Monocytogenes Serotype Research Articles

Outbreak of Listeriosis Likely Associated with Baker's Yeast Products, Switzerland, 2022-2024.

We traced back a nationwide outbreak of human listeriosis in Switzerland to a persisting production line contamination of a factory producing baker's yeast with Listeria monocytogenes serotype 1/2a sequence type 3141. We used whole-genome sequencing to match clinical isolates to isolates from product samples.

Modeling Behavior of Salmonella spp. and Listeria monocytogenes in Raw and Processed Vegetables.

Given the persistent occurrence of foodborne illnesses linked to both raw and processed vegetables, understanding microbial behavior in these foods under distribution conditions is crucial. This study aimed to develop predictive growth models for Salmonella spp. and Listeria monocytogenes in raw (mung bean sprouts, onion, and cabbage) and processed vegetables (shredded cabbage salad, cabbage and onion juices) at various temperatures, ranging from 4 to 36 °C. Growth models were constructed and validated using isolated strains of Salmonella spp. (S. Bareilly, S. Enteritidis, S. Typhimurium) and L. monocytogenes (serotypes 1/2a and 1/2b) from diverse food sources. The minimum growth temperatures for Salmonella varied among different vegetable matrices: 8 °C for mung bean sprouts, 9 °C for both onion and cabbage, and 10 °C for ready-to-eat (RTE) shredded cabbage salad. Both pathogens grew in cabbage juice at temperatures above 17 °C, while neither demonstrated growth in onion juice, even at 36 °C. Notably, Salmonella spp. exhibited faster growth than L. monocytogenes in all tested samples. At 8 °C, the lag time (LT) and specific growth rate (SGR) for Salmonella spp. in mung bean sprouts were approximately tenfold longer and threefold slower, respectively, compared to those at 10 °C. A decrease in refrigerator storage temperature by 1 or 2 degrees significantly prevented the growth of Salmonella in raw vegetables. These findings offer valuable insights into assessing the risk of foodborne illness associated with the consumption of raw and processed vegetables and inform management strategies in mitigating these risks.

Fitness Differences Between Listeria monocytogenes Serotypes 1/2a, 4b, and 4bv-1 in Competition for Growth on Lettuce Leaf Sections

Listeria monocytogenes is a foodborne pathogen that lives in nature as a saprophyte. Two of the three most common serotypes that cause foodborne listeriosis are 1/2a and 4b. Within serotype 4b, there is a variant called 4bv-1. In the last decade, several produce-related outbreaks (linked to leafy salad, caramel apples, and stone fruit) were linked to 4bv-1 strains, specifically those of Sequence Type 382. This study assessed the fitness of ST 382 strains on lettuce leaf sections to determine if they are more fit on produce than strains of other serotypes. Strains of serotypes 1/2a, 4b, and ST 382 were inoculated as mixtures onto lettuce and incubated at 4 °C for 7 days or 25 °C for 24 h. Thirty L. monocytogenes colonies resulting from the growth on each lettuce piece were characterized for serotype by multiplex PCR, and the percentages of each serotype recovered were compared. In the individual mixtures with three strains, none of the ST 382 strains showed better fitness for growth on lettuce at either 4 °C or 25 °C. Overall, ST 382 strains showed better recovery from lettuce sections grown at 4 °C than at 25 °C. Statistical analysis of the recovery of twelve strains tested in competition experiments indicated that ST 382 strains were less fit for lettuce growth when competing against the other serotypes. The data indicate that ST 382 strains do not have a competitive fitness advantage on cut lettuce sections.

Complete genome sequence of a Listeria monocytogenes serotype 1 isolated from a critically ill patient in Italy, 2023.

Listeria monocytogenes, a concerning foodborne pathogen, causes severe infections in vulnerable subjects such as pregnant women and the elderly. In this article, we present the complete genome sequence of P4_LIS, an L. monocytogenes isolated from a patient with invasive bacteria infection.

LISTERIA MONOCYTOGENES INFECTION IN FREE-RANGING RED FOXES (VULPES VULPES) AND EURASIAN LYNX (LYNX LYNX) IN SWITZERLAND.

Listeria monocytogenes is an ubiquitous environmental saprophytic bacterium causing listeriosis in domestic animals, humans, and occasionally wildlife. In animals, this foodborne zoonotic disease mainly occurs in ruminants and it is rare in carnivores. Seven red foxes (Vulpes vulpes) and one Eurasian lynx (Lynx lynx) were diagnosed with listeriosis between 2010 and 2021 at the Institute for Fish and Wildlife Health, Bern, Switzerland. Necropsy and histopathology revealed meningitis (six of seven red foxes), hepatitis (six of seven red foxes), pneumonia (five of seven red foxes), splenitis (two of seven red foxes) and splenomegaly (the Eurasian lynx, two of seven red foxes). Listeria monocytogenes was isolated from either lung, spleen, liver, or kidney of all animals. Serotyping detected L. monocytogenes serotype 1/2a in five red foxes and the Eurasian lynx and serotype 4b in two red foxes. Six red foxes were positive for canine distemper virus (CDV) by polymerase chain reaction, whereas the Eurasian lynx and one red fox were negative. One red fox that was positive for CDV and listeriosis was also diagnosed with salmonellosis. The identified L. monocytogenes serotypes are among the three most frequently isolated serotypes (1/2a, 1/2b, and 4b) from food or the food production environment and those that cause most listeriosis cases in humans and animals. Coinfection with CDV in six red foxes questions the role of CDV as potential predisposing factor for septicemic listeriosis. The detection of listeriosis in the regionally endangered Eurasian lynx and in carnivores highly abundant in urban settings, such as red foxes, reinforces the importance of wildlife health surveillance in a One Health context and adds the Eurasian lynx to the list of carnivores susceptible to the disease. Further investigations are required to assess the prevalence and epidemiology of L. monocytogenes in free-ranging carnivores and its interaction with CDV.

Detection and Characterization of Zoonotic Pathogens in Game Meat Hunted in Northwestern Italy.

Wildlife can represent a reservoir of zoonotic pathogens and a public health problem. In the present study, we investigated the spread of zoonotic pathogens (Salmonella spp., Yersinia enterocolitica, Listeria monocytogenes, Shiga-toxin-producing Escherichia coli (STEC), and hepatitis E virus (HEV)) considering the presence of virulence and antibiotic resistance genes in game meat from animals hunted in northwest Italy. During two hunting seasons (2020 to 2022), samples of liver and/or muscle tissue were collected from chamois (n = 48), roe deer (n = 26), deer (n = 39), and wild boar (n = 35). Conventional microbiology and biomolecular methods were used for the detection, isolation, and characterization of the investigated pathogens. Two L. monocytogenes serotype IIa strains were isolated from wild boar liver; both presented fosfomycin resistance gene and a total of 22 virulence genes were detected and specified in the text. Eight Y. enterocolitica biotype 1A strains were isolated from chamois (2), wild boar (5), and deer (1) liver samples; all showed streptogramin and beta-lactam resistance genes; the virulence genes found were myfA (8/8 strains), ymoA (8/8), invA (8/8), ystB (8/8), and ail (4/8). Our data underscore the potential role of wildlife as a carrier of zoonotic and antibiotic-resistant pathogens in northwest Italy and a food safety risk for game meat consumers.

Effect of low doses of biocides on the susceptibility of Listeria monocytogenes and Salmonella enterica to various antibiotics of clinical importance

The use of subinhibitory concentrations of biocides in food processing environments requires special attention because it is related to potential increases in antibiotic resistance. In this study, we determined the effect of exposure to low doses of four biocides (sodium hypochlorite, SHY; benzalkonium chloride, BZK; peracetic acid, PAA; trisodium phosphate, TSP) on the resistance to 10 antibiotics and on the hydrophobicity of the cellular surface of a strain of Listeria monocytogenes serotype 1/2 b (LM) and a strain of Salmonella enterica serotype Agona (SA), both of meat origin. The cultures were exposed at 37 °C in tryptone soya broth to increasing concentrations of the biocides, starting with half the minimum inhibitory concentration (MIC/2) and incrementing by 1.5 times the concentration until growth was no longer observed, calculating the MIC of the antibiotics before (control cultures) and after exposure. After exposure to TSP, LM was able to grow in the presence of a concentration of the biocide 2.53 times higher than the MIC of unexposed cultures. No adaptation was observed for SHY, BZK or PAA. SE demonstrated adaptation to BZK (it tolerated a concentration 1.13 times higher than the MIC for the unadapted strain) and PAA (2.53 times). LM cultures presented increased resistance (from susceptibility to reduced susceptibility, from susceptibility to resistance, or from reduced susceptibility to resistance) to erythromycin (strains exposed to BZK, PAA and TSP), chloramphenicol (all compounds), tetracycline (BZK) and fosfomycin (all compounds). Regarding SA, after exposure its resistance to gentamicin (all compounds) and enrofloxacin (BZK, PAA and TSP) increased. The cell surface hydrophobicity (determined through the microbial adhesion to solvents -MATS- test) increased (LM exposed to PAA and TSP; SA exposed to BZK) or decreased (SA exposed to PAA) after contact with the biocides. These findings suggest that the use of biocides at subinhibitory concentrations can contribute to the increase in bacterial resistance to antibiotics, in addition to modifying the hydrophobicity of the cellular surface, which is related to the capacity of bacteria to form biofilm.

Targeting Novel LPXTG Surface Proteins with Monoclonal Antibodies for Immunomagnetic Separation of Listeria monocytogenes.

The Gram-positive bacterium Listeria monocytogenes causes a significantly high percentage of fatalities among human foodborne illnesses. Surface proteins, specifically expressed from a wide range of L. monocytogenes serotypes under selective enrichment culture conditions, can serve as targets for the isolation of this pathogen using antibody-based methods to facilitate molecular detection. In this study, monoclonal antibodies (MAbs), previously raised against the L. monocytogenes LPXTG surface proteins LMOf2365_0639 and LMOf2365_0148, were investigated for their ability to isolate L. monocytogenes from bacterial samples with immunomagnetic separation (IMS). Only 1 out of 35 MAbs against LMOf2365_0639, M3644, was capable of capturing L. monocytogenes. Among all the 24 MAbs examined against LMOf2365_0148, 4 MAbs, M3686, M3697, M3699, and M3700, were capable of capturing L. monocytogenes cells specifically from abbreviated primary selective enrichment cultures in either Palcam or LEB/UVM1 media or from mixed samples containing target and nontarget bacteria. MAb M3686 showed a unique specificity with the capability to capture strains of seven L. monocytogenes serotypes (1/2a, 1/2b, 1/2c, 3a, 4a, 4b, and 4d). These promising MAbs were subsequently characterized by quantitative measurements of antigen-binding affinity using surface plasmon resonance analysis and epitope mapping using overlapping recombinant polypeptides. The usefulness of these MAbs to LMOf2365_0148 in bacterial capture was consistent with their high affinities with KD constants in the nanomolar range and can be explored further for the development of an automated IMS method suitable for routine isolation of L. monocytogenes from food and environmental samples.

Phenotypic and genotypic characterization of Listeria monocytogenes in clinical ruminant cases in Korea

Listeria monocytogenes, a foodborne human and veterinary pathogen, is associated with high mortality rates in ruminants. However, no studies have investigated the antimicrobial resistance of L. monocytogenes isolates from clinical ruminant cases. This study aimed to determine the phenotypic and genotypic characteristics of L. monocytogenes isolates from clinical cases of Korean ruminants. We collected 24 L. monocytogenes isolates from aborted bovine fetuses and goats presenting with listeriosis-related symptoms. The isolates were subjected to PCR serogrouping, conventional serotyping, virulence gene detection, and antimicrobial susceptibility testing. Furthermore, pulsed-field gel electrophoresis and multilocus sequence typing were used to classify and compare genetic diversity among the isolates, including human L. monocytogenes isolates. The most prevalent L. monocytogenes serotypes were 4b (Ⅳb), 1/2a (Ⅱa; Ⅱc), and 1/2b (Ⅱb). All isolates harbored the virulence genes; however, llsX-encoding listeriolysin were identified only in serotypes 4b and 1/2b. All isolates, including two found in humans, formed three genetically diverse pulsed-field gel electrophoresis clusters according to serotype, lineage, and sequence type. The most prevalent sequence type was ST1, followed by ST365 and ST91. The isolates from ruminants with listeriosis were resistant to oxacillin and ceftriaxone and showed diverse lineage, serotype (serogroup), and sequence type characteristics. Considering that the atypical sequence types exhibited clinical manifestations and histopathological lesions, further study is needed to elucidate the pathogenicity of genetically diverse ruminant L. monocytogenes isolates. Furthermore, continuous monitoring of antimicrobial resistance is required to prevent the emergence of L. monocytogenes strains resistant to common antimicrobials.

Effect of acidic conditions on the growth and expression of two virulence genes of Listeria monocytogenes serotype 4b

In this work, the effect of the usual acidic conditions of dry-cured fermented foods (pH values between 4.5 and 6), on the growth and expression of the virulence genes, hly and inlA, of Listeria monocytogenes serotype 4b, was evaluated. To analyse the expression of the inlA gene, a novel real-time PCR (qPCR) method using SYBR® Green methodology was developed. L. monocytogenes levels increased as the pH did and they were kept constant throughout incubation time at pH 4.5. However, a significant increase in the relative expression of the virulence genes was detected in most of the acidic conditions in all the incubation times. The most pronounced upregulation of the relative expression of the virulence genes was found at pH 4.5. The efficient inlA-based qPCR method could be of interest to check changes in the expression of such virulence gene of this pathogenic bacterium in acidic environments.

Design and 3D printing of an electrochemical sensor for Listeria monocytogenes detection based on loop mediated isothermal amplification

The aim of this work is the design and 3D printing of a new electrochemical sensor for the detection of Listeria monocytogenes based on loop mediated isothermal amplification (LAMP). The food related diseases involve a serious health issue all over the world. Listeria monocytogenes is one of the major problems of contaminated food, this pathogen causes a disease called listeriosis with a high rate of hospitalization and mortality. Having a fast, sensitive and specific detection method for food quality control is a must in the food industry to avoid the presence of this pathogen in the food chain (raw materials, facilities and products). A point-of-care biosensor based in LAMP and electrochemical detection is one of the best options to detect the bacteria on site and in a very short period of time. With the numerical analysis of different geometries and flow rates during sample injection in order to avoid bubbles, an optimized design of the microfluidic biosensor chamber was selected for 3D-printing and experimental analysis.For the electrochemical detection, a novel custom gold concentric-3-electrode consisting in a working electrode, reference electrode and a counter electrode was designed and placed in the bottom of the chamber. The LAMP reaction was optimized specifically for a primers set with a limit of detection of 1.25 pg of genomic DNA per reaction and 100% specific for detecting all 12 Listeria monocytogenes serotypes and no other Listeria species or food-related bacteria. The methylene blue redox-active molecule was tested as the electrochemical transducer and shown to be compatible with the LAMP reaction and very clearly distinguished negative from positive food samples when the reaction is measured at the end-point inside the biosensor.

Serotype-identifying ions in Listeria monocytogenes using matrix-associated laser desorption ionization-time of flight mass spectrometry

Listeria monocytogenes is a foodborne pathogen that can cause a potentially life-threatening infection, and almost all cases of human listeriosis are caused by L. monocytogenes isolates in serotypes 1/2a, 1/2b, 1/2c, and 4b. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In the current study, we examined the potential of MALDI–TOF MS for rapid identification of the foodborne pathogen L. monocytogenes and to identify high-risk serotypes. To achieve this, MALDI–TOF MS was applied to 50 L monocytogenes strains. All strains were identified as L. monocytogenes species based on pattern matching against reference spectra for the species. Importantly, 83 specific mass ions were consistently and uniquely found in high-risk L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b. These 83 mass ions were also unique to specific combinations of these serotypes, which enabled specific identification of these four serotypes using MALDI Biotyper analysis. Hence, this method shows potential for using MALDI-TOF MS for the rapid identification of L. monocytogenes species and to discriminate high-risk L. monocytogenes serotypes through specific serotype-specific biomarker ions.

Comparison between immunofluorescence and immunohistochemistry for Listeria monocytogenes detection in formalin-fixed paraffin-embedded tissues

ABSTRACT: Listeria monocytogenes is a bacterium that infect humans and animals and causes a zoonotic disease characterized by encephalitis, septicemia or abortion. In addition, listeriosis leads to significant economic losses due to animal death and sacrifice. This research compared the technique of immunofluorescence (IF) and immunohistochemistry (IHC) for the diagnosis of L. monocytogenes in formalin-fixed and paraffin-embedded (FFPE) tissues. A total of 30 tissue blocks from 15 animals with history and/or lesions compatible with listeriosis were selected. For both IHC and IF, the same diluted (1:200) polyclonal primary antibody was used against L. monocytogenes serotypes 1 and 4. For IHC, a polymer secondary antibody conjugated to peroxidase (HRP) was used. For IF, samples were incubated with a fluorescein-labeled anti-rabbit IgG secondary antibody. Each sample was classified according to the presence and percentage of immunolabeling area. From 30 samples, 10 were positive at least for one technique, whereas eight samples were positive for both IHC and IF with similar score. There was strong immunolabeling in tissue samples from bovines experimentally infected with L. monocytogenes ATCC 7644, as well as in nervous tissues from naturally infected ruminants. Additionally, IF did not show any difference in sensitivity when compared to IHC. Using processed biological materials for IF, instead of fresh tissues, is a quite unique technique, since there are few protocols described. Therefore, this study demonstrated that both techniques are efficient to detect L. monocytogenes in FFPE tissues.

Ecology of Listeria monocytogenes and Listeria species in India: the occurrence, resistance to biocides, genomic landscape and biocontrol.

Listeria monocytogenes, the causative agent of listeriosis, has been implicated in increasing foodborne outbreaks worldwide. The disease is manifested in various forms ranging from severe sepsis in immune-compromised individuals, febrile gastroenteritis, still birth, abortions and meningoencephalitis. In India, data from studies on the detection and molecular epidemiological analysis of L. monocytogenes are only recently emerging. The presence of Listeria in different ecological niches has been recorded from India, including foods, soil, vegetables, mangrove swamps, seafood, freshwater fishes, clinical cases, and also insects. The organism has also been isolated from women with spontaneous abortions, miscarriage or recurrent obstetric history, aborted foetuses, animal clinical cases and wildlife samples. A novel species of Listeria has also been characterized. Listeria monocytogenes strains isolated from clinical, environmental, and foods showed biofilm-forming abilities. Listeria monocytogenes serotype 4b isolates of ST328, a predominant and unique ST observed in India, was repeatedly isolated from different sources, times, and geographical locations. Here, we reviewed the occurrence of Listeria in different sources in India, its resistance to biocides, and provide epidemiological analysis on its genomic landscape.

Control of Listeria monocytogenes growth and virulence in a traditional soft cheese model system based on lactic acid bacteria and a whey protein hydrolysate with antimicrobial activity

“Torta del Casar” is a Spanish soft-ripened cheese made with sheep's raw milk and subjected to a short ripening process, which favors the growth of pathogenic microorganisms including Listeria monocytogenes. The development of strategies to control pathogens and minimize health risks associated with the presence of L. monocytogenes in these products is of great interest. In this regard, the anti-Listeria activity of a whey protein hydrolysate (ProH) alone or combined with six lactic acid bacteria strains isolated from cheese was evaluated in this study as a biocontrol strategy using a “Torta del Casar” cheese-based medium. The most active combinations of lactic acid bacteria assayed induced a reduction higher than two logarithmic units in the growth of L. monocytogenes (serotype 4b) compared to their respective control when they were co-inoculated in “Torta del Casar” cheese-based medium at 7 °C for 7 days. In addition, the observed downregulation of some key virulence genes of L. monocytogenes suggests that the strain Lactiplantibacillus plantarum B2 alone and combined with the strain Lactiplantibacillus spp. B4 are good candidates to be used as biocontrol agents against L. monocytogenes growth in traditional soft cheeses based on raw milk during their storage at refrigeration temperatures.

Current State of Salmonella, Campylobacter and Listeria in the Food Chain across the Arab Countries: A Descriptive Review.

Foodborne infections caused by bacterial pathogens are a common cause of human illness in the Middle East, with a substantial burden of economic loss and public health consequences. This review aims at elucidating recent literature on the prevalence of Non-Typhoidal Salmonella (NTS), Campylobacter and Listeria monocytogens in the food chain in the Arab countries, and to consolidate available evidence on the public health burden and the status of antimicrobial resistance (AMR) among the concerned three pathogens. The reviewed evidence points to a scarcity of understanding of the magnitude of NTS in the food chain in the Arab countries. Additionally, not much work has been done at the molecular characterization level to address the source-attribution of NTS in the Arab World. Very few surveys have been done on Campylobacter in the food chain in the Gulf Cooperation Council (GCC) countries. There is a gap in quantitative (counts/numbers) surveillance efforts for Campylobacter in the chicken meat supply across all Arab countries, despite the availability of some qualitative (presence/absence) surveillance data. While there are several reports on L. monocytogenes in animal-sourced foods, notably in North African Arab countries, fewer are published on L. monocytogenes in plant-sourced foods. Information on the L. monocytogenes serotypes and strain diversity circulating in the Arab region is widely lacking. Antibiotic resistance in the three pathogens is not fully understood across the Arab region, despite some reports indicating varying trends at the human–food interface. The literature evidence presented in this review stresses that Salmonella, Campylobacter and L. monocytogenes continue to challenge food safety and public health in the Arab countries.

Transcriptomic Response of L. monocytogenes to Co-Culture with S. cerevisiae

The aim of the present study was to assess the transcriptomic response of L. monocytogenes during co-culture with three S. cerevisiae strains. For this purpose, BHI broth was inoculated with 7 log CFU·mL−1 L. monocytogenes serotype 4b strain LQC 15257, isolated from a strawberry sample and 4 log CFU·mL−1 S. cerevisiae strains Y32, Y34 and Y37, isolated from spontaneous olive fermentation. Sampling took place after 24 and 48 h incubation at 5 and 20 °C. RNA was extracted, stabilized and the transcription of virulence associated genes prfA, sigB, hly, plcA, plcB, inlA, inlB, inlC and inlJ, was assessed by RT-qPCR. Co-culture with the yeast strains mostly affected the transcription of sigB and inlJ, the upregulation of which during growth at 5 °C for 24 h, reached 10.13 and 9.76 log2(fold change), respectively. Similarly, the effect that incubation time had on the relative transcription of the genes under study was dependent on the co-cultivating yeast strain. On the other hand, the effect of the yeast strain was less pronounced when the relative transcription of the genes under study was assessed between 20 °C and 5 °C. In that case, incubation temperature seemed to have an important effect since, in the 79.2% of the samples analyzed, upregulation was evident, irrespective of yeast strain presence. These results highlight the complex trophic relationships that take place during co-existence between L. monocytogenes and S. cerevisiae.

Relationship of Growth Conditions to Desiccation Tolerance of Salmonella enterica, Escherichia coli, and Listeria monocytogenes

Growth on solid media as sessile cells is believed to increase the desiccation tolerance of Salmonella enterica. However, the reasons behind increased resistance have not been well explored. In addition, the same effect has not been examined for other foodborne pathogens such as pathogenic Escherichia coli or Listeria monocytogenes. The purpose of this research was twofold: first, to determine the role of oxygenation during growth on the desiccation resistance of S. enterica, E. coli, and L. monocytogenes, and second, to determine the effect of sessile versus planktonic growth on the desiccation resistance of these pathogens. Three different serotypes each of Salmonella, E. coli, and L. monocytogenes were cultured in Trypticase soy broth with 0.6% yeast extract (TSBYE), with (aerobic) shaking or on TSBYE with agar under either aerobic or anaerobic conditions and harvested in the stationary phase. After adding cell suspensions to cellulose filter disks, pathogen survival was determined by enumeration before drying (0 h) and after drying for 24 h. Results showed statistical differences in harvested initial populations before drying. For Salmonella, a correlation was found between high initial population and greater survival on desiccation (P = 0.05). In addition, statistical differences (P ≤ 0.05) between survival based on growth type were identified. However, differences found were not the same for the three pathogens, or between their serotypes. In general, Salmonella and E. coli desiccation resistance followed the pattern of aerobic agar media ≥ liquid media ≥ anaerobic agar media. For L. monocytogenes serotypes, resistance to desiccation was not statistically different based on mode of growth. These results indicate growth on solid media under aerobic conditions is not always necessary for optimal desiccation survival, but may be beneficial when the desiccation resistance of the test serotype is unknown.

Phylogeny of the Bacillus altitudinis Complex and Characterization of a Newly Isolated Strain with Antilisterial Activity

Bacillus strain UTK D1-0055 was isolated from a laboratory environment and appeared to have antilisterial activity. The genome was sequenced, the strain was identified as Bacillus altitudinis, and a high-quality complete annotated genome was produced. The taxonomy was evaluated for this and related Bacillus species (B. aerophilus, B. pumilus, B. safensis, B. stratosphericus, and B. xiamenensis) because the taxonomy is unclear and contains errors in public databases such as NCBI. The included strains grouped into seven clusters based on average nucleotide identity. Strains designated as B. aerophilus, B. altitudinis, and B. stratosphericus grouped together in the cluster containing the B. altitudinis type strain, suggesting that these three species should be considered a single species, B. altitudinis. The antimicrobial activity of UTK D1-0055 was evaluated against a panel of 15 Listeria strains (nine Listeria monocytogenes serotypes, Listeria innocua, and Listeria marthii), other foodborne pathogens (six Salmonella enterica serotypes and Escherichia coli), and three representative fungi (Saccharomyces cerevisiae, Botrytis cinerea, and Hyperdermium pulvinatum). Antibacterial activity was observed against all Listeria strains, but no antibacterial effects were found against the other tested bacterial and fungal strains. Biosynthetic gene clusters were identified in silico that may be related to the observed antibacterial activity, and these clusters included genes that putatively encode bacteriocins and nonribosomally synthesized peptides. The B. altitudinis strain identified in this investigation had a broad range of antilisterial activity, suggesting that it and other related strains may be useful for biocontrol in the food industry.

Listeria environmental sampling tests are compatible with polymorphic locus sequence typing.

Food processors invest significant resources into environmental sampling to detect contamination with potential pathogens, particularly Listeria monocytogenes. To facilitate these efforts, multiple environmental sampling tests (ESTs) have been developed and commercialized that minimize workload, turnaround time, and cost while providing convenient colorimetric detection. For presumptive-positive ESTs, we hypothesized that a relatively minor additional investment could provide, in addition to species confirmation, valuable strain typing data for tracking pathogen spread through a facility, identifying harborage sites, and distinguishing sporadic from persistent or resident contaminants. This hypothesis is based on the demonstrated compatibility of polymorphic locus sequence typing (PLST) with crude samples including food enrichments. Five Listeria ESTs were tested here: broth-based InSite (Hygiena), Path-Chek (Mericon), and Pathfinder (Hardy Diagnositics); and gel-based Petrifilm (3M) and HardyChrom (Hardy Diagnostics). ESTs were inoculated with strains representing two common L. monocytogenes serotypes and nonpathogenic Listeria innocua. Following incubation, broths or suspended colonies were heat treated to inactivate bacteria. Lysates or purified DNAs were prepared and used as templates in PCRs targeting the previously described PLST loci LmiMT1 and LisMT2. Single clear products were obtained from all inoculated ESTs; uninoculated controls were negative. PCR products were subjected to Sanger sequencing, yielding high-quality chromatograms. Phylogenetic analysis confirmed identities to previously determined sequences and revealed relatedness to serotype-matched strains represented in GenBank databases. PRACTICAL APPLICATION: Multiple environmental sampling tests have been commercialized in recent years to facilitate the proactive detection of pathogens, particularly Listeria monocytogenes, within food processing facilities. Coupling a positive detection test with strain typing would enhance its value by providing data that can be used to track pathogen spread through a facility, identify harborage sites, and distinguish sporadic from resident contamination.