Processing Environmental Samples Research Articles

Different approaches to processing environmental DNA samples in turbid waters have distinct effects for fish, bacterial and archaea communities

Coastal lagoons are an important habitat for endemic and threatened species in California that have suffered impacts from urbanization and increased drought. Environmental DNA has been promoted as a way to aid in the monitoring of biological communities, but much remains to be understood on the biases introduced by different protocols meant to overcome challenges presented by unique systems under study. Turbid water is one methodologic challenge to eDNA recovery in these systems as it quickly clogs filters, preventing timely processing of samples. We investigated biases in community composition produced by two solutions to overcome slow filtration due to turbidity: freezing of water prior to filtration (for storage purposes and long-term processing), and use of sediment (as opposed to water samples). Bias assessments of community composition in downstream eDNA analysis was conducted for two sets of primers, 12S (fish) and 16S (bacteria and archaea). Our results show that freezing water prior to filtration had different effects on community composition for each primer, especially for the 16S, when using a filter of larger pore size (3 μm). Nevertheless, pre-freezing water samples can still be a viable alternative for storage and processing of turbid water samples when focusing on fish communities (12S). The use of sediment samples as an alternative to processing water samples should be done with caution, and at minimum the number of biological replicates and/or volume sampled should be increased.

Processing environmental DNA samples in turbid waters from coastal lagoons

A recommendation of: Rachel Turba, Glory H. Thai, and David K Jacobs Different approaches to processing environmental DNA samples in turbid waters have distinct effects for fish, bacterial and archaea communities. https://doi.org/10.1101/2022.06.17.495388

Microbial Profiling of Biltong Processing Using Culture-Dependent and Culture-Independent Microbiome Analysis

Biltong is a South African air-dried beef product that does not have a heat lethality step, but rather relies on marinade chemistry (low pH from vinegar, ~2% salt, spices/pepper) in combination with drying at ambient temperature and low humidity to achieve microbial reduction during processing. Culture-dependent and culture-independent microbiome methodologies were used to determine the changes in the microbial community at each step during biltong processing through 8 days of drying. Culture-dependent analysis was conducted using agar-based methods to recover viable bacteria from each step in the biltong process that were identified with 16S rRNA PCR, sequencing, and BLAST searching of the NCBI nucleotide database. DNA was extracted from samples taken from the laboratory meat processing environment, biltong marinade, and beef samples at three stages of processing (post-marinade, day 4, and day 8). In all, 87 samples collected from two biltong trials with beef obtained from each of three separate meat processors (n = six trials) were amplified, sequenced with Illumina HiSeq, and evaluated with bioinformatic analysis for a culture-independent approach. Both culture-dependent and independent methodologies show a more diverse population of bacteria present on the vacuum-packaged chilled raw beef that reduces in diversity during biltong processing. The main genera present after processing were identified as Latilactobacillus sp., Lactococcus sp., and Carnobacterium sp. The high prevalence of these organisms is consistent with extended cold-storage of vacuum-packaged beef (from packers, to wholesalers, to end users), growth of psychrotrophs at refrigeration temperatures (Latilactobacillus sp., Carnobacterium sp.), and survival during biltong processing (Latilactobacillus sakei). The presence of these organisms on raw beef and their growth during conditions of beef storage appears to 'front-load' the raw beef with non-pathogenic organisms that are present at high levels leading into biltong processing. As shown in our prior study on the use of surrogate organisms, L. sakei is resistant to the biltong process (i.e., 2-log reduction), whereas Carnobacterium sp. demonstrated a 5-log reduction in the process; the recovery of either psychrotroph after biltong processing may be dependent on which was more prevalent on the raw beef. This phenomenon of psychrotrophic bloom during refrigerated storage of raw beef may result in a natural microbial suppression of mesophilic foodborne pathogens that are further reduced during biltong processing and contributes to the safety of this type of air-dried beef.

Aptasensor for the detection of Methicillin resistant Staphylococcus aureus on contaminated surfaces

There is mounting evidence that contaminated hospital environment plays a crucial role in the transmission of nosocomial pathogens such as MRSA. The institution of infection control protocols is predicated on the early laboratory detection of the pathogen from relevant samples.Processing of environmental samples for the presence of bacterial contaminants in the clinical environment is poorly standardized when compared with analysis of clinical samples. The various laboratory methods available for processing environmental samples are difficult to standardized and most require a long turnaround time before results are available.In this study, we present a report of the performance of a novel pathogen aptasensor swab designed to qualitatively and quantitatively detect MRSA, on contaminated non-absorbable surfaces. The visual detection limit of the MRSA aptasensor swab was less than 100 CFU/ml and theoretically using a standard curve, was 2 CFU/ml. A relatively short turnaround time of 5 min was established for the assay while the linear range of quantitation was 102–105 CFU/ml. Engineered aptasensor targets MRSA selectively and binds to none of the other tested bacterial pathogen, on a multi-contaminated surface. This novel detection tool was easy to use and relatively cheap to produce.

Isolation, Screening and Time Course Study of Amylase-producing Fungi from Garri Processing Environment

Amylases (E.C.3.2.1.1) are enzymes which catalyze the breakdown of -1,4 glycosidic linkages of starch to simple sugars and different monomeric products. Microbial amylases are essential enzymes compared with animals and plants amylases in industrial applications. Thus, in southern Nigeria, garri processing waste-water from mills poses a serious environmental challenge and this situation could be exploited by utilizing microorganisms colonizing the area to manufacture microbial products. This present study was aimed at evaluating the potential of fungi isolated from garri processing environment for amylase secretion and conduct a time course study of the enzyme production. Fungi were isolated from the soil and waste water from garri processing sites using Standard Microbiological Procedures. The five fungi isolated from garri processing environmental samples were screened using Lugols iodine. Three fungal isolates were then selected on the hydrolysis of starch in qualitative zonation agar plates but one isolate named B among them was better in starch hydrolysis related to highest clear zone plates. After qualitative screening, the three hyper production amylase extracted fungi were identified according to the morphological characteristics. The hyper producing amylase isolate B was then identified as Aspergillus flavus (RCBBR_AEAFUN2) and recorded as a novel strain in southern Nigeria according to molecular characteristics, which was selected for further studies. The current results of time course study showed that the maximum growth (2.453 g) occurred after 24 h of incubation at pH 4.5 while maximum enzyme production (2.3 U/ml/min) was obtained after 96 h of incubation at pH 3.5. Therefore, the hyper producing amylase from garri processing sites, Aspergillus flavus (RCBBR_AEAFUN2) novel strain has great prospects for future biotechnological and industrial applications and help in ameliorating the environmental pollution posed by the waste-water.

Microbiological quality and HACCP of nono production in a Fulani settlement, Zaria Kaduna state, Nigeria

This research studied the frequency of occurrence of Escherichia coli, Bacillus cereus, yeasts and moulds in some retail uncontrolled fermented milk samples (nono), and a hazard analysis was conducted to trace the sources of contamination during processing and suggest critical control measures. Fifty (50) nono samples purchased from Fulani milkmaids in Samaru market of Zaria town, Kaduna, Nigeria, were analyzed for presence of E. coli, B. cereus, Yeasts and Moulds. The traditional processing of nono by 3 producers of some of the market samples analyzed was studied by observing processing steps, processing environment, collecting raw and fermented milk samples, cow dung, hand swabs, swabs of milk contact surfaces, and exposing sterile plates to milking environment. Nono processing for all 3 producers involved spontaneous fermentation of fresh unpasteurized milk in calabashes at ambient temperature (25±40C) for 24 hours, fat separation from fermented milk, addition of water, whisking, packaging in calabashes and storing at ambient temperature before sale. Mean plate counts of market nono samples were at levels considered unsatisfactory for E. coli (≥ 102) in 76 %of samples, potentially hazardous for B. cereus (≥104) in 78% of samples and exceeded satisfactory limits for yeasts and moulds (<10) in all samples (100%). E. coli, B. cereus, yeasts and moulds were present in all 9 milk samples collected before and after fermentation during traditional processing. Yeasts and moulds were isolated from all samples (100%) during traditional processing except for wooden spoons (78%) and hand swabs (78%). Substandard practices like using unclean hands in manual milking and use of unpasteurized milk observed during traditional processing could explain presence of these microorganisms in market samples, as was also detected in raw and fermented milk during traditional processing. Findings of this study support that of previous researchers which established the impact of substandard processing techniques and hygiene on food quality.

Phenotypic and genotypic drug resistance profile of Salmonella serovars isolated from poultry farm and processing units located in and around Mumbai city, India.

Background and Aim:The extensive use of antimicrobials in poultry has led to an increase in bacterial multidrug resistance, and the emergence of multidrug-resistant nontyphoidal Salmonella is a global problem. This study was performed to detect antibiotic-resistant Salmonella serovars in poultry farming and processing environment.Materials and Methods:A total of 956 various samples, comprising 432 farm origin, 324 poultry processing stage wise and environmental, and 154 product processing stages and environmental samples, were collected from poultry farms and processing units located in and around Mumbai city. Of a total of 71 recovered isolates, 42 randomly selected Salmonella isolates were subjected for antibiotic susceptibility testing by disk diffusion method and serotyping. A total of 31 serotypically confirmed isolates were characterized for the presence of tetA, tetB, blaTEM, and CTX-M gene.Results:Higher resistance was recorded against Doxycycline (100%), followed by Oxytetracycline (97.62%), Neomycin (88.10%), Erythromycin (83.33%), Tetracycline (78.57%), and Ceftizoxime (35.71%). Resistance from 0.00 to 26.19 percent was found to antimicrobials, namely Norfloxacin (26.19%), Ampicillin (21.43%), Azithromycin (21.43%), Ciprofloxacin (19.05%), Colistin (4.76%), Streptomycin (16.67%), Cefotaxime (14.19%), Enrofloxacin (14.29%), Amoxyclav (14.29%), Gentamicin (7.14%), Chloramphenicol (4.76%), Amikacin (4.76%), and Ceftazidime (0.0%). Results demonstrate that the Salmonella Virchow dominated and all serotypes were found to carry Tetracycline resistance gene tetA, 5 isolates were found to be positive for blaTEM, whereas none of the isolates were carrying tetB and CTX-M gene.Conclusion:This study revealed that there is a significant rise of Tetracycline resistance with the presence of tetA gene in Salmonella spp. which indicates selective pressure for adopting resistance against tetracycline group of antibiotics.

DNA metabarcoding of airborne pollen: new protocols for improved taxonomic identification of environmental samples

Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. Sample preparation and DNA extraction are of fundamental importance for obtaining an optimal DNA yield. Currently, there is no standard procedure for these steps, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols for processing environmental samples for pollen DNA extraction and for metabarcoding analysis and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen collected by gravimetric (Tauber trap) and volumetric (Hirst-type trap) samplers. Protocols were tested across an increasing complexity of samples, from pure single-species pollen to environmental multi-species samples. A short fragment (about 150 base pairs) of the chloroplast trnL gene was amplified using universal primers for plants. After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison with a custom-made reference database including chloroplast DNA sequences from most of the anemophilous taxa occurring in the study area (Trentino, northern Italy), representing 46 plant families. Using the classical morphological pollen analysis as a benchmark, we show that DNA metabarcoding is efficient and applicable even in complex samples, provided that protocols for sample preparation, DNA extraction, and metabarcoding analysis are carefully optimized.

Rapid extraction and assay of uranium from environmental surface samples

Environmental sampling to detect trace nuclear signatures is key component of international nuclear treaty enforcement. Herein, we explored rapid chemical extraction methods coordinated with measurement systems to provide faster, simpler assay of low level uranium from environmental samples. A key problem with the existing analytical method for processing environmental surface samples is the requirement for complete digestion of sample and sampling material. This is a time-consuming and labor-intensive process that limits laboratory throughput, elevates analytical costs, and increases background levels. Promising extraction methods were competitively evaluated for their potential to quickly and efficiently remove different chemical species of uranium from standard surface sampling material. A preferred combination of carbonate and peroxide solutions is shown to give rapid and complete form of uranyl compound extraction and dissolution. This simplified and accelerated extraction process is demonstrated with standard sampling material to be compatible with standard inductive coupled plasma mass spectrometry methods for uranium isotopic assay as well as rapid screening techniques such as X-ray fluorescence (XRF). Rapid extraction of the entire swipe is shown to allow efficient XRF assay of all collected material for simple, fast, nanogram-level XRF assay of the sample. The new methods have direct application in the support of nuclear safeguards treaty enforcement efforts as well as health and safety monitoring. The general approach described may have applications beyond uranium to other trace analytes of nuclear forensic interest (e.g., rare earth elements and plutonium) as well as heavy metals for environmental and industrial hygiene monitoring.

Microbial Performance of Food Safety Control and Assurance Activities in a Fresh Produce Processing Sector Measured Using a Microbial Assessment Scheme and Statistical Modeling

Current approaches such as inspections, audits, and end product testing cannot detect the distribution and dynamics of microbial contamination. Despite the implementation of current food safety management systems, foodborne outbreaks linked to fresh produce continue to be reported. A microbial assessment scheme and statistical modeling were used to systematically assess the microbial performance of core control and assurance activities in five Kenyan fresh produce processing and export companies. Generalized linear mixed models and correlated random-effects joint models for multivariate clustered data followed by empirical Bayes estimates enabled the analysis of the probability of contamination across critical sampling locations (CSLs) and factories as a random effect. Salmonella spp. and Listeria monocytogenes were not detected in the final products. However, none of the processors attained the maximum safety level for environmental samples. Escherichia coli was detected in five of the six CSLs, including the final product. Among the processing-environment samples, the hand or glove swabs of personnel revealed a higher level of predicted contamination with E. coli , and 80% of the factories were E. coli positive at this CSL. End products showed higher predicted probabilities of having the lowest level of food safety compared with raw materials. The final products were E. coli positive despite the raw materials being E. coli negative for 60% of the processors. There was a higher probability of contamination with coliforms in water at the inlet than in the final rinse water. Four (80%) of the five assessed processors had poor to unacceptable counts of Enterobacteriaceae on processing surfaces. Personnel-, equipment-, and product-related hygiene measures to improve the performance of preventive and intervention measures are recommended.

Enhanced detection of pathogenic enteric viruses in coastal marine environment by concentration using methacrylate monolithic chromatographic supports paired with quantitative PCR

Currently, around 50% of the world's population lives in towns and cities within 100 km of the coast. Monitoring of viruses that are frequently present in contaminated coastal environments, such as rotavirus (RoV) and norovirus (NoV), which are also the major cause of human viral gastroenteritis, is essential to ensure the safe use of these water bodies. Since exposure to as few as 10–100 particles of RoV or NoV may induce gastrointestinal disease, there is a need to develop a rapid and sensitive diagnostic method for their detection in coastal water samples. In this study, we evaluate the application of methacrylate monolithic chromatographic columns, commercially available as convective interaction media (CIM®), to concentrate pathogenic enteric viruses from saline water samples prior to virus quantification by one-step reverse transcription quantitative PCR (RT-qPCR). Using RoV and NoV as model enteric viruses, we present our results on the most effective viral concentration conditions from saline water matrices using butyl (C4) hydrophobic interaction monolithic support (CIM® C4). C4 monolithic columns exhibit a good capacity to bind both RoV and NoV and both viruses can be eluted in a single step. Our protocol using a 1 ml C4 column enables processing of 400 ml saline water samples in less than 60 min and increases the sensitivity of RoV and NoV detection by approximately 50-fold and 10-fold respectively. The protocol was also scaled up using larger capacity 8 ml C4 columns to process 4000 ml of seawater samples with concentration factors of 300-fold for RoV and 40-fold for NoV, without any significant increase in processing time. Furthermore, C4 monolithic columns were adapted for field use in an on-site application of RoV concentration from seawater samples with performance equivalent to that of the reference laboratory setup. Overall, the results from successful deployment of CIM C4 columns for concentration of rotavirus and norovirus in seawater samples reiterate the utility of monolithic supports as efficient, scalable and modular preparative tools for processing environmental water samples to enhance viral detection using molecular methods.

Tracing sources of Listeria contamination in traditional Italian cheese associated with a US outbreak: investigations in Italy.

In 2012 a US multistate outbreak of listeriosis was linked to ricotta salata imported from Italy, made from pasteurized sheep's milk. Sampling activities were conducted in Italy to trace the source of Listeria monocytogenes contamination. The cheese that caused the outbreak was produced in a plant in Apulia that processed semi-finished cheeses supplied by five plants in Sardinia. During an 'emergency sampling', 179 (23·6%) out of 758 end-products tested positive for L. monocytogenes, with concentrations from <10 c.f.u./g to 1·1 × 106 c.f.u./g. Positive processing environment samples were found in two out of four processing plants. A 'follow-up sampling' was conducted 8 months later, when environmental samples from three out of six plants tested positive for L. monocytogenes and for Listeria spp. PFGE subtyping showed 100% similarity between US clinical strains and isolates from ricotta salata, confirming the origin of the outbreak. The persistence of strains in environmental niches of processing plants was demonstrated, and is probably the cause of product contamination. Two PFGE profiles from clinical cases of listeriosis in Italy in 2011, stored in the MSS-TESSy database, were found to have 100% similarity to one PFGE profile from a US clinical case associated with the consumption of ricotta salata, according to the US epidemiological investigation (sample C, pulsotype 17). However, they had 87% similarity to the only PFGE profile found both in the US clinical case and in 14 ricotta cheese samples collected during the emergency sampling (sample B, pulsotype 1). Sharing of molecular data and availability of common characterization protocols were key elements that connected the detection of the US outbreak to the investigation of the food source in Italy. Simultaneous surveillance systems at both food and human levels are a necessity for the efficient rapid discovery of the source of an outbreak of L. monocytogenes.

Preparedness for a nuclear accident: removal of radioiodine from soil by chemical processing

Processing environmental samples for analysis can result in loss of target or bystander radionuclides. Removal of carrier-free 131I from soil samples by chemical processes was evaluated in the presence and absence of iodide carrier, H2O2, and AgNO3. Soil samples spiked with 131I and 134Cs tracer were boiled under reflux for 30 min in HNO3 and combinations of aforementioned reagents. Spectroscopic analysis revealed the greatest removal of radioiodine (45 ± 1 %) was achieved using AgNO3 and carrier; the lowest (7 ± 1 %) was achieved by refluxing with H2O2 and HNO3. The results quantify the effect of iodide carrier and suggest the preferential interaction of H2O2 with iodide instead of soil organic matter.

Contamination pathways of spore-forming bacteria in a vegetable cannery

Spoilage of low-acid canned food during prolonged storage at high temperatures is caused by heat resistant thermophilic spores of strict or facultative bacteria. Here, we performed a bacterial survey over two consecutive years on the processing line of a French company manufacturing canned mixed green peas and carrots. In total, 341 samples were collected, including raw vegetables, green peas and carrots at different steps of processing, cover brine, and process environment samples. Thermophilic and highly-heat-resistant thermophilic spores growing anaerobically were counted. During vegetable preparation, anaerobic spore counts were significantly decreased, and tended to remain unchanged further downstream in the process. Large variation of spore levels in products immediately before the sterilization process could be explained by occasionally high spore levels on surfaces and in debris of vegetable combined with long residence times in conditions suitable for growth and sporulation. Vegetable processing was also associated with an increase in the prevalence of highly-heat-resistant species, probably due to cross-contamination of peas via blanching water. Geobacillus stearothermophilus M13-PCR genotypic profiling on 112 isolates determined 23 profile-types and confirmed process-driven cross-contamination. Taken together, these findings clarify the scheme of contamination pathway by thermophilic spore-forming bacteria in a vegetable cannery.

Unraveling the unseen players in the ocean - a field guide to water chemistry and marine microbiology.

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Evaluation of the 3M™ Petrifilm™ Salmonella express system for the detection of Salmonella species in selected foods: collaborative study.

The 3M™ Petriflm™ Salmonella Express (SALX) System is a simple, ready-to-use chromogenic culture medium system for the rapid qualitative detection and biochemical confirmation of Salmonella spp. in food and food process environmental samples. The 3M Petrifilm SALX System was compared using an unpaired study design in a multilaboratory collaborative study to the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) Microbiology Laboratory Guidebook (MLG) 4.07 (2013) Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products and Carcass and Environmental Sponges for raw ground beef and the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA/BAM) Chapter 5, Salmonella (2011) reference method for dry dog food following the current AOAC validation guidelines. For this study, a total of 17 laboratories located throughout the continental United States evaluated 1872 test portions. For the 3M Petrifilm SALX System, raw ground beef was analyzed using 25 g test portions, and dry dog food was analyzed using 375 g test portions. For the reference methods, 25 g test portions of each inatrix were analyzed. The two matrices were artificially contaminated with Salmonella at three inoculation levels: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2-2 CFU/test portion), and a high inoculum level (2-5 CFU/test portion). Each inoculation level was statistically analyzed using the probability of detection statistical model. For the raw ground beef and dry dog food test portions, no significant differences at the 95% confidence interval were observed in the number of positive samples detected by the 3M Petrifilm SALX System versus either the USDA/FSIS-MLG or FDA/BAM methods.

Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1.

Absence of growth ofListeria monocytogenesin naturally contaminated Cheddar cheese

Each cheese producer is responsible by the legislation for the number of Listeria monocytogenes in cheese and is required to prove that numbers will not exceed 100cfu/g throughout the shelf-life of the cheese. Even in the case of hard-cheese such as Cheddar cheese, the absence of growth of List. monocytogenes during ripening has to be demonstrated to comply with EU legislation. Studies dedicated to assessing List. monocytogenes growth throughout cheese shelf-life are generally based on artificially contaminated cheeses. Contrary to the majority of works, the current study focused on the growth of List. monocytogenes in naturally contaminated raw milk farmhouse Cheddar cheeses during a five-month ripening period. List. monocytogenes growth was assessed by direct count and its presence was detected by enrichment in two naturally contaminated cheese batches. In order to track routes of contamination, 199 processing environment samples from inside and outside the processing facility were taken, and their analysis for the presence of List. monocytogenes was performed on four occasions over a 9-month period. List. monocytogenes isolates were differentiated using PFGE and serotyping. List. monocytogenes never exceeded 20cfu/g in the cheeses and could not be detected after five months of ripening. Eleven pulsotypes were identified. One pulsotype was found in the yard outside the processing facility, in a vat, on the processing area floor and in a cheese. This indicated that the outside environment constitutes a potential source of contamination of the processing environment and of the cheese. These results demonstrate that this farmhouse Cheddar cheese does not support List. monocytogenes growth and suggests that the efforts to reduce processing environment contamination are worthwhile.

Organic Acid Resistance and Toxin Gene Profiles of B. cereus Isolated from Red Pepper Powder

AbstractThe objectives of the study were to determine the diversity of organic acid resistance as well as the toxin gene profiles of Bacillus cereus isolated from red pepper powder and to analyze the inhibition kinetics of unstressed type culture and resistant food isolates against organic acids.Among 68 B. cereus isolated from red pepper and processing environmental samples (n = 180), 1.4–16.7% of the cells could survive at 0.5% of acetic, malic and tartaric acid. Parameter α of the Weibull model for type strain were up to threefold lower than for resistant food isolate at 0.2% or lower concentration of organic acid treatment. All the B. cereus isolates were positive for three genes in the nonhemolytic enterotoxin complex (nheA, nheB and nheC), entFM and bceT genes. The ces gene‐encoding emetic toxin was detected in nine (13.2%) of the 68 isolates tested.Practical ApplicationsBacillus cereus is an etiological agent of foodborne illness in Korea. The food products under risk for B. cereus food poisoning are pasteurized (dairy foods or cooked chilled foods) and dry foods. When B. cereus has been exposed to sublethal acid treatment conditions, adapted or resistant strains have developed. Acid resistance of the strain cause a food safety problem because it may result in enhanced protection for cells which are subsequently exposed to lethal heat or hydrogen peroxide stress. The rate of acid resistance and the kinetic parameters of wild type food isolates against organic acid treatment can be used to develop intervention strategies and in risk assessment to assure food safety. A diversity of toxin gene profiles which include diarrheal and emetic toxins also provide useful information in risk assessment of food poisoning caused by B. cereus.