Studies on the drug resistance profile of Enterococcus faecium distributed from poultry retailers to hospitals.

The multidrug resistant Enterococcus faecium (MEF) strains originating from farm animals are proliferating at a substantial pace to impact downstream food chains and could reach hospitals. This study was conducted to elucidate the drug susceptibility profile of MEF strains collected from poultry products in Ann Arbor, MI area and clinical settings from Michigan State Lab and Moffitt Cancer Center (MCC) in Florida. Presumptive positive Enterococcus isolates at species level were identified by Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) analysis. The antibiotic susceptibility profile for both poultry and clinical strains was determined by the Thermo Scientific's Sensititre conform to the National Committee for Clinical Laboratory Standards (NCCLS) and validated via quantitative real-time PCR (qPCR) methods. Out of 50 poultry samples (Turkey: n = 30; Chicken: n = 20), 36 samples were positive for Enterococcus species from which 20.83% were identified as E. faecium. All the E. faecium isolates were multidrug resistant and displayed resistance to the last alternative drug, quinupristin/dalfopristin (QD) used to treat vancomycin resistant E. faecium (VRE) in hospitals. Results indicate the presence of MEF strains in food animals and clinical settings that are also resistant to QD.

Klebsiella michiganensis sp. nov., a new bacterium isolated from a tooth brush holder.

Isolate W14(T) recovered from a household tooth brush holder was found to be gram-negative, a facultative anaerobic, non-motile, capsulated, and a non-endospore-forming straight rod. Based on phylogenetic analysis with 16S rRNA gene sequence, isolate W14(T) was affiliated to the genus Klebsiella. The closest phylogenetic relative was K. oxytoca with 99 % similarity in the 16S rRNA gene sequence. The major whole-cell fatty acids were C(16:0) (31.23 %), C(18:1ω6c)/C(18:1ω7c) (21.10 %), and C(16:1ω7c)/C(16:1ω6c) (19.05 %). The sequence similarities of isolate W14(T) based on rpoB, gyrA, and gyrB were 97, 98, and 98 % with K. oxytoca, and 97, 93, and 90 % with K. mobilis (=Enterobacter aerogenes), respectively. The ribotyping pattern showed a 0.46 similarity with K. oxytoca ATCC 13182(T) and 0.24 with K. mobilis ATCC 13048(T). The DNA G+C content of isolate W14(T) was 54.6 mol%. The DNA-DNA relatedness was 55.7 % with K. oxytoca ATCC 13182(T). Using the identification technology of MALDI-TOF mass spectrometry, the top matches for this isolate were K. oxytoca ATCC 13182(T) (Match Factor Score 1.998) and K. mobilis (Score 1.797). On the basis of phenotypic, biochemical, chemotaxonomic, and molecular studies, isolate W14(T) could be differentiated from other members of the genus Klebsiella including K. mobilis. Therefore, it is proposed that isolate W14(T) (=ATCC BAA-2403(T)=DSM 25444(T)) should be classified as the type strain of a novel species of the genus Klebsiella, K. michiganensis sp. nov.

Laboratory validation of an ozone device for recreational water treatment.

Obtaining an accurate assessment of a treatment system's antimicrobial efficacy in recreational water is difficult given the large scale and high flow rates of the water systems. A laboratory test system was designed to mimic the water conditions and potential microbial contaminants found in swimming pools. This system was utilized to evaluate the performance of an in situ ozone disinfection device against four microorganisms: Cryptosporidium parvum, bacteriophage MS2, Enterococcus faecium, and Pseudomonas aeruginosa. The sampling regimen evaluated the antimicrobial effectiveness in a single pass fashion, with samples being evaluated initially after exposure to the ozone unit, as well as at points downstream from the device. Based on the flow dynamics and log reductions, cycle threshold (Ct) values were calculated. The observed organism log reductions were as follows: >6.7 log for E. faecium and P. aeruginosa; >5.9 log for bacteriophage MS2; and between 2.7 and 4.1 log for C. parvum. The efficacy results indicate that the test system effectively functions as a secondary disinfection system as defined by the Centers for Disease Control and Prevention's Model Aquatic Health Code.

Microbial siderophores: a mini review.

Iron is one of the major limiting factors and essential nutrients of microbial life. Since in nature it is not readily available in the preferred form, microorganisms produce small high affinity chelating molecules called siderophores for its acquisition. Microorganisms produce a wide variety of siderophores controlled at the molecular level by different genes to accumulate, mobilize and transport iron for metabolism. Siderophores also play a critical role in the expression of virulence and development of biofilms by different microbes. Apart from maintaining microbial life, siderophores can be harnessed for the sustainability of human, animals and plants. With the advent of modern molecular tools, a major breakthrough is taking place in the understanding of the multifaceted role of siderophores in nature. This mini review is intended to provide a general overview on siderophore along with its role and applications.

Validation of the Reveal® 3-D for Gluten Assay for Detection of Gluten in Clean-in-Place Rinses and Stainless Steel Environmental Surfaces: AOAC Performance Tested MethodSM 122201.

BACKGROUND: Reveal® 3-D for Gluten is an immunochromatographic assay for the qualitative detection of gluten in environmental samples. The test uses monoclonal antibodies reactive to prolamins in wheat. OBJECTIVE: The objective of the study was to validate the Reveal 3-D test for detection of gluten in clean-in-place rinse and swabs from a stainless steel surface. METHODS: Elements of the study included food selectivity and interference testing, matrix testing, an assay robustness study, and reagent stability/lot-to-lot consistency testing. Wheat flour was used as the spiking material for all matrixes. RESULTS: In selectivity and interference testing, nine target matrixes all tested positive and 36 of 39 non-target matrixes tested negative. Almond flour, sesame flour, and cornstarch produced positive results as 100% commodities; reactivity can be eliminated with dilution or by testing without use of food extraction buffer, which is not a standard part of the environmental testing method. With a gluten spike at 9.3 mg/kg, chestnut flour, guar gum, and xanthan gum as 100% commodities inhibited the ability of the assay to detect gluten when tested without dilution. In quaternary ammonium clean-in-place rinse and swabs from stainless steel, 100% positive results were obtained at levels of 2.8 mg/kg and 4.7 µg/100 cm2, respectively. Results of independent laboratory testing of swabs from stainless steel supported those of internal trials. Robustness testing showed that introducing variations to three operating parameters simultaneously had no adverse effect on assay performance. In the reagent stability study, data supported kit expiration dating of 11 months. CONCLUSION: Results of the current study show that the Reveal test is an accurate and reliable method for qualitative detection of gluten in select clean-in-place rinse and environmental samples. HIGHLIGHTS: The Reveal test was able to detect gluten at levels of 2.8 ppm in clean-in-place rinse and 4.7 µg/100 cm2 in swabs from stainless steel.

The microbiology of metalworking fluids.

Metalworking fluids (MWFs) are complex mixtures of chemicals and are indispensable materials in industry. They are used as cooling and lubricating agents in different machining process such as grinding, milling, and cutting. The quality of MWFs is affected by physical, chemical, and microbial contaminates. In particular, MWFs are highly vulnerable to microbial contamination, which may act both as potential pathogens and deteriorgens. Microbial contamination is of major concern due to potential health hazards such as skin dermatitis and hypersensitivity pneumonitis. The contaminated MWFs can exhibit high degrees of microbial loading, ranging from 10(4) to 10(10) colony-forming units (CFU)/ml. Wide varieties of microorganisms are reported to colonize MWFs. Traditional culturing techniques are not only laborious and time consuming but also underestimate the actual distribution of the microorganisms present in the contaminated MWFs. Therefore, rapid molecular methods such as real-time PCR and fluorescent in situ hybridization are implemented to monitor the microbial load. In industry, biocides are presently used to control microbial contamination. However, it has its own disadvantages and therefore, in recent years, alternative methods such as UV irradiation were evaluated to reduce microbial contamination in MWFs. Microbes inhabiting the MWF are also capable of forming biofilm which is detrimental to the MWF system. Biofilm is resistant to common disinfectant methods, and thus further research and development is required to effectively control its formation within MWF systems. This review is intended to discuss the overall microbiological aspects of MWF.

Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization.

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 10(3) cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (± 1.1) × 10(6) and 5.0 (± 2.3) × 10(6) cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.

Molecular cloning of Brevundimonas diminuta for efficacy assessment of reverse osmosis devices.

Brevundimonas diminuta is the test organism specified in the United States Environmental Protection Agency's (USEPA) reverse osmosis (RO) treatment device verification protocol. As non-selective growth medium is employed, enumeration of B. diminuta may be impaired due to interference by indigenous heterotrophic bacteria. Thus the microbial removal capability of the filtration system may be incorrectly assessed. As these treatment devices are used in emergency situations, the health of the public could be compromised. The objective of this study was to develop selective approaches for enumerating viable B. diminuta in test water. Two molecular approaches were investigated: expression of a kanamycin resistance gene and expression of a fluorescent protein gene. The USEPA protocol specifies a 0.3 µm cell size, so the expression of the selective markers were assessed following growth on media designed to induce this small cell diameter. The kan(R) strain was demonstrated to be equivalent to the wild type in cell dimension and survival following exposure to the test water. The kan(R) strain showed equivalent performance to the wild type in the RO protocol indicating that it is a viable alternative surrogate. By utilizing this strain, a more accurate validation of the RO system can be achieved.

Are we aware of microbial hotspots in our household?

Household microorganisms are found in unexpected places. Therefore, the authors conducted a study to investigate the microbial hotspots and reveal the misconceptions regarding the most contaminated objects in the household. In the authors' study, 26 daily use objects in 22 households were sampled to determine the levels of heterotrophic plate count (HPC), coliforms, E. coli, yeast and mold, and Staphylococcus aureus. High microbial concentration was found in the kitchen area and the dish sponge was the most contaminated item in the household, followed by the toothbrush holder. Coliforms were most prevalent in the kitchen on items such as sponges, sinks, and cutting boards. Yeast and molds were found on leather, fabric, porcelain, and laminate, and S. aureus was found on personal objects and pet's items. Overall, HPC and the presence of coliforms were significantly related to surface type (p < .05). In the kitchen, cleaning frequency (p < .03) and type of cleaning (p < .0003) had significant effects on HPC. The authors' study provides information that will help the general population to make an educated decision in developing a proper and routine cleaning regime in their homes. This baseline data might contribute to designing appropriate sanitation guidelines or standards that will help to implement proper sanitation practices in households and to conducting further research in the area of foodborne and household communicable diseases.

Soleris® Automated System for the Rapid Detection of Burkholderia cepacia Complex in Cosmetic Products.

BACKGROUND: Burkholderia cepacia complex (Bcc) has emerged as an important opportunistic pathogen with rising concern in pharmaceuticals and cosmetic products. The Bcc supplement (S2-BCC-S) was purposely developed and used with the Pseudomonas vial (PD-109) for the detection of Bcc through the Soleris® Next Generation automated instrument system. OBJECTIVE: This study aimed to evaluate the performance of the Soleris Bcc testing method for cosmetic products. METHOD: Inclusivity and exclusivity were assessed with the Soleris Bcc method and the United States Pharmacopeia (USP) method in three enrichment broths. Matrix testing was conducted using 28 cosmetic products to compare the equivalency of the Soleris Bcc method to that of the USP reference method. Repeatability of the Soleris Bcc assay, method robustness, product stability, and lot-to-lot consistency of the Soleris reagents were also assessed. RESULTS: Both the Soleris Bcc and the USP methods supported the growth of all 26 inclusivity strains, except the USP method missed one inclusivity strain in one broth. For exclusivity, 0-6% was presumptive positive with the Soleris Bcc method, and 42-48% was presumptive positive with the reference method. Kappa index was 0.96 for the matrix testing, indicating a good agreement between the Soleris Bcc assay and the reference method for testing Bcc in cosmetics. Repeatability results showed the coefficient of variation was less than 4%. The robustness and ruggedness study yielded detection times within 1 h differences when small variations were introduced. The lot-to-lot study showed consistent results among four lots of the Bcc reagents. CONCLUSIONS: The automated Soleris method was successfully demonstrated to be robust, sensitive, and specific for Bcc detection in cosmetic products. HIGHLIGHTS: The Soleris Bcc method is user-friendly. It shows the results in real time and generates the report automatically. Implementation of this method for detection of Bcc in cosmetics would save significant time and resources.

Emergency Response Validation of the Soleris® Direct Yeast and Mold Method for Detection of Yeast and Mold in Dried Cannabis Flower: AOAC Performance Tested MethodSM 051301.

BACKGROUND: Soleris® Direct Yeast and Mold (DYM) is a growth-based, automated method for detection of yeast and mold in select foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested. OBJECTIVE: The objective of this study was to validate the method for testing of dried cannabis flower. The validation was conducted under the Emergency Response Validation program of the AOAC Research Institute. METHOD: The study included inclusivity and exclusivity testing, in particular testing of yeast and mold species associated with cannabis, and a matrix study in which Soleris method presumptive results were compared with Soleris confirmed results using Dichloran Rose-Bengal Chloramphenicol (DRBC) agar for confirmation. Samples at four different levels of natural yeast and mold contamination were tested at two test thresholds. RESULTS: In inclusivity testing, all 63 yeast and mold strains tested produced positive results within the specified test duration of 72 h. In exclusivity testing, 36 of 37 strains tested produced no detection within 72 h. In matrix testing, there were no significant differences between Soleris presumptive and confirmed results for any contamination level or test threshold as determined by probability of detection analysis. CONCLUSIONS: Results indicate that the Soleris method is an effective procedure for detection of yeast and mold in dried cannabis flower. HIGHLIGHTS: With the Soleris method, results are available within 72 h compared with the 5-7 days required for microbiological culture methods.

Validation of the Soleris® Coliform Method for Detection of Coliform Bacteria in Dried Cannabis Flower: AOAC Performance Tested MethodSM 010302.

BACKGROUND: The Soleris® Coliform Vial is a growth-based, automated method for detection of coliform bacteria in foods and other consumer products such as nutraceuticals and cosmetics. The method was granted AOAC Performance Tested MethodSM certification for select foods after successful completion of a validation study. OBJECTIVE: The objective of the current study was to validate the Soleris coliform method for use with dried cannabis flower (>0.3% delta 9-tetrahydrocannabinol). METHODS: A comparative matrix study was performed in which naturally contaminated dried cannabis flower was tested with the Soleris coliform method and with the U.S. Food and Drug Administration Bacteriological Analytical Manual solid medium method. Multiple lots of dried cannabis flower were obtained, pre-screened for coliforms, and blended to produce test materials at four different contamination levels ranging from 4.5 to 1600 CFU/g. Each material was tested at three different Soleris detection threshold levels determined by the dilution used to inoculate the Soleris vials. Probability of detection analysis was performed to determine if differences in the number of positive results obtained with the two methods were significant. RESULTS: For all four dried cannabis flower materials, at all three Soleris test thresholds, there were no significant differences in the number of positive results obtained with the Soleris and cultural plating methods as determined by probability of detection analysis at P < 0.05. CONCLUSION: The Soleris coliform test is an accurate method for detection of coliform bacteria in dried cannabis flower. HIGHLIGHTS: The Soleris coliform method provides cannabis industry QC personnel with an effective method for analysis of dried cannabis flower and produces results in 18-24 h.

Validation of the Soleris® NF-TVC Method for Detection of Aerobic, Mesophilic Microorganisms in Dried Cannabis Flower: AOAC Performance Tested MethodSM 071203.

BACKGROUND: Soleris® Non-Fermenting Total Viable Count (NF-TVC) is a growth-based, automated method for semiquantitative detection of aerobic, mesophilic microorganisms in foods and other consumer products such as nutraceuticals and cosmetics. The method was granted AOAC Performance Tested MethodSM status for select foods after successful completion of a validation study. OBJECTIVE: The objective of the current study was to validate the Soleris NF-TVC method for use with dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)]. METHODS: The validation consisted of a comparative matrix study in which naturally contaminated dried cannabis flower was tested with the Soleris NF-TVC method and with the AOAC Official Methods of AnalysisSM  966.23 dilution plating method. Multiple lots of dried cannabis flower were obtained, pre-screened for total aerobic, mesophilic viable count levels, and blended to produce test materials at four different levels of contamination ranging from 1.0 × 103 to 2.2 × 105 CFU/g. Each material was tested at three different Soleris detection threshold levels determined by the dilution used to inoculate the Soleris vials. Probability of detection analysis was performed to determine if differences in the number of positive results obtained with the two methods were significant. RESULTS: For all four dried cannabis flower materials, at all three Soleris test thresholds, there were no significant differences in performance comparing the Soleris and reference dilution plating methods as determined by probability of detection analysis at P < 0.05. CONCLUSIONS: It is concluded that the Soleris NF-TVC method is an accurate and effective method for detection of aerobic, mesophilic microorganisms in dried cannabis flower. HIGHLIGHTS: The Soleris NF-TVC method provides cannabis industry quality control personnel with an effective method for analysis of dried cannabis flower and produces results in 24-48 h.

Validation of the Soleris®Enterobacteriaceae Method for Detection of Enterobacteriaceae in Dried Cannabis Flower: AOAC Performance Tested MethodSM 121901.

BACKGROUND: The Soleris®Enterobacteriaceae vial is a growth-based, automated method for detection of bacteria of the family Enterobacteriaceae in foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner, in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested. The Soleris method was granted AOAC Performance Tested MethodSM (PTM) status for select foods after successful completion of a validation study (PTM 121901). OBJECTIVE: The objective of this study was to validate the method for the detection of Enterobacteriaceae in dried cannabis flower [>0.3% delta-9-tetrahydrocannabinol (THC)]. METHODS: The matrix study included comparison of Soleris method presumptive results to confirmation from the Soleris vials, and comparison of the Soleris confirmed results to those of the ISO 21528-2:2017 colony count method. Test materials at four different levels of contamination ranging from 7.8 to 3500 CFU/g were tested at three dilutions, corresponding to test thresholds. RESULTS: Probability of detection analysis at P < 0.05 showed there were no significant differences between Soleris presumptive and confirmed results, and no significant differences between Soleris confirmed and ISO 21528-2:2017 results. CONCLUSION: The results provided evidence that the Soleris Enterobacteriaceae test is an accurate method for detection of Enterobacteriaceae in dried cannabis flower. HIGHLIGHTS: The Soleris Enterobacteriaceae method provides cannabis industry QC personnel with an effective method for analysis of dried cannabis flower and produces results in 20-24 h.

Validation of Modifications to the Soleris®E. coli Method for Detection and Threshold Determination of Escherichia coli in Select Foods: Level 3 Modification to AOAC Performance Tested MethodSM 101101.

BACKGROUND: Soleris®E. coli is an automated, growth-based method for detection and semi-quantitative determination of Escherichia coli in foods. The method can be used in dilute-to-specification (threshold) or presence/absence modes. OBJECTIVE: The objective of the study was to validate four modifications to the method: (1) a change in the vial detection window plug composition from agar to agarose to improve plug consistency and robustness, (2) a change in pre-enrichment incubation time for presence/absence testing from 6 h to 18-24 h, (3) a change in vial incubation temperature from 44.5 to 43.5°C, and (4) incorporation of a simple direct-from-vial confirmation test as an alternative to traditional procedures. METHODS: Elements of the study included inclusivity/exclusivity testing, matrix testing in comparison to the ISO 7251:2005 reference method, reagent stability/lot-to-lot consistency testing, and an independent laboratory study. RESULTS: In inclusivity testing, all 55 Escherichia coli strains tested produced positive results. In exclusivity testing, 30 of 31 strains of other bacterial species produced negative results, the sole exception being a strain of Enterobacter cloacae. In internal and independent laboratory matrix testing of mozzarella cheese, condensed milk, pasteurized liquid egg, and frozen green beans, results showed no significant differences in performance of the Soleris and reference methods with two exceptions, one in which the Soleris method produced more positive results, and one in which the reference method produced more positive results. CONCLUSION: Performance characteristics of the modified Soleris E. coli method are consistent with those of the original validated method. HIGHLIGHTS: The Soleris E. coli method offers improvements in ease of use and reagent robustness.

Evaluation of molecular techniques for identification and enumeration of Raoultella terrigena ATCC 33257 in water purifier efficacy testing.

Raoultella terrigena ATCC 33257, a representative of the coliform group, is commonly used as a challenge organism in water purifier efficacy testing. In addition to being time consuming, traditional culturing techniques and metabolic identification systems (including automated systems) also fail to accurately differentiate this organism from its closely related neighbors belonging to the Enterobacteriaceae group. Molecular-based techniques, such as real-time quantitative polymerase chain reaction (qPCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting, are preferred methods of detection because of their accuracy, reproducibility, specificity, and sensitivity, along with shorter turnaround time. ERIC-PCR performed with the 1R primer set demonstrated stable unique banding patterns (~800, ~300 bp) for R. terrigena ATCC 33257 different from patterns observed for R. planticola and R. ornithinolytica. The primer pair developed from gyraseA (gyrA) sequence of R. terrigena for the SYBR Green qPCR assay using the AlleleID(®) 7.0 primer probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cells and 4.7 fg with genomic DNA. The primer pair was successful in determining the concentration (5.5 ± 0.3 × 10(6) CFU/ml) of R. terrigena from water samples spiked with equal concentration of Escherichia coli and R. terrigena. Based on these results from the ERIC-PCR and the SYBR Green qPCR assay, these molecular techniques can be efficiently used for rapid identification and quantification of R. terrigena during water purifier testing.

Validation of the One Broth One Plate for Salmonella Method for Detection of Salmonella Spp. in Select Food and Environmental Samples: AOAC Performance Tested MethodSM 102002.

BACKGROUND: One Broth One Plate for Salmonella (OBOP Salmonella) is a rapid and simple method for detection of Salmonella spp. in food and environmental samples using traditional culture methodology. The method utilizes single-step enrichment followed by plating to a selective/differential, chromogenic agar. OBJECTIVE: The purpose of the validation study was to measure the effectiveness of the OBOP Salmonella method in comparison to reference culture procedures. METHOD: Performance of the OBOP Salmonella method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5 reference method for queso fresco, smoked salmon, cantaloupe, chocolate, black pepper, chili powder, dry pet food, and sponge samples from a stainless steel surface, or to that of the U.S. Department of Agriculture Microbiology Laboratory Guidebook Chapter 4.10 method for raw ground turkey, chicken carcass rinse, and pasteurized liquid egg. Inclusivity/exclusivity, robustness, and stability/lot-to-lot consistency testing was also performed. RESULTS: In the matrix study, there were no statistically significant differences in performance between the OBOP Salmonella and reference methods, as determined by probability of detection analysis (P < 0.05), for any of the matrixes examined. All 104 Salmonella spp. strains produced positive results in inclusivity testing, and all 33 non-salmonellae exclusivity strains tested negative with the OBOP Salmonella method. CONCLUSIONS: Results of the validation study show that the OBOP Salmonella method is a reliable procedure for detection of Salmonella spp. in select matrixes. The method is simple to perform, requires no specialized equipment, and produces results in as little as 37 h. HIGHLIGHTS: The OBOP Salmonella method was awarded AOAC PTMSM (#102002) for detection of Salmonella in queso fresco, smoked salmon, cantaloupe, chocolate, black pepper, chili powder, dry pet food, sponge samples on a stainless steel surface, raw ground turkey, chicken carcass rinse, and pasteurized liquid egg. The method is also approved by MicroVal® for a broad range of foods under certification number 2019LR88.

Overview of Portable Assays for the Detection of Mycotoxins, Allergens, and Sanitation Monitoring.

BACKGROUND: Many food recalls are related to the presence of undeclared allergens and microorganisms in food products. To reduce these occurrences, portable diagnostic assay kits are available to quantitate mycotoxins, to detect allergens and gluten in foods and on environmental surfaces, and for sanitation monitoring. OBJECTIVE: This article reviews diagnostic kits that can detect sources of contamination in food and ingredients as well as on surfaces and clean-in-place rinses. METHOD: Mycotoxins and gluten were detected using lateral flow diagnostic assays. Sanitation monitoring of surfaces was completed using a chemiluminescent assay to detect adenosine 5'-triphosphate disodium salt hydrate (ATP) and another assay to detect protein. RESULTS: Gluten was detected at 10 ppm in spiked commodities and on wet and dry surfaces at 2.5 µg/100cm2. Deoxynivalenol was quantitated in dry distillers grains plus solubles and mean results were within two SDs of those determined by HPLC. The chemiluminescent assay had an LOD of 6 fmol of ATP and was able to detect a 1:10 000 dilution of orange juice from surfaces. The protein assay detected 5 µg of bovine serum albumin (BSA) directly applied to the sampler, 100 µg of BSA on surfaces, and detected 1:10 dilutions of Greek yogurt and raw beef from surfaces. CONCLUSIONS: Portable diagnostic kits evaluated in this work provided accurate, rapid, and sensitive results for detection of mycotoxins, gluten, proteins, and ATP. These methods can be used in facilities with minimal training and provide results that are important to ensure food safety. HIGHLIGHTS: Portable methods to detect gluten, mycotoxins, proteins, and ATP are presented.

Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids.

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer-probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer-probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 x 10(3) and 3.9 x 10(6) CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 x 10(1) to 1.4 x 10(5) CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer-probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.

Selective enumeration strategies for Brevundimonas diminuta from drinking water.

Brevundimonas diminuta is used as a control organism for validating the efficiency of water filtration systems. Since these protocols use nonselective growth media, heterotrophic plate count bacteria (HPCs) indigenous to the water distribution system may interfere with B. diminuta enumeration, thus leading to inaccurate assessment of the filter's microbial reduction capability. This could negatively impact public health as unsafe drinking water may be produced. This study was conducted to evaluate different potential routes for selective enumeration of B. diminuta in drinking water. B. diminuta's biochemical and molecular relationships to HPCs recovered from a laboratory drinking-water system were investigated. Of the 24 HPC morphotypes recovered, members of the Alpha- and Betaproteobacteria were most commonly identified. Based on comparisons of catabolic profiles (generated by the Biolog system) using principal component analysis, B. diminuta possessed similar metabolic patterns to several of the Alphaproteobacteria (Sphingomonas and Caulobacter), indicating that development of a selective medium based solely on carbon source was not feasible. Antibiotic susceptibility profiles revealed that the HPCs were least resistant to kanamycin, making it a candidate for future selective applications. Sequence comparisons of partial 16S rRNA sequences did not reveal any distinct similarities. However, basic local alignment search tool (BLAST) alignments of the gyrB and rpoD sequences for B. diminuta did show uniqueness, with the next closest match being to Caulobacter (88% and 79% similarity, respectively). Future investigation will focus on applying molecular assays, such as fluorescent in situ hybridization and quantitative real-time polymerase chain reaction (PCR), and incorporating an antibiotic marker or expressed fluorescent protein into the wild-type strain of B. diminuta for selective enumeration of B. diminuta.