M. Guarddon

Prevalence and antimicrobial resistance patterns of Salmonella from different raw foods in Mexico.

The presence of Salmonella was determined in 116 samples of poultry meat, 81 samples of pork, 73 samples of beef, 33 samples of cheese, 61 samples of fish, and 78 samples of vegetables collected from retail stores and supermarkets in Hidalgo State (Mexico). Ninety-three Salmonella strains isolated from raw foods were characterized, and MICs were determined for 10 antimicrobials. Salmonella was detected in 35.3% of poultry meat, 30.3% of cheese, 21.8% of vegetable, 17.3% of pork, and 15.1% of beef samples, but no Salmonella was detected in fish samples. Significantly higher counts were obtained in chicken meat (P = 0.0001), pork (P = 0.0116), cheese (P = 0.0228), and vegetables (P = 0.0072) obtained from retail stores compared with those samples obtained from supermarkets. Salmonella isolates had high levels of resistance to ampicillin (66.7% of isolates), tetracycline (61.3%), and chloramphenicol (64.5%) and low levels of resistance to cefotaxime (0%), gentamicin (3.2%), and kanamycin (4.3%). Higher levels of quinolone resistance were found in isolates from poultry meat and vegetables compared with that in other foods tested. High levels of multiresistant strains were found in all foods tested except fish, ranging from 100% of pork samples to 47.1% of vegetable samples. The present study revealed that Salmonella prevalence was higher in foods from retail stores than in foods from supermarkets. Resistance rates observed for Salmonella were largely comparable to those reported in other countries for most antimicrobials, although resistance to chloramphenicol tended to be higher.

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R(2) values of 0.9969 and 0.9958 respectively. Linear correlations between the log(10) input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10(1) CFU/mL to 1.65 × 10(6) CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.

Real-time polymerase chain reaction for the quantitative detection of tetA and tetB bacterial tetracycline resistance genes in food

A new, rapid, sensitive and specific method was developed to directly detect and quantify tetA and tetB in food. Both tet genes are two of the most frequently present tetracycline resistance genes in gram-negative bacteria. A set of primers and Taqman probes was designed for each gene. The standard curves were performed using Escherichia coli BM13 (C600 RifR)/RP4 and E. coli NCTC 50365, which carry tetA and tetB, respectively. Meat and fish samples inoculated with these reference strains were used as a matrix to construct the standard curves for the analysis of 20 samples of chicken meat and 10 samples of hake (Merlucius merlucius). The limits of detection in pure culture were 5 cfu/mL (0.7 log cfu/mL) in the case of tetA, 50 cfu/mL (1.7log cfu/mL) for tetB and 5×10(2)cfu/g (2.7 log cfu/g) for both genes in food samples. The results obtained by real-time quantitative polymerase chain reaction (qPCR) were compared to counts of tetracycline-resistant bacteria obtained by plating extracts of poultry and hake samples in culture media supplemented with 16 mg/L of tetracycline. Counts of tetracycline-resistant bacteria obtained by qPCR showed a positive correlation, especially interesting when compared with microbiological counts of tetracycline-resistant Enterobacteriaceae in poultry meat (r=0.5509) and with tetracycline-resistant mesophilic aerobic bacteria in hake samples (r=0.7146). The obtained results demonstrate that this method could be a useful tool for the direct quantification of the amount of bacterial strains that carry tetA and/or tetB genes in food samples.

Quantitative detection of tetracycline-resistant microorganisms in conventional and organic beef, pork and chicken meat

The use of antimicrobials has increased the number of resistant bacteria to these drugs; however, the organic production has restricted the use of these compounds. The objectives of this work were to assess counts of tetracycline-resistant bacteria using conventional microbiology, to compare these results with those obtained for tet(A) and tet(B) genes by qPCR and to investigate both genes in conventional and organic meat. Counts of mesophilic aerobic bacteria were higher in organic beef, while chicken meat obtained higher counts for Enterobacteriaceae. Only tet(B) was higher in conventional pork and chicken meat than in their organic counterparts. The tet(A) gene was found in almost 100% of samples and tet(B) gene changed according to the type of meat. The presence of tet genes suggests that they are widely distributed, especially tet(A), in food of animal origin, even in organic meat samples obtained from animals in which the use of antimicrobials is restricted.

Direct Quantification and Distribution of Tetracycline-Resistant Genes in Meat Samples by Real-Time Polymerase Chain Reaction

The evolution of antimicrobial-resistant bacteria has become a threat to food safety and methods to control them are necessary. Counts of tetracycline-resistant (TR) bacteria by microbiological methods were compared with those obtained by quantitative PCR (qPCR) in 80 meat samples. TR Enterobacteriaceae counts were similar between the count plate method and qPCR (P= 0.24), whereas TR aerobic mesophilic bacteria counts were significantly higher by the microbiological method (P < 0.001). The distribution of tetA and tetB genes was investigated in different types of meat. tetA was detected in chicken meat (40%), turkey meat (100%), pork (20%), and beef (40%) samples, whereas tetB was detected in chicken meat (45%), turkey meat (70%), pork (30%), and beef (35%) samples. The presence of tetracycline residues was also investigated by a receptor assay. This study offers an alternative and rapid method for monitoring the presence of TR bacteria in meat and furthers the understanding of the distribution of tetA and tetB genes.

Presence and antimicrobial resistance of Escherichia coli isolated from foodstuffs in Hidalgo State (Mexico)

The presence of Escherichia coli in foods taken from the grocery stores and the supermarkets in Hidalgo State (Mexico) was determined for 73 samples of poultry meat, 60 samples of pork, 86 samples of beef, and 66 samples of vegetables. A total of 352 E. coli strains were isolated, identified, and analyzed by an agar disk diffusion assay for their resistance to 10 antimicrobials. Poultry meat and vegetables taken from groceries showed significantly higher counts (P = 0.0002 and P = 0.0461, respectively) when compared with the samples taken from supermarkets. Compared with the isolates recovered from other foods, E. coli isolated from chicken meat had higher levels of antimicrobial resistance against all antimicrobials tested, with the exceptions of nitrofurantoin resistance of isolates from pork and streptomycin resistance in isolates from pork and beef. In addition, the E. coli isolates from samples taken from the groceries showed higher resistance rates than the isolates from samples taken from the supermarkets for the cases of pork isolates resistance to ampicillin (P = 0.0497), chloramphenicol (P = 0.0075), doxycycline (P = 0.002), and streptomycin (P = 0.0094) and beef isolates resistance against ampicillin (P = 0.0048), streptomycin (P = 0.002), and sulfisoxazole (P = 0.003). The present study revealed that the observed resistance rates correlated well with those reported in the national surveillance programmes of developed countries, with the exception of isolates from chicken meat, which have higher resistance rates. Also, from a microbiological safety point of view, samples taken from supermarkets were in a much better conditions than those obtained from the groceries.

Assessment of food safety using a new real‐time PCR assay for detection and quantification of virulence factors of enterococci in food samples

Development of Taqman MGB real‐time PCR (q‐PCR) assays for the quantitative detection of virulence factor genes in pure culture and food samples with regard to food safety assessment.

Assessment of Tetracyclines Residues and Tetracycline Resistant Bacteria in Conventional and Organic Baby Foods

Children are very vulnerable to bacterial infections and they are sometimes subject to antimicrobials for healing. The presence of resistance genes may counteract effects of antimicrobials. This work has thereby compared the amount of tetracycline resistance genes, tet(A) and tet(B), between conventional and organic meat-based or vegetable-based baby foods and used the quantification of these genes to assess the presence of tetracycline residues in these samples. Counts of bacteria harboring the tet(A) gene were higher than those containing tet(B), and there was no difference between the organic and the conventional samples. Samples with detectable amounts of tetracycline residues were also positive for the presence of tet genes, and when the presence of the genes was not detected, the samples were also negative for the presence of residues. The percentages of tetracycline residues were higher in organic samples than in conventional ones. It cannot be concluded that organic formulas are safer than conventional ones for the studied parameters.