Toward the Adoption of Loop-Mediated Isothermal Amplification for Salmonella Screening at the National Antimicrobial Resistance Monitoring System's Retail Meat Sites.

The National Antimicrobial Resistance Monitoring System (NARMS) is a One Health program in the United States that collects data on antimicrobial resistance in enteric bacteria from humans, animals, and the environment. Salmonella is a major pathogen tracked by the NARMS retail meat arm but currently lacks a uniform screening method. We evaluated a loop-mediated isothermal amplification (LAMP) assay for the rapid screening of Salmonella from 69 NARMS retail meat and poultry samples. All samples were processed side by side for culture isolation using two protocols, one from NARMS and the other one described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). Overall, 10 (14.5%) samples screened positive by the Salmonella LAMP assay. Of those, six were culture-confirmed by the NARMS protocol and six by the BAM method with overlap on four samples. No Salmonella isolates were recovered from samples that screened negative with LAMP. These results suggested 100% sensitivity for LAMP in reference to culture. Antimicrobial susceptibility testing and whole-genome sequencing analysis confirmed identities of these isolates. Using the BAM protocol, all Salmonella isolates were recovered from samples undergoing Rappaport-Vassiliadis medium selective enrichment and presumptive colonies (n = 130) were dominated by Hafnia alvei (44.6%), Proteus mirabilis (22.3%), and Morganella morganii (9.9%) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This method comparison study clearly demonstrated the benefit of a rapid, robust, and highly sensitive molecular screening method in streamlining the laboratory workflow. Fourteen NARMS retail meat sites further verified the performance of this assay using a portion of their routine samples, reporting an overall specificity of 98.8% and sensitivity of 90%. As of July 2022, the vast majority of NARMS retail meat sites have adopted the Salmonella LAMP assay for rapid screening of Salmonella in all samples.

A novel gene, lstC, of Listeria monocytogenes is implicated in high salt tolerance.

Listeria monocytogenes, causative agent of human listeriosis, has been isolated from a wide variety of foods including deli meats, soft cheeses, cantaloupes, sprouts and canned mushrooms. Standard control measures for restricting microbial growth such as refrigeration and high salt are often inadequate as L. monocytogenes grows quite well in these environments. In an effort to better understand the genetic and physiological basis by which L. monocytogenes circumvents these controls, a transposon library of L. monocytogenes was screened for changes in their ability to grow in 7% NaCl and/ or at 5 °C. This work identified a transposon insertion upstream of an operon, here named lstABC, that led to a reduction in growth in 7% NaCl. In-frame deletion studies identified lstC which codes for a GNAT-acetyltransferase being responsible for the phenotype. Transcriptomic and RT-PCR analyses identified nine genes that were upregulated in the presence of high salt in the ΔlstC mutant. Further analysis of lstC and the genes affected by ΔlstC is needed to understand LstC's role in salt tolerance.

Listeria monocytogenes mutants with altered growth phenotypes at refrigeration temperature and high salt concentrations.

Listeria monocytogenes can survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion between secA2 and iap and showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression of iap. Additionally, a hypothetical gene (met), containing a putative S-adenosylmethionine-dependent methyltransferase domain, downstream of iap had downregulated expression. In-frame deletion mutants of iap and met were created in LS411. The LS560 (LS411 Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of the gbu operon, showed reduced expression of the gbu transcript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of the gbu operon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence in L. monocytogenes.

Interlaboratory performance and quantitative PCR data acceptance metrics for NIST SRM® 2917.

Surface water quality quantitative polymerase chain reaction (qPCR) technologies are expanding from a subject of research to routine environmental and public health laboratory testing. Readily available, reliable reference material is needed to interpret qPCR measurements, particularly across laboratories. Standard Reference Material® 2917 (NIST SRM® 2917) is a DNA plasmid construct that functions with multiple water quality qPCR assays allowing for estimation of total fecal pollution and identification of key fecal sources. This study investigates SRM 2917 interlaboratory performance based on repeated measures of 12 qPCR assays by 14 laboratories (n = 1008 instrument runs). Using a Bayesian approach, single-instrument run data are combined to generate assay-specific global calibration models allowing for characterization of within- and between-lab variability. Comparable data sets generated by two additional laboratories are used to assess new SRM 2917 data acceptance metrics. SRM 2917 allows for reproducible single-instrument run calibration models across laboratories, regardless of qPCR assay. In addition, global models offer multiple data acceptance metric options that future users can employ to minimize variability, improve comparability of data across laboratories, and increase confidence in qPCR measurements.

A National Antimicrobial Resistance Monitoring System Survey of Antimicrobial-Resistant Foodborne Bacteria Isolated from Retail Veal in the United States.

ABSTRACT: Little is known about the prevalence of antimicrobial-resistant (AMR) bacteria in veal meat in the United States. We estimated the prevalence of bacterial contamination and AMR in various veal meats collected during the 2018 U.S. National Antimicrobial Resistance Monitoring System (NARMS) survey of retail outlets in nine states and compared the prevalence with the frequency of AMR bacteria from other cattle sources sampled for NARMS. In addition, we identified genes associated with resistance to medically important antimicrobials and gleaned other genetic details about the resistant organisms. The prevalence of Campylobacter, Salmonella, Escherichia coli, and Enterococcus in veal meats collected from grocery stores in nine states was 0% (0 of 358), 0.6% (2 of 358), 21.1% (49 of 232), and 53.5% (121 of 226), respectively, with ground veal posing the highest risk for contamination. Both Salmonella isolates were resistant to at least one antimicrobial agent as were 65.3% (32 of 49) of E. coli and 73.6% (89 of 121) of Enterococcus isolates. Individual drug and multiple drug resistance levels were significantly higher (P < 0.05) in E. coli and Enterococcus from retail veal than in dairy cattle ceca and retail ground beef samples from 2018 NARMS data. Whole genome sequencing was conducted on select E. coli and Salmonella from veal. Cephalosporin resistance (blaCMY and blaCTX-M), macrolide resistance (mph), and plasmid-mediated quinolone resistance (qnr) genes and gyrA mutations were found. We also identified heavy metal resistance genes ter, ars, mer, fieF, and gol and disinfectant resistance genes qac and emrE. An stx1a-containing E. coli was also found. Sequence types were highly varied among the nine E. coli isolates that were sequenced. Several plasmid types were identified in E. coli and Salmonella, with the majority (9 of 11) of isolates containing IncF. This study illustrates that veal meat is a carrier of AMR bacteria.

Chaperone action of a versatile small heat shock protein from Methanococcoides burtonii, a cold adapted archaeon.

The Methanococcoides burtonii small heat shock protein (Mb-sHsp) is an alphaB-crystallin homolog that delivers protein stabilizing and protective functions to model enzymes, presumably reflecting its role as a molecular chaperone in vivo. Although the gene encoding Mb-shsp was cloned from a cold-adapted microorganism, the Mb-sHsp is an efficient protein chaperone at temperatures far above the optimum growth temperature of M. burtonii. We show that Mb-sHsp can prevent aggregation in E. coli cell free extracts at 60 degrees C for 4 h and can stabilize bovine liver glutamate dehydrogenase for 3 h at 50 degrees C. Surface plasmon resonance was used to determine the binding affinity of Mb-sHsp for denatured proteins. Mb-sHsp bound tightly to denatured lysozyme but not to the native form. When Mb-Cpn and Mg(2+)-ATP were added to the reaction, bound lysozyme was released from Mb-sHsp establishing that Mb-Cpn is able to off-load folding intermediates from Mb-sHsp. In addition, Mb-sHsp and Mb-Cpn also function cooperatively to protect an enzyme substrate. Through characterization of these M. burtonii chaperones, we were able to reconstitute a key heat shock regulated protein folding function of this cold adapted organism in vitro.

An exceptionally stable Group II chaperonin from the hyperthermophile Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus (Pf) grows optimally at 100 degrees C and encodes single genes for the Group II chaperonin (Cpn), Pf Cpn and alpha-crystallin homolog, the small Heat shock protein (sHsp). Recombinant Pf Cpn is exceptionally thermostable and remained active in high ionic strength, and up to 3M guanidine hydrochloride (Gdn-HCl). Pf Cpn bound specifically to denatured lysozyme and ATP addition resulted in protection of lysozyme from aggregation and inactivation at 100 degrees C. While complexed to heat inactivated lysozyme, Pf Cpn showed enhanced thermostability and ATPase activity, and increased the optimal temperature for ATPase activity from 90 to 100 degrees C. Protein substrate binding also stabilized the 16-mer oligomer of Pf Cpn in 3M Gdn-HCl and activated ATPase hydrolysis in 3-5M Gdn-HCl. In addition, Pf Cpn recognized and refolded the non-native lysozyme released from Pf sHsp, consistent with the inferred functions of these chaperones as the primary protein folding pathway during cellular heat shock.

Genomic characterization of novel Listeria monocytogenes serotype 4b variant strains.

Over 90% of the human listeriosis cases are caused by Listeria monocytogenes serotypes 1/2a, 1/2b and 4b strains. As an alternative to antigen-antibody based serotyping, a PCR-based method for serogrouping has been developed and validated. In this communication, we report an in-depth analysis of five 4b variant strains, four clinical isolates from Australia and one environmental isolate from USA. Although these five strains were serotype 4b by classical serotyping method, the serogrouping PCR profiles of these strains show the presence of a 1/2a-3a specific amplicon in addition to the standard 4b-4d-4e specific amplicons. These strains were further analyzed by pulsed field gel electrophoresis, binary gene typing, multi-locus variable-number-tandem-repeat analysis and a high density pan-genomic Listeria microarray. Using these sub-typing results, the clinical isolates were grouped into two distinct genomic groups- one of which could be part of an unidentified outbreak. The microarray results when compared with our database of other 4b outbreak isolates indicated that the serotype 4b variant strains represent very different genotypic profiles than the known reported 4b outbreak strains representing major epidemic clones. The acquisition of serotype 1/2a gene clusters by the 4b variant strains appears to be independent in origin, spanning large areas of geographical and temporal space and may indicate predisposition of some 4b strains towards accepting DNA from related organisms.

Whole-Genome Sequencing Identifies an Atypical Listeria monocytogenes Strain Isolated from Pet Foods.

Four Listeria isolates, including an atypical strain, were isolated from various pet foods and sequenced. We report here the draft genome sequences of these isolates and a comparative genomic analysis with a closely related human clinical isolate. An analysis of the atypical strain identified a frameshift mutation in the prfA gene.

Recent developments in molecular sub-typing of Listeria monocytogenes.

As a vast majority of the human listeriosis cases are caused by serotypes 1/2a, 1/2b and 4b strains, it is imperative that strains from clinical as well as from food and environment are further characterised so that accurate and timely epidemiological determination of sources of the contamination can be established to minimise the disease burden. Recent developments in the field of genomics provide a great opportunity to use these tools towards the development of molecular sub-typing techniques with a greater degree of discrimination spanning the entire length of the genome. This brief review summarises a few of these DNA-based techniques with an emphasis on DNA microarray and other whole genome sequencing-based approaches and their usefulness in Listeria monocytogenes sub-typing and outbreak investigations.

Genome Sequences of Listeria monocytogenes Serotype 4b Variant Strains Isolated from Clinical and Environmental Sources.

Listeria monocytogenes strains that show a novel PCR serotyping profile (IVb-v1) have been reported recently. Here, we announce the draft genome sequences of five L. monocytogenes IVb-v1 strains isolated from the United States and Australia that harbor a 6.3-kb DNA cassette characteristic of serotype 1/2a strains.

High density microarray analysis reveals new insights into genetic footprints of Listeria monocytogenes strains involved in listeriosis outbreaks.

Listeria monocytogenes, a foodborne bacterial pathogen, causes invasive and febrile gastroenteritis forms of listeriosis in humans. Both invasive and febrile gastroenteritis listeriosis is caused mostly by serotypes 1/2a, 1/2b and 4b strains. The outbreak strains of serotype 1/2a and 4b could be further classified into several epidemic clones but the genetic bases for the diverse pathophysiology have been unsuccessful. DNA microarray provides an important tool to scan the entire genome for genetic signatures that may distinguish the L. monocytogenes strains belonging to different outbreaks. We have designed a pan-genomic microarray chip (Listeria GeneChip) containing sequences from 24 L. monocytogenes strains. The chip was designed to identify the presence/absence of genomic sequences, analyze transcription profiles and identify SNPs. Analysis of the genomic profiles of 38 outbreak strains representing 1/2a, 1/2b and 4b serotypes, revealed that the strains formed distinct genetic clusters adhering to their serotypes and epidemic clone types. Although serologically 1/2a and 1/b strains share common antigenic markers microarray analysis revealed that 1/2a strains are further apart from the closely related 1/2b and 4b strains. Within any given serotype and epidemic clone type the febrile gastroenteritis and invasive strains can be further distinguished based on several genetic markers including large numbers of phage genome, and intergenic sequences. Our results showed that the microarray-based data can be an important tool in characterization of L. monocytogenes strains involved in both invasive and gastroenteritis outbreaks. The results for the first time showed that the serotypes and epidemic clones are based on extensive pan-genomic variability and the 1/2b and 4bstrains are more closely related to each other than the 1/2a strains. The data also supported the hypothesis that the strains causing these two diverse outbreaks are genotypically different and this finding might be important in understanding the pathophysiology of this organism.

Genomic characterization of Listeria monocytogenes strains involved in a multistate listeriosis outbreak associated with cantaloupe in US.

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.

Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size.

Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-beta-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions--perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including alpha B-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.

Stabilization of Taq DNA polymerase at high temperature by protein folding pathways from a hyperthermophilic archaeon, Pyrococcus furiosus.

Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 100 degrees C, encodes three protein chaperones, a small heat shock protein (sHsp), a prefoldin (Pfd), and a chaperonin (Cpn). In this study, we report that the passive chaperones sHsp and Pfd from P. furiosus can boost the protein refolding activity of the ATP-dependent Cpn from the same hyperthermophile. The thermo-stability of Taq polymerase was significantly improved by combinations of P. furiosus chaperones, showing ongoing protein folding activity at elevated temperatures and during thermal cycling. Based on these results, we propose that the protein folding apparatus in the hyperthermophilic archaeon, P. furiosus can be utilized to enhance the durability and cost effectiveness of high temperature biocatalysts.

Small heat shock proteins from extremophiles: a review.

Many microorganisms from extreme environments have been well characterized, and increasing access to genomic sequence data has recently allowed the analysis of the protein families related to stress responses. Heat shock proteins appear to be ubiquitous in extremophiles. In this review, we focus on the family of small heat shock proteins (sHSPs) from extremophiles, which are alpha-crystallin homologues. Like the alpha-crystallin eye lens proteins, sHSPs act as molecular chaperones and prevent aggregation of denatured proteins under heat and desiccation stress. Many putative sHSP homologues have been identified in the genomic sequences of all classes of extremophiles. Current studies of shsp gene expression have revealed mechanisms of regulation and activity distinct from other known hsp gene regulation systems. Biochemical studies on sHSPs are limited to thermophilic and hyperthermophilic organisms, and the only two available crystal structures of sHSPs from Methanocaldococcus jannaschii, a hyperthermophilic archaeon and a mesophilic eukaryote, have contributed significantly to an understanding of the mechanisms of action of sHSPs, although many aspects remain unclear.