal health. Arcobacter butzleri and Arcobacter cryaerophilus are the species most frequently isolated from food of animal origin and from other samples. The aim of this study was to evaluate the susceptibility of arcobacters isolated in the Czech Republic. No information about antibiotic susceptibility and multidrug resistance of arcobacters isolated in the Czech Republic is available in the literature before now. METHODS The antimicrobial resistance of A. butzleri (n=80) and A. cryaerophilus (n=20) isolated from meat of animal origin, water sources and clinical samples was examined by the disk diffusion method. RESULTS Arcobacters were resistant to one or more antimicrobial agents in 99% (99/100) of tested isolates. Most of the Arcobacter isolates were resistant to β-lactam antibiotics, i.e. ampicillin (81.0%), amoxicillin/clavulanic acid (28.0%), cefalotin (73.0%) and aztreonam (93.0%). Arcobacters were also frequently resistant to lincosamides, i.e. clindamycin (98.0%). Of the aminoglycosides, amikacin, gentamicin and tobramycin were evaluated to be the most effective antibiotics among those tested against arcobacters. CONCLUSIONS These results demonstrate substantial resistance in Arcobacter isolates to 18 antimicrobial agents commonly used in medical and veterinary medicine. Multidrug resistance was found in 93.8% (75/80) of A. butzleri isolates and 70.0% (14/20) of A. cryaerophilus isolates.

Occurrence of virulence-associated genes in Arcobacter butzleri and Arcobacter cryaerophilus isolates from foodstuff, water, and clinical samples within the Czech Republic

Bacteria of the Arcobacter (A.) genus, originating mainly from food and water, are dreaded germs for humans as well as animals. However, the virulence of these bacteria has not been fully elucidated yet. This study looked at the occurrence of eight virulence-associated factors (ciaB, cj1349, pldA, irgA, hecA, tlyA, mviN, hecB) in a total of 80 isolates of Arcobacter butzleri and 22 isolates of A. cryaerophilus. The isolates were derived from food, water, and clinical samples. A polymerase chain reaction using specific primers was used to detect these virulence-associated genes. The presence of all genes in the isolates of A. butzleri (98.8% ciaB, 95.0% cj1349, 98.8% pldA, 22.5% irgA, 31.3% hecA, 95.0% tlyA, 97.5% mviN, 38.8% hecB) and A. cryaerophilus (95.5% ciaB, 0.0% cj1349, 9.1% pldA, 0.0% irgA, 0.0% hecA, 31.8% tlyA, 90.9% mviN, 0.0% hecB) was monitored. Among the tested isolates, there were 13 isolates (12.7%) of A. butzleri, in which the presence of all eight virulence-associated genes was recorded in the genome. In contrast, in one A. cryaerophilus strain, none of the observed genes were detected. The presence of ciaB and mviN genes was significantly more frequent in A. cryaerophilus isolates than other genes (P < 0.05). In general, more virulence-associated genes have been detected in A. butzleri isolates compared to A. cryaerophilus. The most common gene combination (ciaB, cj1349, pldA, tlyA, mviN) was detected in case of 39 isolates. In 50.0% of A. butzleri isolates derived from clinical samples, all eight virulence-associated genes were significantly more frequently detected (P < 0.05). The tlyA gene occurred significantly more frequent in A. butzleri isolates from meat and water samples and irgA and hecB genes in clinical samples. Therefore, our study provides information about occurrence of virulence-associated genes in genome of Arcobacter isolates. These findings could be hazardous to human health, because the presence of virulence-associated genes is the assumption for potential dangerousness of these bacteria. Our results indicate high incidence of virulence-associated genes in Arcobacter genomes and hence potentially pathogenic properties of the studied strains.

Presence of Arcobacter species in pet cats and dogs in the Czech Republic

This study was conducted to evaluate the occurrence of the genus Arcobacter in cats and dogs in the Czech Republic. These animals may be carriers of the bacteria and potential sources of human infection. Oral smears were collected from animals using smear swabs and brushes. Based on previous studies, commercially available DNA kits were used for DNA isolation. Samples were analysed using polymerase chain reaction (PCR) and evaluated using gel electrophoresis. Overall, 178 oral smears were tested, of which 108 were from dogs and 70 were from cats. Out of all smears, five were positive, of which four samples were from dogs and one from a cat. In all five positive cases, PCR confirmed the presence of Arcobacter butzleri. In follow-up sampling, the presence of Arcobacter butzleri was demonstrated in two samples from a dog.

Influence of chosen microbes and some chemical substances on the production of aflatoxins.

Aflatoxins are produced as secondary metabolites by A. flavus, A. parasiticus, A. nomius and A. tamarii . The aflatoxin biosynthetic pathway involves several enzymatic steps and genes ( apa-2 , ver- 1) that appear to be regulated by the aflR gene in these fungi. The aim of this work was the detection of aflatoxins by the HPLC method and the ascertainment of factors influencing their production. A. parasiticus CCM F-108, A. parasiticus CCF 141, A. parasiticus CCF 3137 and two isolates A. flavus were used. These toxigenic isolates were recovered from spice (strain 1) and wraps (strain 2). The gene for the production of aflatoxin B1 for each species of fungi was detected using an optimized PCR method. Rhodotorula spp. * , Lactococcus lactis subsp. lactis CCM 1881, Flavobacterium spp. and fungal strain Pythium oligandrum * were tested for inhibition of aflatoxins production and fungal growth. Having used the HPLC detection, various preservatives (propionic acid, citric acid, potassium sorbate) were tested from the viewpoint of their influence on the growth of aflatoxigenic fungi followed by the production of aflatoxins. The growth of A. flavus and A. parasiticus and aflatoxin production in Potato Dextrose Agar supplemented with propionic acid (1000-2000-3000 mg/kg), citric acid (2000-3000-4000 mg/kg) and potassium sorbate (500-800-1000 mg/kg) was tested by Agar Dilution Method. After 72 h of incubation was evaluated growth of fungi, all samples were frozen for later extraction and aflatoxins quantification by HPLC. Effect of peptone and sucrose additions were studied in yeast extract (2%) supplemented with peptone (5-10-15%) or sucrose (15%). Growth inhibition of Aspergillus by Pythium oligandrum was tested on wood surface. As shown, the highest inhibition effect on the aflatoxins production was obtained when propionic acid was applied in concentrations since 1000 mg/kg. A total inhibition of the fungi growth and aflatoxins production was observed in all samples containing peptone in the concentration range tested. Significant limitation of the growth and production of aflatoxins was also observed in the presence of other microorganisms such like Pythium oligandrum and Rhodotorula spp.