loop-mediated-isothermal-amplification-for-screening-salmonella

Loop-Mediated Isothermal Amplification for Screening <em>Salmonella</em> in Animal Food and Confirming <em>Salmonella</em> from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

U.S. Food and Drug Administration

A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar aadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII, aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in Citrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coli O157:H7.

Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates.

Point mutations in the topoisomerase (DNA gyrase A) gene are known to be associated with fluoroquinolone resistance in Campylobacter. Recent studies have shown that an efflux pump encoded by cmeABC is also involved in decreased susceptibilities to fluoroquinolones, as well as other antimicrobials. Genome analysis suggests that Campylobacter jejuni contains at least nine other putative efflux pumps. Using insertional inactivation and site-directed mutagenesis, we investigated the potential contributions of these pumps to susceptibilities to chloramphenicol, ciprofloxacin, erythromycin, and tetracycline in C. jejuni and Campylobacter coli. Insertional inactivation of cmeB resulted in 4- to 256-fold decreases in the MICs of chloramphenicol, ciprofloxacin, erythromycin, and tetracycline, with erythromycin being the most significantly affected. In contrast, inactivation of all other putative efflux pumps had no effect on susceptibility to any of the four antimicrobials tested. Mutation of gyrA at codon 86 (Thr-Ile) caused 128- and 64-fold increases in the MICs of ciprofloxacin and nalidixic acid, respectively. The replacement of the mutated gyrA with a wild-type gyrA allele resulted in a 32-fold decrease in the ciprofloxacin MIC and no change in the nalidixic acid MIC. Our findings indicate that CmeABC is the only efflux pump among those tested that influences antimicrobial resistance in Campylobacter and that a point mutation (Thr-86-Ile) in gyrA directly causes fluoroquinolone resistance in Campylobacter. These two mechanisms work synergistically in acquiring and maintaining fluoroquinolone resistance in Campylobacter species.

Role of efflux pumps and topoisomerase mutations in fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli.

Rapid and high-throughput identification and serotyping of Shiga toxin-producing Escherichia coli (STEC) O serogroups is important for detecting, investigating, and controlling STEC infection outbreaks and removing hazardous products from commerce. A Luminex microbead-based suspension array has been developed to identify the 11 most clinically relevant STEC serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. Here we present results of a blinded multilaboratory collaborative study involving 10 participants from nine laboratories using 55 unknown strains. From the total 495 analyses, two false-positive and three false-negative results were obtained, indicating the assay to be a rapid, high-throughput, and robust method for identifying clinically relevant STEC serogroups.

Multilaboratory validation of a luminex microbead-based suspension array for the identification of the 11 most clinically relevant Shiga toxin-producing Escherichia coli O serogroups.

Abstract The presence and antimicrobial susceptibility of foodborne pathogens and indicator organisms in animal feed are not well understood. In this study, a total of 201 feed ingredient samples (animal byproducts, n=122; plant byproducts, n=79) were collected in 2002 and 2003 from representative rendering plants and the oilseed (or cereal grain) industry across the United States. The occurrence and antimicrobial susceptibility of four bacterial genera (Salmonella, Campylobacter, Escherichia coli, and Enterococcus) were determined. Salmonella isolates were further characterized by serotyping and pulsed-field gel electrophoresis (PFGE). None of the samples yielded Campylobacter or E. coli O157:H7, whereas Salmonella, generic E. coli, and Enterococcus were present in 22.9%, 39.3%, and 86.6% of samples, respectively. A large percentage (47.8%) of Salmonella-positive samples harbored two serovars, and the vast majority (88.4%) of Enterococcus isolates were E. faecium. Animal byproducts had a significantly higher Salmonella contamination rate (34.4%) than plant byproducts (5.1%) (p<0.05). Among 74 Salmonella isolates recovered, 27 serovars and 55 PFGE patterns were identified; all were pan-susceptible to 17 antimicrobials tested. E. coli isolates (n=131) demonstrated similar susceptibility to these antimicrobials except for tetracycline (15.3% resistance), sulfamethoxazole (7.6%), streptomycin (4.6%), ampicillin (3.8%), and nalidixic acid (1.5%). Enterococcus isolates (n=362) were also resistant to five of 17 antimicrobials tested, ranging from 1.1% to penicillin to 14.6% to tetracycline. Resistance rates were generally higher among isolates recovered from animal byproducts. Taken together, our findings suggest that diverse populations of Salmonella, E. coli, and Enterococcus are commonly present in animal feed ingredients, but antimicrobial resistance is not common. Future large-scale studies to monitor these pathogenic and indicator organisms in feed commodities is warranted.

Retrospective Analysis of Salmonella, Campylobacter, Escherichia coli, and Enterococcus in Animal Feed Ingredients.

Most Campylobacter infections are self-limiting but antimicrobial treatment (e.g., macrolides, fluoroquinolones) is necessary in severe or prolonged cases. Susceptibility testing continues to play a critical role in guiding therapy and epidemiological monitoring of resistance. The methods of choice for Campylobacter recommended by the Clinical and Laboratory Standards Institute (CLSI) are agar dilution and broth microdilution, while a disk diffusion method was recently standardized by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Macrolides, quinolones, and tetracyclines are among the common antimicrobials recommended for testing. Molecular determination of Campylobacter resistance via DNA sequencing or PCR-based methods has been performed. High levels of resistance to tetracycline and ciprofloxacin are frequently reported by many national surveillance programs, but resistance to erythromycin and gentamicin in Campylobacter jejuni remains low. Nonetheless, variations in susceptibility observed over time underscore the need for continued public health monitoring of Campylobacter resistance from humans, animals, and food. 

Antimicrobial resistance in campylobacter: susceptibility testing methods and resistance trends.

Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 10(5) to 10(6) CFU per 25 g (i.e., 10(3) to 10(4) CFU per g) in produce, except for strains harboring the stx2c, eae-β, and eae-θ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis.

Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce.

Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study.

Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce.

Salmonella is among the most significant pathogens causing food and feed safety concerns. This study examined the rapid detection of Salmonella in various types of food and feed samples by coupling loop-mediated isothermal amplification (LAMP) with a novel reporter, bioluminescent assay in real-time (BART). Performance of the LAMP-BART assay was compared to a conventional LAMP and the commercially available 3M Molecular Detection Assay (MDA) Salmonella.

Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time.

Methicillin-resistant Staphylococcus aureus (MRSA) has been detected in retail meats, although large-scale studies are scarce. We conducted a one-year survey in 2010-2011 within the framework of the National Antimicrobial Resistance Monitoring System. Among 3520 retail meats collected from eight U.S. states, 982 (27.9%) contained S. aureus and 66 (1.9%) were positive for MRSA. Approximately 10.4% (107/1032) of S. aureus isolates, including 37.2% (29/78) of MRSA, were multidrug-resistant (MDRSA). Turkey had the highest MRSA prevalence (3.5%), followed by pork (1.9%), beef (1.7%), and chicken (0.3%). Whole-genome sequencing was performed for all 66 non-redundant MRSA. Among five multilocus sequence types identified, ST8 (72.7%) and ST5 (22.7%) were most common and livestock-associated MRSA ST398 was assigned to one pork isolate. Eleven spa types were represented, predominately t008 (43.9%) and t2031 (22.7%). All four types of meats harbored t008, whereas t2031 was recovered from turkey only. The majority of MRSA (84.8%) possessed SCCmec IV and 62.1% harbored Panton-Valentine leukocidin. Pulsed-field gel electrophoresis showed that all ST8 MRSA belonged to the predominant human epidemic clone USA300, and others included USA100 and USA200. We conclude that a diverse MRSA population was present in U.S. retail meats, albeit at low prevalence.

MRSA and multidrug-resistant Staphylococcus aureus in U.S. retail meats, 2010-2011.

Distillers grains are co-products of the corn ethanol industry widely used in animal feed. We examined the effects of erythromycin, penicillin, and virginiamycin at low concentrations reflective of those detected in distillers grains on bacterial resistance selection. At 0.1 µg/ml erythromycin, macrolide-resistant mutants were induced in one Campylobacter coli and one Enterococcus faecium strain, while these strains plus three additional C. coli, one additional E. faecium, and one C. jejuni also developed resistance when exposed to 0.25 µg/ml erythromycin. At 0.5 µg/ml erythromycin, a total of eight strains (four Campylobacter and four Enterococcus) obtained macrolide-resistant mutants, including two strains from each genus that were not selected at lower erythromycin concentrations. For penicillin, three of five E. faecium strains but none of five Enterococcus faecalis strains consistently developed resistance at all three selection concentrations. Virginiamycin at two M:S ratios did not induce resistance development in four out of five E. faecium strains; however, increased resistance was observed in the fifth one under 0.25 and 0.5 µg/ml virginiamycin selections. Although not yet tested in vivo, these findings suggest a potential risk of stimulating bacterial resistance development in the animal gut when distillers grains containing certain antibiotic residues are used in animal feed.

Effects of low concentrations of erythromycin, penicillin, and virginiamycin on bacterial resistance development in vitro.

Loop-mediated isothermal amplification (LAMP) has emerged as a promising alternative to PCR for pathogen detection in food testing and clinical diagnostics. This study aimed to validate a Salmonella LAMP method run on both turbidimetry (LAMP I) and fluorescence (LAMP II) platforms in representative animal food commodities. The U.S. Food and Drug Administration (FDA)'s culture-based Bacteriological Analytical Manual (BAM) method was used as the reference method and a real-time quantitative PCR (qPCR) assay was also performed. The method comparison study followed the FDA's microbiological methods validation guidelines, which align well with those from the AOAC International and ISO. Both LAMP assays were 100% specific among 300 strains (247 Salmonella of 185 serovars and 53 non-Salmonella) tested. The detection limits ranged from 1.3 to 28 cells for six Salmonella strains of various serovars. Six commodities consisting of four animal feed items (cattle feed, chicken feed, horse feed, and swine feed) and two pet food items (dry cat food and dry dog food) all yielded satisfactory results. Compared to the BAM method, the relative levels of detection (RLODs) for LAMP I ranged from 0.317 to 1 with a combined value of 0.610, while those for LAMP II ranged from 0.394 to 1.152 with a combined value of 0.783, which all fell within the acceptability limit (2.5) for an unpaired study. This also suggests that LAMP was more sensitive than the BAM method at detecting low-level Salmonella contamination in animal food and results were available 3days sooner. The performance of LAMP on both platforms was comparable to that of qPCR but notably faster, particularly LAMP II. Given the importance of Salmonella in animal food safety, the LAMP assays validated in this study holds great promise as a rapid, reliable, and robust method for routine screening of Salmonella in these commodities.

Validation of a Salmonella loop-mediated isothermal amplification assay in animal food.

Loop-mediated isothermal amplification (LAMP) has become a powerful alternative to polymerase chain reaction (PCR) for pathogen detection in clinical specimens and food matrices. Nontyphoidal Salmonella is a zoonotic pathogen of significant food and feed safety concern worldwide. The first study employing LAMP for the rapid detection of Salmonella was reported in 2005, 5 years after the invention of the LAMP technology in Japan. This review provides an overview of international efforts in the past decade on the development and application of Salmonella LAMP assays in a wide array of food and feed matrices. Recent progress in assay design, platform development, commercial application, and method validation is reviewed. Future perspectives toward more practical and wider applications of Salmonella LAMP assays in food and feed testing are discussed.

Article	Year
Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates. Applied and environmental microbiology\| PubMed ID: 11282605	2001
Role of efflux pumps and topoisomerase mutations in fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli. Antimicrobial agents and chemotherapy\| PubMed ID: 16048946	2005
Multilaboratory validation of a luminex microbead-based suspension array for the identification of the 11 most clinically relevant Shiga toxin-producing Escherichia coli O serogroups. Journal of food protection\| PubMed ID: 23643130	2013
Retrospective Analysis of Salmonella, Campylobacter, Escherichia coli, and Enterococcus in Animal Feed Ingredients. Foodborne pathogens and disease\| PubMed ID: 23692074	2013
Antimicrobial resistance in campylobacter: susceptibility testing methods and resistance trends. Journal of microbiological methods\| PubMed ID: 23827324	2013
Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce. Applied and environmental microbiology\| PubMed ID: 24509927	2014
Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce. Food microbiology\| PubMed ID: 25475319	2015
Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time. BMC microbiology\| PubMed ID: 27316515	2016
MRSA and multidrug-resistant Staphylococcus aureus in U.S. retail meats, 2010-2011. Food microbiology\| PubMed ID: 27889161	2017
Effects of low concentrations of erythromycin, penicillin, and virginiamycin on bacterial resistance development in vitro. Scientific reports\| PubMed ID: 28887450	2017
Validation of a Salmonella loop-mediated isothermal amplification assay in animal food. International journal of food microbiology\| PubMed ID: 29121500	2018
Loop-Mediated Isothermal Amplification for Salmonella Detection in Food and Feed: Current Applications and Future Directions. Foodborne pathogens and disease\| PubMed ID: 29902082	2018

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