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Enterotoxigenic Clostridium perfringens is one of the main causes of foodborne illness in Canada. The use of a conventional bacterial culture approach to isolate enterotoxigenic C. perfringens from poultry meat is common. This approach is based on the phenotype attributable to a double hemolysis phenomenon, whereas few enterotoxigenic strains of C. perfringens produce it, which further complicates the study of the reservoirs of this important pathogen. The objectives of the current study were to validate the ability of a digoxigenin-labeled probe to detect the C. perfringens cpe gene and to validate the use of either a filtration or a direct plating approach, combined with colony hybridization to detect enterotoxigenic C. perfringens. Pure DNA and pure colonies of enterotoxigenic C. perfringens and broiler chicken carcass rinsate samples were subjected to colony hybridization. The results showed that the synthesized DNA probe can detect the cpe gene from both DNA and pure colonies of enterotoxigenic C. perfringens, and from colonies grown from carcass rinsates artificially contaminated with enterotoxigenic C. perfringens. Our study suggests that this isolation method is a promising tool for a better understanding of the epidemiology of this zoonotic pathogen.

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Hermetia illucens larvae (black soldier fly larvae, BSFL) convert efficiently organic waste to high quality biomass. To gain knowledge on the specific functions of gut microbes in this process it is a prerequisite to culture members of the core gut microbiota. Two different cultivation strategies were applied here for this purpose, a dilution-to-extinction cultivation and direct plating using six different media to culture aerobic heterotrophic bacteria. A total of 341 isolates were obtained by the dilution-to-extinction cultivation and 138 isolates by direct plating from guts of BSFL reared on chicken feed. Bacterial isolates were phylogenetically identified at the genus level by 16S rRNA gene sequencing (phylotyping) and differentiated at the strain level by genomic fingerprinting (genotyping). The main proportion of isolates was assigned to Proteobacteria, Firmicutes (Bacilli), and Actinobacteria. Predominant genera discussed in literature as member of a potential BSFL core gut microbiota, Providencia, Proteus, Morganella, Enterococcus, Bacillus, and members of the family Enterobacteriaceae, were isolated. A high intra-phylotype diversity was obtained by genomic fingerprinting which was especially enhanced by the dilution-to-extinction cultivation. This study showed that the application of different cultivation strategies including a dilution-to-extinction cultivation helps to culture a higher diversity of the BSFL gut microbiota and that genomic fingerprinting gives a better picture on the genetic diversity of cultured bacteria which cannot be covered by a 16S rRNA gene sequence based identification alone.

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Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May to October 2016 and 2017. Abundances and detection frequencies for total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus were compared using the standard most-probable-number-PCR (MPN-PCR) assay and a direct-plating (DP) method on CHROMagar Vibrio for total (tlh+ ) and pathogenic (tdh+ and trh+ ) V. parahaemolyticus genes and total (vvhA) and pathogenic (vcgC) V. vulnificus genes. The colony overlay procedure for peptidases (COPP) assay was evaluated for total Vibrionaceae DP had high false-negative rates (14 to 77%) for most PCR targets and was deemed unsatisfactory. Logistic regression models of the COPP assay showed high concordances with MPN-PCR for tdh+ and trh+V. parahaemolyticus and vvhA+V. vulnificus in oysters (85.7 to 90.9%) and seawater (81.1 to 92.7%) when seawater temperature and salinity were factored into the model, suggesting that the COPP assay could potentially serve as a more rapid method to detect vibrios in oysters and seawater. Differences in total Vibrionaceae and pathogenic Vibrio abundances between state sampling sites over different collection years were contrasted for oysters and seawater by MPN-PCR. Abundances of tdh+ and trh+V. parahaemolyticus were ∼8-fold higher in Delaware oysters than in Maryland oysters, whereas abundances of vcgC+V. vulnificus were nearly identical. For Delaware oysters, 93.5% were both tdh+ and trh+, compared to only 19.2% in Maryland. These results indicate that pathogenic V. parahaemolyticus was more prevalent in the Delaware Bay than in the Chesapeake Bay.IMPORTANCE While V. parahaemolyticus and V. vulnificus cause shellfish-associated morbidity and mortality among shellfish consumers, current regulatory assays for vibrios are complex, time-consuming, labor-intensive, and relatively expensive. In this study, the rapid, simple, and inexpensive COPP assay was identified as a possible alternative to MPN-PCR for shellfish monitoring. This paper shows differences in total Vibrionaceae and pathogenic vibrios found in seawater and oysters from the commercially important Delaware and Chesapeake Bays. Vibrio parahaemolyticus isolates from the Delaware Bay were more likely to contain commonly recognized pathogenicity genes than those from the Chesapeake Bay.

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Characterization of the microbiota of chickens is of current interest. The goals of the current study were to apply anaerobic isolation methods to comprehensively isolate and identify bacteria from the gastrointestinal tract of chickens and their environment. Bacterial communities within the drinking water were dominated by Escherichia, whereas communities in litter were more representative of the cecum. The crop and small intestine (jejunum and ileum) were dominated by Lactobacillus and Enterococcus spp., and the cecum was dominated by Proteus spp. The collection of bacteria isolated was dominated by Enterococcus spp., Escherichia/Shigella spp., Lactobacillus spp., and Proteus spp.; however, many rare taxa were observed. These included members of the Clostridiales and Clostridium spp., which were commonly isolated from the ileum and cecum. Bacteria isolated by enrichment and direct plating differed. The selective de Man-Rogosa-Sharpe agar was commonly associated with the isolation of Lactobacillus spp. and yielded the lowest diversity of all methods utilized. Increased diversity and frequency of Clostridium spp. was observed in enrichments of blood and mucus or by plating on Columbia agar supplemented with 10% blood and gentamicin. The bacteria isolated from this study provide source material for genomic and functional studies in chicken hosts.

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AIMS: Three cultivation methods were used to study the prevalence and abundance of Vibrio cholerae in Eastern Austrian bathing waters and to elucidate the main factors controlling their distribution. METHODS AND RESULTS: Vibrio cholerae abundance was monitored at 36 inland bathing sites with membrane filtration (MF), a standard most probable number (MPN) approach and direct plating (DP). Membrane filtration yielded the most reliable and sensitive results and allowed V. cholerae detection at 22 sites with concentrations up to 39 000 CFU per 100 ml, all belonging to serogroups other than O1 and O139 and not coding for cholera toxin and toxin coregulated pilus. Direct plating turned out as an easy method for environments with high V. cholerae abundances, conductivity was the only significant predictor of V. cholerae abundance in the bathing waters at warm water temperatures. CONCLUSIONS: Vibrio cholerae nonO1/nonO139 are widely prevalent in Eastern Austrian bathing waters. Instead of the standard MPN approach, MF and DP are recommended for V. cholerae monitoring. Conductivity can be used as a first easy-to-measure parameter to identify potential bathing waters at risk. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio cholerae nonO1/nonO139 infections associated with bathing activities are an increasing public health issue in many countries of the northern hemisphere. However, there are only limited data available on the prevalence and abundance of V. cholerae in coastal and inland bathing waters. For monitoring V. cholerae prevalence and abundance, reliable and simple quantification methods are needed. Moreover, prediction of V. cholerae abundance from environmental parameters would be a helpful tool for risk assessment. This study identified the best culture-based quantification methods and a first quick surrogate parameter to attain these aims.

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Screening for multidrug-resistant Enterobacteriaceae is performed in many institutions as part of infection control measures. However, the sensitivity of current standard diagnostics is modest. Furthermore, patients are usually screened by rectal swabs (mostly rayon based), which have been shown to be sub-optimal for the recovery of Enterobacteriaceae. Therefore, it is likely that many patients colonised with multidrug-resistant Enterobacteriaceae remain undetected. The present study aimed to analyse if the detection of multidrug-resistant Enterobacteriaceae can be improved when screening with rayon swabs is done in combination with an additional pre-enrichment step. The detection of third-generation cephalosporin-resistant Enterobacteriaceae (3GCREB) was assessed in 514 rectal samples by the standard diagnostic approach (direct plating of swabs on selective ESBL agar) and after pre-enrichment in 5 mL of a semi-selective MacConkey broth. The recovery rate of 3GCREB and extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E), patient characteristics and isolate characteristics were evaluated for both diagnostic approaches. Overall, by pre-enrichment, the detection of 3GCREB carriers increased by 22.8% (13/57, p = 0.004) and the detection of ESBL-E carriers by 21.4% (9/42, p = 0.01). This study demonstrates the low sensitivity of rectal screening by direct plating and the improvement by pre-enrichment. We believe that it is no longer acceptable to refrain from pre-enrichment as, with the standard approach, more than 20% of 3GCREB and ESBL-E carriers remain undetected.

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A precise and accurate method for enumeration of low level of Listeria monocytogenes in foods is critical to a variety of studies. In this study, paired comparison of most probable number (MPN) and direct plating enumeration of L. monocytogenes was conducted on a total of 1730 outbreak-associated ice cream samples that were naturally contaminated with low level of L. monocytogenes. MPN was performed on all 1730 samples. Direct plating was performed on all samples using the RAPID'L.mono (RLM) agar (1600 samples) and agar Listeria Ottaviani and Agosti (ALOA; 130 samples). Probabilistic analysis with Bayesian inference model was used to compare paired direct plating and MPN estimates of L. monocytogenes in ice cream samples because assumptions implicit in ordinary least squares (OLS) linear regression analyses were not met for such a comparison. The probabilistic analysis revealed good agreement between the MPN and direct plating estimates, and this agreement showed that the MPN schemes and direct plating schemes using ALOA or RLM evaluated in the present study were suitable for enumerating low levels of L. monocytogenes in these ice cream samples. The statistical analysis further revealed that OLS linear regression analyses of direct plating and MPN data did introduce bias that incorrectly characterized systematic differences between estimates from the two methods.

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AIMS: To perform a comparative study for determining the optimum culture method (direct plating or enrichment) and medium (ampicillin dextrin agar (ADA), starch ampicillin agar (SAA), bile salts irgasan brilliant green modified (BIBG-m)) for recovering Aeromonas species from water and shellfish samples. METHODS AND RESULTS: By direct culture, Aeromonas was detected in 65% (13/20) of the water samples and in 54·5% (6/11) of the shellfish samples. However, when a pre-enrichment step was included, the number of positive water samples increased to 75% (15/20) and the ones of shellfish to 90·1% (10/11). The enriched culture significantly favoured (P < 0·05) the isolation of Aeromonas allosaccharophila from water, Aeromonas salmonicida from shellfish and Aeromonas caviae from both types of samples. The most specific (P < 0·05) culture medium for detecting Aeromonas from water was ADA. However, no differences were observed in the case of shellfish samples (P > 0·05). Isolation of Aeromonas media from water was favoured (P < 0·05) in the ADA medium, while SAA enhanced (P < 0·05) the isolation of Aer. salmonicida from shellfish. CONCLUSIONS: The culture method and medium used influenced the recovery of some Aeromonas species from water and shellfish samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This fact should be considered in future prevalence studies to avoid overestimating the above mentioned Aeromonas species.

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In Southeast Asian countries, including Korea, China, and Japan, the considerable amounts of raw fish have been annually consumed in the manner of live fish fillets without minimally thermal processing, increasing the risks of causing food-borne diseases. This study investigated the occurrence of total aerobic bacteria (TAB), coliform, Vibrio parahaemolyticus, Salmonella enterica serovar spp., Listeria monocytogenes, and Staphylococcus aureus in jacopevers and plaices. Total 200 live fishes were collected from randomly selected restaurants located in Anseong-si, and then they were microbiologically monitored. TAB ranged from 3.09 to 3.21 Log10 CFU/g in jacopever and plaice. Coliform in the levels of 1.54 Log10 CFU/g were detected in samples. Out of 100 jacopevers, a single jacopever (1%) exhibited the prevalence of S. aureus in the edible part, though none of pathogenic bacteria were detected. These results will be useful for understanding the microbial prevalence in the domestic living jacopevers and plaices.

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O monitoramento da contaminação fúngica do arroz é imprescindível para assegurar a qualidade e segurança desse cereal. Atualmente, para avaliar a qualidade microbiológica dos alimentos, diferentes métodos têm sido propostos. Duas diferentes metodologias, plaqueamento direto e blotter test, foram comparadas quanto à eficiência em efetuar a detecção de fungos em arroz branco polido irradiado. Para realizar o blotter test, foram utilizadas placas de Petri contendo três folhas de papéis de filtro esterilizados, umedecidas em água destilada esterilizada e acrescidas com 5 mL de ágar água 0,5%. Na técnica de plaqueamento direto, os grãos foram plaqueados em meio de cultura DRBC. As amostras foram incubadas a 25ºC por sete dias e analisadas em microscópio estereoscópico. Os gêneros fúngicos presentes no arroz irradiado foram Penicillium, Aspergillus, Cladosporium, Fusarium e Trichoderma, com a predominância de Penicillium sp. e Aspergillus sp., cujas frequências foram, respectivamente, de 5,2% e 5,6% no plaqueamento direto e de 34,5% e 5,6% no blotter test. Observou-se que a irradiação gama diminuiu consideravelmente o número de grãos contaminados, sendo 96,7% pela metodologia de plaqueamento direto e até 100% pelo blotter test. O blotter test possibilitou efetuar maior contagem dos gêneros fúngicos presentes no arroz, constando-se que o método de detecção escolhido pode interferir na quantificação fúngica presente nesse cereal.

Monitoring the fungal contamination of rice is essential for ensuring the product quality and safety. Different methods have been proposed for assessing the microbiological quality of foods. Two methodologies based direct plating and blotter test were compared for detecting fungi in irradiated polished white rice. For performing the blotter test, the Petri dishes containing three sterile filter paper sheets were moistened with sterile distilled water, and 5 mL of 0.5% water-agar were added onto them. For carrying out the direct plating technique, the rice samples were plated onto DRBC medium. The samples were incubated at 25ºC for seven days, and analyzed under a stereomicroscope. The fungi genera found in irradiated rice were:Penicillium, Aspergillus, Cladosporium, Fusarium and Trichoderma, being Penicillium sp. and Aspergillus sp. the mostly predominant, with the prevalence of 5.2% and 5.6% by direct plating and 34.5% and 5.6% by blotter test, respectively. The gamma-ray irradiation significantly decreased the proportions of contaminated grains, being 96.7% by means of direct plating technique and 100% by blotter test. The blotter test showed highest efficiency in fungal genera counting found in rice samples, which evidences that the chosen detection methodology may interfere on quantifying the fungi contaminants in cereals.

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A direct plating procedure was developed for the enumeration of Listeria spp. and Listeria monocytogenes in foods. Both naturally contaminated foods and foods spiked with L. innocua , L. seeligeri , and L monocytogenes were studied. The enhanced hemolysis agar (EHA) developed by Cox and modified in our study resulted in two types of agar, referred to as listeria enumeration agar (LEA) no. 1 and 2, used for products of lighter and heavier background microbial populations, respectively. On LEA plates, total Listeria spp. counts were determined by fluorescence caused by the breakdown of 4-methylumbelliferyl-ß-d-glucoside contained in EHA. L. monocytogenes counts were determined by picking a representative number of hemolytic colonies and stabbing them into a xylose agar plate to distinguish L. monocytogenes from L seeligeri . Contamination levels of >200 Listeria cells per g of food can be accurately quantified by this procedure with >80% recovery. Counts of <200 Listeria cells per g of food were considered estimates. When the level of contamination was < 100 Listeria cells per g of food, the recovery was <58%. Occasionally, with low-level inocula, Listeria was not detected. Nevertheless, when the procedure was combined with incubation of the enrichment mixture (used for the 1:10 direct plating dilution) and subsequent streaking, Listeria contamination could still be detected and the level, therefore, was determined to be between 1 and 150/g.