Fate of Clostridium botulinum and incidence of pathogenic clostridia in biogas processes.

AIMS: This study aimed to assess the sanitary situation in agricultural biogas plants (BP) regarding pathogenic Clostridium spp. METHODS AND RESULTS: The incidence of Clostridium botulinum, Clostridium difficile, Clostridium novyi, Clostridium haemolyticum, Clostridium septicum and Clostridium chauvoei was investigated in 154 plant and animal substrates, digester sludges and digestates from full-scale BP using a method combining microbial enrichment with Real-Time PCR. The investigated clostridia were absent in the samples, except for Cl. novyi that was barely present (3·9%) and Cl. difficile that was more frequently detected (44·8%). Clostridium botulinum exposed to lab-scale digesters in sentinel chambers was reduced with D-values of 34·6 ± 11·2 days at 38°C and 1·0 ± 0·2 days at 55°C. CONCLUSIONS: These findings indicate minor relevance of clostridial pathogens in BP and an improved sanitary quality of the digestion product compared to untreated substrates concerning Cl. botulinum. However, the frequent detection of Cl. difficile opens questions on the durability of this organism in manure digestion lines. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study providing data on the reduction of Cl. botulinum during biogas processes that scientifically invalidate contrary claims by some media in the public. Furthermore, the results improve the fragmentary knowledge on the prevalence of several clostridial pathogens in agricultural biogas production.

Occurrence of zoonotic clostridia and Yersinia in healthy cattle.

Zoonotic pathogens are a frequent cause of disease worldwide. This study was designed to determine the occurrence of Clostridium difficile, Clostridium botulinum, and Yersinia enterocolitica in cattle in southern Bavaria, Germany. The study population included 49 farms; 34 were dairy farms (30 also fattening beef cattle) and 15 were solely beef cattle farms. Fecal and dust samples were collected from summer 2011 to summer 2012 and analyzed using a combination of enrichment procedures and real-time PCR. For the detection of C. difficile, samples were screened for the presence of the tpi gene and toxin genes tcdA, tcdB, and cdtA. Samples also were screened for genes for C. botulinum toxins A through F and for the ail gene of Y. enterocolitica. Of 506 samples, C. difficile genes were found in 29 samples (5.7%): 25 samples from dairy farms and 4 samples from beef cattle farms. Toxin genes were identified in 17 samples, with toxigenic profiles of A(+)B(+)CDT(-), A(+)B(-)CDT(+), and A(+)B(+)CDT(+). C. botulinum toxin genes were not detected in fecal samples from cattle, but the gene for toxin B was detected in 1 (0.8%) of 125 dust samples. Y. enterocolitica genes were found in 6 (1.6%) of 382 fecal samples from three dairy farms and one beef cattle farm. This study revealed that C. difficile and Y. enterocolitica are rare on cattle farms in Bavaria, Germany. In contrast to results of previous studies, C. botulinum was not detected in fecal samples but was found very rarely in dust samples from the cattle environment.

Presence of virulence genes, adhesion and invasion of Arcobacter butzleri.

AIMS: The pathogenic potential of Arcobacter butzleri isolates was investigated by detecting the presence of putative virulence genes and analysing the adhesive and invasive capabilities in cell cultures of human cell lines. METHODS AND RESULTS: The presence of ten putative virulence genes in 52 A. butzleri isolates was determined by PCR. The genes ciaB, mviN, pldA, tlyA, cj1349 and cadF were detected in all, whilst irgA (15%), iroE (60%), hecB (44%) and hecA (13%) were detected only in few A. butzleri isolates. On HT-29 cells, four of six isolates adhered to and three of them were able to invade, whilst all six isolates adhered to and invaded Caco-2 cells with higher degrees. The genes ciaB, cadF and cj1349 of all six isolates were sequenced, but no considerable changes of the amino acids in putative functional domains were observed. CONCLUSION: Selected A. butzleri isolates adhere to and invade HT-29 and Caco-2 cells, which emphasize their human pathogenic potential. The efficiency of invasion depends on the eukaryotic cell line and individual bacterial strain used. We could not show any functional correlation between the amino acid sequence of CadF, CiaB or Cj1349 and the adhesive and invasive phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: We have shown that some A. butzleri strains invade various cell lines. This underlines their pathogenic potential and hints at their relevance in human disease.

Prevalence of extended-spectrum ß-lactamase-producing Escherichia coli on Bavarian dairy and beef cattle farms.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli strains are believed to be widely distributed among humans and animals; however, to date, there are only few studies that support this assumption on a regional or countrywide scale. Therefore, a study was designed to assess the prevalence of ESBL-producing E. coli in dairy cows and beef cattle in the southern part of Bavaria, Germany. The study population included 30 mixed dairy and beef cattle farms and 15 beef cattle farms. Fecal samples, boot swabs, and dust samples were analyzed for ESBL-producing E. coli using selective media. PCR was performed to screen for CTX-M and ampC resistance genes. A total of 598 samples yielded 196 (32.8%) that contained ESBL-producing E. coli, originating from 39 (86.7%) of 45 farms. Samples obtained from mixed farms were significantly more likely to be ESBL-producing E. coli positive than samples from beef cattle farms (fecal samples, P < 0.001; boot swabs, P = 0.014; and dust samples, P = 0.041). A total of 183 isolates (93.4%) of 196 ESBL-producing E. coli-positive strains harbored CTX-M genes, CTX-M group 1 being the most frequently found group. Forty-six additional isolates contained ampC genes, and 5 of the 46 isolates expressed a blaCMY-2 gene. The study shows that ESBL-producing E. coli strains are commonly found on Bavarian dairy and beef cattle farms. Moreover, to our knowledge, this is the first report of the occurrence of blaCMY-2 in cattle in Germany.

Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

Occurrence of thermotolerant Campylobacter spp. on eggshells: a missing link for food-borne infections?

We analyzed the prevalence of thermotolerant Campylobacter spp. compared that of to Salmonella spp. in raw yolk and on eggshells. A total of 2,710 eggs were investigated for each bacterium. Viable bacteria were found in 4.1% (Campylobacter spp.) and 1.1% (Salmonella spp.) of the eggshell samples, whereas the egg yolk samples were negative for both bacteria.

Detection and differentiation of Vibrio spp. in seafood and fish samples with cultural and molecular methods.

Vibrio spp. as natural inhabitants of sea- and brackwater of both tropical and temperate regions of the world are commonly found in different kinds of seafood. Even among the three main human pathogenic species Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus most of the isolates from seafood do not carry the different virulence factors responsible for foodborne infections. Therefore, the risk assessment of Vibrio spp. in seafood is currently based mainly on the knowledge of the genetic setting of foodborne strains. For the detection and differentiation of Vibrio spp. (V. parahaemolyticus, V. cholerae and V. vulnificus) three probe-based multiplex real-time PCR systems were developed and validated. One real-time PCR system simultaneously detects V. parahaemolyticus, V. cholerae and V. vulnificus on genus level combined with an Internal Amplification Control. The detection limit for the system was between 1cfu/mL and 10cfu/mL in pure culture and in different artificially contaminated sample material, e. g. prawns or Alaska Pollock. The other two PCR systems were implemented for the detection of different virulence genes of V. parahaemolyticus and V. cholerae isolates. The molecular detection systems were applied for the investigation of 338 raw and cooked seafood and fish samples for the presence of the different Vibrio spp. The collected data indicate that the PCR systems can be useful for rapid detection and differentiation of Vibrio spp. in different food matrices as basis for a preventive consumer protection policy.

Prevalence of emetic Bacillus cereus in different ice creams in Bavaria.

In this study, 809 samples of ice cream from different sources were investigated by using cultural methods for the presence of presumptive Bacillus cereus. Isolates from culture-positive samples were examined with a real-time PCR assay targeting a region of the cereulide synthetase gene (ces) that is highly specific for emetic B. cereus strains. The samples were collected from ice cream parlors and restaurants that produced their own ice cream and from international commercial ice cream companies in different regions of Bavaria during the summer of 2008. Presumptive B. cereus was found in 508 (62.7%) ice cream samples investigated, and 24 (4.7%) of the isolates had the genetic background for cereulide toxin production. The level of emetic B. cereus in the positive samples ranged from 0.1 to 20 CFU/g of ice cream.