Prevalence of Salmonella Typhimurium and Salmonella Enteritidis isolated from poultry meat: virulence and antimicrobial-resistant genes.

Salmonellosis, a zoonotic disease, is one of the leading causes of foodborne illness worldwide. It is responsible for most infections caused by consumption of contaminated food. In recent years, a significant increase in the resistance of these bacteria to common antibiotics has been observed, posing a serious threat to global public health. The aim of this study was to investigate the prevalence of virulent antibiotic-resistant Salmonella spp. strains in Iranian poultry markets. A total of 440 chicken meat samples were randomly selected from meat supply and distribution facilities in Shahrekord and tested for bacteriological contamination. After culturing and isolating the strains, identification was performed using the classical bacteriological method and PCR. To determine antibiotic resistance, a disc diffusion test was performed according to the recommendations of the French Society of Microbiology. PCR was used to detect resistance and virulence genes. Only 9% of the samples were positive for Salmonella. These were Salmonella typhimurium isolates. All Salmonella typhimurium serotypes tested positive for the rfbJ, fljB, invA and fliC genes. Resistance to TET, cotrimoxazole, NA, NIT, piperacillin/tazobactam and other antibiotics was found in 26 (72.2%), 24 (66.7%), 22 (61.1%) and 21 (58.3%) isolates, respectively. The sul1, sul2 and sul3 genes were present in 20, 12 and 4 of 24 cotrimoxazole-resistant bacteria, respectively. Chloramphenicol resistance was found in six isolates, but more isolates tested positive for the floR and cat two genes. In contrast, 2 (33%) of the cat three genes, 3 (50%) of the cmlA genes and 2 (34%) of the cmlB genes were all positive. The results of this investigation showed that Salmonella typhimurium is the most common serotype of the bacterium. This means that most of the antibiotics commonly used in the livestock and poultry industries are ineffective against most Salmonella isolates, which is important for public health.

Investigation of Virulence and Antibiotic-Resistance of Bacillus cereus Isolated from Various Spices.

Spices and herbs are potential vectors for virulent and pathogenic micro-organisms, which cause illness in consumers, contribute to spoilage, and reduce the durability of foodstuffs. The present study aims to provide relevant data about virulence and antibiotic resistance of Bacillus cereus isolated from various spices. A total of 200 samples of 8 types of spices (black pepper, chilli, white pepper, cumin, cinnamon, turmeric, curry powder, and sumac) were collected from various markets, retail shops, and sucuk production premises located in the Isfahan province of Iran. Presumptive B. cereus strains were obtained using Bacara Agar plates after enrichment in saline peptone water and final colonies were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enterotoxin (HBL) and nonhaemolytic enterotoxin (NHE) production were assessed using the Duopath® Cereus Enterotoxins Test kit. The Kirby-Bauer disc diffusion method was applied as antibiotics susceptibility test. PCR was used to detect Emetic toxin gene (CES and CER) and enterotoxigenic toxin gene (cytK, nheA, hblC, and entFM). Results show a significant prevalence of B. cereus (42%) in spices. However, the spices meet food safety recommendations (<104 cfu/g). Antibiotics susceptibility test show alarming rate of resistance to beta-lactam antibiotics specially ampicillin (83.33%) and penicillin (82.14%). Concerning the toxin producing capacity more than half of the isolates (51.19%) produce NHE toxin and 27.38% produce HBL toxin. The most abundant gene were nheA, nheB, and nheC and a combination of 4 genes (entFM, nheA, hblC, and cytK) was detected in many isolates. In conclusion, the presence of multidrug resistant B. cereus strains carrying diarrhoeal toxin-encoding genes in spices intended for human consumption represents a serious health hazard. These results indicate the need for regular surveillance of the occurrence of B. cereus strains in spices and food products in Iran.

Helicobacter pylori Strains Isolated from Raw Poultry Meat in the Shahrekord Region, Iran: Frequency and Molecular Characteristics.

Even though Helicobacter pylori (H. pylori) is a serious pathogen, its origin is unknown. Poultry (chicken, turkey, quail, goose, and ostrich) is consumed as a regular protein source by many people across the world; therefore, sanitary ways of delivering poultry for food are important for global health. As a result, the distribution of the virulence genes cagA, vacA, babA2, oipA, and iceA in H. pylori isolates in poultry meat, as well as their antibacterial resistance, was investigated. A Wilkins Chalgren anaerobic bacterial medium was used to cultivate 320 samples of raw poultry meat. Disk diffusion and multiplex-PCR were used to investigate both antimicrobial resistance and genotyping patterns. H. pylori was found in 20 of 320 (6.25 %) raw chicken meat samples. The highest incidence of H. pylori was found in chicken raw meat (15%), whereas no isolate was recovered from goose or quail raw meat (0.00%). Resistance to ampicillin (85%), tetracycline (85%), and amoxicillin (75%) were the most commonly detected in the tested H. pylori isolates. The percentage of H. pylori isolates with a multiple antibiotic resistance (MAR) index value of more than 0.2 was 17/20 (85%). The most prevalent genotypes detected were VacA (75%), m1a (75%), s2 (70%) and m2 (65%), and cagA (60%). The most typically detected genotype patterns were s1am1a (45 %), s2m1a (45 %), and s2 m2 (30%). babA2, oipA+, and oipA- genotypes were found in 40%, 30%, and 30% of the population, respectively. In summary, fresh poultry meat was polluted by H. pylori, with the babA2, vacA, and cagA genotypes being more prevalent. The simultaneous occurrence of vacA, cagA, iceA, oipA, and babA2 genotypes in antibiotic-resistant H. pylori bacteria raises a serious public health concern regarding the consumption of raw poultry. Future research should evaluate antimicrobial resistance among H. pylori isolates in Iran.

Examining the Extent of Contamination, Antibiotic Resistance, and Genetic Diversity of Clostridioides (Clostridium) difficile Strains in Meat and Feces of Some Native Birds of Iran.

Clostridioides (Clostridium) difficile (C. difficile) is one of the essential enteropathogens in humans and livestock and is a severe health threat, according to the Centre for Disease Control and Prevention. Also, antimicrobials are one of the most critical risk factors for C. difficile infection (CDI). The present study examined the infection, antibiotic resistance, and genetic diversity of the C. difficile strains in the meat and feces of some native birds (chicken, duck, quail, and partridge) in the Shahrekord region, Iran, from July 2018 to July 2019. Samples were grown on CDMN agar after an enrichment step. To determine the toxin profile, the tcdA, tcdB, tcdC, cdtA, and cdtB genes were detected via multiplex PCR. The antibiotic susceptibility of these isolates was examined using the disk diffusion method and followed based on MIC and epsilometric test. 300 meat samples of chicken, duck, partridge, and quail and 1100 samples of bird feces were collected from six traditional farms in Shahrekord, Iran. Thirty-five meat samples (11.6%) and 191 fecal samples (17.36%) contained C. difficile. Moreover, five toxigenic samples isolated had 5, 1, and 3 tcdA/B, tcdC, and cdtA/B genes. Out of the studied strains isolated from the 226 samples, two isolates belonging to ribotype RT027 and one isolated RT078 profile related to native chicken feces were observed from chicken sample. The antimicrobial susceptibility testing showed that all the strains are resistant to ampicillin, 28.57% are resistant to metronidazole, and 100% were susceptible to vancomycin. Based on the results, it can be concluded that the raw meat of birds might be a source of resistant C. difficile that poses a hygienic threat to the consumption of native bird meat. Nevertheless, further studies are essential to understand additional epidemiological features of C. difficile in bird meat.

Distribution of Virulence Genes in Campylobacter spp. Isolated from Agaricus Mushrooms in Iran.

The white button mushroom (Agaricus) is a significant nutritional and therapeutic species utilized in the human diet and could transmit various bacterial infections. Campylobacter species are the most common cause of foodborne illness across the world. The present study has been planned to determine the frequency of virulence genes and antibiotic susceptibility test in Campylobacter spp. recovered from Agaricus mushroom. In this study, 740 Agaricus mushroom samples were gathered randomly from various markets from June 2020 to December 2020. Confirmation of Campylobacter spp. using biochemical analyses and 23S rRNA-based PCR was performed. The agar dilution technique was used to determine resistance to antibiotics using gentamicin (GM10µg), ciprofloxacin (CIP5µg), nalidixic acid (NA30µg), tetracycline (TE30µg), ampicillin (AM10µg), amoxicillin+ clavulanic acid (AMC30µg), erythromycine (E15µg), azithromycin (AZM15µg), clindamycin (CC2µg), and chloramphenicol (C30µg). Multiplex PCR was utilized to determine the prevalence of the recR, dnaJ, wlaN, virBll, cdtC, cdtB, cdtA, flaA, cadF, pidA, ciaB, ceuE, and cgtB genes. Campylobacter spp. were detected in 74 out of 740 Agaricus mushroom samples (10%). According to the data, Agaricus mushroom samples included 32 (4.32%) C. jejuni, 11 (1.48%) C. coli, and 31 (4.18%) other Campylobacter spp. Antimicrobial resistance was most common in C. jejuni isolates. C. jejuni isolates also had the lowest resistance rate to gentamycin, ciprofloxacin, and nalidixic acid. C. coli isolates were reported to have the highest antimicrobial resistance to ciprofloxacin, ampicillin, and erythromycine. Resistance to gentamycin and amoxicillin+ clavulanic acid was likewise lowest among C. coli strains. The flaA and ciaB genes were found in 100% of B-lactams-susceptible C. jejuni and C. coli strains. When examining the relationship between antibiotic resistance and the existence of virulence genes, it was observed that there is a statistically significant relationship (p < 0.001) between bacterial resistance and virulence genes. Our findings indicated that changes in resistance patterns in Campylobacter strains have emerged from multiple treatment approaches in Agaricus mushrooms.

Campylobacter spp. isolated from poultry in Iran: Antibiotic resistance profiles, virulence genes, and molecular mechanisms.

Campylobacter spp. genera is one of the most common causes of microbial enteritis worldwide. The objective of this work was to investigate the antimicrobial resistance (AMR) patterns, virulence genes, and genetic variation of thermophilic Campylobacter species collected from chicken meat samples in Iran. A total of 255 meat specimens were taken and transferred to the laboratory. Culture methods were utilized to identify the Campylobacter genus, and PCR and sequencing were performed to confirm the organisms. Antimicrobial susceptibility evaluation was performed using broth microdilution for six antimicrobials [ciprofloxacin (CIP), nalidixic acid (NAL), sitafloxacin (SIT), erythromycin (ERY), tetracycline (TET), and gentamicin (GEN)]. By using PCR, AMR and virulence genes were detected. The detection rate of Campylobacter spp. was 64 (25.09%) out of 255 meat samples, with C. jejuni and C. coli accounting for 41 (64.06%) and 14 (21.87%), respectively. Other Campylobacter isolates accounted for 14.06% of the total (nine samples). The antibiotic susceptibility of all Campylobacter isolates was tested using six antibiotics, and all (100%) were resistant to CIP and NAL. However, TET resistance was observed in 93.9% and 83.3% of C. jejuni and C. coli isolates, respectively. Four (8.2%) C. jejuni isolates were multidrug-resistant (MDR), while none of the C. coli isolates were MDR. Two of the four MDR isolates were resistant to CIP, NAL, TET, and ERY, whereas the other two isolates were resistant to CIP, NAL, TET, and GEN. The values of the Minimum Inhibitory Concentration (MIC) were as follows: CIP, 64-256 µg/ml; NAL, 128-512 µg/ml; TET, 2-1024 µg/ml; SIT, 0.25-1 µg/ml; ERY, 1-32 µg/ml; and GEN, 1-256 µg/ml. recR, dnaJ, cdtC, cdtB, cdtA, flaA, ciaB, cadF, and pidA were discovered in more than 50% of C. jejuni isolates, although wlaN, virbll, cgtB, and ceuE were found in <50%. flaA, cadF, pidA, and ciaB were discovered in more than 50% of the C. coli samples, whereas recR, cdtC, cdtB, cdtA, and cgtB were found in less than half. For C. coli, the percentages for wlaN, dnaJ, virbll, and ceuE were all zero. The results of this study show Campylobacter isolates obtained from poultry have higher resistance to quinolones and TET, pathogenicity potential, and varied genotypes.

Prevalence, antimicrobial resistance, virulence gene profile and molecular typing of Campylobacter species isolated from poultry meat samples.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are the most significant Campylobacter species responsible for severe gastrointestinal disorders. Raw poultry meat is considered a source of Campylobacter transmission to the human population. OBJECTIVES: The present study was aimed to assess the prevalence rate, antibiotic resistance properties, virulence characters and molecular typing of C. jejuni and C. coli strains isolated from raw poultry meat samples. METHODS: Three hundred and eighty raw poultry meat samples were collected and analysed for the presence of Campylobacter spp. using the microbial culture. Species identification was done using the Polymerase Chain Reaction. Disk diffusion was developed to assess the antimicrobial resistance pattern of isolates. The distribution of virulence and antimicrobial resistance genes was determined by PCR. Enterobacterial Repetitive Intergenic Consensus-PCR was used for molecular typing. RESULTS: Campylobacter species were isolated from 6.25% of examined samples. C. jejuni and C. coli contamination rates were found to be 57.44% and 48.14%, respectively. C. jejuni strains harboured the highest resistance rate against serythromycin (42.59%), ampicillin (38.88%), ciprofloxacin (33.33%), chloramphenicol (31.48%) and tetracycline (31.48%). C. coli isolates harboured the highest resistance rate against ampicillin (73.07%), ciprofloxacin (73.07%), erythromycin (65.38%) and chloramphenicol (50%). AadE1 (44.44%), blaOXA-61 (42.59%) and tet(O) (35.18%) were the most commonly detected resistance genes in C. jejuni and cmeB (34.61%) and blaOXA-61 (34.61%) were the most commonly detected among C. coli strains. The most frequent virulence factors among the C. jejuni isolates were flaA (100%), ciaB (100%), racR (83.33%), dnaJ (81.48%), cdtB (81.48%), cdtC (79.62%) and cadF (74.07%). The most frequent virulence factors among the C. coli isolates were flaA (100%), ciaB (100%), pldA (65.38%) and cadF (61.53%). CONCLUSIONS: The majority of C. jejuni and C. coli strains had more than 80% similarities in their ERIC-PCR pattern, which may show their common source of transmission. The role of goose and quebec meat samples as reservoirs of virulent and antimicrobial resistant Campylobacter spp. was determined.

Antibiotic resistance's Genotypic and Phenotypic Characteristics and the Frequency of Virulence Factors in P. aeruginosa Isolates Isolated from Water Samples in Iran.

Pseudomonas aeruginosa is a pathogenic bacterium that can contaminate water. In this study, 430 water samples were evaluated for P. aeruginosa, antibiotic resistance, and the abundance of virulence factors. P. aeruginosa was isolated from 28 (6.51%) water samples. Among the types of water, well and spring water showed the highest P. aeruginosa with, respectively, 20 (15.6%) and 5 (8.06%) positive samples per type of samples. Drinking water and mineral water showed minor contamination with P. aeruginosa. The prevalence of antibiotic resistance against meropenem, imipenem, erythromycin, gentamicin, chloramphenicol, and enrofloxacin was zero. The lowest and highest prevalence of antibiotic resistance was observed in drinking water and well water, respectively. The most abundant genes encoding antibiotic resistance in the P. aeruginosa were blaTEM , blaCTX-M , and blaSHV . This study also showed that the most abundant virulence genes in the Pseudomonas aeruginosa strain isolated from water were algD (15 = 3.49%), lasB (11 = 2.56%), toxA (10 = 2.32%), and exoS (7 = 1.63%). This study suggests that water may be a source of P. aeruginosa and contribute to releasing resistance genes through the food chain. Cross-contamination is the water transfer process that can cause contamination with P. aeruginosa in water. Therefore, hygienic principles can be effective in reducing water contamination.

Genotyping, antibiotic resistance and prevalence of Arcobacter species in milk and dairy products.

BACKGROUND: Arcobacter spp. has been considered an emerging foodborne pathogen and a hazard to human health. The dairy chain has been isolated from different sources; nevertheless, data on Arcobacter occurrence in raw milk and dairy products in Iran are still scant. OBJECTIVE: The present study investigates the prevalence, antimicrobial susceptibility and the presence of virulence genes of Arcobacters species isolated from milk and dairy products. METHODS: Then, a total of 350 raw milk samples and 400 dairy product samples were collected from dairy supply centers in Isfahan, Iran. Presumptive Arcobacter strains were obtained by enriching samples in Oxoid Arcobacter enrichment broth (AEB) followed by the filtration of enrichment product through 0.45-µm pore size membrane filters laid onto non-selective blood at 30°C under microaerophilic conditions. Molecular identification of Arcobacter cryaerophilus and A. butzleri was performed by Polymerase chain reaction (PCR) amplification of the 16S rRNA gene, followed by sequencing. The disc diffusion method was used to determine the antimicrobial susceptibility of isolates. Targeted resistance and virulence genes were detected using multiplex PCR. RESULTS: The results show a low recovery rate of Arcobacter spp. in milk. Arcobacters were found in all types of milk, except raw camel milk, but were absent from all dairy products. Arcobacter butzleri was the predominant species in raw milk. Detection of virulence genes shows that all virulence genes targeted were found among A. butzleri, and six (cadF, cj1349, irgA, mviN, pldA, tlyA) were found among A. cryaerophilus. All A. butzleri strains and some A. cryaerophilus strains isolated from milk were resistant to amoxicillin-clavulanic acid and tetracycline. All A. cryaerophilus isolates from milk were susceptible to gentamycin, streptomycin, erythromycin and ciprofloxacin. The distribution of resistance genes in Arcobacter strains in milk shows that all isolates carried tet(O) and blaOXA-61 genes. CONCLUSIONS: In conclusion, the results indicate a low recovery rate of Arcobacter spp. in milk and milk products. However, a significant number of Arcobacter strains with putative virulence genes may be potential pathogens for humans and an overall increase in Arcobacter resistance to first-line antibiotics. These results highlight the need for regular surveillance of Arcobacter strains in milk and milk products in Iran.

Accuracy of the amount of trans-fatty acids in traffic light labelling of traditional sweets distributed in Isfahan, Iran.

BACKGROUND: High consumption of trans-fatty acids (TFAs) is introduced as dietary risk factor of cardiovascular diseases (CVDs). The accuracy of the information shown on the traffic light (TL) labelling has a significant influence on consumers to reduce TFA content in foods. This study is conducted aiming to determine the TFA content in traditional sweets distributed in Isfahan, Iran. Furthermore, the accuracy of the amount of TFAs on TL was considered by comparing it with the experimentally analyzed values. METHODS: In this cross-sectional study, a total of 99 Iranian traditional sweets with a TL label were randomly collected from confectionary shops located in Isfahan. TFAs were analyzed by gas chromatography (GC). RESULTS: TFAs were detected in all samples with the total average of 1.6 ± 0.3% in total fat (range of 0.040 ± 0.001 to 7.900 ± 1.100%). More than half of the samples had less than 2% of TFAs in the total fat. Overall, 81.8% of the studied products with TL labelling showed a discrepancy in the TFAs in the values analyzed in laboratory. CONCLUSION: In the present study, the discrepancy of TFAs in the experimentally measured values with TL food labelling was observed in more than 80% of Iranian traditional sweets. Most of the samples contained less than 2% of TFAs that is defined as a limit in Iran Food and Drug Administration (IFDA). These findings could be alarming for the consumers of this kind of products.

Phenotypic and Genotypic Assessment of Antibiotic Resistance of Staphylococcus aureus Bacteria Isolated from Retail Meat.

BACKGROUND: Resistant Staphylococcus aureus (S. aureus) bacteria are determined to be one of the main causes of foodborne diseases. PURPOSE: This survey was done to assess the genotypic and phenotypic profiles of antibiotic resistance of S. aureus bacteria isolated from retail meat. METHODS: Four-hundred and eighty-five retail meat samples were collected and examined. S. aureus bacteria were identified using culture and biochemical tests. The phenotypic profile of antibiotic resistance was examined using the disk diffusion method. The genotypic pattern of antibiotic resistance was determined using the polymerase chain reaction. RESULTS: Forty-eight out of 485 (9.89%) raw retail meat samples were contaminated with S. aureus. Raw retail buffalo meat (16%) had the highest incidence of S. aureus, while raw camel meat (4%) had the lowest. S. aureus bacteria exhibited the uppermost incidence of resistance toward tetracycline (79.16%), penicillin (72.91%), gentamicin (60.41%), and doxycycline (41.666%). The incidence of resistance toward chloramphenicol (8.33%), levofloxacin (22.91%), rifampin (22.91%), and azithromycin (25%) was lower than other examined antibiotics. The most routinely detected antibiotic resistance genes were blaZ (58.33%), tetK (52.08%), aacA-D (33.33%), and ermA (27.08%). Cat1 (4.16%), rpoB (10.41%), msrA (12.50%), grlA (12.50%), linA (14.58%), and dfrA1 (16.66%) had the lower incidence rate. CONCLUSION: Raw meat of animals may be sources of resistant S. aureus which pose a hygienic threat about the consumption of raw meat. Nevertheless, further investigations are essential to understand supplementary epidemiological features of S. aureus in retail meat.

Vegetables and Restaurant Salads as a Reservoir for Shiga Toxigenic Escherichia coli: Distribution of Virulence Factors, O-Serogroups, and Antibiotic Resistance Properties.

Close contact of vegetables with soil, polluted water, and animal manure and unsanitary conditions during processing of restaurant salads led us to study the distribution of virulence factors, O-serogroups, and antibiotic resistance properties in Shiga toxigenic Escherichia coli (STEC) isolated from vegetables and salads. Samples of vegetables and salad (n = 420) were collected and evaluated for the presence of E. coli using culture and a PCR assay. Total prevalence of E. coli in studied samples was 49.5%. E. coli was found in 49.6% of vegetable samples and 49% of salad samples. Leek and traditional salad had the highest incidence of E. coli. Significant differences in the incidence of E. coli were found between the hot and cold seasons. Of the 149 E. coli isolates from vegetable samples, 130 (87%) were STEC, and of the 59 E. coli isolates from salad samples, 50 (84%) were STEC. The most commonly detected virulence factors were stx1 and eaeA. A significant difference was found between the frequency of the attaching and effacing and the enterohemorrhagic E. coli subtypes. Serogroups O26 (46% of isolates), O157 (14%), O121 (10%), and O128 (9%) were the most commonly detected serogroups among the STEC strains. The tetA, sul1, aac(3)-IV, dfrA1, blaSHV, and CITM antibiotic resistance genes were found in 96, 47.7, 90, 51, 27, and 93% of isolates, respectively. The highest levels of resistance were found against ampicillin (96.6% of isolates), tetracycline (87%), and gentamicin (90%). This study shows the importance of vegetables and salads as potential sources of E. coli infection.

Mercury and arsenic in green tiger shrimp from the Persian Gulf.

Mercury and arsenic pollution has been recognized as a potential environmental and public health problem for over 40 years. The major source of exposure to mercury and arsenic for humans is the ingestion of sea food. Concentrations of mercury and arsenic in the muscle samples of green tiger shrimp (Penaeus semisulcatus) from the Persian Gulf were determined by cold vapor atomic absorption spectrometry. Concentrations of metals in muscle samples were 49-115 µg kg(-1) for mercury, 115-131 µg kg(-1)for arsenic, with means of 82.7 and 251.6 µg kg(-1), respectively. Statistical analysis showed no significant difference of mercury and arsenic concentration in shrimp muscles between selected stations (Bushehr, Khuzestan, and Hormozgan provinces). No significant difference was found among heavy metal contents of male and female shrimps. The results of this study indicate that the green tiger shrimp from the Persian Gulf have concentrations well below the maximum permissible levels for mercury (500 µg kg(-1)) and arsenic (6000 µg kg(-1)), according to international standards, with no health risk of consumers.