Landscape and Meteorological Factors Affecting Prevalence of Three Food-Borne Pathogens in Fruit and Vegetable Farms

Produce-related outbreaks have been traced back to the preharvest environment. A longitudinal study was conducted on five farms in New York State to characterize the prevalence, persistence, and diversity of food-borne pathogens in fresh produce fields and to determine landscape and meteorological factors that predict their presence. Produce fields were sampled four times per year for 2 years. A total of 588 samples were analyzed for Listeria monocytogenes, Salmonella, and Shiga toxin-producing Escherichia coli (STEC). The prevalence measures of L. monocytogenes, Salmonella, and STEC were 15.0, 4.6, and 2.7%, respectively. L. monocytogenes and Salmonella were detected more frequently in water samples, while STEC was detected with equal frequency across all sample types (soil, water, feces, and drag swabs). L. monocytogenes sigB gene allelic types 57, 58, and 61 and Salmonella enterica serovar Cerro were repeatedly isolated from water samples. Soil available water storage (AWS), temperature, and proximity to three land cover classes (water, roads and urban development, and pasture/hay grass) influenced the likelihood of detecting L. monocytogenes. Drainage class, AWS, and precipitation were identified as important factors in Salmonella detection. This information was used in a geographic information system framework to hypothesize locations of environmental reservoirs where the prevalence of food-borne pathogens may be elevated. The map indicated that not all croplands are equally likely to contain environmental reservoirs of L. monocytogenes. These findings advance recommendations to minimize the risk of preharvest contamination by enhancing models of the environmental constraints on the survival and persistence of food-borne pathogens in fields.     

Spatial and Temporal Factors Associated with an Increased Prevalence of Listeria monocytogenes in Spinach Fields in New York State

While rain and irrigation events have been associated with an increased prevalence of foodborne pathogens in produce production environments, quantitative data are needed to determine the effects of various spatial and temporal factors on the risk of produce contamination following these events. This study was performed to quantify these effects and to determine the impact of rain and irrigation events on the detection frequency and diversity of Listeria species (including L. monocytogenes) and L. monocytogenes in produce fields. Two spinach fields, with high and low predicted risks of L. monocytogenes isolation, were sampled 24, 48, 72, and 144 to 192 h following irrigation and rain events. Predicted risk was a function of the field''s proximity to water and roads. Factors were evaluated for their association with Listeria species and L. monocytogenes isolation by using generalized linear mixed models (GLMMs). In total, 1,492 (1,092 soil, 334 leaf, 14 fecal, and 52 water) samples were collected. According to the GLMM, the likelihood of Listeria species and L. monocytogenes isolation from soil samples was highest during the 24 h immediately following an event (odds ratios [ORs] of 7.7 and 25, respectively). Additionally, Listeria species and L. monocytogenes isolates associated with irrigation events showed significantly lower sigB allele type diversity than did isolates associated with precipitation events (P = <0.001), suggesting that irrigation water may be a point source of L. monocytogenes contamination. Small changes in management practices (e.g., not irrigating fields before harvest) may therefore reduce the risk of L. monocytogenes contamination of fresh produce.     

Ecology and Transmission of Listeria monocytogenes Infecting Ruminants and in the Farm Environment

A case-control study involving 24 case farms with at least one recent case of listeriosis and 28 matched control farms with no listeriosis cases was conducted to probe the transmission and ecology of Listeria monocytogenes on farms. A total of 528 fecal, 516 feed, and 1,012 environmental soil and water samples were cultured for L. monocytogenes. While the overall prevalence of L. monocytogenes in cattle case farms (24.4%) was similar to that in control farms (20.2%), small-ruminant (goat and sheep) farms showed a significantly (P < 0.0001) higher prevalence in case farms (32.9%) than in control farms (5.9%). EcoRI ribotyping of clinical (n = 17) and farm (n = 414) isolates differentiated 51 ribotypes. L. monocytogenes ribotypes isolated from clinical cases and fecal samples were more frequent in environmental than in feed samples, indicating that infected animals may contribute to L. monocytogenes dispersal into the farm environment. Ribotype DUP-1038B was significantly (P < 0.05) associated with fecal samples compared with farm environment and animal feedstuff samples. Ribotype DUP-1045A was significantly (P < 0.05) associated with soil compared to feces and with control farms compared to case farms. Our data indicate that (i) the epidemiology and transmission of L. monocytogenes differ between small-ruminant and cattle farms; (ii) cattle contribute to amplification and dispersal of L. monocytogenes into the farm environment, (iii) the bovine farm ecosystem maintains a high prevalence of L. monocytogenes, including subtypes linked to human listeriosis cases and outbreaks, and (iv) L. monocytogenes subtypes may differ in their abilities to infect animals and to survive in farm environments.     

Risk Factors Associated with Salmonella and Listeria monocytogenes Contamination of Produce Fields

Identification of management practices associated with preharvest pathogen contamination of produce fields is crucial to the development of effective Good Agricultural Practices (GAPs). A cross-sectional study was conducted to (i) determine management practices associated with a Salmonella- or Listeria monocytogenes-positive field and (ii) quantify the frequency of these pathogens in irrigation and nonirrigation water sources. Over 5 weeks, 21 produce farms in New York State were visited. Field-level management practices were recorded for 263 fields, and 600 environmental samples (soil, drag swab, and water) were collected and analyzed for Salmonella and L. monocytogenes. Management practices were evaluated for their association with the presence of a pathogen-positive field. Salmonella and L. monocytogenes were detected in 6.1% and 17.5% of fields (n = 263) and 11% and 30% of water samples (n = 74), respectively. The majority of pathogen-positive water samples were from nonirrigation surface water sources. Multivariate analysis showed that manure application within a year increased the odds of a Salmonella-positive field (odds ratio [OR], 16.7), while the presence of a buffer zone had a protective effect (OR, 0.1). Irrigation (within 3 days of sample collection) (OR, 6.0), reported wildlife observation (within 3 days of sample collection) (OR, 6.1), and soil cultivation (within 7 days of sample collection) (OR, 2.9) all increased the likelihood of an L. monocytogenes-positive field. Our findings provide new data that will assist growers with science-based evaluation of their current GAPs and implementation of preventive controls that reduce the risk of preharvest contamination.     

Reservoirs of Listeria Species in Three Environmental Ecosystems

Soil and water are suggested to represent pivotal niches for the transmission of Listeria monocytogenes to plant material, animals, and the food chain. In the present study, 467 soil and 68 water samples were collected in 12 distinct geological and ecological sites in Austria from 2007 to 2009. Listeria was present in 30% and 26% of the investigated soil and water samples, respectively. Generally, the most dominant species in soil and water samples were Listeria seeligeri, L. innocua, and L. ivanovii. The human- and animal-pathogenic L. monocytogenes was isolated exclusively from 6% soil samples in regions A (mountainous region) and B (meadow). Distinct ecological preferences were observed for L. seeligeri and L. ivanovii, which were more often isolated from wildlife reserve region C (Lake Neusiedl) and from sites in proximity to wild and domestic ruminants (region A). The higher L. monocytogenes detection and antibiotic resistance rates in regions A and B could be explained by the proximity to agricultural land and urban environment. L. monocytogenes multilocus sequence typing corroborated this evidence since sequence type 37 (ST37), ST91, ST101, and ST517 were repeatedly isolated from regions A and B over several months. A higher L. monocytogenes detection and strain variability was observed during flooding of the river Schwarza (region A) and Danube (region B) in September 2007, indicating dispersion via watercourses.     

Distribution and Characteristics of Listeria monocytogenes Isolates from Surface Waters of the South Nation River Watershed, Ontario, Canada

Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 ± 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.     

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.     

Retail Survey of Brazilian Milk and Minas Frescal Cheese and a Contaminated Dairy Plant To Establish Prevalence, Relatedness, and Sources of Listeria monocytogenes Isolates

A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, “brand F” of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil.     

Comparison of the Prevalences and Diversities of Listeria Species and Listeria monocytogenes in an Urban and a Rural Agricultural Watershed

Foods and related processing environments are commonly contaminated with the pathogenic Listeria monocytogenes. To investigate potential environmental reservoirs of Listeria spp. and L. monocytogenes, surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, were examined over 18 months. Presumptive Listeria spp. were cultured from 72 and 35% of rural and urban water samples, respectively, with 24% of the positive samples containing two or three different Listeria spp. The L. innocua (56%) and L. welshimeri (43%) groups were predominant in the rural and urban watersheds, respectively. Analysis by the TaqMan assay showed a significantly (P < 0.05) higher prevalence of L. monocytogenes of 62% versus 17% by the culture-based method. Both methods revealed higher prevalences in the rural watershed and during the fall and winter seasons. Elevated Escherichia coli (≥100 CFU/100 ml) levels were not associated with the pathogen regardless of the detection method. Isolation of Listeria spp. were associated with 70 times higher odds of isolating L. monocytogenes (odds ratio = 70; P < 0.001). Serogroup IIa was predominant (67.7%) among the 285 L. monocytogenes isolates, followed by IVb (16.1%), IIb (15.8%), and IIc (0.4%). L. monocytogenes was detected in cow feces and raw sewage but not in septic tank samples. Pulsotyping of representative water (n = 54) and local human (n = 19) isolates suggested genetic similarities among some environmental and human L. monocytogenes isolates. In conclusion, temperate surface waters contain a diverse Listeria species population and could be a potential reservoir for L. monocytogenes, especially in rural agricultural watersheds.     

Anthropogenic impact on the presence of L. monocytogenes in soil, fruits, and vegetables

The aim of this study was to determine the prevalence of Listeria sp. and Listeria monocytogenes in soil samples with reference to type of fertilizers (natural and artificial) and distance from places intensively exploited by men, as well as to determine the relationship between the presence of L. monocytogenes in the soil and in fruits and vegetables. The examined 1,000 soil samples originated from 15 different areas, whilst 140 samples of fruits and 210 samples of vegetables were collected from those areas. L. monocytogenes was isolated only from 5.5 % of all soil samples coming exclusively from meadows intensively grazed by cattle (27.8 %) and areas near food processing plants (25 %) and wild animal forests (24 %). Listeria sp. and L. monocytogenes were not present on artificially fertilized areas and wastelands. L. monocytogenes was detected in 10 % of samples of strawberry, 15 % of potato samples, and 5 % of parsley samples. Our data indicate that Listeria spp. and particularly L. monocytogenes were found in the soil from (1) arable lands fertilized with manure, (2) pasture (the land fertilized with feces of domestic animals), and (3) forests (again, the land fertilized with feces of animals, not domestic but wild). The bacteria were not detected in the soil samples collected at (1) artificially fertilized arable lands and (2) wastelands (the lands that were not fertilized with manure or animal feces). Moreover, a correlation was determined in the presence of L. monocytogenes between soil samples and samples of the examined fruits and vegetables.     

The Prevalence of Listeria monocytogenes in the Environment of Dairy Farms

The prevalence of Listeria monocytogenes in the environment of dairy farms was surveyed from December 1993 to June 1994 in one city of Hokkaido. L. monocytogenes was isolated from 3 out of 5 farms investigated. Serovar 4b organism was isolated from the brain stem of a cow from one farm which was clinically diagnosed as having listeriosis. The same serovar of L. monocytogenes was also isolated from the rectal contents of a healthy cow, straw on the floor, straw in the barn, and silage scattered around the silo from the same farm. At another farm, with no reported cases of bovine listeriosis, serovar 1/2 organism was isolated from the same types of samples as the above mentioned farm except from straw on the floor. The difference in the isolation rates of the organism from straw on the floor between the two farms (22%: 5/23 vs 0%: 0/24) is considered to be caused by the different feeding methods of silage between the two farms.     

Listeria monocytogenes is common in wild birds in Helsinki region and genotypes are frequently similar with those found along the food chain

Aims: To evaluate the prevalence and genetic diversity of Listeria monocytogenes in wild birds and to compare the genotypes with isolates previously collected from foods and food processing environments. Methods and Results: Samples of wild birds’ faeces (n = 212) were collected from a municipal landfill site and from urban areas in the Helsinki region and analysed by two‐step enrichment and plating onto L. monocytogenes‐selective agar. The overall prevalence of L. monocytogenes in bird faeces was 36% (95% CI 30–43%), and prevalence on the landfill site was significantly higher. All isolates were analysed with pulsed‐field gel electrophoresis and compared with the L. monocytogenes profiles in an existing collection. Similar pulsotypes were found in birds and in isolates collected along the food chain. Conclusions: Birds commonly carry L. monocytogenes, and strains are frequently similar with those detected in foods and food processing environments. Thus, birds may disseminate L. monocytogenes in nature and may also contaminate foods when entering the food processing environments and outdoor market places. Significance and Impact of the Study: Populations of L. monocytogenes in wild birds and along the food processing chain overlap. Our findings add to the epidemiological data on this significant foodborne pathogen.     

Estimation of Microbial Contamination of Food from Prevalence and Concentration Data: Application to Listeria monocytogenes in Fresh Vegetables

A normal distribution and a mixture model of two normal distributions in a Bayesian approach using prevalence and concentration data were used to establish the distribution of contamination of the food-borne pathogenic bacteria Listeria monocytogenes in unprocessed and minimally processed fresh vegetables. A total of 165 prevalence studies, including 15 studies with concentration data, were taken from the scientific literature and from technical reports and used for statistical analysis. The predicted mean of the normal distribution of the logarithms of viable L. monocytogenes per gram of fresh vegetables was −2.63 log viable L. monocytogenes organisms/g, and its standard deviation was 1.48 log viable L. monocytogenes organisms/g. These values were determined by considering one contaminated sample in prevalence studies in which samples are in fact negative. This deliberate overestimation is necessary to complete calculations. With the mixture model, the predicted mean of the distribution of the logarithm of viable L. monocytogenes per gram of fresh vegetables was −3.38 log viable L. monocytogenes organisms/g and its standard deviation was 1.46 log viable L. monocytogenes organisms/g. The probabilities of fresh unprocessed and minimally processed vegetables being contaminated with concentrations higher than 1, 2, and 3 log viable L. monocytogenes organisms/g were 1.44, 0.63, and 0.17%, respectively. Introducing a sensitivity rate of 80 or 95% in the mixture model had a small effect on the estimation of the contamination. In contrast, introducing a low sensitivity rate (40%) resulted in marked differences, especially for high percentiles. There was a significantly lower estimation of contamination in the papers and reports of 2000 to 2005 than in those of 1988 to 1999 and a lower estimation of contamination of leafy salads than that of sprouts and other vegetables. The interest of the mixture model for the estimation of microbial contamination is discussed.     

Ruminant-associated Listeria monocytogenes isolates belong preferentially to dairy-associated hypervirulent clones: a longitudinal study in 19 farms

Studies have shown that ruminants constitute reservoirs of Listeria monocytogenes, but little is known about the epidemiology and genetic diversity of this pathogen within farms. Here we conducted a large-scale longitudinal study to monitor Listeria spp. in 19 dairy farms during three consecutive seasons (N = 3251 samples). L. innocua was the most prevalent species, followed by L. monocytogenes. Listeria monocytogenes was detected in 52.6% of farms and more frequently in cattle (4.1%) and sheep (4.5%) than in goat farms (0.2%). Lineage I accounted for 69% of L. monocytogenes isolates. Among animal samples, the most prevalent sublineages (SL) and clonal complexes (CC) were SL1/CC1, SL219/CC4, SL26/CC26 and SL87/CC87, whereas SL666/CC666 was most prevalent in environmental samples. Sixty-one different L. monocytogenes cgMLST types were found, 28% common to different animals and/or surfaces within the same farm and 21% previously reported elsewhere in the context of food and human surveillance. Listeria monocytogenes prevalence was not affected by farm hygiene but by season: higher prevalence was observed during winter in cattle, and during winter and spring in sheep farms. Cows in their second lactation had a higher probability of L. monocytogenes faecal shedding. This study highlights dairy farms as a reservoir for hypervirulent L. monocytogenes.     

Modelling and predicting the simultaneous growth of Listeria monocytogenes and spoilage micro-organisms in cold-smoked salmon

Aims: To evaluate and model the simultaneous growth of Listeria monocytogenes and spoilage micro-organisms in cold-smoked salmon. Methods and Results: Growth kinetics of L. monocytogenes, lactic acid bacteria (LAB), Enterobacteriaceae, enterococci and Photobacterium phosphoreum were determined in two series of challenge tests with sliced and vacuum-packed cold-smoked salmon (SVP-CSS). The product contained a high level of smoke components and at 2°C levels of L. monocytogenes increased <100-fold in 193 days. Without the addition of spoilage micro-organisms, L. monocytogenes reached ca 10⁸ CFU g⁻¹ at 5, 10, 17·5 and 25°C. Inoculation with spoilage micro-organisms reduced this level to 10²–10⁴ CFU g⁻¹. LAB dominated the spoilage microfora of SVP-CSS and competition between LAB and L. monocytogenes in SVP-CSS was appropriately described by a simple expansion of the Logistic model. This interaction model aided in predicting the growth of L. monocytogenes in naturally contaminated SVP-CSS when it was used in combination with expanded versions of existing secondary models for L. monocytogenes and LAB. Conclusions: Temperature, water activity/NaCl, simultaneous growth of LAB, smoke components and to a lesser extent lactate and pH control growth of L. monocytogenes in SVP-CSS. These factors must be included in mathematical models to predict growth of the pathogen in this product. Significance and Impact of the Study: The suggested predictive model can be used to support assessment and management of the human health risk due to L. monocytogenes in SVP-CSS.     

Prevalence and Relative Risk of Cronobacter spp., Salmonella spp., and Listeria monocytogenes Associated with the Body Surfaces and Guts of Individual Filth Flies

Although flies are important vectors of food-borne pathogens, there is little information to accurately assess the food-related health risk of the presence of individual flies, especially in urban areas. This study quantifies the prevalence and the relative risk of food-borne pathogens associated with the body surfaces and guts of individual wild flies. One hundred flies were collected from the dumpsters of 10 randomly selected urban restaurants. Flies were identified using taxonomic keys before being individually dissected. Cronobacter spp., Salmonella spp., and Listeria monocytogenes were detected using the PCR-based BAX system Q7. Positive samples were confirmed by culture on specific media and through PCR amplification and sequencing or ribotyping. Among collected flies were the housefly, Musca domestica (47%), the blowflies, Lucilia cuprina (33%) and Lucilia sericata (14%), and others (6%). Cronobacter species were detected in 14% of flies, including C. sakazakii, C. turicensis, and C. universalis, leading to the proposal of flies as a natural reservoir of this food-borne pathogen. Six percent of flies carried Salmonella enterica, including the serovars Poona, Hadar, Schwarzengrund, Senftenberg, and Brackenridge. L. monocytogenes was detected in 3% of flies. Overall, the prevalence of food-borne pathogens was three times greater in the guts than on the body surfaces of the flies. The relative risk of flies carrying any of the three pathogens was associated with the type of pathogen, the body part of the fly, and the ambient temperature. These data enhance the ability to predict the microbiological risk associated with the presence of individual flies in food and food facilities.     

Generic Escherichia coli Contamination of Spinach at the Preharvest Stage: Effects of Farm Management and Environmental Factors

The objective of this study was to determine the effects of farm management and environmental factors on preharvest spinach contamination with generic Escherichia coli as an indicator of fecal contamination. A repeated cross-sectional study was conducted by visiting spinach farms up to four times per growing season over a period of 2 years (2010 to 2011). Spinach samples (n = 955) were collected from 12 spinach farms in Colorado and Texas as representative states of the Western and Southwestern United States, respectively. During each farm visit, farmers were surveyed about farm-related management and environmental factors using a questionnaire. Associations between the prevalence of generic E. coli in spinach and farm-related factors were assessed by using a multivariable logistic regression model including random effects for farm and farm visit. Overall, 6.6% of spinach samples were positive for generic E. coli. Significant risk factors for spinach contamination with generic E. coli were the proximity (within 10 miles) of a poultry farm, the use of pond water for irrigation, a >66-day period since the planting of spinach, farming on fields previously used for grazing, the production of hay before spinach planting, and the farm location in the Southwestern United States. Contamination with generic E. coli was significantly reduced with an irrigation lapse time of >5 days as well as by several factors related to field workers, including the use of portable toilets, training to use portable toilets, and the use of hand-washing stations. To our knowledge, this is the first report of an association between field workers'' personal hygiene and produce contamination with generic E. coli at the preharvest level. Collectively, our findings support that practice of good personal hygiene and other good farm management practices may reduce produce contamination with generic E. coli at the preharvest level.     

Occurrence ofListeria monocytogenes in bovine milk in Hyderabad, Pakistan

In the present study 200 milk samples (50 each from cow, buffalo, market and mastitic milk) were collected from Hyderabad. Among the isolated presumptiveListeria spp., all the isolates were identified asListeria monocytogenes and showed a prevalence of 6, 8, 10 and 12%, respectively. Through serotyping analysis all isolates were confirmed asListeria genus, since they agglutinated with polyvalent ‘O’ serum; 12 of the 18L. monocytogenes isolates were ascribed to serotype 1, and 6 to serotype 4. Furthermore, the pathogenicity was investigated in rabbit and mice through Anton and inoculation tests, respectively. The organism produces keratoconjunctivitis in rabbit while it causes death and presence of necrotic foci in liver, spleen and kidneys of mice.Listeria monocytogenes was found sensitive to ampicillin, chloramphenicol, ciprofloxacin, gentamycin and kanamycin.     

Co-Culture with Listeria monocytogenes within a Dual-Species Biofilm Community Strongly Increases Resistance of Pseudomonas putida to Benzalkonium Chloride

Biofilm formation is a phenomenon occurring almost wherever microorganisms and surfaces exist in close proximity. This study aimed to evaluate the possible influence of bacterial interactions on the ability of Listeria monocytogenes and Pseudomonas putida to develop a dual-species biofilm community on stainless steel (SS), as well as on the subsequent resistance of their sessile cells to benzalkonium chloride (BC) used in inadequate (sub-lethal) concentration (50 ppm). The possible progressive adaptability of mixed-culture biofilms to BC was also investigated. To accomplish these, 3 strains per species were left to develop mixed-culture biofilms on SS coupons, incubated in daily renewable growth medium for a total period of 10 days, under either mono- or dual-species conditions. Each day, biofilm cells were exposed to disinfection treatment. Results revealed that the simultaneous presence of L. monocytogenes strongly increased the resistance of P. putida biofilm cells to BC, while culture conditions (mono-/dual-species) did not seem to significantly influence the resistance of L. monocytogenes biofilm cells. BC mainly killed L. monocytogenes cells when this was applied against the dual-species sessile community during the whole incubation period, despite the fact that from the 2nd day this community was mainly composed (>90%) of P. putida cells. No obvious adaptation to BC was observed in either L. monocytogenes or P. putida biofilm cells. Pulsed field gel electrophoresis (PFGE) analysis showed that the different strains behaved differently with regard to biofilm formation and antimicrobial resistance. Such knowledge on the physiological behavior of mixed-culture biofilms could provide the information necessary to control their formation.