Log Most Probable Number Research Articles

Low-Level Clostridial Spores' Milk to Limit the Onset of Late Blowing Defect in Lysozyme-Free, Grana-Type Cheese.

The growth of clostridial spores during ripening leads to late blowing (LB), which is the main cause of spoilage in Grana Padano Protected Designation of Origin (PDO) cheese and other hard, long-ripened cheeses such as Provolone, Comté, and similar cheeses. This study aimed to verify the cause-effect relationship between the level of clostridial butyric spores (BCS) in milk and the onset of the LB defect. To this end, experimental Grana-type cheeses were produced without lysozyme, using bulk milk with different average BCS content. The vat milk from the so-called "virtuous" farms (L1) contained average levels of BCS of 1.93 ± 0.61 log most probable number (MPN) L-1, while the vat milk from farms with the highest load of spores (L2), were in the order of 2.99 ± 0.69 log MPN L-1. Cheeses after seven months of ripening evidenced a strong connection between BCS level in vat milk and the occurrence of LB defect. In L2 cheeses, which showed an average BCS content of 3.53 ± 1.44 log MPN g-1 (range 1.36-5.04 log MPN g-1), significantly higher than that found in L1 cheeses (p < 0.01), the defect of LB was always present, with Clostridium tyrobutyricum as the only clostridial species identified by species-specific PCR from MPN-positive samples. The L1 cheeses produced in the cold season (C-L1) were free of defects whereas those produced in the warm season (W-L1) showed textural defects, such as slits and cracks, rather than irregular eyes. A further analysis of the data, considering the subset of the cheesemaking trials (W-L1 and W-L2), carried out in the warm season, confirmed the presence of a climate effect that, often in addition to the BCS load in the respective bulk milks (L1 vs. L2), may contribute to explain the significant differences in the chemical composition and some technological parameters between the two series of cheeses. Metagenomic analysis showed that it is not the overall structure of the microbial community that differentiates L1 from L2 cheeses but rather the relative distribution of the species between them. The results of our trials on experimental cheeses suggest that a low-level BCS in vat milk (<200 L-1) could prevent, or limit, the onset of LB in Grana-type and similar cheeses produced without lysozyme.

Microbiological Profile, Prevalence, and Characterization of Salmonella enterica in Peanuts, Pecans, Raisins, Sun-Dried Tomatoes, and Chocolate Sprinkles Sold in Bulk in Markets in Querétaro, Mexico

In Mexico, the prevalence of Salmonella enterica in low-water-activity foods and its link to outbreaks are unknown. The aim of this study was to determine the microbiological profile and the prevalence of S. enterica in several low-water-activity foods, including peanuts, pecans, raisins, sun-dried tomatoes, and chocolate sprinkles, purchased in retail establishments in Querétaro, Mexico. Seventy samples of each food item sold in bulk were purchased. Aerobic plate count, molds, yeasts, total coliforms, Escherichia coli, and Staphylococcus aureus were quantified in 10-g samples. The prevalence of S. enterica in 25-g samples was determined. From positive samples, S. enterica isolates (60) were characterized based on their antimicrobial susceptibility to 14 antibiotics, the presence-absence of 13 virulence genes, and serotype. The concentration of aerobic plate count, molds, yeasts, total coliforms, and E. coli ranged from 3.1 to 5.2 log CFU g-1, from 2.0 to 2.4 log CFU g-1, from 2.0 to 3.0 log CFU g-1, from 0.6 to 1.1 log most probable number (MPN) g-1, and from 0.5 to 0.9 log MPN g-1, respectively. S. aureus was not detected in any sample (<10 CFU g-1). The prevalence of S. enterica in chocolate sprinkles, raisins, peanuts, pecans, and sun-dried tomatoes was 26, 29, 31, 40, and 52%, respectively. Most isolates (68.3%) were resistant to at least one antibiotic. Chromosome-associated virulence genes were found in all isolates, and only one strain had sopE, and 98.3% of the isolates were grouped in the same virulotype. Among the isolates, the most frequent serotype was Tennessee (51 of 60). According to the characteristics evaluated, we grouped the isolates into 24 clusters. The elevated prevalence of S. enterica highlights the role of low-water-activity food items sold in bulk at markets as potential vehicles for pathogen transmission. Regardless of the low variability among S. enterica isolates, their characterization could be helpful to elucidate which strains are circulating in these foods for improving epidemiological surveillance.

Microbiological Profile, Incidence, and Behavior of Salmonella on Seeds Traded in Mexican Markets

Consumption of seeds has increased in recent years due to their high nutrient content. However, Salmonella outbreaks associated with the consumption of low-water-activity food items have also increased, although these food items do not support microbial growth. The main goal of this study was to quantify microbial indicators and to determine the prevalence and content of Salmonella in chia, amaranth, and sesame seeds obtained from Mexican retail outlets. In addition, the behavior of this pathogen on seeds was evaluated. One hundred samples of each product (chia, amaranth, and sesame seeds) were collected from Queretaro City markets. Aerobic plate count, coliforms, and Escherichia coli bacteria were quantified, and the presence and number of Salmonella pathogens were also determined. Chia, amaranth, and sesame seeds (1 kg each) were inoculated with a cocktail of five Salmonella strains (∼6 log CFU mL-1) and stored at ambient temperature, and then populations of Salmonella were quantified. The median aerobic plate count contents in chia, amaranth, and sesame seeds were 2.1, 2.4, and 3.8 log CFU g-1, respectively, and the content of coliforms on the seeds ranged from 0.48 to 0.56 log most probable number (MPN) per g. E. coli was present at low concentrations in the three types of seeds. Salmonella was detected in chia (31%), amaranth (15%), and sesame (12%) seeds, and the population ranged from 0.48 to 0.56 log MPN g-1. Salmonella levels decreased through 240 days of storage, showing inactivation rates of 0.017, 0.011, and 0.016 log CFU h-1 in chia, amaranth, and sesame seeds, respectively. The high prevalence of Salmonella in the seeds highlights potential risks for consumers, particularly given that seeds are generally consumed without treatments guaranteeing pathogen inactivation.

Reduction of Salmonella contamination on the surface of chicken skin using bacteriophage

BackgroundEnteric infections caused by Salmonella spp. remain a major public health burden worldwide. Chickens are known to be a major reservoir for this zoonotic pathogen. The presence of Salmonella in poultry farms and abattoirs is associated with financial costs of treatment and a serious risk to human health. The use of bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization of chickens could be reduced. In a prior study, phages Eϕ151 and Tϕ7 significantly reduced broiler chicken caecal colonization by S. Enteritidis and S. Typhimurium respectively.MethodsSalmonella-free Ross broiler chickens were orally infected with S. Enteritidis P125109 or S. Typhimurium 4/74. After 7 days of infection, the animals were euthanased, and 25cm2 sections of skin were collected. The skin samples were sprayed with a phage suspension of either Eϕ151 (S. Enteritidis), Tϕ7 phage suspension (S. Typhimurium) or SM buffer (Control). After incubation, the number of surviving Salmonellas was determined by direct plating and Most Probable Number (MPN). To determine the rate of reduction of Salmonella numbers on the skin surface, a bioluminescent S. Typhimurium DT104 strain was cultured, spread on sections of chicken breast skin, and after spraying with a Tϕ11 phage suspension, skin samples were monitored using photon counting for up to 24 h.ResultsThe median levels of Salmonella reduction following phage treatment were 1.38 log10 MPN (Enteritidis) and 1.83 log10 MPN (Typhimurium) per skin section. Treatment reductions were significant when compared with Salmonella recovery from control skin sections treated with buffer (p < 0.0001). Additionally, significant reduction in light intensity was observed within 1 min of phage Tϕ11 spraying onto the skin contaminated with a bioluminescent Salmonella recombinant strain, compared with buffer-treated controls (p < 0.01), implying that some lysis of Salmonella was occurring on the skin surface.ConclusionsThe results of this study suggest that phages may be used on the surface of chicken skin as biocontrol agents against Salmonella infected broiler chicken carcasses. The rate of bioluminescence reduction shown by the recombinant Salmonella strain used supported the hypothesis that at least some of the reduction observed was due to lysis occurred on the skin surface.

Assessment of the microbiological quality and safety of minimally processed vegetables sold in Piracicaba, SP, Brazil

The present study shows the results of the microbiological quality and safety of minimally processed vegetables sold in supermarkets and grocery stores located in the city of Piracicaba, SP, Brazil. A total of 100 samples were collected and submitted to enumeration of total coliforms, coliforms at 45°C and generic Escherichia coli using the standard most probable number (MPN) method, in addition to enumeration of total Enterobacteriaceae by plating on MacConkey agar. Moreover, colonies of Enterobacteriaceae were randomly selected and submitted to identification on a MALDI-TOF MS Biotyper™. Samples were also tested for Salmonella spp. according to the ISO 6579:2002 method. The mean count of total coliforms was 2·9±0·5 log MPN per g. For coliforms at 45°C, 20 samples were positive (mean 1·5±1·0 log MPN per g). Generic E. coli was detected in 16 samples (mean 1·4±0·9 log MPN per g) and only one was positive for Salmonella. The mean count of total Enterobacteriaceae was 6·5±1·2 log CFU per g and the most frequent genera identified by MALDI-TOF were Enterobacter (25·9%), Pantoea (9·6%) and Rahnella (9·0%). Overall, results point to poor microbiological quality of a few samples, indicating hygiene failure during their processing. This can pose health risks to consumers, mainly because these products were labelled as sanitized and marketed as ready-to-eat. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the microbiological quality and safety of minimally processed vegetables sold in the city of Piracicaba, SP, Brazil, were evaluated through the detection of Salmonella spp., generic Escherichia coli and other Enterobacteriaceae. The results obtained point to poor microbiological quality and safety of a few samples, as generic E. coli was detected in 16 out of 100 samples and Salmonella was isolated from one of them. This indicates hygiene failures during their processing and health risk to consumers, since these products are usually sold as ready-to-eat and typically require no further heat treatment before consumption.

Persistence of Escherichia coli O157:H7 and Listeria monocytogenes on the exterior of three common food packaging materials

An estimated 179 million cases of acute gastroenteritis (AGE) occur each year in the United States. Escherichia coli O157:H7 and Listeria monocytogenes, common etiologic agents of AGE, both have low-infectious doses and high environmental resistance. Surfaces contaminated with either pathogen, such as the exterior surface of a food package, could serve as a vehicle for AGE transmission. The aim of this study was to determine the persistence of E. coli O157:H7 and L. monocytogenes on three common food packaging materials–oriented polyethylene terephthalate (OPET), oriented polypropylene (OPP), and nylon-6. Coupons (5 × 5 cm2) from each material were sterilized under ultraviolet light for 5 min, inoculated with ca. 7 log CFU of a three-strain-mixture of green fluorescent protein (GFP)-labeled E. coli O157:H7, then incubated according to the Technical Association of the Pulp and Paper Industry standards. Surviving E. coli O157:H7 cells were recovered in saline at 0, 0.25, 0.5, 1, 2, 3, 5, 7, 14, 15 days then enumerated on tryptic soy broth (TSB) supplemented with ampicillin using the three-tube most probable number (MPN) method. The same procedure using a three-strain mixture of rifampin-resistant (Rif)-L. monocytogenes was followed except TSB was supplemented with rifampin. E. coli O157:H7 and L. monocytogenes both survived on OPET, OPP, and nylon-6 for 15 days. The log reductions for E. coli O157:H7 were 5.7 log MPN, 5.3 log MPN, and 5.1 log MPN across the three materials respectively. The log reduction for L. monocytogenes was 6.5 log MPN, 6.3 log MPN, and 4.8 log across the same three materials. The number of surviving E. coli cells was significantly higher (P > 0.05) than L. monocytogenes at sampling times 1, 2, 3, 5, 7, 14, and 15 days. No significant difference was shown between the two bacteria between 0 and 0.5 days. Survival of both bacteria across all three materials was not significantly different (P > 0.05). These results suggest that a highly contaminated (107 CFU/coupon) exterior surface of a food package could be a potential vehicle for the transmission of bacteria associated with AGE.

Application of Bacteriocin-Like Inhibitory Substances (BLIS)-Producing Probiotic Strain of Lactobacillus plantarum in Control of Staphylococcus aureus in White-Brined Cheese Production

The aim of this study was to investigate the antimicrobial activity of an autochthonous probiotic strain of bacteriocin-like inhibitory substances (BLIS)-producing Lactobacillus plantarum, previously isolated from a Tulum cheese and satisfied technological criteria as adjunct culture in cheese production, in reducing Staphylococcus aureus during production and ripening of white-brined cheeses. Cheeses were manufactured in two trials from pasteurized milk artificially contaminated with S. aureus to the mean level of 6.243 log MPN mL-1. Lb. plantarum BG33 was added at 1% as adjunct to the starter culture. The study was also carried out with control group cheeses produced without the adjunct culture. S. aureus counts were monitored for up to 90 days by BAM’s 5-tube MPN method and each positive tube of MPN (most probable number) method was confirmed by PCR amplification of a 400 bp fragment of the nuc gene, which encodes the thermostable nuclease of S. aureus. The capacity of Lb. plantarum BG33 to reduce S. aureus count was found as 0.9 log unit on the 18th day of ripening. After 39 and 59 days of ripening, Lb. plantarum BG33 lowered S. aureus count by 1.9 and 2.0 log units, respectively, when compared to control group cheeses in which it was lowered by 0.5 and 1.0 log units, respectively. As a result, the BLIS activity of Lb. plantarum BG33 throughout ripening of white-brined cheese could make it useful as bioprotective adjunct culture in white-brined cheese production to prevent S. aureus growth which is an important foodborne pathogen in respect of safe cheese production. inhibitory substances (BLIS)-producing Lactobacillus plantarum, previously isolated from a Tulum cheese and satisfied technological criteria as adjunct culture in cheese production, in reducing Staphylococcus aureus during production and ripening of white-brined cheeses. Cheeses were manufactured in two trials from pasteurized milk artificially contaminated with S. aureus to the mean level of 6.243 log MPN mL-1. Lb. plantarum BG33 was added at 1% as adjunct to the starter culture. The study was also carried out with control group cheeses produced without the adjunct culture. S. aureus counts were monitored for up to 90 days by BAM’s 5-tube MPN method and each positive tube of MPN (most probable number) method was confirmed by PCR amplification of a 400 bp fragment of the nuc gene, which encodes the thermostable nuclease of S. aureus. The capacity of Lb. plantarum BG33 to reduce S. aureus count was found as 0.9 log unit on the 18th day of ripening. After 39 and 59 days of ripening, Lb. plantarum BG33 lowered S. aureus count by 1.9 and 2.0 log units, respectively, when compared to control group cheeses in which it was lowered by 0.5 and 1.0 log units, respectively. As a result, the BLIS activity of Lb. plantarum BG33 throughout ripening of white-brined cheese could make it useful as bioprotective adjunct culture in white-brined cheese production to prevent S. aureus growth which is an important foodborne pathogen in respect of safe cheese production.

Application of Bacteriocin-Like Inhibitory Substances (BLIS)- Producing Probiotic Strain of Lactobacillus plantarum in Control of Staphylococcus aureus in White-Brined Cheese Production

The aim of this study was to investigate the antimicrobial activity of an autochthonous probiotic strain of bacteriocin-like inhibitory substances (BLIS)-producing Lactobacillus plantarum, previously isolated from a Tulum cheese and satisfied technological criteria as adjunct culture in cheese production, in reducing Staphylococcus aureus during production and ripening of white-brined cheeses. Cheeses were manufactured in two trials from pasteurized milk artificially contaminated with S. aureus to the mean level of 6.243 log MPN mL-1. Lb. plantarum BG33 was added at 1% as adjunct to the starter culture. The study was also carried out with control group cheeses produced without the adjunct culture. S. aureus counts were monitored for up to 90 days by BAM’s 5-tube MPN method and each positive tube of MPN (most probable number) method was confirmed by PCR amplification of a 400 bp fragment of the nuc gene, which encodes the thermostable nuclease of S. aureus. The capacity of Lb. plantarum BG33 to reduce S. aureus count was found as 0.9 log unit on the 18th day of ripening. After 39 and 59 days of ripening, Lb. plantarum BG33 lowered S. aureus count by 1.9 and 2.0 log units, respectively, when compared to control group cheeses in which it was lowered by 0.5 and 1.0 log units, respectively. As a result, the BLIS activity of Lb. plantarum BG33 throughout ripening of white-brined cheese could make it useful as bioprotective adjunct culture in white-brined cheese production to prevent S. aureus growth which is an important foodborne pathogen in respect of safe cheese production.

Microbiological contamination of young children's hands in rural Bangladesh: Associations with child age and observed hand cleanliness as proxy.

BackgroundHands are a route of transmission for fecal-oral pathogens. This analysis aimed to assess associations between hand E. coli contamination and child age and determine if observed hand cleanliness can serve as a proxy for E. coli contamination on young children’s hands.MethodsTrained field workers collected hand rinse samples from children aged 1–14 months in 584 households in rural Bangladesh and assessed the visual cleanliness of child hands (fingernails, finger pads and palms). Samples were analyzed using the IDEXX most probable number (MPN) methodto enumerate E. coli. We assessed if child age (immobile children aged 1–4 months vs. mobile children aged 5–14 months) is associated with log10 E. coli counts on hands using generalized estimating equations (GEE). We estimated the log10 difference in hand E. coli counts associated with the cleanliness of different hand parts using a multivariable GEE model.We calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for dirty fingernails, fingerpads, palms and overall hands (the three observed parts combined) against binary E. coli presence on hands.ResultsE. coli was detected on 43% of child hands. Children in the mobile age range had 0.17 log10 MPN higher E. coli on hands than those in the immobile age range (Δlog10 = 0.17, 95% CI = 0.02, 0.32, p = 0.03). Children with visible dirt particles on finger pads had 0.46 log10 MPN higher E. coli on hands than those with clean finger pads (Δlog10 = 0.46, 95% CI = 0.05, 0.87, p = 0.03). Dirty fingernails indicated binary E. coli presence with 81% sensitivity and 26% specificity while dirty fingerpads and palms indicated E. coli presence with 29% sensitivity and 75–77% specificity. The PPV was 45–48% and NPV 59–65% for all three types of observations.ConclusionHand contamination with E. coli was prevalent among young children in rural Bangladesh, with higher levels of contamination among mobile children. Studies should assess if strategies to remove animal feces from the courtyard, provide designated hygienic play spaces for children and deliver targeted messaging to mothers to wipe or wash children’s hands after contact with animals and animal feces reduce child hand contamination. Visible hand cleanliness was a poor predictor of E. coli presence on young children’s hands so other low-cost field measurements are needed to accurately detect fecal contamination on hands.

Prevalence and concentration of stx+ E. coli and E. coli O157 in bovine manure from Florida farms.

Fresh produce outbreaks due to Shiga toxin-producing Escherichia coli (STEC) continue to occur in the United States (US). Manure-amended soils can pose a public health risk when used for growing raw agricultural commodities. Knowing the prevalence and concentration of STEC in untreated biological soil amendments of animal origin (BSAAO) is important to help guide the most appropriate pre-harvest interval(s) following application to limit risks from these soil amendments. Bovine manure samples were collected from 12 farms in Florida, including samples from piles, lagoons, barns, and screened solids. Two methods were used to detect stx1/2 and rfbE genes in samples. A prevalence rate of 9% for stx1 and/or stx2 and 19% for rfbE was observed from the 518 bovine manure samples evaluated. A most probable number (MPN) assay was performed on stx+ samples when applicable. The geometric mean for stx+ samples (n = 20) was 3.37 MPN g-1 (0.53 log MPN g-1) with a maximum value of 6,800 MPN g-1 (3.83 log MPN g-1). This research was part of a larger nationwide geographical study on the prevalence and concentration of STEC in bovine manure to help guide regulations on feasible pre-harvest intervals for the application of untreated BSAAO.

Effect of a Yeast Cell Wall Preparation on Cecal and Ovarian Colonization With Salmonella enteritidis in Commercial Layers

SUMMARY Salmonella enteritidis (S.E.) was the most common serotype in human infections in 2016. Various yeast products have been shown to inhibit colonization of chickens by Salmonella and could be a useful adjunct in an S.E. control strategy. This study sought to determine whether feeding a yeast cell wall product (Celmanax SCP) at 100 ppm to commercial layer pullets either from 1 d to 17 wk or from 10 to 17 wk would affect S.E. colonization of both the digestive tract and ovaries. At 16 wk of age, the pullets were orally challenged with 1.8 × 109 CFU S.E. and then ceca and ovaries were assayed at 1 wk post challenge for S.E. prevalence and load. The colonization level was very high in the ceca with many counts too numerous to count (>106 most probable number (MPN)/g, TNTC). A censored regression model was used to account for the large number of TNTC samples from each treatment group. Both of the Celmanax SCP-treated groups had an estimated marginal mean log10 MPN that was numerically lower than that of the untreated challenged positive control group by about 1.5 log10, and while this difference approached significance, it was not significant at the 5% level (P = 0.056). Although the proportion of isolates that exceeded the upper limit of the MPN assay was numerically lower for the group fed Celmanax SCP from day 1 (25.5%) than for the group fed Celmanax SCP from 10 wk (43.8%), or the untreated challenged positive control (47.9%), this difference again approached but did not achieve significance at the 5% level (P = 0.060). Also, there was no significant difference between challenged treatment groups with respect to ovary Salmonella prevalences (P = 0.322).

GENES DE VIRULENCIA E RESISTí­Å NCIA ANTIMICROBIANA DE Vibrio parahaemolyticus EM ÁREAS DE OSTREICULTURA

Este estudo teve como objetivo quantificar Vibrio spp. e avaliar o perfil de suscetibilidade antimicrobiana e fatores de virulência em cepas de Vibrio parahaemolyticus em amostras de ostras em dois estuários do Baixo Sul da Bahia. As coletas foram realizadas no perí­­odo de junho de 2015 a janeiro de 2016, com coletas de ostras provenientes de bancos naturais (E1) e coletas de água e ostras provenientes de cultivo (E2). Para a quantificação de Vibrio spp. usou-se o número mais provável (NMP) e a caracterização de V. parahaemolyticus o marcador (espécie-especí­­fica) gene tl. A patogenicidade foi observada com o teste de Kanagawa e presença dos genes tdh, trh e ure. Para o perfil de suscetibilidade antimicrobiana foram usados 15 antimicrobianos (método disco difusão), produção de enzimas β-lactamases e presença dos genes blaTEM, blaSHV e blaCTX-M. A densidade máxima de Vibrio spp. em ostras de cultivo foi de log 4,70 NMP g-1 e de extrativismo log 6,10 NMP g-1, sendo a temperatura a variável que mais influenciou na presença dos microrganismos na área de cultivo (E2). O gene tl foi encontrado em 71% dos isolados, sem a presença dos genes tdh, trh e ure. Todas as cepas de V. parahaemolyticus foram Kanagawa negativo. Elevada resistência antimicrobiana foi observada aos antimicrobianos betalactí­¢micos (cefalotina - 72% e ampicilina - 60%) e aminoglicosí­­deos (amicacina - 64%), com multirresistência em 88% das cepas de V. parahaemolyticus, e dessas, em 68% a resistência era mediada por plasmí­­deos. Fenotipicamente não foi observada produção de enzimas β-lactamases, embora tenha sido observado a presença de genes blaTEM. O caráter de multirresistência sobretudo por plasmí­­deos em cepas de V. parahaemolyticus aumenta a preocupação de bactérias autóctones do meio marinho, pois compromete o controle de infecção por esta bactéria em moluscos bivalves.

Temperature-humidity Index (THI) Is Associated with Gestation Length in Thoroughbred Mares

The objective of this study was to determine the relationship between Salmonella levels (presence and numbers) in chicken spleens and in mechanically separated chicken (MSC) at a commercial processing plant. Composite spleen and MSC samples were collected from 18 flocks at a commercial poultry processing plant. A total of 180 samples of each type were collected and tested for Salmonella using the most probable number (MPN) and enrichment methods. Overall, Salmonella was detected in 15.6% and 27.8% of spleen and MSC samples, respectively. The mean log MPN was 0.95 and 0.85 for the spleen and MSC samples, respectively. There was a significant relationship between Salmonella presence in spleen and MSC samples. However, the log MPN numbers in MSC samples were not significantly related to those in spleen samples. Salmonella presence in composite spleen samples (an indication of systemic infection in chickens) may predict MSC contamination with this pathogen, at the flock level.

Development and validation of a neural network model for predicting growth of Salmonella Newport on diced Roma tomatoes during simulated salad preparation and serving: extrapolation to other serotypes

SummaryA study was undertaken to model growth of Salmonella on tomatoes for developing and validating a predictive model for use in risk assessment. Cylindrical portions (0.14 g) of Roma tomato pulp were inoculated with a low dose (0.89 log MPN) of Salmonella Newport. The inoculated tomato portions were incubated for 0–8 h at 16–40 °C in 2 °C increments to obtain most probable number (MPN) data for model development and validation. A multiple‐layer feedforward neural network model with two hidden layers of two nodes each was developed. The proportion of residuals in an acceptable prediction zone (pAPZ) from −1 (fail‐safe) to 0.5 log (fail‐dangerous) was 0.93 (194/209) for dependent data and 0.96 (86/90) for independent data for interpolation. A pAPZ ≥0.7 indicated that the model provided acceptable predictions. Thus, the model was successfully validated. It was also validated for extrapolation to seven other Salmonella serotypes.

Prevalence and Association of Escherichia coli and Diarrheagenic Escherichia coli in Stored Foods for Young Children and Flies Caught in the Same Households in Rural Bangladesh

.Consumption of contaminated stored food can cause childhood diarrhea. Flies carry enteropathogens, although their contribution to food contamination remains unclear. We investigated the role of flies in contaminating stored food by collecting food and flies from the same households in rural Bangladesh. We selected 182 households with children ≤ 24 months old that had stored foods for later feeding at room temperature for ≥ 3 hours. We collected food samples and captured flies with fly tapes hung by the kitchen. We used the IDEXX Quanti-Tray System (Colilert-18 media; IDEXX Laboratories, Inc., Westbrook, ME) to enumerate Escherichia coli with the most probable number (MPN) method. Escherichia coli–positive IDEXX wells were analyzed by polymerase chain reaction for pathogenic E. coli genes (eae, ial, bfp, ipaH, st, lt, aat, aaiC, stx1, and stx2). Escherichia coli was detected in 61% (111/182) of food samples, with a mean of 1.1 log10 MPN/dry g. Fifteen samples (8%) contained pathogenic E. coli; seven (4%) had enteropathogenic E. coli (EPEC) genes (eae and/or bfp); and 10 (5%) had enteroaggregative E. coli genes (aat and/or aaiC). Of flies captured in 68 (37%) households, E. coli was detected in 41 (60%, mean 2.9 log10 MPN/fly), and one fly (1%) had an EPEC gene (eae). For paired fly-food samples, each log10 MPN E. coli increase in flies was associated with a 0.31 log10 MPN E. coli increase in stored food (95% confidence interval: 0.07, 0.55). In rural Bangladesh, flies possibly a likely route for fecal contamination of stored food. Controlling fly populations may reduce contamination of food stored for young children.

Impact of storm runoff on Salmonella and Escherichia coli prevalence in irrigation ponds of fresh produce farms in southern Georgia.

To examine Salmonella and Escherichia coli in storm runoff and irrigation ponds used by fresh produce growers, and compare Salmonella serovars with those found in cases of human salmonellosis. We collected water before and after rain events at two irrigation ponds on farms in southern Georgia, USA, and collected storm runoff/storm flow within the contributing watershed of each pond. Salmonella and E.coli concentrations were higher in ponds after rain events by an average of 0·46 (P<0·01) and 0·61 (P<0·05) log10 most probable number (MPN) per 100ml respectively. Salmonella concentrations in storm runoff from fields and forests were not significantly higher than in ponds before rain events, but concentrations in storm flow from streams and ditches were higher by an average of 1·22 log10 MPN per 100ml (P<0·001). Eighteen Salmonella serovars were identified from 155 serotyped isolates, and eight serovars were shared between storm runoff/storm flow and ponds. Seven of the serovars, including five of the shared serovars, were present in cases of human illness in the study region in the same year. However, several serovars most commonly associated with human illness in the study region (e.g. Javiana, Enteritidis, and Montevideo) were not found in any water samples. Salmonella and E.coli concentrations in irrigation ponds were higher, on average, after rain events, but concentrations of Salmonella were low, and the ponds met FDA water quality standards based on E.coli. Some similarities and notable differences were found between Salmonella serovars in water samples and in cases of human illness. This study directly examined storm runoff/storm flow into irrigation ponds and quantified increases in Salmonella and E.coli following rain events, with potential implications for irrigation pond management as well as human health.

Assessment of pit latrines in a peri-urban community in KwaZulu-Natal (South Africa) as a source of antibiotic resistant E. coli strains.

Due to the frequent use of antibiotics and recurring illnesses related to multidrug-resistant (MDR) bacteria in South Africa, we determined if MDR Escherichia coli were present in pit latrine fecal sludge samples obtained from a peri-urban community in KwaZulu-Natal, South Africa. The abundance of E. coli in pit latrine samples was established using a most probable number (MPN) method with species confirmation done using biochemical tests and polymerase chain reaction (PCR). Forty-four randomly selected E. coli pit latrine isolates were further characterized, using the European committee on antimicrobial susceptibility testing (EUCAST) disk diffusion method to establish antibiotic resistance profiles for these E. coli isolates. The resulting MPN values for E. coli ranged from one to 6.2 log10 MPN per gram of fresh pit latrine fecal sludge. While only 3 out of 44 E. coli pit latrine isolates showed no resistance to any of the 12 tested antibiotics, most isolates were resistant to two or more antibiotics. The majority of isolates showed resistance to at least one of the two tested aminoglycosides, one isolate showed resistance to the carbapenem ertapenem, and although resistance was not detected for tigecycline four pit latrine E. coli isolates showed intermediate resistance to this antibiotic. However, about 14% of the E. coli pit latrine isolates were categorized as MDR, all of which showed resistance to four or more antibiotics. The presence of MDR E. coli strains in pit latrine samples demonstrates that these facilities are potential sources for MDR bacteria.

Survival of Escherichia coli on Lettuce under Field Conditions Encountered in the Northeastern United States

Although wildlife intrusion and untreated manure have been associated with microbial contamination of produce, relatively few studies have examined the survival of Escherichia coli on produce under field conditions following contamination (e.g., via splash from wildlife feces). This experimental study was performed to estimate the die-off rate of E. coli on preharvest lettuce following contamination with a fecal slurry. During August 2015, field-grown lettuce was inoculated via pipette with a fecal slurry that was spiked with a three-strain cocktail of rifampin-resistant nonpathogenic E. coli. Ten lettuce heads were harvested at each of 13 time points following inoculation (0, 2.5, 5, and 24 h after inoculation and every 24 h thereafter until day 10). The most probable number (MPN) of E. coli on each lettuce head was determined, and die-off rates were estimated. The relationship between sample time and the log MPN of E. coli per head was modeled using a segmented linear model. This model had a breakpoint at 106 h (95% confidence interval = 69, 142 h) after inoculation, with a daily decrease of 0.70 and 0.19 log MPN for 0 to 106 h and 106 to 240 h following inoculation, respectively. These findings are consistent with die-off rates obtained in similar studies that assessed E. coli survival on produce following irrigation. Overall, these findings provide die-off rates for E. coli on lettuce that can be used in future quantitative risk assessments.

Assessment of three indigenous South African herbivores as potential reservoirs and vectors of antibiotic-resistant Escherichia coli

Due to limited data available on the presence of antibiotic-resistant (ABR) bacteria in faeces of wild herbivores in South Africa, this study analysed resistance patterns for Escherichia coli isolates from wildebeest, zebra and giraffe in addition to pet and farm pig faeces. Total and faecal coliforms and E. coli were quantified in faecal matter using a most probable number (MPN) guideline procedure. Antibiotic resistance profiles against 12 selected antibiotics representing seven classes were determined for 30 randomly selected E. coli isolates from each animal using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion procedure. While log10 MPN values per gram of animal faeces for total/faecal coliforms ranged from 4.51/4.11 to 5.70/5.50, the E. coli MPN values were in a range of 3.43–5.14. The proportion of ABR E. coli isolates ranged from 43% (giraffe) to 93% (zebra). About 47% of E. coli isolates from zebra faeces were categorized as multidrug-resistant (MDR), while for wildebeest and giraffe, no MDR isolates were detected. In comparison, 10% of E. coli isolates from pet pig and about 7% from farm pig faeces were categorized as MDR. Although most MDR isolates were resistant to at least one β-lactam antibiotic, only one MDR isolate from farm pig faeces was resistant to both norfloxacin and ciprofloxacin, the two fluoroquinolones tested. However, no resistance was detected to the tested carbapenems and tigecycline. The results of this study indicate that indigenous South African herbivores may serve as potential reservoirs and vectors for the dissemination of ABR E. coli strains.

Effectiveness of the Thermal Treatments Used for Curd Stretching in the Inactivation of Shiga Toxin-Producing O157 and O26 Escherichia coli.

The kneading treatment of the fresh curd in hot water is a critical control point in the manufacturing of mozzarella. Factors such as the ratio between hot water and curd mass, the rheological properties, and the mixing and kneading activity affect the processing time and the internal temperature of the curd. The aim of this study was to investigate the effect of thermal treatments on the fate of Shiga toxin-producing Escherichia coli (STEC). Nine curd samples (weight 160–270 g) were artificially contaminated with O157 or O26 STEC and stretched in hot water (90–95°C) for 5–10 min. Depending on the heating process and spinning, different nonisothermal profiles were recorded. Observed reductions of O157 and O26 STEC varied between 1.01 and more than 5.38 log⁡MPN (Most Probable Number)/g at the end of the temperature treatments. Further, nonisothermal log-linear tail models were developed to compare observed reductions for O157 and O26 VTEC under variable temperature conditions. Results obtained showed that the comparison of predictions provided by the dynamic model with observations described well the linear inactivation pattern since nonsignificant differences were denoted at all profiles tested. The dynamic model developed can be useful to evaluate the effectiveness of the thermal treatments used in the manufacturing of mozzarella in the inactivation of STEC.