Linking intraspecies variability of Salmonella enterica isolates under acidic conditions to genotype.

There is a lack of information about Salmonella enterica strains under acidic conditions and their association with their genome. This study characterized intraspecies variability in the growth of 167 S. enterica isolates under two acid conditions (pH 4 and 5) and linked to the whole genome sequencing (WGS) data. A total of 1002 curves for each condition were obtained using turbidimetry measurements, and Baranyi and Roberts model was used to estimate the maximum rate of change (rcmax; OD600 nm h-1). Strains were categorized into slow, intermediate, and fast; and associations with their WGS data were performed. Huge variability in r c max ¯ $\overline {{\mathrm{r}}{{{\mathrm{c}}}_{{\mathrm{max}}}}} $ was observed at both conditions (pH 5 = 0.016-0.066 OD600nm h-1 and pH 4 = 0.003-0.028 OD600nm h-1). The majority of isolates was classified as intermediate r c max ¯ $\overline {{\mathrm{r}}{{{\mathrm{c}}}_{{\mathrm{max}}}}} $ (59.5% at pH 5 and 46.1% at pH 4). Strains classified as fast had a low frequency of allABCD genes at both pHs, and any of them having the presence of pefABCD, spvBCR, aadA2, dfrA12, and gyrA_D87G genes were linked to virulence or antimicrobial resistance. This study suggests that strains with fast capacity for growth under acidic conditions could have a fitness cost in their virulence or resistance potential. PRACTICAL APPLICATION: Data presented in this study could be used to select representative strains to evaluate the exposure assessment in different food items, mainly the growth and survival in acidic foods.

Genomic diversity of Salmonella enterica isolated from raw chicken at retail establishments in Mexico.

The genomic diversity of circulating non-typhoidal Salmonella in raw chicken was investigated in three states of central Mexico. A total of 192 S. enterica strains from chicken meat samples collected at supermarkets, fresh markets, and butcher shops were analyzed by whole-genome sequencing. The serovar distribution, occurrence of genes encoding for antimicrobial resistance, metal resistance, biocide resistance, plasmids and virulence factors, and clonal relatedness based on single nucleotide polymorphism (SNP) analysis were investigated. Serovars Infantis, Schwarzengrund and Enteritidis predominated among twenty identified. The distribution of serovars and proportion of AMR genes was different according to the state, year, season, and retail establishment (p < 0.001). Genes encoding metals resistance were identified in all the strains. A total of 145 virulence genes were identified and strains were classified into 32 virulotypes; serovars Infantis, Typhimurium, and Enteritidis showed the highest number of virulence genes. The strains matched 34 SNP clusters in the NCBI Pathogen Detection server and 59 %, which corresponded to Infantis, Schwarzengrund, Saintpaul, and Enteritidis, were associated with five major clusters and matched with chicken, environmental and clinical isolates from at least three countries. These results provide useful information to understand the epidemiology of Salmonella, conduct microbial risk assessment, and design risk-based control measures.

Genomic analysis of the MLST population structure and antimicrobial resistance genes associated with Salmonella enterica in Mexico.

Salmonella enterica is one of the most commonly reported foodborne pathogens by public health agencies worldwide. In this study, the multilocus sequence typing (MLST) population structure and frequency of antimicrobial resistance (AMR) genes were evaluated in S. enterica strains from Mexico (n = 2561). The most common sources of isolation were food (44.28%), environment (27.41%), animal-related (24.83%), and human (3.48%). The most prevalent serovars were Newport (8.51%), Oranienburg (7.03%), Anatum (5.78%), Typhimurium (5.12%), and Infantis (4.57%). As determined by the 7-gene MLST scheme, the most frequent sequence types were ST23, ST64, and ST32. The core genome MLST scheme identified 132 HC2000 and 195 HC900 hierarchical clusters, with the HC2000_2 cluster being the most prevalent in Mexico (n = 256). A total of 78 different AMR genes belonging to 13 antimicrobial classes were detected in 638 genomic assemblies of S. enterica. The most frequent class was aminoglycosides (31.76%), followed by tetracyclines (12.53%) and sulfonamides (11.91%). These results can help public health agencies in Mexico prioritize their efforts and resources to increase the genomic sequencing of circulating Salmonella strains. Additionally, they provide valuable information for local and global public health efforts to reduce the impact of foodborne diseases and AMR.

Risk ranking of food categories associated with Salmonella enterica contamination in the central region of Mexico.

To prevent and control foodborne diseases, there is a fundamental need to identify the foods that are most likely to cause illness. The goal of this study was to rank 25 commonly consumed food products associated with Salmonella enterica contamination in the Central Region of Mexico. A multicriteria decision analysis (MCDA) framework was developed to obtain an S. enterica risk score for each food product based on four criteria: probability of exposure to S. enterica through domestic food consumption (Se); S. enterica growth potential during home storage (Sg); per capita consumption (Pcc); and food attribution of S. enterica outbreak (So). Risk scores were calculated by the equation Se*W1 +Sg*W2 +Pcc*W3 +So*W4 , where each criterion was assigned a normalized value (1-5) and the relative weights (W) were defined by 22 experts' opinion. Se had the largest effect on the risk score being the criterion with the highest weight (35%; IC95% 20%-60%), followed by So (24%; 5%-50%), Sg (23%; 10%-40%), and Pcc (18%; 10%-35%). The results identified chicken (4.4 ± 0.6), pork (4.2 ± 0.6), and beef (4.2 ± 0.5) as the highest risk foods, followed by seed fruits (3.6 ± 0.5), tropical fruits (3.4 ± 0.4), and dried fruits and nuts (3.4 ± 0.5), while the food products with the lowest risk were yogurt (2.1 ± 0.3), chorizo (2.1 ± 0.4), and cream (2.0 ± 0.3). Approaches with expert-based weighting and equal weighting showed good correlation (R2  = 0.96) and did not show significant differences among the ranking order in the top 20 tier. This study can help risk managers select interventions and develop targeted surveillance programs against S. enterica in high-risk food products.

Genotypic and phenotypic quantitative microbial risk assessment model of human salmonellosis related to the consumption of chicken meat in the central region of Mexico.

Chicken meat is often associated withSalmonella entericacontamination worldwide. This study proposes a risk assessment model for human salmonellosis linked to the domestic consumption of chicken meat in the central region of Mexico, incorporating genotypic and phenotypic data. SixS. entericagroups were used, considering the presence of specific virulence genes and multidrug resistance (MDR). Sixteen exposure scenarios were established considering retail point (RP1 = fresh market/butcher shop; RP2 = mini-super/supermarket), transportation, home storage, cooking, and cross-contamination. The model predicted a mean annual salmonellosis cases of 66,754 due to chicken consumption (CI95% 10775-231606). The mean probability of illness (Pill) among the exposure scenarios ranged from 2.5 × 10-9 to 3.7 × 10-6, 7.7 × 10-8 to 1.1 × 10-4, and 6.7 × 10-4 to 7.8 × 10-2 for low, moderate, and high virulence groups. Exposure scenarios with the highest Pill were not responsible for most cases due to their low frequency of occurrence. The high virulence/ MDR group was responsible for most cases (66.5 %), despite the low S. enterica prevalence (RP1 0.5 % and RP2 5.0 %). The years lost due to disability (YLD) value for MDR was 2.6 × higher than for non-MDR. Spearman rank showed that the inputs with higher influence on the variability of salmonellosis depended on the type of exposure scenario. For example, the cooking temperature and time had the most significant influence in the scenarios where S. enterica can survive after cooking. Including the microbial genotypic and phenotypic characteristics in risk assessment modeling highlights the importance of focusing on high-virulent and MDR strains, which are not the most frequent but represent the highest public health risk.

Detection, quantification, and characterization of Salmonella enterica in mango, tomato, and raw chicken purchased in the central region of Mexico.

To estimate human exposure to Salmonella enterica, it is essential to understand the pathogen distribution and characteristics. Prevalence and concentration of S. enterica were determined in mango, tomato, and raw chicken samples purchased in three states (Aguascalientes, Querétaro, and Guadalajara) located in the central region of Mexico during two seasons. In addition, S. enterica isolates were characterized by absence/presence of 13 virulence genes (chromosomal, prophage, and plasmid) and resistance to 14 antibiotics. A total of 300 samples of mango, 272 of tomato, and 354 of raw chicken were analyzed. The mean of the prevalence (24.9%) and concentration (-0.61 Log MPN/g) of S. enterica in chicken was higher than in mango (1.3%, -1.7 Log MPN/g) and tomato (1.1%, -1.7 Log MPN). Among S. enterica isolates (284), there were 7 different virulotypes, belonging 68.7% of isolates to V2; there was high variability in the presence of mobile genetic elements. The occurrence of specific mobile elements ranged from 81.4% to 11.3% among isolates. Among the isolates, 91.5% were resistant to at least one antibiotic with ampicillin being the most frequent; 54.9% of isolates were multidrug resistant. Data from this study can be used for quantitative microbial risk assessment of S. enterica related to mango, tomato, and raw chicken consumption in the central region of Mexico. PRACTICAL APPLICATION: Data on the prevalence and concentration of Salmonella enterica obtained in this study can be used to estimate the exposure assessment for the consumption of mango, tomato, and chicken in the central region of Mexico. In addition, the characteristics of the S. enterica isolates could be used to select representative strains for future studies to evaluate the intraspecies variability.

A new classification criterion for the biofilm formation index: A study of the biofilm dynamics of pathogenic Vibrio species isolated from seafood and food contact surfaces.

The bacterial biofilm formation index (BFI) is measured by a microtiter plate assay, and it is typically performed at 72 hr. However, the dynamics of biopolymer formation change during this incubation period. The aims of this study were to follow the biofilm formation dynamics of Vibrio strains isolated from samples of seafood and food contact surfaces (FCS) and to propose a new BFI classification criterion. Samples from seafood (136) and FCS (14) were collected from retail markets in Queretaro, Mexico. The presence of Vibrio spp. was determined, the strains were isolated, and the six major pathogenic species (V. cholerae, V. alginolyticus, V. fluvialis, V. parahaemolyticus, V. vulnificus, V. mimicus) were identified by PCR. The BFI of the isolates was determined by the microtiter plate method. Fifty-one strains were isolated and identified as V. alginolytivcus (25), V. vulnificus (12), V. cholerae (7), V. parahaemolyticus (6), and V. mimicus (1). A quantitative classification criterion of biofilm formation was proposed based on the following factors: BFI dynamics (no formation, continuous increase, and increase followed by decrease), time of maximum BFI (early: 24 hr; late: 48 to 72 hr), and degree of BFI (none, weak, moderate, and strong). A numerical value was assigned to each factor to correlate the resulting BFI profile with a risk level. Thirteen BFI profiles were observed, having risk level values from 0 to 10. Vibrio alginolyticus, V. cholerae, and V. vulnificus showed the highest BFI profile diversities, which included the riskiest profiles. The proposed BFI criterion describes the dynamics of bacterial biopolymer formation and associates them with the possible risk implications. PRACTICAL APPLICATION: In food processing environments, the presence of bacterial biofilms that could include foodborne pathogens might favor cross-contamination due to direct contact or biofilm dispersal into food products. The new quantitative classification criterion for biofilm formation considers their production dynamics over time, the biofilm quantity, and the level of biofilm dispersal. These characteristics are represented by a numerical value that reflects the level of risk associated with the presence of a biofilm-producing strain on a food contact surface.

Salmonella enterica in Mexico 2000-2017: Epidemiology, Antimicrobial Resistance, and Prevalence in Food.

In Mexico, information of Salmonella enterica cases linked to food consumption is scarce. The objective of this article was to assess how S. enterica affect public health in Mexico. To conduct this study, data on the epidemiology of nontyphoidal S. enterica (NTS), Salmonella Typhi, and Salmonella Paratyphi A collected from 2000 to 2017 through the National Epidemiological Surveillance System of Mexico (Sistema Nacional de Vigilancia Epidemiológica de Mexico [SINAVE]) were used. Geographical distribution, season, age groups, and gender were variables considered to analyze S. enterica incidence. An estimation of cases caused by S. enterica in Mexico was calculated while considering data underestimation and the proportion of foodborne diseases. Information of the prevalence of the pathogen in food and the antimicrobial resistance of isolates from food and human cases were obtained from published studies. Outbreaks of S. enterica derived from imported Mexican products in the Unites States are discussed. In 2017, the numbers of reported cases of NTS (92,013) were two and seven times higher than the reported cases of Salmonella Typhi (45,280) and Salmonella Paratyphi A (12, 458). The NTS incidence was higher in lower socioeconomic Mexican regions. The gaps in the surveillance system make it impossible to establish a reliable tendency among age groups, geographical distribution, and gender. In 2017, the estimated frequency of NTS foodborne cases was 49 times higher than that reported in SINAVE, whereas for Salmonella Typhi and Salmonella Paratyphi A it was 23 times. Fresh meat showed the highest prevalence of S. enterica, and most of their isolates had multidrug resistance. Salmonella Typhimurium was the most common serotype isolated from human cases and food. Food safety agencies in Mexico need to prioritize efforts and resources to establish guidelines to ensure the absence of S. enterica in food.

Food Consumer Behavior and Salmonella Exposure Self-Perception in the Central Region of Mexico.

In Mexico, there is limited information regarding food consumer behaviors at home. The knowledge of food consumption and food handling practices can help to determine more realistically the exposure to different hazards, such as Salmonella spp. on the population. The main goal of this study was to characterize the food consumption habits of five food groups (fruits and vegetables, fresh meat, dairy products, processed meat products, and seafood), food handling practices, and Salmonella exposure self-perception of the population of the central region of Mexico using an online survey. A total of 1,199 surveys were analyzed. The group of fruits and vegetables is the most consumed and one of the most related to the consumer self-perception of suffering from salmonellosis. Mexican consumers regularly use refrigeration as the principal food storage method for the five food groups (42.2% to 90.8%), and generally, they store the products between 2 and 3 days (26.5% to 38.1%). A total of 86.4% of consumers reported that they always wash their hands prior to food preparation. A total of 16.9% and 13.0% use the same cutting board and knife, respectively, to cut more than one product without applying sanitization. Men, people between 20 and 24 years, and people between 60 and 64 years, had the highest risk food handling practices at home. Finally, age (P < 0.0001), education level (P = 0.0004), and suffering from gastrointestinal diseases (P = 0.0005) showed significant effects with self-perception of having salmonellosis. PRACTICAL APPLICATION: Data presented in this study could be used in future risk and exposure assessment research to evaluate the public health risk from the consumption of contaminated food. In addition, it could be used by government agencies to design education campaigns in food safety area.

Antimicrobial effect of nisin electrospun amaranth: pullulan nanofibers in apple juice and fresh cheese.

Nisin-loaded amaranth protein isolate:pullulan (API:PUL) nanofibers were prepared by the electrospinning method. The nisin release kinetic was evaluated at pH 3.4 and 6.1 and the antimicrobial effectiveness of the electrospun mats was evaluated in apple juice and fresh cheese. The nisin API:PUL fibers with 120 nm average diameter reached 81.49% and 43.85% nisin release after 12 h at pH 3.4 and 6.1, respectively. The encapsulation of nisin in electrospun fibers allowed complete bactericidal activity against Salmonella Typhimurium, L. monocytogenes and L. mesenteroides inoculated in apple juice after 48, 20 and 48 h, respectively. When nisin API:PUL fibers were applied to fresh cheese, microorganism inactivation was complete after 142, 120 and 170 h, respectively. The results demonstrated that nisin API:PUL electrospun fibers significantly reduce the bacterial population and can be used in food products for microbiological safety.

Characterization of Bacterial Communities in Mexican Artisanal Raw Milk "Bola de Ocosingo" Cheese by High-Throughput Sequencing.

The dynamics of bacteria community of "Bola de Ocosingo" cheese, a Mexican artisanal raw milk cheese was investigated by high-throughput sequencing (454 pyrosequencing). Dairy samples (raw milk, curd, cheese at 50 and 110 days of ripening) were collected at dry (March-June) and rainy season (August-November) from three producers located in Chiapas, Mexico. In general, raw milk contained high bacterial diversity which was reduced throughout cheese manufacture. However, in two productions an important increase during cheese ripening was observed probably due to cross-contamination. Species such as Streptococcus thermophilus, Lactococcus lactis, Lactobacillus helveticus, L. delbrueckii and L. plantarum from which potential probiotic strains may be obtained, predominated during processing, varying its prevalence from one producer to another. Furthermore, low proportions of Escherichia coli/Shigella flexnerii were detected in almost all processes, however, could not be recovered by traditional methodology, indicating presence of non-cultivable cells. This work provides insights into bacteria communities of Bola de Ocosingo cheese for starter culture development, many of which are reported to provide health related benefits, and the usefulness of high-throughput sequencing to evidence cross-contamination during processing.

Microbiological Quality and Incidence of Salmonella on Cherry Tomatoes at Retail in Querétaro, México.

Multiple outbreaks related to Salmonella in tomatoes require an evaluation of the risk associated with cherry tomatoes due to the increase in its production, consumption, and marketing in Mexico's central region. The purpose of this study was to determine the microbial quality of cherry tomatoes obtained from two retail sale points (supermarkets and local markets). Cherry tomato samples (333) were collected from four supermarkets and from four local markets, and the contents of aerobic plate count, molds and yeasts, total coliforms, and Escherichia coli were quantified; the presence of Salmonella was simultaneously determined. The median values of the microbial populations were obtained, and the data were analyzed per the sampling site by using the Wilcoxon and Kruskal-Wallis tests. The median of aerobic plate count content in tomatoes obtained from supermarkets ranged between 2.2 and 4.4 log CFU/g, and in markets from 2.9 to 4.8 log CFU/g. For molds and yeasts, the tomatoes from supermarkets (2.0 to 4.1 log CFU/g) and markets (1.5 to 4.5 log CFU/g) showed similar contents. Regardless of the sampling site, the values of total coliforms were very low, ranging from 1.0 to 1.8 log CFU/g. E. coli was detected in 5.4 and 20.1% of samples from supermarkets and markets, respectively; in both sites, the content was low (0.3 to 5.8 most probable number per g). The incidence of Salmonella was 14.1% in supermarkets and 7.8% in local markets. The results obtained from this investigation highlight the elevated risk for consumer health associated with the ingestion of cherry tomatoes.

An Improve Protocol for PCR Using LM1 and LM2 Primers for Listeria monocytogenes Detection in Food Matrices.

Several studies have observed that a conventional PCR protocol using primers LM1 and LM2 for the identification of gene hlyA Listeria monocytogenes generates non-specific PCR amplifications and false positives. For this reason in this study, we provide a modified PCR protocol that improves the specificity of the LM1 and LM2 primers.

Microbiological profile and incidence of Salmonella and Listeria monocytogenes on hydroponic bell peppers and greenhouse cultivation environment.

The aim of this study was to generate information regarding the microbiological profile, including Salmonella and Listeria monocytogenes incidence, of hydroponically grown bell peppers and materials associated with their production in greenhouses located in Mexico. Samples of coconut fiber (24), knives (30), drippers (20), conveyor belts (161), pepper transportation wagons (30), air (178), water (16), nutrient solution for plant irrigation (78), and bell pepper fruits (528) were collected during one cycle of production (2009 to 2010) for the quantification of microbial indicators (aerobic plate counts [APC], molds, coliforms, and Escherichia coli) and the detection of Salmonella and L. monocytogenes. With regard to surfaces (conveyor belts and wagons) and utensils (knives and drippers), the APC, coliform, and mold counts ranged from 3.0 to 6.0, from 1.4 to 6.3, and from 3.6 to 5.2 log CFU/100 cm(2) or per utensil, respectively. The air in the greenhouse contained low median levels of APC (1.2 to 1.4 log CFU/100 liters) and molds (2.2 to 2.5 log CFU/100 liters). The median content of APC and coliforms in water were 0.5 log CFU/ml and 0.3 log MPN/100 ml, respectively. The median content of coliforms in nutrient solution ranged from 1.8 to 2.4 log MPN/100 ml, and E. coli was detected in 18 samples (range, <0.3 to 1.2 log MPN/100 ml). On bell pepper analyzed during the study, populations (median) of APC, coliforms, and molds were 5.4, 3.6, and 5.8 log CFU per fruit, respectively; E. coli was detected in 5.1% of the samples (range, 0.23 to 1.4 log MPN per fruit). Salmonella was isolated from only one sample (1.6%) of conveyor belt located at the packing area and in four bell pepper samples (3%). L. monocytogenes was not detected. This information could help producers to establish effective control measures to prevent the presence of foodborne pathogens in bell peppers based on a scientific approach.

High-throughput sequencing of microbial communities in Poro cheese, an artisanal Mexican cheese.

The bacterial diversity and structure of Poro cheese, an artisanal food, was analysed by high-throughput sequencing (454 pyrosequencing) in order to gain insight about changes in bacterial communities associated with the cheese-making process. Dairy samples consisting of milk, fermented whey, curd and ripened cheese (during 7 and 60 d) were collected from three manufacturers located in the state of Tabasco, México during dry (March-June) and rainy (August-November) seasons. Independently of producer and season, raw milk samples displayed the highest diversity in bacterial communities. In raw milk, genera found were Macrococcus, Staphylococcus, Enterococcus, Streptococcus, Lactobacillus and Enhydrobacter. Diversity in whey, curd and cheese was lower, principally containing Streptococcus and Lactobacillus; however, bacteria such as Staphylococcus, Acinetobacter, Chryseobacterium, Bacillus, Sediminibacter, Lactococcus and Enterococcus were occasionally present. After curdling step, the most dominant and abundant species were Streptococcus thermophilus and Lactobacillus delbrueckii.

Food antimicrobials nanocarriers.

Natural food antimicrobials are bioactive compounds that inhibit the growth of microorganisms involved in food spoilage or food-borne illness. However, stability issues result in degradation and loss of antimicrobial activity. Nanoencapsulation allows protection of antimicrobial food agents from unfavorable environmental conditions and incompatibilities. Encapsulation of food antimicrobials control delivery increasing the concentration of the antimicrobials in specific areas and the improvement of passive cellular absorption mechanisms resulted in higher antimicrobial activity. This paper reviews the present state of the art of the nanostructures used as food antimicrobial carriers including nanoemulsions, nanoliposomes, nanoparticles, and nanofibers.

New insights into the bactericidal activity of chitosan-Ag bionanocomposite: the role of the electrical conductivity.

The relationship between electrical conductivity, structure and antibacterial properties of chitosan-silver nanoparticles (CS/AgnP) biocomposites has been analyzed. To test the film's antimicrobial activity, Gram-positive and Gram-negative bacteria were studied. The interactions between silver nanoparticles with chitosan suggest the formation of silver ions which plays a major role in nanocomposite's bactericidal potency. In CS/AgnP biocomposites, the bactericide effectiveness increases by increasing AgnP concentrations up to 3 wt%, which is close to the electrical percolation threshold of ca. 3 wt%. As the AgnP concentration increases above this threshold, the bactericidal potency is greatly diminished. The elucidated correlation between electrical conductivity and antibacterial activity could be useful in the design of other nanocomposites that involve polymeric-based matrices.