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.

Effect of storage temperature and time on the behavior of Salmonella, Listeria monocytogenes, and background microbiota on whole fresh avocados (Persea americana var Hass).

Avocados are popular fruits; however, contamination of whole fresh avocados and avocado products with foodborne pathogens has raised concern about their safety. Recalls and import alerts of avocado products due to contamination with Listeria monocytogenes cause important economic losses. The behavior of Salmonella, L. monocytogenes, and background microbiota on whole fresh avocados at 5 and 25 °C as affected by temperature and time of storage was investigated. Whole fresh avocados were inoculated by immersion in suspensions containing six rifampicin-resistant strains of Salmonella or L. monocytogenes, and stored at 5 °C for 48 d, or at 25 °C for 11 d. At selected sampling times, avocados were removed from storage and pathogens enumerated. The log counts of both pathogens at each temperature were fitted to the Weibull distribution nonlinear model to estimate kinetic parameters including the time for the first 1-log reduction (δ), the shape of the curve (ρ), and the time for two (2-D) and three (3-D) log reductions. Salmonella and L. monocytogenes initial populations (approx. 7 log CFU/avocado) decreased during storage at 5 and 25 °C; L. monocytogenes mean counts were higher than those observed for Salmonella (P < 0.05). L. monocytogenes showed a lower rate of decline at 5 °C when compared to Salmonella. In general, the ability of both pathogens to survive on the surface of avocados stored at room temperature was similar. Salmonella and L. monocytogenes counts decline over time on the epicarp of whole avocados; however, if the initial number of cells is large enough, the pathogens could be present for large periods of time. Simultaneously, psychrotrophic microorganisms (PM), aerobic plate count (APC), coliforms (C) and yeasts/molds (Y/M) were enumerated from non-inoculated avocados stored at 5 and 25 °C. Initial mean counts for PM, APC, C and Y/M ranged from 6.1 to 6.6 log CFU/avocado and showed no change (P > 0.05) during storage at both temperatures. Good agricultural and handling practices from farm to fork are crucial to prevent or minimize contamination of whole avocados; otherwise, if large numbers of pathogens contaminate the fruit, they could survive and be transferred to the pulp, or to other ready to eat foods, representing a risk for consumers.

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.

Thermal and Chemical Treatments To Reduce Salmonella on Alfalfa (Medicago sativa) and Broccoli (Brassica oleracea var. italica) Seeds before and during the Sprouting Process: A Hurdle Approach.

Sprouts are vehicles of foodborne diseases caused by pathogens such as Salmonella. The aim of this study was to evaluate thermal and chemical treatments applied as a hurdle approach to reduce Salmonella in alfalfa (Medicago sativa) and broccoli (Brassica oleracea var. italica) seeds before and during their germination. Seeds, inoculated and then dried at 55°C for 48 h, were subjected to a chemical treatment and a thermal shock with (i) 75 mM caprylic acid at 70°C for 5 s, (ii) 0.04% CaO at 70°C for 5 s, or (iii) 1% H2O2 at 70°C for 5 s. After each treatment, seeds were immersed in water at 3°C for 5 s. Next, the imbibition process was carried out with 0.016% H2O2 at pH 3.0. Finally, the seeds were transferred to a rotary drum-type germinator and were sprayed with the same chemical solution that was applied before the imbibition process, for 20 s at intervals of 5 min for 40 min at 3 rpm. All chemical treatments reduced Salmonella at least 5 log CFU/g on both seeds. Germination rates between 90 and 93% were obtained after application of thermal and chemical treatments. Salmonella was not detected after the imbibition stage when caprylic acid and H2O2 treatments were applied. However, during the germination process of both seeds, Salmonella counts of >6 log CFU/g were obtained despite all treatments being applied at different stages of the sprouting process. These results demonstrated that thermal and chemical treatments used as a hurdle approach to control Salmonella on alfalfa and broccoli seeds significantly reduced the pathogen concentration on seeds >5 log but were ineffective to eliminate Salmonella and to control its growth during the sprouting process. The production of safe sprouts continues to be a major challenge for industry.

Salmonella, Listeria monocytogenes, and Indicator Microorganisms on Hass Avocados Sold at Retail Markets in Guadalajara, Mexico.

Hass avocados may become contaminated with Salmonella and Listeria monocytogenes at the farm and the packing facility or later during transportation and at retail. In Mexico, avocados are frequently sold in bulk at retail markets, where they are stored at room temperature for several hours or days and exposed to potential sources of microorganisms. These conditions may favor the entry, adhesion, survival, and biofilm formation of Salmonella and L. monocytogenes. The aim of this study was to determine the occurrence of Salmonella, L. monocytogenes, and other Listeria species and the levels of indicator microorganisms on the surface of avocados sold at retail markets. A total of 450 samples (Persea americana var. Hass) were acquired from retail markets located in Guadalajara, Mexico. One group of 225 samples was evaluated for the presence of Salmonella and for enumeration of aerobic plate counts, yeasts and molds, Enterobacteriaceae, coliforms, and Escherichia coli. The other 225 samples were processed for isolation of L. monocytogenes and other Listeria species. Microbial counts (log CFU per avocado) were 4.3 to 9.0 for aerobic plate counts, 3.3 to 7.1 for yeasts and molds, 3.3 to 8.2 for Enterobacteriaceae, 3.3 to 8.4 for coliforms, and 3.3 to 6.2 for E. coli. Eight samples (3.5%) were positive for Salmonella. Listeria spp. and L. monocytogenes were detected in 31 (13.8%) and 18 (8.0%) of 225 samples, respectively. Listeria innocua, Listeria welshimeri, and Listeria grayi were isolated from 7.6, 1.3, and 0.9% of samples. These results indicate that avocados may carry countable levels of microorganisms and could be a vehicle for transmission of Salmonella and L. monocytogenes.

Genetic diversity and antimicrobial resistance of Salmonella serotypes recovered throughout the beef production chain and from patients with salmonellosis.

Salmonella is one of the major foodborne pathogens worldwide. The antimicrobial resistance (AMR) of this foodborne pathogen has raised a great concern in recent years. Studies on the frequency and characterization of Salmonella serotypes can help to improve our knowledge on the epidemiology of this pathogen. The purpose of this study was to compare the serotypes, AMR and genetic profiles of Salmonella isolates recovered from raw beef throughout the beef production chain and from human feces associated with clinical cases of salmonellosis. The serotype, AMR and pulsed-field gel electrophoresis profile of 243 Salmonella enterica isolates recovered from beef carcasses (n = 78), ground beef (n = 135), and human feces from clinical cases of salmonellosis (n = 30) were compared. Forty-three different Salmonella serotypes were identified and regardless of the source, the top five corresponded to Typhimurium, Give, Group B (partially serotyped), Infantis and Anatum. Twelve serotypes from beef carcasses were also found in ground beef, showing their presence throughout the beef production chain. Salmonella Typhimurium, Infantis, Anatum and Montevideo were the only serotypes identified in all sample types. Resistance to tetracyclines was the most frequent (41.2%) followed by resistance to aminoglycosides (37%), folate pathway inhibitors (21%), quinolones (20.2%), phenicols (17.1%), penicillins (15.6%) and cephems (7%). Multidrug resistance was observed in 28.8% of the isolates, and those from human feces showed resistance to a larger number of antimicrobials. Great concern arises from the resistance and reduced susceptibility observed to quinolones and cephalosporins because these drugs are the first line of treatment for invasive Salmonella infections. Twenty-seven distinct pulse-types were detected among 238 isolates. Clustering analysis for the most frequent serotypes identified groups of isolates with similar AMR profiles. Multidrug resistance spreading throughout the food production chain should be continually monitored and its importance emphasized.

Occurrence, serotype diversity, and antimicrobial resistance of salmonella in ground beef at retail stores in Jalisco state, Mexico.

The occurrence, serotype diversity, and antimicrobial resistance of Salmonella bacteria in commercial ground beef at retail establishments were investigated. Salmonella was isolated from 135 (56.7%) of 238 ground beef samples collected at the same number of butcher's shops located in three municipalities of Jalisco State, Mexico, during an 11-month period. The isolation frequency differed by municipality (P < 0.05) and was higher (P < 0.05) during the warm season (68.5%) than during the cold season (43.2%). Overall, 25 serotypes and 8 serogroups were identified among 135 Salmonella isolates; predominant were Salmonella group B (9.6%), Salmonella Anatum (8.9%), Salmonella Agona (6.7%), Salmonella Infantis (6.7%), and Salmonella Typhimurium (5.9%). All Salmonella isolates were tested for susceptibility to 11 antimicrobial drugs of human and veterinary use. Resistance to tetracycline was the most commonly observed (40.7%), followed by resistance to streptomycin (35.6%), trimethoprim-sulfamethoxazole (20.7%), and nalidixic acid (19.3%). Thirty-seven Salmonella isolates (27.4%) were multidrug resistant, and the majority corresponded to Salmonella Group B, Salmonella Anatum, and Salmonella Typhimurium. Three Salmonella isolates were resistant to seven different antimicrobials. The frequency of Salmonella in ground beef samples (56.7%) was higher than that observed in our previous investigation on beef carcasses (15.4%) at small abattoirs in the same region of Mexico. This may be a result of increasing contamination at these two points of the raw-beef production chain or may be an effect of the grinding process that facilitates a more-homogeneous pathogen distribution in the product. Poor hygiene, temperature abuse, and practices allowing cross-contamination during ground beef fabrication at these retail establishments increase the consumer's exposure to Salmonella.

Frequency of Salmonella and Listeria monocytogenes in five commercial brands of chicken eggs using a combined method of enrichment and nested-PCR.

Eggs or egg-based foods, either raw or undercooked, have been identified as vehicles of Salmonella outbreaks. The low numbers of Salmonella organisms in eggs makes it difficult to detect them in frequency studies. The nested-PCR (n-PCR) technique shows more sensitivity and specificity than bacteriological culture methods (BCMs). A preenrichment method followed by enrichment and n-PCR is a good alternative for the investigation of Salmonella and Listeria monocytogenes in eggs. A total of 2,650 chicken eggs representing five commercial brands were purchased from 10 grocery stores. Ten eggs of each brand were combined in order to obtain 265 pooled samples (53 per brand). The shells and yolks of 100 pooled samples were analyzed for Salmonella, while the shells of 65 pooled samples were analyzed for L. monocytogenes, using BCM and a combined method of enrichment and n-PCR (CM-n-PCR). Sixteen eggshell pooled samples tested positive for Salmonella by CM-n-PCR, compared with only two by BCM. Three egg yolk pooled samples tested positive for this pathogen by CM-n-PCR; none tested positive by BCM. Three eggshell pooled samples tested positive for L. monocytogenes by CM-n-PCR and none by BCM. In Mexico, as in other countries, official methods for detection of Salmonella and L. monocytogenes in foods are based on standard bacteriological culture techniques. The inclusion of more sensitive methods such as the one used in the present investigation would increase the probability of detecting positive samples, particularly in those foods in which a very low number of cells is expected.

Frequency and antimicrobial resistance of Salmonella serotypes on beef carcasses at small abattoirs in Jalisco State, Mexico.

The prevalence and antimicrobial resistance of Salmonella serotypes on beef carcasses from four small abattoirs in Jalisco State, Mexico, were investigated during a 10-month period. Following U.S. Department of Agriculture Food Safety and Inspection Service protocols, Salmonella was isolated from 78 (15.4%) beef carcasses (n = 505) after the final carcass water wash. Isolation frequency differed by establishment (P < 0.05) and was higher (P < 0.05) during the wet season (May through September) for all establishments. Thirteen Salmonella serotypes and four serogroups (partially serotyped isolates) were identified. The most prevalent were Salmonella enterica Give (24.4%), Salmonella Typhimurium (17.9%), and Salmonella Group B (14.1%). Antimicrobial susceptibility was tested against 11 drugs, and results indicated that 46.2% of the isolates were resistant to tetracycline, 42.3% were resistant to streptomycin, 23.1% were resistant to chloramphenicol, 21.8% were resistant to trimethoprim-sulfamethoxazole, and 19.2% were resistant to gentamicin. No resistance to ceftriaxone or ciprofloxacin was observed, and 33% of the isolates were resistant to three or more antimicrobials. Although Salmonella Give was the most prevalent serotype, 95% of the isolates of this serotype were susceptible to all antimicrobials tested. Antimicrobial resistance was more common in Salmonella Typhimurium, and 93% (13 of 14) of the isolates of this serotype were resistant to at least five antimicrobials. The frequency of multidrug-resistant Salmonella isolates differed among establishments (P < 0.05) and may be related to the origin of the cattle presented for harvesting. These findings highlight the need for control measures to reduce Salmonella prevalence on beef carcasses in small abattoirs in Mexico and for strategies to ensure the cautious use of antimicrobials in animal production to prevent and control the spread of antimicrobial-resistant foodborne pathogens.

Fluorescent protein-marked Escherichia coli biotype I strains as surrogates for enteric pathogens in validation of beef carcass interventions.

The efficacy of antimicrobial interventions implemented in slaughter establishments to reduce enteric pathogens on beef carcasses should optimally be validated under commercial operation conditions. This study was conducted to identify surrogate organisms for enteric pathogens that could be used to validate beef carcass interventions. The growth, resistance, and attachment properties of nonpathogenic fluorescent protein-marked Escherichia coli strains were compared with those of E. coli O157:H7 and Salmonella strains. Growth curves were obtained based on growth in tryptic soy broth at 37 degrees C. In general, growth parameters were not different among potential surrogates and target pathogens (P > 0.05). Thermal resistance was compared in phosphate-buffered saline (pH 7.4) at 55, 60, and 65 degrees C, and D-values of potential surrogates were not different (P > 0.05) or were higher (P < 0.05) than those of the target pathogens. Acid resistance was tested in phosphate-buffered saline acidified with L-lactic acid at pH 2.5, 3.0, and 3.5, and log reductions (CFU per milliliter) were not different (P > 0.05) among potential surrogates and E. coli O157:H7 strains; however, some Salmonella serotypes were less acid resistant than were surrogates (P < 0.05). The cell surface hydrophobicity was different (P < 0.05) among surrogates and some E. coli O157:H7 strains, but the strength of attachment to beef carcasses was not different (P > 0.05) among all microorganisms. Log reductions (CFU per square centimeter) after application of hot water washes and 2% L-lactic acid sprays on beef carcasses were not different (P > 0.05) among surrogates and pathogens. The nonpathogenic E. coli strains evaluated in this study could be used as surrogates for E. coli O157:H7 and Salmonella to validate hot water and lactic acid interventions on beef carcasses.

Effects of nitroethane and monensin on ruminal fluid fermentation characteristics and nitrocompound-metabolizing bacterial populations.

Nitroethane is a potent inhibitor of ruminal CH 4 production, a digestive inefficiency resulting in the loss of 2-15% of a ruminant's gross energy intake and an important emission source of this greenhouse gas. To assess the effect of nitroethane on methanogenesis and characterize ruminal adaptation observed with low treatment doses to this inhibitor, ruminal microbes were cultured in vitro with supplements of water (controls), 4.5 and 9 mM nitroethane, and 0.09 mM monensin, with or without 9 mM nitroethane. All treatments decreased CH 4 production >78% compared to controls; however, differential effects of treatments were observed on CO 2, butyrate isobutyrate, and valerate production. Treatments did not affect H 2 accumulation or acetate and propionate production. Most probable numbers of nitrometabolizing bacteria were increased with 4.5 and 9 mM nitroethane compared to numbers recovered from controls or monensin-containing treatments, which may explain ruminal adaptation to lower nitroethane treatments.

Reduction of Escherichia coli O157:H7 and Salmonella on baby spinach, using electron beam radiation.

The effect of low-dose electron beam (e-beam) radiation on the reduction of Escherichia coli O157:H7 and Salmonella in spinach was studied. Fresh baby spinach (Spinacia oleracea) was inoculated with a bacterial cocktail containing multiple strains of rifampin-resistant E. coli O157:H7 and rifampin-resistant Salmonella. Inoculated samples were exposed to e-beam radiation from a linear accelerator and tested for counts of both E. coli O157:H7 and Salmonella. Irradiated spinach was also stored for 8 days at 4 degrees C, and counts were made at 2-day intervals to determine if there was any effect of radiation on the survival trend of both pathogens. When no pathogens were detected on plates, additional enrichment plating was conducted to verify total destruction. Respiration rates were measured on spinach samples exposed to e-beam radiation. Each dose of e-beam radiation significantly reduced the numbers of E. coli O157:H7 and Salmonella from initial levels of 7 log CFU/g. Treatment by e-beam radiation at a dose of 0.40 kGy resulted in a reduction in populations of E. coli O157:H7 and Salmonella of 3.7 and 3.4 log cycles, respectively. At 0.70 kGy, both pathogens were reduced by 4 log. All doses above 1.07 kGy showed reductions greater than 6 log and decreased to undetectable levels when stored for 8 days. The respiration rate of spinach showed no changes after irradiation up to 2.1 kGy. These results suggest that low-dose e-beam radiation may be a viable tool for reducing microbial populations or eliminating E. coli O157:H7 and Salmonella from spinach without product damage.