Development of multiplex real-time PCR with Internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter spp. and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of Cronobacter and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 10³ to 10⁸ CFU/ml for both Salmonella and Cronobacter. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 10³ CFU/ml for Salmonella and Cronobacter without enrichment. The commercial PIF was then inoculated with Salmonella and Cronobacter at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. At 12 h post-enrichment, we could detect Salmonella and Cronobacter at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of Cronobacter and Salmonella in PIF.     

Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes

Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10² cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28°C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat

In this study, a combined enrichment/real-time PCR method for the rapid detection of Salmonella on fresh meat carcasses, was designed, developed and validated in-house following requirements outlined in ISO 16140:2003. The method included an 18 h non-selective enrichment in buffered peptone water (BPW) and a 6 h selective enrichment in Rappaport Vasilliadis Soya (RVS) broth, based on the traditional culture method, ISO 6579:2002. The real-time PCR assay included an internal amplification control (IAC), was 100% specific and was sensitive to one cell equivalent. The alternative method was validated against the traditional culture method and relative accuracy of 94.9%, sensitivity of 94.7% and specificity of 100% were determined using 150 fresh meat carcass swabs. This alternative method had a detection limit of 1–10 CFU/100 cm² for fresh meat carcass swabs and was performed in 26 h. Following further inter-laboratory studies, this alternative method could be suitable for implementation in testing laboratories for the analysis of carcass swabs.     

Development and validation of a duplex real-time PCR method for the simultaneous detection of celery and white mustard in food

The developed duplex real-time PCR method allows the simultaneous detection of traces of potentially allergenic white mustard (Sinapis alba) and celery roots (Apium graveolens var. rapaceum), celery stalks (A. g. var. dulce) and leaf celery (A. g. var. secalinum). The duplex assay does not show any cross-reactivity with 64 different biological species, including various members of the Brassicaceae and Apiaceae family. In raw model sausages spiked with white mustard and celery roots, the LOD was found to be 0.001% white mustard and 0.005% celery. In model sausages brewed at 75–78 °C for 15 min the LOD was found to be 0.005% white mustard and 0.005% celery. The duplex real-time PCR assay was applied to check if commercial food products are labelled in compliance with the legal regulations.     

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R² > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.     

Aerobic bacterial, coliform, Escherichia coli and Staphylococcus aureus counts of raw and processed milk from selected smallholder dairy farms of Zimbabwe

A cross sectional study was conducted to enumerate total viable bacteria (TBC), coliforms, Escherichia coli and Staphylococcus aureus in raw (n = 120) and processed (n = 20) milk from individual farms from three smallholder dairy schemes of Zimbabwe between October, 2009 and February, 2010. Data on management factors were collected using a structured questionnaire. A standard pour plate technique was used to enumerate total viable bacteria, while for coliforms, E. coli and S. aureus, counts were assessed by the spread plate technique. The association of total viable bacterial counts and management factors was assessed using univariable and a linear regression model. The log₁₀ TBC for raw milk differed significantly (P < 0.05) amongst the schemes with the lowest (5.6 ± 4.7 log₁₀ cfu/ml) and highest (6.7 ± 5.8 log₁₀ cfu/ml) recorded from Marirangwe and Nharira respectively. The mean log₁₀ of TBC of processed milk (6.6 ± 6.0 log₁₀ cfu/ml) were marginally higher than those of raw milk (6.4 ± 5.6 log₁₀ cfu/ml) but not significant (P > 0.05). The coliform, E. coli and S. aureus counts for raw milk significantly differed (P < 0.05) amongst the study areas. The variation in TBC, coliforms, E. coli and S. aureus counts amongst the schemes could be attributed to differences in milking hygiene where farms with more access to training and monitoring of microbiological quality of milk had lower counts. Linear regression analysis revealed dairy scheme, delivery time and season of milking as independently associated with increased TBC of raw milk. The high TBC of raw and processed milk generally indicated low levels of milking hygienic practices, and high level of post-processing contamination, respectively. The high TBC, coliform, E. coli and S. aureus counts of both raw and processed milk may present a public health hazard. Thus, educating the farmers on general hygienic practices, quickening the delivery of milk to collection centres, or availing cooling facilities on-farm will improve the microbiological quality and safety of milk.     

Short communication: An evaluation of the effectiveness of Lactobacillus buchneri 40788 to alter fermentation and improve the aerobic stability of corn silage in farm silos

The objective of this study was to determine if the effects of inoculation with Lactobacillus buchneri 40788 were detectable when applied to whole-plant corn stored in farm silos. Corn silage was randomly sampled from farms in Wisconsin, Minnesota, and Pennsylvania, and was untreated (n = 15) or treated with an inoculant (n = 16) containing L. buchneri 40788 alone or this organism combined with Pediococcus pentosaceus during May and June 2007. Corn silage that was removed from the silo face during the morning feeding was sampled, vacuum-packed, and heat sealed in polyethylene bags and shipped immediately to the University of Delaware for analyses. Silage samples were analyzed for dry matter (DM), nutrient composition, fermentation end-products, aerobic stability, and microbial populations. The population of L. buchneri in silages was determined using a real-time quantitative PCR method. Aerobic stability was measured as the time after exposure to air that it took for a 2°C increase above an ambient temperature. The DM and concentrations of lactic and acetic acids were 35.6 and 34.5, 4.17 and 4.85, and 2.24 and 2.41%, respectively, for untreated and inoculated silages and were not different between treatments. The concentration of 1,2-propanediol was greater in inoculated silages (1.26 vs. 0.29%). Numbers of lactic acid bacteria determined on selective agar were not different between treatments. However, the numbers of L. buchneri based on measurements using real-time quantitative PCR analysis were greater and averaged 6.46 log cfu-equivalents/g compared with 4.89 log cfu-equivalent for inoculated silages. There were fewer yeasts and aerobic stability was greater in inoculated silages (4.75 log cfu/g and 74 h of stability) than in untreated silages (5.55 log cfu/g and 46 h of stability). This study supports the effectiveness of L. buchneri 40788 on dairy farms.     

Development and validation of a novel real-time PCR method for the detection of celery (Apium graveolens) in food

The paper presents a novel real-time PCR method allowing the detection of traces of celery (Apium graveolens) in complex food matrices. The method is based on the amplification of a sequence of the gene coding for the Apium graveolens NADPH-dependent mannose-6-phosphate reductase. It allows the detection of three commonly used celery varieties, celery roots (Apium graveolens var. rapaceum), celery stalks (Apium graveolens var. dulce) and leaf celery (Apium graveolens var. secalinum) and does not show any cross-reactivity with 64 biological species, including ten members of the Apiaceae family. The limit of detection, determined by analysing serially diluted celery extracts, is 10 pg celery DNA for all three celery varieties. In spiked model sausages, the LOD is 0.005% celery. The real-time PCR method was applied to 26 commercial food products. Celery DNA was found in one out of ten samples without any information about the presence of celery.     

Detection of Cronobacter species in powdered infant formula by probe-magnetic separation PCR

Cronobacter species are opportunistic foodborne pathogens associated with serious infections in preterm neonates and infants. Based on the epidemiological research, infant formula products are considered to be the main source of infections from this organism. Therefore, accurate methods are required for detection of Cronobacter species. In this study, the specific probe and primers for detection of this organism were designed and verified. The probe-magnetic beads were prepared for sequence capture, followed by PCR assay to detect the target gene. This probe-magnetic separation PCR assay could detect as few as 10³ cfu/mL of Cronobacter in artificially contaminated infant formulas in less than 4 h. The combination of magnetic beads and PCR showed the potential for the detection of Cronobacter in infant formulas and may have applications in the dairy industry.     

Identification of hare meat by a species-specific marker of mitochondrial origin

Meat species identification in food has gained increasing interest in recent years due to public health, economic and legal concerns. Following the consumer trend towards high quality products, game meat has earned much attention. The aim of the present work was to develop a DNA-based technique able to identify hare meat. Mitochondrial cytochrome b gene was used to design species-specific primers for hare detection. The new primers proved to be highly specific to Lepus species, allowing the detection of 0.01% of hare meat in pork meat by polymerase chain reaction (PCR). A real-time PCR assay with the new intercalating EvaGreen dye was further proposed as a specific and fast tool for hare identification with increased sensitivity (1 pg) compared to end-point PCR (10 pg). It can be concluded that the proposed new primers can be used by both species-specific end-point PCR or real-time PCR to accurately authenticate hare meat.     

Angiotensin-converting enzyme inhibitory activity of milk fermented by wild and industrial Lactococcus lactis strains

Angiotensin I-converting enzyme inhibitory (ACEI) activity was evaluated and compared in <3 KDa water-soluble extracts (WSE) isolated from milk fermented by wild and commercial starter culture Lactococcus lactis strains after 48 h of incubation. The highest ACEI activities were found in WSE from milk inoculated with wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures. On the other hand, the lowest ACEI activities were found in WSE from milk inoculated with wild strains isolated from vegetables. Moreover, the IC₅₀ values (concentration that inhibits 50% activity) of WSE from artisanal dairy products were the lowest, indicating that these fractions were the most effective in inhibiting 50% of ACE activity. In fact, a strain isolated from artisanal cheese presented the lowest IC₅₀ (13 μg/mL). Thus, it appears that wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures showed good potential for the production of fermented dairy products with ACEI properties.     

Short communication: Prevalence of methicillin resistance in coagulase-negative staphylococci and Staphylococcus aureus isolated from bulk milk on organic and conventional dairy farms in the United States

The objective of this study was to evaluate the presence of methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus spp. in bulk tank milk samples from 288 organic and conventional dairy farms located in New York, Wisconsin, and Oregon from March 2009 to May 2011. Due to recent publications reporting the presence mecC (a mecA homolog not detected by traditional mecA-based PCR methods), a combination of genotypic and phenotypic approaches was used to enhance the recovery of methicillin-resistant organisms from bulk tank milk. In total, 13 isolates were identified as methicillin resistant: Staph. aureus (n = 1), Staphylococcus sciuri (n = 5), Staphylococcus chromogenes (n = 2), Staphylococcus saprophyticus (n = 3), Staphylococcus agnetis (n = 1), and Macrococcus caseolyticus (n = 1). The single methicillin-resistant Staph. aureus isolate was identified from an organic farm in New York, for an observed 0.3% prevalence at the farm level. The methicillin-resistant coagulase-negative staphylococci prevalence was 2% in the organic population and 5% in the conventional population. We did not identify mecC in any of the isolates from our population. Of interest was the relatively high number of methicillin-resistant Staph. sciuri recovered, as the number of isolates from our study was considerably higher than those recovered from other recent studies that also assessed milk samples. Our research suggests that the presence of a potential methicillin-resistant Staphylococcus reservoir in milk, and likely the dairy farm population in the United States, is independent of the organic or conventional production system.     

Spoilage characteristics of Brochothrix thermosphacta and campestris in chilled vacuum packaged lamb,and their detection and identification by real time PCR

The spoilage potential of Brochothrix campestris and Brochothrix thermosphacta was investigated in vacuum-packed lamb. Striploins (n = 338) were inoculated and stored for twelve weeks at temperatures − 1.5, 0, 2 and 7 °C. Growth around 5–6 log₁₀ CFU/cm² was recorded after six weeks at 0, 2 and 7 °C, and ~ 3 log₁₀ CFU/cm² after nine weeks at − 1.5 °C. B. campestris was shown to cause spoilage by nine weeks at temperatures above 0 °C by the presence of green drip and unacceptable odours. Molecular based assays for the detection and differentiation of B. thermosphacta and B. campestris were developed and validated. A TaqMan assay was designed to target a unique single-nucleotide polymorphism in the Brochothrix 16 s rRNA gene with a sensitivity of < 7 CFU per reaction. Secondly a specific PCR was designed for B. campestris targeting the structural genes, brcA and brcB. These testing regimes offer a rapid and cost effective method for the detection and screening of Brochothrix species in meat products and processing environments.     

Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk

A multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) was developed and validated for simultaneous detection of Salmonella strains and Shigella strains in milk. In this system, two sets of LAMP primers were designed to specifically target invA of Salmonella spp. and ipaH of Shigella spp. Under isothermal conditions at 63 °C, ladder pattern of DNA bands could be amplified within 60 min in the presence of genomic DNAs of Salmonella strains and Shigella strains, which could be distinguished between Salmonella spp. and Shigella spp. simultaneously based on the different ladder pattern of DNA bands and subsequent restriction enzyme analysis. The overall analysis time was approximately 20 h including the enrichment of the bacterial cells, which greatly saved detection time. The sensitivity of mLAMP was found to be 100 fg DNA/tube with genomic DNAs of Salmonella strains and Shigella strains, comparatively, multiplex PCR was 1 pg DNA/tube. The mLAMP allowed the detection of milk sample artificially contaminated by Salmonella strains and Shigella strains at initial inoculation levels of approximate 5 CFU/10 mL. In conclusion, the mLAMP described here can potentially facilitate simultaneous monitoring of Salmonella and Shigella in a large number of food samples, which could be used as a primary screening method and as a supplement to classical detection method.     

Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella

Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5 ng–50 fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 10⁴ CFU/g, this was improved to 10 CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food.     

Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.     

Prevalence of Mycobacterium avium subspecies paratuberculosis in Swiss raw milk cheeses collected at the retail level

A total of 143 raw milk cheese samples (soft cheese, n = 9; semihard cheese, n = 133; hard cheese, n = 1), collected at the retail level throughout Switzerland, were tested for Mycobacterium avium ssp. paratuberculosis (MAP) by immunomagnetic capture plus culture on 7H10-PANTA medium and in supplemented BAC-TEC 12B medium, as well as by an F57-based real-time PCR system. Furthermore, pH and water activity values were determined for each sample. Although no viable MAP cells could be cultured, 4.2% of the raw milk cheese samples tested positive with the F57-based real-time PCR system, providing evidence for the presence of MAP in the raw material. As long as the link between MAP and Crohn's disease in humans remains unclear, measures designed to minimize public exposure should also include a focus on milk products.     

Herd-level relationship between antimicrobial use and presence or absence of antimicrobial resistance in gram-negative bovine mastitis pathogens on Canadian dairy farms

Concurrent data on antimicrobial use (AMU) and resistance are needed to contain antimicrobial resistance (AMR) in bacteria. The present study examined a herd-level association between AMU and AMR in Escherichia coli (n = 394) and Klebsiella species (n = 139) isolated from bovine intramammary infections and mastitis cases on 89 dairy farms in 4 regions of Canada [Alberta, Ontario, Québec, and Maritime Provinces (Prince Edward Island, Nova Scotia, and New Brunswick)]. Antimicrobial use data were collected using inventory of empty antimicrobial containers and antimicrobial drug use rate was calculated to quantify herd-level AMU. Minimum inhibitory concentrations (MIC) were determined using Sensititre National Antimicrobial Resistance Monitoring System (NARMS) gram-negative MIC plate (Trek Diagnostic Systems Inc., Cleveland, OH). Isolates were classified as susceptible, intermediate, or resistant. Intermediate and resistant category isolates were combined to form an AMR category, and multivariable logistic regression models were built to determine herd-level odds of AMR to tetracycline, ampicillin, cefoxitin, chloramphenicol, trimethoprim-sulfamethoxazole combination, sulfisoxazole, streptomycin and kanamycin in E. coli isolates. In the case of Klebsiella species isolates, logistic regression models were built for tetracycline and sulfisoxazole; however, no associations between AMU and AMR in Klebsiella species were observed. Ampicillin-intermediate or -resistant E. coli isolates were associated with herds that used intramammarily administered cloxacillin, penicillin-novobiocin combination, and cephapirin used for dry cow therapy [odds ratios (OR) = 26, 32, and 189, respectively], and intramammary ceftiofur administered for lactating cow therapy and systemically administered penicillin (OR = 162 and 2.7, respectively). Use of systemically administered penicillin on a dairy farm was associated with tetracycline and streptomycin-intermediate or -resistant E. coli isolates (OR = 5.6 and 2.8, respectively). Use of cephapirin and cloxacillin administered intramammarily for dry cow therapy was associated with increasing odds of having at least 1 kanamycin-intermediate or -resistant E. coli isolate at a farm (OR = 8.7 and 9.3, respectively). Use of systemically administered tetracycline and ceftiofur was associated with cefoxitin-intermediate or -resistant E. coli (OR = 0.13 and 0.16, respectively); however, the odds of a dairy herd having at least 1 cefoxitin-intermediate or -resistant E. coli isolate due to systemically administered ceftiofur increased with increasing average herd parity (OR = 3.1). Association between herd-level AMU and AMR in bovine mastitis coliforms was observed for certain antimicrobials. Differences in AMR between different barn types and geographical regions were not observed.