Pathogenic vibrios in environmental, seafood and clinical sources in Germany.

Bacteria of the family Vibrionaceae naturally occur in marine and estuarine environments. Only few species of Vibrionaceae are associated with human cases of gastroenteritis, ear and wound infections, caused by ingestion of seafood or contact with Vibrio containing water. Increasing consumption of seafood (fish, fishery products and shellfish) poses a possible source of Vibrio infections in Germany. Additionally, there is a growing concern that abundances of pathogenic vibrios may increase in German coastal waters as a result of e.g. climate change resulting in probably rising surface water temperatures. According to the One Health concept the VibrioNet consortium started in 2010 to investigate the occurrence and relevance of non-cholera vibrios of human concern in Germany. Vibrios from environmental, seafood and clinical sources were analyzed with the aim to find connections between different reservoirs or sources and to identify potential ways of transmission of these pathogens to assess the risk of infections associated with them. Potentially pathogenic strains mostly belong to the species Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae. Investigations on imported seafood and mussels from primary production areas confirmed the frequent occurrence of these species. Moreover, studies of German coastal waters and sediments showed the presence and seasonality of these marine bacteria. So far the incidence of clinical cases of vibriosis in Germany is low. Between 1994 and 2013 thirteen cases of Vibrio spp. associated wound infections and/or septicaemia have been reported. However, the high prevalence of vibrios in aquatic environments and aquatic organisms is of concern and demands continued control of food and surveillance for clinical infections with pathogenic vibrios.

Detection of resistance to macrolides in thermotolerant campylobacter species by fluorescence in situ hybridization.

The resistance of enteritis-causing Campylobacter strains to erythromycin is an emerging problem. We therefore evaluated fluorescence in situ hybridization (FISH) for the rapid detection of resistance using 74 campylobacter isolates. FISH showed specificity and sensitivity of 100% for the detection of high-level resistance.

Predominant strains of thermophilic Campylobacter spp. in a German poultry slaughterhouse.

Campylobacter causes bacterial diarrhoea in man and is a common foodborne pathogen, that has been associated mainly with poultry carcasses and processed poultry products as well as with drinking water. Genotyping of Campylobacter spp. from poultry was done in order to prove if predominant stable strains in the food chain are present. The influence of the slaughter process on the stability should be determined. Thermophilic Campylobacter spp. from eight poultry flocks were isolated from cloacal swabs, carcasses and offal at different abattoir processing steps to determine their stability. DNA-fingerprinting was done using Pulsed-Field Gel Electrophoresis (PFGE) with two enzymes (SmaI and KpnI) and ribotyping. More than 150 Campylobacter strains were ribotyped and these data were combined with the results of PFGE. Molecular typing showed that strains found in cloacal swabs before processing could also be isolated from carcasses and offal at different processing steps representing predominating stable strains. Strains with varying molecular pattern could additionally be detected at different processing steps. Both genotyping methods identified in agreement flock-specific strains. These remained stable through the slaughter of poultry and were not altered through the slaughter process. Despite the known genetic variability of thermophilic Campylobacter, stable predominant strains could be identified in the poultry slaughter process and those strains can thus enter the food chain.

Quantification of Campylobacter on the surface and in the muscle of chicken legs at retail.

The objective of this study was to determine the prevalence and numbers of Campylobacter on the skin and in the muscle of chicken legs at retail to examine the external and internal contamination for an exposure assessment. Furthermore, the study assessed seasonal influence on Campylobacter contamination in chicken legs. Of the 140 examined skin samples, 66% were positive, and the internal contamination of 115 sampled chicken legs was 27%. The enumeration of Campylobacter on the surface of positive chicken legs revealed a median of 2.4 log CFU/g of skin, and the quantification of Campylobacter in the muscle gave results mainly under the detection limit of the most-probable-number method (<0.3 MPN Campylobacter per g). The external contamination was significantly higher than the internal. In both skin and muscle samples, Campylobacter jejuni had a much higher incidence than Campylobacter coli. However, with regard to the specification of Campylobacter on the surface of chicken legs, C. coli was isolated at higher colony counts than C. jejuni. During the 1-year study, two peaks of Campylobacter contamination occurred, one in the early springtime (February and March, 100 and 90%, respectively) and the second during the warmer months in the summer (July and August, both 90%). Furthermore, a positive correlation between prevalence and numbers of Campylobacter on chicken legs was observed.

Quantification of campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens.

Numerous outbreak investigations and case-control studies for campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a risk factor for infection and illness. There is currently extremely limited quantitative data on the levels of Campylobacter cross-contamination in the kitchen, hindering risk assessments for the pathogen commodity combination of Campylobacter and chicken meat. An exposure assessment needs to quantify the transfer of the bacteria from chicken to hands and the kitchen environment and from there onto ready-to-eat foods. We simulated some typical situations in kitchens and quantified the Campylobacter transfer from naturally contaminated chicken parts most commonly used in Germany. One scenario simulated the seasoning of five chicken legs and the reuse of the same plate for cooked meat. In another, five chicken breast filets were cut into small slices on a wooden board where, without intermediate cleaning, a cucumber was sliced. We also investigated the transfer of the pathogen from chicken via hands to a bread roll. The numbers of Campylobacter present on the surfaces of the chicken parts, hands, utensils, and ready-to-eat foods were detected by using Preston enrichment and colony counting after surface plating on Karmali agar. The mean transfer rates from legs and filets to hands were 2.9 and 3.8%. The transfer from legs to the plate (0.3%) was significantly smaller (P < 0.01) than the percentage transferred from filets to the cutting board and knife (1.1%). Average transfer rates from hands or kitchen utensils to ready-to-eat foods ranged from 2.9 to 27.5%.

International outbreak of Salmonella Oranienburg due to German chocolate.

BACKGROUND: This report describes a large international chocolate-associated Salmonella outbreak originating from Germany. METHODS: We conducted epidemiologic investigations including a case-control study, and food safety investigations. Salmonella (S.) Oranienburg isolates were subtyped by the use of pulsed-field gel electrophoresis (PFGE). RESULTS: From 1 October 2001 through 24 March 2002, an estimated excess of 439 S. Oranienburg notifications was registered in Germany. Simultaneously, an increase in S. Oranienburg infections was noted in other European countries in the Enter-net surveillance network. In a multistate matched case-control study in Germany, daily consumption of chocolate (matched odds ratio [MOR]: 4.8; 95% confidence interval [CI]: 1.3-26.5), having shopped at a large chain of discount grocery stores (MOR: 4.2; CI: 1.2-23.0), and consumption of chocolate purchased there (MOR: 5.0; CI: 1.1-47.0) were associated with illness. Subsequently, two brands from the same company, one exclusively produced for that chain, tested positive for S. Oranienburg. In two other European countries and in Canada chocolate from company A was ascertained that also contained S. Oranienburg. Isolates from humans and from chocolates had indistinguishable PFGE profiles. No source or point of contamination was identified. Epidemiological identification of chocolate as a vehicle of infections required two months, and was facilitated by proxy measures. CONCLUSIONS: Despite the use of improved production technologies, the chocolate industry continues to carry a small risk of manufacturing Salmonella-containing products. Particularly in diffuse outbreak-settings, clear associations with surrogates of exposure should suffice to trigger public health action. Networks such as Enter-net have become invaluable for facilitating rapid and appropriate management of international outbreaks.

Sequence-based typing of flaB is a more stable screening tool than typing of flaA for monitoring of Campylobacter populations.

Different typing schemes for Campylobacter spp. were evaluated with 70 outbreak and sporadic isolates. The discriminatory indexes were 0.944 (by pulsed-field gel electrophoresis), 0.920 (by genotyping of the flagellin A gene), 0.902 (by genotyping of flaB), and 0.886 (by multilocus sequence typing). Cross-classification gave 94.77 or 95.82% (PFGE-flaA or PFGE-flaB) concordance. flaA was overdiscriminatory in three cases, most probably due to intragenomic recombination.

[Suitability of SSCP-PCR analysis for molecular detection of quinolone resistance in Campylobacter jejuni]. / Eignung der SSCP-PCR zum molekularbiologischen Nachweis der Chinolonresistenz bei Campylobacter jejuni.

Foodborne infections with Campylobacter spp. are increasing, especially antibiotic resistant strains are emerging. Quinolone resistant isolates can cause failure of therapy in severe clinical infections. Molecular characterisation is needed for the detection of resistant variants of C. jejuni. Therefore 23 isolates from poultry and human medicine as well as three control strains were tested for their minimal inhibitory concentration, their Single-Strand-Conformation-Polymorphism (SSCP)-PCR pattern (a method for the detection of resistance determining point mutations), and their sequence of the quinolone resistance determining region (QRDR). Six different SSCP types could be identified: two types for quinolone resistant isolates and other types containing so called silent mutations without influence on the resistance. A genotypic monitoring of the quinolone resistance in C. jejuni can be useful for the early detection of new resistance variants. As a screening method for detection of point mutations in the QRDR the SSCP-PCR can be applied. Compared to other genotypic methods the SSCP-PCR is less time and cost consuming and needs only standard technical equipment.

Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2001-2002 from poultry and humans in Berlin, Germany.

The susceptibilities of 430 Campylobacter jejuni strains and 79 C. coli strains to six antimicrobial agents were tested and analyzed. The two sets of strains originated from retail market chicken and turkey samples and from humans, respectively, in Berlin, Germany. Two groups of isolates, one dating from 1991 and the other dating from 2001-2002, were tested. Of the Campylobacter sp. isolates recovered from humans in 2001-2002, 45.1% were resistant to ciprofloxacin, 37.8% were resistant to tetracycline, 12.8% were resistant to ampicillin, and 50.0% were resistant to trimethoprim-sulfamethoxazole. All isolates were susceptible to gentamicin, while the overall rate of resistance to erythromycin was 6.1%. During the 10 years between the two sampling times, the rates of resistance to ciprofloxacin (P<0.001), ampicillin (P=0.035), and tetracycline (P=0.01) increased significantly among strains isolated from humans. Furthermore, among human C. coli strains the rate of resistance to erythromycin rose from 7.1% in 1991 to 29.4% in 2001-2002. In comparison, Campylobacter sp. isolates from poultry already had high rates of resistance in 1991. Different rates of resistance to tetracycline among isolates from chickens and turkeys suggested the development of resistance during antimicrobial treatment in food animals. Thus, discrepancies in the antimicrobial resistance rates among Campylobacter isolates originating from poultry and humans support the hypothesis that at least some of the resistant Campylobacter strains causing infection in humans come from sources other than poultry products.

Comparison of broth microdilution, E Test, and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli.

A standardized broth microdilution method was compared to the E test and an agar dilution method for the antimicrobial susceptibility testing of Campylobacter jejuni and C. coli isolates. A group of 47 human clinical isolates, 37 isolates from retail poultry, and 29 isolates from living turkeys (total, 113 isolates) was included in the study. These encompassed 92 C. jejuni and 21 C. coli strains. The MICs of six antimicrobial agents were determined by the broth microdilution and E test methods, and the strains of human origin were additionally tested by the agar dilution method. In general, broth microdilution MICs agreed within 1 log(2) MIC increment with 90.0% of E test results and 78.7% of agar dilution test results. The agar dilution method gave much lower gentamicin MICs than the broth microdilution method, but the data were significantly (P < 0.01) correlated and there was 100% agreement in the sensitivities and specificities in the comparison of the tests. The broth microdilution method had the highest sensitivity for analysis of the susceptibilities of Campylobacter to nalidixic acid and trimethoprim-sulfamethoxazole. The MICs of ciprofloxacin and erythromycin complied numerically by all three methods. The classification of the results and the correlation of the data demonstrated a high degree of agreement. All methods were equally suitable for the testing of the sensitivity of Campylobacter to tetracycline. Thus, the broth microdilution method appears to be an easy and reliable method for determination of the MICs of antibiotics for C. jejuni and C. coli, and it may offer an interesting alternative to MIC determination by the agar dilution technique or the E test.