Molecular characterization of Escherichia coli isolates recovered from broilers with cellulitis.

Avian cellulitis in broilers, caused by avian pathogenic Escherichia coli, is a major cause for carcass rejections during meat inspection, resulting in significant economic losses. In this study, we analysed E. coli isolates obtained from broiler chickens affected by cellulitis for their genetic relatedness and antimicrobial resistance phenotype and genotype. The objective was to determine whether there is a clonal spread or whether these clinical isolates differ. For this purpose, E. coli was isolated from swab samples collected from diseased broilers across 77 poultry farms in Germany, resulting in 107 isolates. These isolates were subjected to serotyping, PCR-based phylotyping and macrorestriction analysis with subsequent pulsed-field gel-electrophoresis for typing purposes. In addition, the presence of virulence genes associated with avian pathogenic E. coli (APEC) was investigated by PCR. Antimicrobial susceptibility of the isolates was examined by the disk diffusion method according to CLSI guidelines and subsequently, the presence of corresponding resistance genes was investigated by PCR. Typing results revealed that a significant proportion of the isolates belonged to serotype O78:K80, which is one of the major APEC serotypes. Phylogenetic grouping showed that phylogenetic group D was most commonly represented (n = 49). Macrorestriction analysis showed overall heterogenous results, however, some clustering of closely related isolates was observed. The level of antimicrobial resistance was high, with 83.8% of isolates non-susceptible to at least one class of antimicrobial agents and 40% of isolates showing resistance to at least three classes. The most frequently observed resistance was to ampicillin, mediated by blaTEM (n = 56). However, few isolates were non-susceptible to ciprofloxacin (n = 8) and none of the isolates was resistant to 3rd generation cephalosporins or carbapenems. Overall, the results show that genetically diverse APEC associated with avian cellulitis can be found among and within German poultry farms. While most isolates were antimicrobial resistant, resistance levels to high(est) priority critically important antimicrobials were low.

Occurrence of Antimicrobial-Resistant Escherichia coli in Marine Mammals of the North and Baltic Seas: Sentinels for Human Health.

Antimicrobial resistance is a global health threat that involves complex, opaque transmission processes in the environment. In particular, wildlife appears to function as a reservoir and vector for antimicrobial-resistant bacteria as well as resistance genes. In the present study, the occurrence of antimicrobial-resistant Escherichia coli was determined in marine mammals and various fish species of the North and Baltic Seas. Rectal or faecal swabs were collected from 66 live-caught or stranded marine mammals and 40 fish specimens. The antimicrobial resistance phenotypes and genotypes of isolated E. coli were determined using disk diffusion tests and PCR assays. Furthermore, isolates were assigned to the four major phylogenetic groups of E. coli. Additionally, post mortem examinations were performed on 41 of the sampled marine mammals. The investigations revealed resistant E. coli in 39.4% of the marine mammal samples, while no resistant isolates were obtained from any of the fish samples. The obtained isolates most frequently exhibited resistance against aminoglycosides, followed by ß-lactams. Of the isolates, 37.2% showed multidrug resistance. Harbour porpoises (Phocoena phocoena) mainly carried E. coli isolates belonging to the phylogenetic group B1, while seal isolates were most frequently assigned to group B2. Regarding antimicrobial resistance, no significant differences were seen between the two sampling areas or different health parameters, but multidrug-resistant isolates were more frequent in harbour porpoises than in the sampled seals. The presented results provide information on the distribution of antimicrobial-resistant bacteria in the North and Baltic Seas, and highlight the role of these resident marine mammal species as sentinels from a One Health perspective.

High Rates of Multidrug-Resistant Escherichia coli in Great Cormorants (Phalacrocorax carbo) of the German Baltic and North Sea Coasts: Indication of Environmental Contamination and a Potential Public Health Risk.

Antimicrobial-resistant bacteria pose a serious global health risk for humans and animals, while the role of wildlife in the dynamic transmission processes of antimicrobial resistance in environmental settings is still unclear. This study determines the occurrence of antimicrobial-resistant Escherichia coli in the free-living great cormorants (Phalacrocorax carbo) of the North and Baltic Sea coasts of Schleswig-Holstein, Germany. For this, resistant E.coli were isolated from cloacal or faecal swabs and their antimicrobial resistance pheno- and genotypes were investigated using disk diffusion tests and PCR assays. The isolates were further assigned to the four major phylogenetic groups, and their affiliation to avian pathogenic E. coli (APEC) was tested. Resistant E. coli were isolated from 66.7% of the 33 samples, and 48.9% of all the resistant isolates showed a multidrug resistance profile. No spatial differences were seen between the different sampling locations with regard to the occurrence of antimicrobial resistance or multidrug resistance. Most commonly, resistance percentages occurred against streptomycin, followed by tetracycline and sulfonamides. More than half of the isolates belonged to the phylogenetic group B1. Of all the isolates, 24.4% were classified as APEC isolates, of which almost 82% were identified as multidrug-resistant. These results add information on the dispersal of antimicrobial-resistant bacteria in wild birds in Germany, thereby allowing conclusions on the degree of environmental contamination and potential public health concerns.

Characterization of Escherichia coli from Edible Insect Species: Detection of Shiga Toxin-Producing Isolate.

Insects as novel foods are gaining popularity in Europe. Regulation (EU) 2015/2283 laid the framework for the application process to market food insects in member states, but potential hazards are still being evaluated. The aim of this study was to investigate samples of edible insect species for the presence of antimicrobial-resistant and Shiga toxin-producing Escherichia coli (STEC). Twenty-one E. coli isolates, recovered from samples of five different edible insect species, were subjected to antimicrobial susceptibility testing, PCR-based phylotyping, and macrorestriction analysis. The presence of genes associated with antimicrobial resistance or virulence, including stx1, stx2, and eae, was investigated by PCR. All isolates were subjected to genome sequencing, multilocus sequence typing, and serotype prediction. The isolates belonged either to phylogenetic group A, comprising mostly commensal E. coli, or group B1. One O178:H7 isolate, recovered from a Zophobas atratus sample, was identified as a STEC. A single isolate was resistant to tetracyclines and carried the tet(B) gene. Overall, this study shows that STEC can be present in edible insects, representing a potential health hazard. In contrast, the low resistance rate among the isolates indicates a low risk for the transmission of antimicrobial-resistant E. coli to consumers.

Wild Boars Carry Extended-Spectrum ß-Lactamase- and AmpC-Producing Escherichia coli.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) represent major healthcare concerns. The role of wildlife in the epidemiology of these bacteria is unclear. The purpose of this study was to determine their prevalence in wild boars in Germany and to characterize individual isolates. A total of 375 fecal samples and 439 nasal swabs were screened for the presence of ESBL-/AmpC-E. coli and MRSA, respectively. The associations of seven demographic and anthropogenic variables with the occurrence of ESBL-/AmpC-E. coli were statistically evaluated. Collected isolates were subjected to antimicrobial susceptibility testing, molecular typing methods, and gene detection by PCR and genome sequencing. ESBL-/AmpC-E. coli were detected in 22 fecal samples (5.9%) whereas no MRSA were detected. The occurrence of ESBL-/AmpC-E. coli in wild boars was significantly and positively associated with human population density. Of the 22 E. coli, 19 were confirmed as ESBL-producers and carried genes belonging to blaCTX-M group 1 or blaSHV-12. The remaining three isolates carried the AmpC-ß-lactamase gene blaCMY-2. Several isolates showed additional antimicrobial resistances. All four major phylogenetic groups were represented with group B1 being the most common. This study demonstrates that wild boars can serve as a reservoir for ESBL-/AmpC-producing and multidrug-resistant E. coli.

Impact of a Combination of UV-C Irradiation and Peracetic Acid Spray Treatment on Brochothrix thermosphacta and Yersinia enterocolitica Contaminated Pork.

Efficient ways of decontamination are needed to minimize the risk of infections with Yersinia (Y.) enterocolitica, which causes gastrointestinal diseases in humans, and to reduce the numbers of Brochothrix (B.) thermosphacta to extend the shelf-life of meat. While many studies have focused on a single treatment of peracetic acid (PAA) or UV-C-irradiation, there are no studies about a combined treatment on meat. Therefore, in the present study, pork was inoculated with either Y. enterocolitica or B. thermosphacta, and was treated with a combination of 2040 mJ/cm2 UV-C irradiation followed by a 2000 ppm PAA spray treatment (30 s). Samples were packed under modified atmosphere and stored for 1, 7, or 14 days. The samples were examined for Y. enterocolitica and B. thermosphacta content, chemical and sensory effects, and meat quality parameters. For Y. enterocolitica, a significant reduction of up to 2.16 log10 cfu/cm2 meat and for B. thermosphacta, up to 2.37 log10 cfu/cm2 meat was seen on day 14 after UV-C/PAA treatment compared to the untreated controls.

Mutations in the Phenicol Exporter Gene fexA Impact Resistance Levels in Three Bacterial Hosts According to Susceptibility Testing and Protein Modeling.

The prototype fexA gene confers combined resistance to chloramphenicol and florfenicol. However, fexA variants mediating resistance only to chloramphenicol have been identified, such as in the case of a Staphylococcus aureus isolate recovered from poultry meat illegally imported to Germany. The effects of the individual mutations detected in the fexA sequence of this isolate were investigated in this study. A total of 11 fexA variants, including prototype fexA and variants containing the different previously described mutations either alone or in different combinations, were generated by on-chip gene synthesis and site-directed mutagenesis. The constructs were inserted into a shuttle vector and transformed into three recipient strains (Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium). Subsequently, minimal inhibitory concentrations (MIC) of florfenicol and chloramphenicol were determined. In addition, protein modeling was used to predict the structural effects of the mutations. The lack of florfenicol-resistance mediating properties of the fexA variants could be attributed to the presence of a C110T and/or G98C mutation. Transformants carrying fexA variants containing either of these mutations, or both, showed a reduction of florfenicol MICs compared to those transformants carrying prototype fexA or any of the other variants. The significance of these mutations was supported by the generated protein models, indicating a substitution toward more voluminous amino-acids in the substrate-binding site of FexA. The remaining mutations, A391G and C961A, did not result in lower florfenicol-resistance compared to prototype fexA.

Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis.

Glaesserella parasuis is a fastidious pathogen that colonizes the respiratory tract of pigs and can lead to considerable economic losses in pig production. Therefore, a rapid detection assay for the pathogen, preferably applicable in the field, is important. In the current study, we developed a new and improved detection method using loop-mediated isothermal amplification (LAMP). This assay, which targets the infB gene, was tested on a collection of 60 field isolates of G. parasuis comprising 14 different serovars. In addition, 63 isolates from seven different closely related species of the family Pasteurellaceae, including A. indolicus, A. porcinus, and A. minor, and a species frequently found in the respiratory tract of pigs were used for exclusivity experiments. This assay showed an analytical specificity of 100% (both inclusivity and exclusivity) and an analytical sensitivity of 10 fg/µL. In further steps, 36 clinical samples were tested with the LAMP assay. An agreement of 77.1 (95% CI: 59.9, 89.6) and 91.4% (95% CI: 75.9, 98.2) to the culture-based and PCR results was achieved. The mean limit of detection for the spiked bronchoalveolar lavage fluid was 2.58 × 102 CFU/mL. A colorimetric assay with visual detection by the naked eye was tested to provide an alternative method in the field and showed the same sensitivity as the fluorescence-based LAMP assay. Overall, the optimized LAMP assay represents a fast and reliable method and is suitable for detecting G. parasuis in the laboratory environment or in the field.

Occurrence of extended-spectrum beta-lactamase-producing Enterobacteriaceae, microbial loads, and endotoxin levels in dust from laying hen houses in Egypt.

BACKGROUND: Poultry houses are often highly contaminated with dust, which might contain considerable amounts of microorganisms and endotoxins. The concentrations of microorganisms and endotoxins in dust from laying hen houses in Egypt are unknown. However, to estimate the risks for birds, the environment, and people working in laying hen houses, it is important to gather information about the composition of these dusts. Here we report the microbial loads, the occurrence of antimicrobial-resistant bacteria, and endotoxin concentrations in dust samples from 28 laying hen farms in Dakahliya Governorate, Egypt, and discuss the results relevant to the literature. RESULTS: Pooled settled dust samples (n = 28) were analyzed for total viable counts of bacteria and fungi (CFU/g), the occurrence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, Salmonella spp., and methicillin-resistant Staphylococcus aureus (MRSA), and endotoxin concentrations (ng/g). The means and standard deviations of total viable counts were 7.10 × 108 ± 2.55 × 109 CFU/g for bacteria and 5.37 × 106 ± 7.26 × 106 CFU/g for fungi. Endotoxin levels varied from 2.9 × 104 to 6.27 × 105 ng/g. None of the tested samples contained Salmonella spp. or MRSA. In contrast, by direct plating, Enterobacteriaceae were found frequently (57%; n = 16), and suspected ESBL-producing Enterobacteriaceae occurred in 21% (n = 6) of the sampled barns. Using an enrichment method, the detection of Enterobacteriaceae and suspected ESBL-producing Enterobacteriaceae increased to 20 and 16 positive barns, respectively. Taking results from both methods into account, Enterobacteriaceae and suspected ESBL-producing Enterobacteriaceae were detected in 23 barns Overall, 100 ESBL suspected isolates (Escherichia coli, n = 64; Enterobacter cloacae, n = 20; and Klebsiella pneumoniae n = 16) were identified to species level by MALDI-TOF MS. Isolates from 20 barns (71% positive barns) were confirmed as ESBL producing Enterobacteriaceae by the broth microdilution test. CONCLUSIONS: Dust in Egyptian laying hen houses contains high concentrations of microorganisms and endotoxins, which might impair the health of birds and farmers when inhaled. Furthermore, laying hens in Egypt seem to be a reservoir for ESBL-producing Enterobacteriaceae. Thus, farmers are at risk of exposure to ESBL-producing bacteria, and colonized hens might transmit these bacteria into the food chain.

Association of intestinal colonization of ESBL-producing Enterobacteriaceae in poultry slaughterhouse workers with occupational exposure-A German pilot study.

BACKGROUND: Bacteria that have acquired antimicrobial resistance, in particular ESBL-producing Enterobacteriaceae, are an important healthcare concern. Therefore, transmission routes and risk factors are of interest, especially for the carriage of ESBL-producing E. coli. Since there is an enhanced risk for pig slaughterhouse employees to carry ESBL-producing Enterobacteriaceae, associated with animal contact as potential risk factor, the present study investigated the occurrence of ESBL-producing Enterobacteriaceae in poultry slaughterhouse employees. Due to the higher level of resistant Enterobacteriaceae in primary poultry production than in pig production, a higher risk of intestinal colonization of poultry slaughterhouse employees was expected. RESULTS: ESBL-producing Enterobacteriaceae were detected in 5.1% (5 of 99) of the fecal samples of slaughterhouse workers. The species of these isolates was confirmed as E. coli. PCR assays revealed the presence of the genes blaCTX-M-15 (n = 2) and blaSHV-12 (n = 3) in these isolates, partly in combination with the ß-lactamase gene blaTEM-135. Participants were divided into two groups according to their occupational exposure and results indicated an increased probability of colonization with ESBL-producing Enterobacteriaceae for the group of 'higher exposure' (OR 3.7, exact 95% CI 0.6-23.5; p = 0.4). For intestinal colonization with ESBL-producing Enterobacteriaceae, a prevalence of 10% (3/30) was observed in the group of 'higher exposure' versus 2.9% (2/69) in the group of 'lower exposure'. Employees in working steps such as 'hanging' poultry in the process of slaughter and 'evisceration' seemed to have a higher risk for intestinal colonization with ESBL-producing Enterobacteriaceae compared to the group of 'lower exposure'. CONCLUSION: This study is the first of its kind to collect data on the occupational exposure of slaughterhouse workers to ESBL-producing Enterobacteriaceae in Europe. The results suggested that colonization with ESBL-producing Enterobacteriaceae is associated with occupational exposure in poultry slaughterhouses. However, the presence of ESBL-producing E. coli isolates in only 5.1% (5/99) of the tested employees in poultry slaughterhouses suggests a lower transmission risk than in pig slaughterhouses.

Curcumin Mitigates Immune-Induced Epithelial Barrier Dysfunction by Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa, a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni-induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10-/- mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni-induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response-represented by TNF-α, IL-1ß, and IL-6 secretion-was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni-triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients.

Characteristics of methicillin-resistant Staphylococcus aureus from broiler farms in Germany are rather lineage- than source-specific.

Methicillin-resistant Staphylococcus aureus (MRSA) are a major concern for public health, and broiler farms are a potential source of MRSA isolates. In this study, a total of 56 MRSA isolates from 15 broiler farms from 4 different counties in Germany were characterised phenotypically and genotypically. Spa types, dru types, SCCmec types, and virulence genes as well as resistance genes were determined by using a DNA microarray or specific PCR assays. In addition, PFGE profiles of isolates were used for analysis of their epidemiological relatedness. While half of the isolates belonged to spa type t011, the other half was of spa types t1430 and t034. On 3 farms, more than 1 spa type was found. The most common dru type was dt10a (n = 19), followed by dt11a (n = 17). Susceptibility testing of all isolates by broth microdilution revealed 21 different resistance phenotypes and a wide range of resistance genes was present among the isolates. Up to 10 different resistance phenotypes were found on individual farms. Resistance to tetracyclines (n = 53), MLSB antibiotics (n = 49), trimethoprim (n = 38), and elevated MICs of tiamulin (n = 29) were most commonly observed. Microarray analysis detected genes for leucocidin (lukF/S), haemolysin gamma (hlgA), and other haemolysines in all isolates. In all t1430 isolates, the egc cluster comprising of genes encoding enterotoxin G, I, M, N, O, U, and/or Y was found. The splitstree analysis based on microarray and PCR gene profiles revealed that all CC9/SCCmec IV/t1430/dt10a isolates clustered apart from the other isolates. These findings confirm that genotypic patterns were specific for clonal lineages rather than for the origin of isolates from individual farms.

Peracetic acid reduces Campylobacter spp. on turkey skin: Effects of a spray treatment on microbial load, sensory and meat quality during storage.

Handling and consumption of Campylobacter-contaminated poultry meat is the most common cause of human campylobacteriosis. While many studies deal with interventions to reduce Campylobacter spp. on chicken carcasses, studies on other poultry species are rare. In the present study, a spray treatment with peracetic acid (PAA) on turkey carcasses was evaluated. For this, parts of breast fillets with skin and Campylobacter (C.) jejuni DSM 4688 (108 cfu/ml) inoculated drumsticks were sprayed for 30 s with PAA (1200 ppm) or water as control solution. Samples were packaged under modified atmosphere and stored at 4°C until analysis on day 1, 6 and 12. The breast fillets were used for determination of the total viable count, sensory and meat quality examination as well as myoglobin content and biogenic amines. The drumsticks were used for C. jejuni counts. PAA had a significant effect in reducing total viable counts on all days by up to 1.2 log10 compared to the untreated control. Treatment with water alone showed no effect. C. jejuni counts were significantly reduced by PAA (0.9-1.3 log10), while water achieved a 0.5 log10 reduction on C. jejuni counts on day 1. No differences in sensory, pH, electrical conductivity and myoglobin content could be found. The skin of the PAA treated fillets had lower redness values than the water control on day 1, whereas on day 12 parts of the water treated muscles were lighter than the untreated control. A lower putrescine content of the water sprayed fillets in comparison to the control sample on day 12 was the only significant difference concerning the biogenic amines. Results from this study indicate that a spray treatment with 1200 ppm PAA would be a useful measure to lower the Campylobacter spp. counts on turkey carcasses without having a negative influence on product quality.

Peracetic acid reduces Campylobacter spp. numbers and total viable counts on broiler breast muscle and drumstick skins during modified atmosphere package storage.

Constant high case numbers of human campylobacteriosis over the last few years show the necessity of efficient strategies to reduce the number of diseases. The aim of this study was to assess the effectiveness of peracetic acid (PAA) as spray application to reduce Campylobacter spp. on chicken meat. For this, the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of 25 Campylobacter jejuni and C. coli isolates were determined. All tested isolates had MICs ranging between 2 to 8 ppm PAA, while MBCs were 1- to 4-fold higher than the MIC. An additional time-kill test, using strain C. jejuni DSM 4688, revealed that after an incubation time of 2 h in medium, supplemented with 1-fold the MIC (4 ppm) of PAA, no surviving C. jejuni cells were detectable. For evaluation of a spraying treatment, C. jejuni DSM 4688 (108 cfu/mL) inoculated chicken drumsticks and native skin-on breast fillets were treated for 30 s with PAA of 1,200 ppm concentration. Samples were packaged in modified atmosphere packages and stored at 4°C until further analysis. On day 1, 6, and 12, the fillets were used for microbial (total viable count), sensory, and physicochemical (color, pH, electrical conductivity) analysis and meat samples for myoglobin redox forms and antioxidant activity were taken. A significant reduction of the total viable counts was seen on day 6 and 12 in comparison to the water control and to the untreated fillets, respectively. Campylobacter jejuni counts on the drumsticks were significantly reduced by PAA application on day 6 and 12 in comparison to the water treatment. Except on day 12, where PAA-treated fillets showed a slightly higher percentage of oxymyoglobin, no significant differences could be found in the sensory and physicochemical measurements as well as in myoglobin and antioxidant activity. Spray application of 1,200 ppm PAA to Campylobacter-contaminated chicken samples led to a significant reduction up to 1.1 log10 of Campylobacter spp. counts without influencing chemical and sensory meat quality parameters.

Variety of Antimicrobial Resistances and Virulence Factors in Staphylococcus aureus Isolates from Meat Products Legally and Illegally Introduced to Germany.

Food products of animal origin can serve as a vehicle for Staphylococcus (S.) aureus, a facultative pathogen involved in a variety of diseases. As a result, international trade and illegal transportation of foodstuffs can facilitate the distribution of S. aureus over long distances. In this study, we investigated S. aureus isolates recovered from meat products confiscated from passengers returning from non-EU countries at two German airports and from samples of legally imported meats from non-EU countries. The aim was to characterize isolates in regard to their genetic relatedness as well as their antimicrobial resistance profiles and major virulence factors in order to assess potential risks associated with these products. The isolates were characterized by spa typing, MLST, macrorestriction analysis, microarray analysis and antimicrobial susceptibility testing. MRSA isolates were further characterized by dru typing. The characteristics of the majority of the isolates indicated a human origin, rather than an association with livestock. The results further revealed a considerable heterogeneity among the MRSA isolates, despite their common origin. Overall, a plenitude of major virulence factors and antimicrobial resistances was detected among the isolates, highlighting the potential risks associated with contaminated meat products and the transportation of such products among different countries.

Yersinia enterocolitica Isolates from Wild Boars Hunted in Lower Saxony, Germany.

Yersiniosis is strongly associated with the consumption of pork contaminated with enteropathogenic Yersinia enterocolitica, which is harbored by domestic pigs without showing clinical signs of disease. In contrast to data on Y. enterocolitica isolated from conventionally reared swine, investigations into the occurrence of Y. enterocolitica in wild boars in Germany are rare. The objectives of the study were to get knowledge about these bacteria and their occurrence in wild boars hunted in northern Germany by isolation of the bacteria from the tonsils, identification of the bioserotypes, determination of selected virulence factors, macrorestriction analysis, multilocus sequence typing (MLST), and testing of antimicrobial susceptibility. Altogether, tonsils from 17.1% of 111 tested wild boars were positive for Y. enterocolitica by culture methods. All but two isolates belonged to biotype (BT) 1A, with the majority of isolates bearing a ystB nucleotide sequence which was revealed to have 85% identity to internal regions of Y. enterocolitica heat-stable enterotoxin type B genes. The remaining Y. enterocolitica isolates were identified to be BT 1B and did not carry the virulence plasmid. However, two BT 1A isolates carried the ail gene. Macrorestriction analysis and results from MLST showed a high degree of genetic diversity of the isolates, although the region where the samples were taken was restricted to Lower Saxony, Germany, and wild boars were shot during one hunting season. In conclusion, most Y. enterocolitica isolates from wild boars investigated in this study belonged to biotype 1A. Enteropathogenic Y. enterocolitica bioserotypes 4/O:3 and 2/O:9, usually harbored by commercially raised pigs in Europe, could not be identified.

Influencing factors and applicability of the viability EMA-qPCR for a detection and quantification of Campylobacter cells from water samples.

In recent years, increasing numbers of human campylobacteriosis cases caused by contaminated water have been reported. As the culture-based detection of Campylobacter is time consuming and can yield false-negative results, the suitability of a quantitative real-time PCR method in combination with an ethidium monoazide pretreatment of samples (EMA-qPCR) for the rapid, quantitative detection of viable Campylobacter cells from water samples was investigated. EMA-qPCR has been shown to be a promising rapid method for the detection of viable Campylobacter spp. from food samples. Application of membrane filtration and centrifugation, two methods frequently used for the isolation of bacteria from water, revealed a mean loss of up to 1.08 log10 cells/ml from spiked samples. Both methods used alone lead to a loss of dead bacteria and accumulation of viable bacteria in the sample as shown by fluorescence microscopy. After filtration of samples, no significant differences could be detected in subsequent qPCR experiments with and without EMA pretreatment compared to culture-based enumeration. High correlations (R(2)= 0.942 without EMA, R(2) = 0.893 with EMA) were obtained. After centrifugation of samples, qPCR results overestimated Campylobacter counts, whereas results from both EMA-qPCR and the reference method were comparable. As up to 81.59% of nonviable cells were detected in pond water, EMA-qPCR failed to detect correct quantities of viable cells. However, analyses of spiked tap water samples revealed a high correlation (R(2) = 0.863) between results from EMA-qPCR and the reference method. After membrane filtration, EMA-qPCR was successfully applied to Campylobacter field isolates, and results indicated an advantage over qPCR by analysing defined mixtures of viable and nonviable cells. In conclusion, EMA-qPCR is a suitable method to detect viable Campylobacter from water samples, but the isolation technique and the type/quality of the water sample impact the results.

Comparative analysis and limitations of ethidium monoazide and propidium monoazide treatments for the differentiation of viable and nonviable campylobacter cells.

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (CT) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R(2)) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 µg/ml) and PMA (51.10 µg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10(4)) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.