Antibiotic resistance in the pathogenic foodborne bacteria isolated from raw kebab and hamburger: phenotypic and genotypic study.

BACKGROUND: In recent years, interest in the consumption of ready-to-eat (RTE) food products has been increased in many countries. However, RTE products particularly those prepared by meat may be potential vehicles of antibiotic-resistance foodborne pathogens. Considering kebab and hamburger are the most popular RTE meat products in Iran, this study aimed to investigate the prevalence and antimicrobial resistance of common foodborne pathogens (Escherichia coli, Salmonella spp., Staphylococcus aureus, and Listeria monocytogenes) in raw kebab and hamburger samples collected from fast-food centers and restaurants. Therefore, total bacterial count (TBC), as well as the prevalence rates and antibiogram patterns of foodborne pathogens in the samples were investigated. Also, the presence of antibiotic-resistance genes (blaSHV, blaTEM, blaZ, and mecA) was studied in the isolates by PCR. RESULTS: The mean value of TBC in raw kebab and hamburger samples was 6.72 ± 0.68 log CFU/g and 6.64 ± 0.66 log CFU/g, respectively. E. coli had the highest prevalence rate among the investigated pathogenic bacteria in kebab (70%) and hamburger samples (48%). Salmonella spp., L. monocytogenes, and S. aureus were also recovered from 58, 50, and 36% of kebab samples, respectively. The contamination of hamburger samples was detected to S. aureus (22%), L. monocytogenes (22%), and Salmonella spp. (10%). In the antimicrobial susceptibility tests, all isolates exhibited high rates of antibiotic resistance, particularly against amoxicillin, penicillin, and cefalexin (79.66-100%). The blaTEM was the most common resistant gene in the isolates of E. coli (52.54%) and Salmonella spp. (44.11%). Fourteen isolates (23.72%) of E. coli and 10 isolates (29.41%) of Salmonella spp. were positive for blaSHV. Also, 16 isolates (55.17%) of S. aureus and 10 isolates (27.27%) of L. monocytogenes were positive for mecA gene. CONCLUSIONS: The findings of this study showed that raw kebab and hamburger are potential carriers of antibiotic-resistance pathogenic bacteria, which can be a serious threat to public health.

A cluster of Shiga Toxin-producing Escherichia coli O157:H7 highlights raw pet food as an emerging potential source of infection in humans.

In August 2017, a cluster of four persons infected with genetically related strains of Shiga toxin-producing Escherichia coli (STEC) O157:H7 was identified. These strains possessed the Shiga toxin (stx) subtype stx2a, a toxin type known to be associated with severe clinical outcome. One person died after developing haemolytic uraemic syndrome. Interviews with cases revealed that three of the cases had been exposed to dogs fed on a raw meat-based diet (RMBD), specifically tripe. In two cases, the tripe had been purchased from the same supplier. Sampling and microbiological screening of raw pet food was undertaken and indicated the presence of STEC in the products. STEC was isolated from one sample of raw tripe but was different from the strain causing illness in humans. Nevertheless, the detection of STEC in the tripe provided evidence that raw pet food was a potential source of human STEC infection during this outbreak. This adds to the evidence of raw pet food as a risk factor for zoonotic transmission of gastrointestinal pathogens, which is widely accepted for Salmonella, Listeria and Campylobacter spp. Feeding RMBD to companion animals has recently increased in popularity due to the belief that they provide health benefits to animals. Although still rare, an increase in STEC cases reporting exposure to RMBDs was detected in 2017. There has also been an increased frequency of raw pet food incidents in 2017, suggesting an increasing trend in potential risk to humans from raw pet food. Recommendations to reduce the risk of infection included improved awareness of risk and promotion of good hygiene practices among the public when handling raw pet food.

Escherichia coli O15 : H7 from Food of Animal Origin in Arsi: Occurrence at Catering Establishments and Antimicrobial Susceptibility Profile.

Escherichia coli O157 : H7 (E. coli O157 : H7) has been found to be the major cause of food-borne diseases and a serious public health problem in the world, with an increasing concern for the emergence and spread of antimicrobial-resistant strains. Hitherto, little is known about the carriage of E. coli O157 : H7 and its antimicrobial susceptibility profile in the food of animal origin in Ethiopia. This study aimed to determine the occurrence and multidrug resistance profile of E. coli O157 : H7 from food of animal origin at different catering establishments in the selected study settings of Arsi Zone. One hundred ninety-two animal origin food items, namely, raw/minced meat (locally known as "Kitfo," "Kurt," and "Dulet"), raw milk, egg sandwich, and cream cake samples were collected and processed for microbiological detection of E. coli O157 : H7. Out of 192 samples, 2.1% (4/192) were positive for E. coli O157 : H7. Two E. coli O157 : H7 isolates were obtained from "Dulet" (6.3%) followed by "Kurt" (3.1%, 1/32) and raw milk (3.1%, 1/32), whereas no isolate was obtained from "Kitfo," egg sandwich, and cream cake samples. Of the 4 E. coli O157 : H7 isolates subjected to 10 panels of antimicrobial discs, 3 (75%) were highly resistant to kanamycin, streptomycin, and nitrofurantoin. Besides, all the isolates displayed multidrug resistance phenotypes, 3 to 5 antimicrobial resistance, amid kanamycin, streptomycin, nitrofurantoin, tetracycline, and chloramphenicol. The occurrence of multidrug-resistant E. coli O157 : H7 isolates from foods of animal origin sampled from different catering establishments reveals that the general sanitary condition of the catering establishments, utensils used, and personnel hygienic practices did not comply with the recommended standards. Thus, this finding calls for urgent attention toward appropriate controls and good hygienic practices in different catering establishments dealing with consuming raw/undercooked foods of animal origin.

A comparison between farmed oysters using floating cages and oysters grown on-bottom reveals more potentially human pathogenic Vibrio in the on-bottom oysters.

Eating raw oysters can come with serious health risks, as oysters can potentially contain bacteria of the Vibrio genus that cause food-borne infections. Vibrio bacteria are concentrated by oysters and, when consumed, infections can result with severe symptoms such as diarrhoea, lesions on the extremities, or even death. Vibrio spp. concentrations are strongly affected by season, location, and other factors such as temperature and salinity. Previous research in North Carolina oysters has been conducted on wild and farmed oysters but not at the same time. Farmed, or aquaculture raised, oysters are considerably different from wild oysters and could possibly pose different health risks. Farmed oysters are handled, raised from seed, and often grown using suspended grow-out systems called 'floating cages'. Therefore, farmed oysters can be grown at the surface of the estuary, while wild oysters typically grow at the bottom of the water column. This project compared the concentrations of Vibrio spp. in suspended, farm-grown oysters and wild oysters at three sites, using a paired approach with farmed and wild oysters sampled in proximity. An important part of this comparison was identifying pathogenicity of the bacteria isolated from the samples. Distinction was made between off- and on-bottom farming. Interestingly, on-bottom oysters had more pathogenic V. vulnificus than off-bottom oysters.

Probiotic and technological features of Enterococcus and Weissella isolates from camel milk characterised by an Argane feeding regimen.

The objectives of this study were to isolate lactic acid bacteria (LAB) from a raw Moroccan camel milk collected after the incorporation of a specific Argane by-products diet, and to investigate their technological properties as well as their probiotic features. The molecular identification of the isolates indicated that they belong to Weissella confusa, Weissella cibaria or Enterococcus durans species. Our results revealed that the tested isolates have a fast acidifying ability (values ranging between 0.045 ± 0.01 to 0.93 ± 0.01 after only 4 h incubation), important proteolysis, autolysis, lipolytic activities and an important diacetyl and exopolysaccharides production. All these isolates demonstrated a high tolerance to gastrointestinal conditions, namely to gastric simulated juice (survival rate ranged between 75.05 ± 7.88 and 85.55 ± 1.77%) and to bile salts (survival rate between 42.79 ± 1.11 and 82.75 ± 1.01%). The autoaggregation, hydrophobicity and antioxidant activity mean values of the isolates were 13.26-41.16%, 13.23-54.47% and 47.57-63.31%, respectively. Importantly, LAB cultures exhibited antibacterial activity against Gram-negative and Gram-positive pathogenic bacteria and none of the tested isolates presented antibiotic resistance, haemolytic or DNase activities. This study revealed interesting properties for LAB isolated and supported their utilization as autochthone starters for camel milk fermentation that represent a challenge process. These results presented as well the probiotic potential for a possible human consumption.

Antimicrobial activity of bacteriocins produced by Enterococcus isolates recovered from Egyptian homemade dairy products against some foodborne pathogens.

The increasing mandate for fresh-like food products and the possible hazards of chemically preserved foods necessitate the search for alternatives. Bacteriocins represent a promising food biopreservative. In the present study, one hundred enterococci isolates recovered from Egyptian raw cow milk and homemade dairy products were screened for bacteriocin production. The overall detection rate was 10%. Three isolates, namely, Enterococcus faecalis (OE-7 and OE-12) and Enterococcus hirae (OE-9), showed the highest antibacterial activity with narrow spectrum against multidrug-resistant (MDR) Gram-positive foodborne bacteria: Enterococcus faecalis and Staphylococcus aureus. The antimicrobial activity was completely abolished by trypsin and proteinase K but not affected by lipase and/or amylase indicating the protein nature of the antimicrobial activity. Optimum conditions for bacteriocin production were cultivation in MRS broth at 37 °C, pH 6-6.5 for 16-24 h. The tested bacteriocins exhibited bactericidal activity on S. aureus subsp. aureus ATCC 25923; such activity was further investigated by transmission electron microscopy that revealed leakage and lysis of treated cells. Characterization of tested bacteriocins revealed high activity in a wide range of pH and temperature, storage stability, and heat resistance. PCR analysis revealed that the tested isolates produced multiple enterocins showing homology with the enterocins L50A, AS-48, and 31. Finally, this study reported potent antibacterial activity of bacteriocins derived from dairy products Enterococci against MDR foodborne and spoilage pathogens. The potency, specificity, and stability of these bacteriocins presented promising perspectives for application as biopreservatives in the food industry. The biopreservation of foods by bacteriocins produced by lactic acid bacteria recovered directly from foods remains an innovative approach.

Growth Kinetics and Spoilage Potential of Co-culturing Acinetobacter johnsonii and Pseudomonas fluorescens from Bigeye Tuna (Thunnus obesus) During Refrigerated Storage.

A. johnsonii and P. fluorescens are the well-known specific spoilage organisms in aquatic products and the study of the interactions between A. johnsonii and P. fluorescens are limited. This study aims to evaluate the growth kinetics, spoilage potential and interactions of A. johnsonii and P. fluorescens isolated from spoiled bigeye tuna (Thunnus obesus) by inoculating into sterile fish slices and stored at 4 °C for 6 days. The growth kinetics of A. johnsonii and P. fluorescens were fitted with Baranyi and Roberts model. The chemical indexes (total volatile base nitrogen (TVB-N), trimethylamine (TMA), pH, proteolytic activity and protein content) of each inoculated block of bigeye tuna were increased during refrigerated storage. Moreover, the higher contents of chemical indexes were observed in co-culture with A. johnsonii and P. fluorescens compared with single culture of A. johnsonii and P. fluorescens. In addition, atomic force microscopy (AFM) observation of co-culturing A. johnsonii and P. fluorescens inoculation into sterile fish slices revealed damage of myofibrillar protein structures and the protein degradation. Based on these parameters, a rapid method to evaluate spoilage potential of A. johnsonii and P. fluorescens was positively correlated with TVB-N value, TMA value and pH value (P < 0.05) by the correlation coefficient. Consequently, spoilage potential of microorganisms became stronger evaluated in a mixed culture than single culture. This paper provides insight for a detection method of interactions of A. johnsonii and P. fluorescens during refrigerated storage.

Comparative Effects of Carum copticum Essential Oil on Bacterial Growth and Shiga-Toxin Gene Expression of Escherichia coli O157:H7 at Abused Refrigerated Temperatures.

Abused refrigerated temperatures are described as unacceptable deviations from the optimal temperature, occurring frequently during transportation of food products. Escherichia coli O157:H7 is a serious contaminant of meats and meat products due to its ability to grow at abused temperatures (> 10 °C). The aim of this study was to evaluate the antibacterial activity of Carum copticum essential oil for the control of Escherichia coli O157:H7 using laboratory media and minced beef at severe abused refrigerated temperature (15 °C). A comparative quantitative reverse transcription real-time PCR was used to assess effects of temperature and Carum copticum essential oil at sub-minimum inhibitory concentrations on bacterial growth and Shiga-toxin gene (stx1A and stx2A) expression. Results indicated that Carum copticum essential oil inhibited growth of E. coli O157:H7 in tryptone soy broth (TSB) media at all sub-MIC values until Hour 48. However, bacterial population increased progressively until Hour 72 at essential oil concentration of 0.75% (ml g-1) and reached 8.6 log CFU g-1 in minced beef. The essential oil at concentration of 0.005% (ml g-1) increased stx gene expression at all times, but increased stx gene expression (0.015%) at Hour 24 in TSB media. The expression rate of stx1A in minced beef decreased progressively (10.39 and 7.67 folds for 0.5 and 0.75%, respectively) and expression of stx2A was variable in minced beef during storage. In conclusion, results from this study have shown that effects of Carum copticum essential oil on growth and virulence gene expression are not necessarily correlated and temperature, essential oil concentration, investigated gene type, and bacterial growth environment (in vivo or in vitro) are effective as well.

Prevalence and virulence of Vibrio species isolated from raw shrimp from retail markets in Reynosa, Mexico.

In this study, the prevalence of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio mimicus, Vibrio vulnificus and Vibrio spp. in shrimp from retail markets in Reynosa, Mexico was determined. A total of 765 isolates, identified as Vibrio spp. (59·1%), V. cholerae (17·8%), V. mimicus (6·7%) and V. parahaemolyticus (4·6%), were obtained; V. vulnificus was not detected. Most of the strains were isolated from supermarkets (48·1%), followed by street vendors (37·3%) and retail stores (14·6%). Moreover, several virulence genes were identified in V. cholerae: toxR (100%), OmpU (76·5%), hlyA (76·5%), VPI (19·9%) and tcpA (5·1%); in V. mimicus: vmh (100%), wzb (74·5%), pilF (54·9%), VPI (43·1%), OmpU (29·4%) and tdh (9·8%); and in V. parahaemolyticus: toxR (100%), tlh (100%), VP1680 (51·4%) and VPI (11·4%). These results show the low safety of this food and the potential risk to consumers' health, since this product in Mexican cuisine is sometimes served raw or semi-cooked. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the prevalence of pathogenic Vibrio cholerae, Vibrio mimicus and Vibrio parahaemolyticus isolated from shrimp that is commercialized in Reynosa city. This could represent a risk to consumers' health, since outbreaks related to shrimp contaminated with Vibrio have been previously reported. Additionally, shrimp fishing has a major role in Mexico's economy.

Manila clam (Ruditapes philippinarum) marketed in Korea as a source of vibrios harbouring virulence and ß-lactam resistance genes.

The present study assessed the occurrence, virulence determinants and antimicrobial susceptibility patterns of Vibrio spp. isolated from live Manila clam (Ruditapes philippinarum). A total of 31 Vibrio spp. including 27 V. diabolicus, two V. fluvialis, one V. alginolyticus and one V. antiquarius were isolated and identified. Phenotypic detection of DNase, lipase, phospholipase, amylase and caseinase activities was 100%; and 87% gelatinase, 45% slime production and 6% haemolysin activities were also observed. The prevalence of toxin-related virulence genes for collagenase (94%), toxR (100%), tlh (68%), and VPI (71%) was detected by PCR. Additionally, two V. fluvialis isolates carried F-toxR and hupo genes. Moreover, 61% of the isolates showed multiple antimicrobial resistance indices >0·2. The resistance rates of ampicillin, piperacillin, colistin sulfate, rifampicin, and cephalothin were 100, 81, 71, 77 and 68% respectively. The prevalence of blaCTX-M (87%), blaTEM (55%) and Int1 (90%) genes was observed, whereas blaSHV, strA-strB, tetA, tetB, and aadA2 gene cassette were reported in varying combinations. However, armA, aac(3)-IIa and quinolone resistance genes (qnrA, qnrB, qnrS) were not amplified. Thus, the virulence along with extended-spectrum ß-lactamases (ESBLs) and other antimicrobial resistance genes in multidrug-resistant Manila clam-borne vibrios may pose a public health threat for consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: In Korea, eating raw seafood is considered a great delicacy. This has negatively affected the public by increasing health issues over the years due to the ingestion of vibrios. For the first time, we could identify Vibrio diabolicus and Vibrio antiquarius in marketed Manila clams in Korea. The prevalent Vibrio diabolicus isolates demonstrated the Vibrio parahaemolyticus and Vibrio cholerae homologous virulence genes (toxR, tlh, and VPI). Additionally, the abundance of extended-spectrum ß-lactamases (ESBLs) and integron (IntI1) harboured by Manila clam-borne vibrios elucidate the potential health risk for consumers and may complicate health treatments in the case of infection.

Prevalence of virulence and extended-spectrum ß-lactamase (ESBL) genes harbouring Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea.

The present study aimed to investigate the incidence, virulence and antibiotic properties in Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea. A total of 32 Vibrio spp. isolates including V. parahaemolyticus (n = 4), V. alginolyticus (n = 11), V. diabolicus (n = 14) and V. harveyi (n = 3) were detected using gyrB sequencing. The phenotypic pathogenicity revealed that the DNase, amylase and phospholipase activities were 100%, while lipase, slime production, gelatinase and caseinase were detected in 72, 88, 88 and 81% of the isolates respectively. The PCR amplification for the detection of V. parahaemolyticus species-specific tdh, tlh, trh and toxR genes were positive in 4 (13%), 16 (50%), 0 (0%) and 4 (13%) isolates respectively. The V. alginolytuicus species-specific tdh, tlh, trh, toxR and vac genes were carried by 15 (47%), 29 (91%), 0 (0%), 15 (47%) and 25 (78%) of the isolates respectively. In addition, multidrug resistance was observed by 27 (84%) isolates, whereas higher resistant rates were observed against ampicillin, piperacillin, streptomycin and cephalothin. The occurrence of blaCTX (78%), blaTEM (40%), blaSHV (22%) and aac(6')-Ib (94%) were prevalent, while strAB, tetB, aphAI-IAB, intl1 and aadA1 gene cassettes were also detected. The results signify the potential health risks resulting from the consumption of raw cockles in Korea. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrios are well known to cause human infections following consumption of raw or undercooked seafood. This phenomenon has undoubtedly increased the number of health issues over the past few years in Korea. Among the identified Vibrio spp., we could detect V. diabolicus and V. harveyi for the first time in marketed cockles in Korea. The presence of species-specific genes (tdh-VA, tlh-VP, tlh-VA and toxR-VA) in V. diabolicus exhibits the close genetic affinity among V. parahaemolyticus and V. alginolyticus. Furthermore, the prevalence of extended-spectrum ß-lactamases (ESBLs) and other antibiotic resistance genes along with multidrug resistance signifies the potential threat for consumers.

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk.

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S-23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated. The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6. Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers' health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.

The effects of varied food acid ratios and egg components on Salmonella Typhimurium culturability from raw egg-based sauces.

Raw egg-based sauces, such as mayonnaise and aioli, are frequently identified as sources of Salmonella during outbreaks of human cases of foodborne gastrointestinal disease. In this study, we surveyed aioli and mayonnaise recipes from different popular food websites to identify potential risk factors that may lead to the survival of Salmonella Typhimurium. In laboratory experiments, different ratios of food acids were used to determine if lemon juice, vinegar, or a combination of both restricted Salmonella Typhimurium culturability. We found that as long as the pH was below 4.2, bacterial culturability was limited. The use of whole egg alone or in combination with egg yolk was also investigated. Sauce preparations containing whole egg exhibited higher pH and supported Salmonella Typhimurium culturability longer than those containing yolk only. Ten restaurant prepared sauces were also obtained to further characterize the effect of preparation variability. Sauce preparations with a pH ≤ 3.8 did not support bacterial culturability after 4 h incubation at any temperature. The higher the pH the longer Salmonella Typhimurium remained culturable. Based on this study, it is recommended that raw egg-based foods are acidified, then stored at room temperature for at least 4 h prior to consumption.

Use of indicator bacteria for monitoring sanitary quality of raw milk cheeses - A literature review.

Many countries use Escherichia coli and coliforms as indicators of sanitary quality of foods and have set limits for cheeses, including raw-milk cheeses. This paper reviewed the scientific literature for E. coli and coliform levels that are found in different types of raw milk, the fate of indicators during the manufacturing and ripening of different cheeses and the indicator levels that have been found in the finished cheeses. These studies from worldwide showed that E. coli and coliforms are found in different types of raw milk but usually at <100 CFU/ml or not found. Instances where raw milk contained indicator levels >1000 CFU/ml have mostly been attributed to unsanitary conditions/production. During cheese-making, indicators present in raw milk will often increase in numbers, but the levels decline as the acidity from lactose fermentation decreases the pH. Except for fresh cheeses that are not aged, indicator levels are further reduced by 2-3 log10 CFU/g or more, during the ripening process. As a result, indicator levels in finished cheeses are often low and within the limits of <10 or <100 CFU/g set by many countries. The cited studies also show that raw milk cheeses that are made with quality raw milk, under hygienic conditions and properly aged, should not contain high levels of indicator bacteria in the final product.

Occurrence and Antimicrobial Susceptibility Profile of Salmonella spp. in Raw and Ready-To-Eat Foods and Campylobacter spp. in Retail Raw Chicken Meat in Transylvania, Romania.

The survey was undertaken to investigate the presence and antimicrobial susceptibility profile of Salmonella spp. in raw and ready-to-eat (RTE) foods, and Campylobacter spp. in the retail raw chicken meat collected in two counties of Transylvania, Romania. A total of 13.1% (51/388) of the examined food samples were found to be Salmonella positive, with a distribution of 14.7% (48/326) in the raw food (i.e., pork, chicken carcass, and shell egg) and 4.8% (3/62) in the RTE samples (i.e., sausages, but not ham and salami), respectively. These differences were statistically significant (p = 0.034). The isolates were serotyped as Salmonella Infantis (n = 19), Salmonella Typhimurium (n = 11) Salmonella Rissen (n = 8), Salmonella Derby (n = 3), Salmonella Enteritidis (n = 3), Salmonella Bredeney (n = 2), Salmonella Brandenburg (n = 1), Salmonella Gloucester (n = 1), Salmonella Goldcoast (n = 1), Salmonella Kottbus (n = 1), and Salmonella Ruzizi (n = 1). Campylobacter strains were present in 29.4% (10/34) of the investigated chicken samples, and the identified species were Campylobacter coli (70%) and C. jejuni (30%). From the 14 tested antimicrobials, the Salmonella isolates were resistant against azithromycin (88.2%), tetracycline (54.9%), sulfamethoxazole (54.9%), ciprofloxacin (45.1%), nalidixic acid (43.1%), ampicillin (35.3%), chloramphenicol (33.3%), tigecycline (25.5%), cefotaxime (13.7%), colistin (13.7%), trimethoprim (7.8%), and gentamicin (2%), resulting in the expression of 21 multidrug-resistant (MDR) profiles. Of 10 Campylobacter isolates, 80% were resistant to ciprofloxacin and nalidixic acid, 40% to tetracycline, and 10% to streptomycin and erythromycin, respectively. Our findings indicate that Romanian isolates of Salmonella spp. and Campylobacter spp., contaminating animal-origin foods, can exhibit MDR patterns, representing a public health risk.

Chicken Meat-Associated Enterococci: Influence of Agricultural Antibiotic Use and Connection to the Clinic.

Industrial farms are unique, human-created ecosystems that provide the perfect setting for the development and dissemination of antibiotic resistance. Agricultural antibiotic use amplifies naturally occurring resistance mechanisms from soil ecologies, promoting their spread and sharing with other bacteria, including those poised to become endemic within hospital environments. To better understand the role of enterococci in the movement of antibiotic resistance from farm to table to clinic, we characterized over 300 isolates of Enterococcus cultured from raw chicken meat purchased at U.S. supermarkets by the Consumers Union in 2013. Enterococcus faecalis and Enterococcus faecium were the predominant species found, and antimicrobial susceptibility testing uncovered striking levels of resistance to medically important antibiotic classes, particularly from classes approved by the FDA for use in animal production. While nearly all isolates were resistant to at least one drug, bacteria from meat labeled as raised without antibiotics had fewer resistances, particularly for E. faecium Whole-genome sequencing of 92 isolates revealed that both commensal- and clinical-isolate-like enterococcal strains were associated with chicken meat, including isolates bearing important resistance-conferring elements and virulence factors. The ability of enterococci to persist in the food system positions them as vehicles to move resistance genes from the industrial farm ecosystem into more human-proximal ecologies.IMPORTANCE Bacteria that contaminate food can serve as a conduit for moving drug resistance genes from farm to table to clinic. Our results show that chicken meat-associated isolates of Enterococcus are often multidrug resistant, closely related to pathogenic lineages, and harbor worrisome virulence factors. These drug-resistant agricultural isolates could thus represent important stepping stones in the evolution of enterococci into drug-resistant human pathogens. Although significant efforts have been made over the past few years to reduce the agricultural use of antibiotics, continued assessment of agricultural practices, including the roles of processing plants, shared breeding flocks, and probiotics as sources for resistance spread, is needed in order to slow the evolution of antibiotic resistance. Because antibiotic resistance is a global problem, global policies are needed to address this threat. Additional measures must be taken to mitigate the development and spread of antibiotic resistance elements from farms to clinics throughout the world.

Did the ban on serving raw beef liver in restaurants decrease Enterohemorrhagic Escherichia coli infection in Japan?: an interrupted time-series analysis.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) is an important pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). After an EHEC outbreak involving uncooked beef, serving raw beef liver dishes at restaurants was completely banned starting on July 1, 2012 in Japan. However, its long-term associations with the incidence rates of EHEC infections have never been assessed by formal interrupted time-series analysis (ITSA). METHODS: A retrospective cohort study to assess the impact of banning raw beef liver provision at restaurants was conducted. The weekly incidence of asymptomatic and symptomatic EHEC infections, the incidence of HUS, and deaths were extracted from the national reportable diseases database from January 2008 to December 2017. ITSA was conducted to evaluate the impact of banning raw beef liver from July 2012. To account for a potential simultaneous external effect, the additional regulation on raw beef red meat handling (implemented in May 2011) and the seasonality were also incorporated into the model. RESULTS: There were 32,179 asymptomatic and 21,250 symptomatic EHEC infections (including 717 HUS cases and 26 deaths) reported during the study period. During the pre-intervention period (before week 27, 2012), there were 0.45 asymptomatic EHEC infections per million-persons per week. The mean post-intervention asymptomatic EHEC infections were 0.51 per million-persons per week. ITSA revealed no baseline trend or change in the intercept and trend (0.002 infections per million-persons per week, 95% Confidence interval - 0.03-0.04, p = 0.93, 1.22, CI -1.96-4.39, p = 0.45, and - 0.006, CI -0.003-0.02, p = 0.68, respectively). For symptomatic EHEC infections, there were 0.30 cases per million per week during the pre-intervention period, and it became 0.33 cases per million per week after the intervention. Time series modeling again did not show a significant baseline trend or changes in the intercept and trend (0.0005, CI -0.02-0.02, p = 0.96, 0.69, CI -1.75-3.12, p = 0.58, and - 0.003, CI -0.02-0.01, p = 0.76, respectively). CONCLUSION: We did not find a statistically significant reduction in the overall incidence rates of both asymptomatic and symptomatic EHEC infections in Japan after implementing measures, including a ban on serving raw beef liver dishes in the restaurant industry.

Suitability of centrifuge water for detecting the presence of Escherichia coli versus finished fresh-cut lettuce testing.

Fresh produce causes most foodborne outbreaks in the USA, and it is also considered a hazardous food product in other areas of the world such as Europe. The outbreaks attributed to fresh produce increase the focus of producers on hygiene to minimize exposure to food hazards. The fresh produce industry has the urgent need to detect if there are production lots contaminated with pathogenic microorganisms before distribution. Although the industry is mostly using end-product testing for the detection of target microorganisms, previous studies have evaluated the suitability of different sampling points within the production line of a fresh-cut processing plant. In the present study, the centrifuge effluent water was assessed as an alternative sampling point to end-product testing. E. coli was selected as an index microorganism of the presence of pathogens. The presence of E. coli was assessed in centrifuge effluent water, and fresh-cut lettuce from a commercial fresh-cut produce processing line (n = 95). The rate of false positives and negatives, as well as the specificity, sensitivity, and efficiency of the alternative method were calculated. The mean population of E. coli in positive water samples was 0.86 log cfu/100 mL, while the mean population of E. coli in positive fresh-cut lettuce samples was 0.23 log cfu/g. The proportion of positive samples in centrifuge effluent water and lettuce was similar (≈20%), and most of the results in both matrices were coincident (81.1%). However, the alternative method was not reliable due to its low sensitivity, as only 47.6% of the lettuce samples positive for E. coli could be matched with positive water samples.

Evaluation of storage conditions and efficiency of a novel microencapsulated Salmonella phage cocktail for controlling S. enteritidis and S. typhimurium in-vitro and in fresh foods.

S. Enteritidis and S. Typhimurium are typically linked to foodborne outbreaks. Phages have continued to expand in various food applications. In this study, microencapsulation is applied for enhancing the stability and efficacy of phages as bio-control agent. Microencapsulated phage cocktail kept in aluminium laminated foil bag (LF) at 4 °C showed the highest survivability with a titer loss of 0.5 log PFU/g after 12 weeks of storage. Titer loss of phage cocktail lysate >4 log PFU/mL was observed after 12 weeks, at 4 °C. Color change of microencapsulated phage cocktail kept in LF at 4 °C did not show any significant difference during storage, and water activity (free water content) at 0.13 was found in these conditions. In-vitro study, S. Enteritidis and S. Typhimurium were decreased 1.79 and 3.63 log CFU/mL, respectively at 37 °C. Whereas, 0.43 and 0.76 log CFU/mL, respectively were observed at 10 °C. In foods, S. Enteritidis and S. Typhimurium were decreased 0.57 and 1.78 log CFU/cm2, respectively in meat. Whereas, 0.86 and 1.2 log CFU/g, respectively were observed in sprout. Foods with/without microencapsulated phage cocktail showed non-significant differences in liking scores after 2 days of storage. Overall, microencapsulated phage cocktail suggests another alternative for phage-based biocontrol with improved stability and efficacy for food application.

Variation of microbial load and biochemical activity of ready-to-eat salads in Cyprus as affected by vegetable type, season, and producer.

Fresh vegetables are important components of an everyday balanced diet making ready to-eat-salads (RTE) a commodity widely consumed. However, in the past few years these products have been linked with outbreaks of salmonellosis and listeriosis; thus the continuous investigation of their safety is an essential requirement. A total of 216 samples of ready-to-eat salads from the Cypriot market were analysed to determine the microbiological quality and safety, along with physicochemical attributes of the salads and identify possible correlations between them. The samples were randomly collected from four retail outlets and correspond to five different salad producing companies. Furthermore, the effects of season, salad producer and type of salad and/or their interactions with the tested parameters were investigated. The results revealed that the higher microbial load among seasons was observed in samples collected during spring. Escherichia coli was found in 11.57% of samples and 2.62% of isolates were found to be able to produce extended spectrum ß-lactamase (ESBL). All samples were found negative for Salmonella enterica, whereas Listeria monocytogenes was present in 3.70% of samples. Higher levels of spoilage bacteria (lactic acid bacteria and Pseudomonas spp.) were detected during winter and spring. Additionally, the %CO2 production was affected by the type of salad, while the interaction between producer and type of salad, affected total phenolic content and antioxidant activity of samples. A positive correlation of phenols and antioxidants with the presence of Staphylococcus spp., Pseudomonas spp., E. coli and Bacillus cereus was observed, suggesting that excessive handling increases microbial load and plant stress.