Colonisation dynamics of Listeria monocytogenes strains isolated from food production environments.

Listeria monocytogenes is a ubiquitous bacterium capable of colonising and persisting within food production environments (FPEs) for many years, even decades. This ability to colonise, survive and persist within the FPEs can result in food product cross-contamination, including vulnerable products such as ready to eat food items. Various environmental and genetic elements are purported to be involved, with the ability to form biofilms being an important factor. In this study we examined various mechanisms which can influence colonisation in FPEs. The ability of isolates (n = 52) to attach and grow in biofilm was assessed, distinguishing slower biofilm formers from isolates forming biofilm more rapidly. These isolates were further assessed to determine if growth rate, exopolymeric substance production and/or the agr signalling propeptide influenced these dynamics and could promote persistence in conditions reflective of FPE. Despite no strong association with the above factors to a rapid colonisation phenotype, the global transcriptome suggested transport, energy production and metabolism genes were widely upregulated during the initial colonisation stages under nutrient limited conditions. However, the upregulation of the metabolism systems varied between isolates supporting the idea that L. monocytogenes ability to colonise the FPEs is strain-specific.

Characterisation of Listeria monocytogenes food-associated isolates to assess environmental fitness and virulence potential.

The ability of Listeria monocytogenes isolates to survive within the food production environment (FPE), as well as virulence, varies greatly between strains. There are specific genetic determinants that have been identified which can strongly influence a strains ability to survive in the FPE and/or within human hosts. In this study, we assessed the FPE fitness and virulence potential, including efficacy of selected hygiene or treatment intervention, against 52 L. monocytogenes strains isolated from various food and food environment sources. Phenotypic tests were performed to determine the minimum inhibitory concentration of cadmium chloride and benzalkonium chloride and the sensitivities to five clinically relevant antibiotics. A genomic analysis was also performed to identify resistance genes correlating to the observed phenotypic resistance profiles, along with genetic determinants of interest which may elude to the FPE fitness and virulence potential. A transposon element containing a novel cadmium resistance gene, cadA7, a Tn916 variant insert in the hypervariable Listeria genomic island 1 region and an LGI2 variant were identified. Resistance to cadmium and disinfectants was prevalent among isolates in this study, although no resistance to clinically important antimicrobials was observed. Potential hypervirulent strains containing full length inlA, LIPI-1 and LIPI-3 were also identified in this study. Cumulatively, the results of this study show a vast array of FPE survival and pathogenicity potential among food production-associated isolates, which may be of concern for food processing operators and clinicians regarding L. monocytogenes strains colonising and persisting within the FPE, and subsequently contaminating food products then causing disease in at risk population groups.

Comparison between cage and free-range egg production on microbial composition, diversity and the presence of Salmonella enterica.

The microbial composition of the food production environment plays an important role in food safety and quality. This study employed both 16 S rRNA gene sequencing technology and culture-based techniques to investigate the bacterial microbiota of an egg production facility comprising of both free-range and conventional cage housing systems. The study also aimed to detect the presence of Salmonella enterica and determine whether its presence was positively or negatively associated with other taxa. Our findings revealed that microbiota profiles of free-range and cage houses differ considerably in relation to the relative abundance and diversity with a number of taxa unique to each system and to individual sampling sites within sheds. Core to each housing system were known inhabitants of the poultry gastrointestinal tracts, Romboutsia and Turicibacter, as well as common spoilage bacteria. Generally, free-range samples contained fewer taxa and were dominated by Staphylococcus equorum, differentiating them from the cage samples. Salmonella enterica was significantly associated with the presence of a taxa belonging to the Carnobacteriaceae family. The results of this study demonstrate that the diversity and composition of the microbiota is highly variable across egg layer housing systems, which could have implications for productivity, food safety and spoilage.

Heat resistance and genomics of spoilage Alicyclobacillus spp. Isolated from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is a spore-forming bacterium of importance to the fruit juice industry due to its remarkable heat resistance and production of guaiacol taint. Whole genome sequencing analysis reveals species demarcation corresponds to the two major genotypic groups to which A. acidoterrestris isolates belong. Heat resistance was significantly different between genotypic groups 1 and 2 with D90 values of 15.5 and 9.3 min, respectively (p < 0.01). Comparison of squalene-hopene cyclase (shc) encoding sequences reveals non-synonymous changes and the alteration of glutamine residues. Glutamine absence may link to the stability reinforcement of the enzyme structure against thermal denaturation. Genomic islands harbouring heavy metal resistance genes are found in the majority of genotypic group 1 genomes (63%) but occurs in only one genome (5%) of genotypic group 2. Distribution of the genomic islands in the genotypic groups 1 and 2 is also consistent with phylogenetic trees and ANI and dDDH values. Subsequently, we propose genotypic group 1 as a new species closely related to A. acidoterrestris that possesses enhanced heat resistance.

High-Throughput Screening of Biofilm Formation of Listeria monocytogenes on Stainless Steel Coupons Using a 96-Well Plate Format.

Listeria monocytogenes is a foodborne pathogen capable of colonizing and persisting in the food production environment (FPE). While there are a variety of factors involved in L. monocytogenes' ability to persist in FPE, the ability to form biofilms has the potential to increase their chance of survival and long-term colonization. Understanding the mechanisms involved in L. monocytogenes ability to form biofilms may potentially help food safety managers optimize control strategies targeting it in the FPE. In this chapter, a high-throughput method to determine L. monocytogenes ability to attach and form biofilms utilizing FPE-grade stainless steel is described. This method provides fast and efficient results, facilitating scaling up to large numbers of isolates to measure their ability to form biofilms, where lower-throughput approaches can then be utilized to further characterize isolates of interest.

Characterization of the biofilm matrix composition of psychrotrophic, meat spoilage pseudomonads.

Psychrotrophic Pseudomonas species are the key spoilage bacteria of aerobically stored chilled meat. These organisms readily form biofilms on meat under refrigerated conditions leading to consumer rejection and associated economic losses. Limited information is available on the matrix composition of the biofilms formed by these bacteria. We quantified and characterized the main components of the matrix of mono-species biofilms of selected Pseudomonas fragi and Pseudomonas lundensis strains using chemical analysis and Raman spectroscopy. The biofilms were grown at 10 °C and 25 °C on nitro-cellulose membranes placed on surface sterilized beef cuts. Extra-cellular polymeric substances of the matrix were extracted in soluble and bound forms and were chemically assessed for total carbohydrates, proteins and extra-cellular DNA. Both Pseudomonas species showed a significant increase in total carbohydrates and total proteins when grown at 10 °C as compared to 25 °C. Extra-cellular DNA did not show a strong correlation with growth temperature. Raman spectra were obtained from planktonic bacteria and membrane grown biofilms at 10 °C and 25 °C. Higher levels of guanine were detected in planktonic cells as compared to biofilm cells. This study suggests that psychrotrophic Pseudomonas species may respond to cold stress by increasing extra-cellular polymer secretions.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Phylogeographic Analysis Reveals Multiple International transmission Events Have Driven the Global Emergence of Escherichia coli O157:H7.

BACKGROUND: Shiga toxin-producing Escherchia coli (STEC) O157:H7 is a zoonotic pathogen that causes numerous food and waterborne disease outbreaks. It is globally distributed, but its origin and the temporal sequence of its geographical spread are unknown. METHODS: We analyzed whole-genome sequencing data of 757 isolates from 4 continents, and performed a pan-genome analysis to identify the core genome and, from this, extracted single-nucleotide polymorphisms. A timed phylogeographic analysis was performed on a subset of the isolates to investigate its worldwide spread. RESULTS: The common ancestor of this set of isolates occurred around 1890 (1845-1925) and originated from the Netherlands. Phylogeographic analysis identified 34 major transmission events. The earliest were predominantly intercontinental, moving from Europe to Australia around 1937 (1909-1958), to the United States in 1941 (1921-1962), to Canada in 1960 (1943-1979), and from Australia to New Zealand in 1966 (1943-1982). This pre-dates the first reported human case of E. coli O157:H7, which was in 1975 from the United States. CONCLUSIONS: Inter- and intra-continental transmission events have resulted in the current international distribution of E. coli O157:H7, and it is likely that these events were facilitated by animal movements (eg, Holstein Friesian cattle). These findings will inform policy on action that is crucial to reduce the further spread of E. coli O157:H7 and other (emerging) STEC strains globally.

Characterisation of the Brochothrix thermosphacta sortase A enzyme.

Gram-positive bacteria utilise class A sortases to coat the surface of their cells with a diversity of proteins that facilitate interactions with their environment and play fundamental roles in cell physiology and virulence. A putative sortase A gene was identified in the genome of the poorly studied meat spoilage bacterium Brochothrix thermosphacta. To understand how this bacterium mediates interactions with its environment, an N-terminal truncated, His-tagged variant of this protein (His6-BtSrtA) was expressed and purified. Catalytic activity of recombinant His6-BtSrtA was investigated, including sorting motif recognition of target proteins and bioconjugation activity. Further, the B. thermosphacta genome was examined for the presence of sortase A (SrtA) protein substrates. His6-BtSrtA readily formed intermediate complexes with LPXTG-tagged proteins. Although the reaction was inefficient, nucleophilic attack of the resultant thioacyl intermediates by tri-glycine was observed. Genome examination identified 11 potential SrtA substrates, two of which contained protein domains associated with adherence of pathogens to host extracellular matrix proteins and cells, suggesting the B. thermosphacta SrtA may be indirectly involved in its attachment to meat surfaces. Thus, further work in this area could provide crucial insight into molecular mechanisms involved in the colonisation of meat by B. thermosphacta.

Novel Biocontrol Methods for Listeria monocytogenes Biofilms in Food Production Facilities.

High mortality and hospitalization rates have seen Listeria monocytogenes as a foodborne pathogen of public health importance for many years and of particular concern for high-risk population groups. Food manufactures face an ongoing challenge in preventing the entry of L. monocytogenes into food production environments (FPEs) due to its ubiquitous nature. In addition to this, the capacity of L. monocytogenes strains to colonize FPEs can lead to repeated identification of L. monocytogenes in FPE surveillance. The contamination of food products requiring product recall presents large economic burden to industry and is further exacerbated by damage to the brand. Poor equipment design, facility layout, and worn or damaged equipment can result in Listeria hotspots and biofilms where traditional cleaning and disinfecting procedures may be inadequate. Novel biocontrol methods may offer FPEs effective means to help improve control of L. monocytogenes and decrease cross contamination of food. Bacteriophages have been used as a medical treatment for many years for their ability to infect and lyse specific bacteria. Endolysins, the hydrolytic enzymes of bacteriophages responsible for breaking the cell wall of Gram-positive bacteria, are being explored as a biocontrol method for food preservation and in nanotechnology and medical applications. Antibacterial proteins known as bacteriocins have been used as alternatives to antibiotics for biopreservation and food product shelf life extension. Essential oils are natural antimicrobials formed by plants and have been used as food additives and preservatives for many years and more recently as a method to prevent food spoilage by microorganisms. Competitive exclusion occurs naturally among bacteria in the environment. However, intentionally selecting and applying bacteria to effect competitive exclusion of food borne pathogens has potential as a biocontrol application. This review discusses these novel biocontrol methods and their use in food safety and prevention of spoilage, and examines their potential to control L. monocytogenes within biofilms in food production facilities.

Salmonella enterica subsp. salamae serovar Sofia, a prevalent serovar in Australian broiler chickens, is also capable of transient colonisation in layers.

1. Salmonella enterica subsp. salamae serovar sofia (S. sofia) is a prevalent strain of Salmonella in Australian broilers and has been isolated from broiler chickens, litter, dust, as well as pre- and post-processing carcasses, and retail chicken portions but has never been reported in commercial Australian layers or eggs. 2. To investigate whether a S. sofia isolate from a broiler could colonise layers, one-month-old Hyline brown layers were orally inoculated with S. sofia and colonisation was monitored for 2-4 weeks. 3. Overall, 30-40% of the chickens shed S. sofia from the cloaca between 6 and 14 d post-inoculation which then declined to 10% by d 21. Necropsy at 2 weeks post-inoculation revealed 80% of birds harboured S. sofia in the caecum, whilst, by 4 weeks post-infection, no chickens were colonised with S. sofia in the gastrointestinal tract, liver or spleen. Additionally, no aerosol 'bird to bird' transfer was evident. 4. This study demonstrated that laying hens can be colonised by broiler-derived S. sofia; however, this colonisation was transient, reaching a peak at 14 d post-inoculation, and was completely cleared by 28 d post-inoculation. The transience of colonisation of S. sofia in layers could be a factor explaining why S. sofia has never been detected when screening for Salmonella serotypes found in Australian laying hens or eggs.

Phenotypic and Genotypic Analysis of Antimicrobial Resistance among Listeria monocytogenes Isolated from Australian Food Production Chains.

The current global crisis of antimicrobial resistance (AMR) among important human bacterial pathogens has been amplified by an increased resistance prevalence. In recent years, a number of studies have reported higher resistance levels among Listeria monocytogenes isolates, which may have implications for treatment of listeriosis infection where resistance to key treatment antimicrobials is noted. This study examined the genotypic and phenotypic AMR patterns of 100 L. monocytogenes isolates originating from food production supplies in Australia and examined this in the context of global population trends. Low levels of resistance were noted to ciprofloxacin (2%) and erythromycin (1%); however, no resistance was observed to penicillin G or tetracycline. Resistance to ciprofloxacin was associated with a mutation in the fepR gene in one isolate; however, no genetic basis for resistance in the other isolate was identified. Resistance to erythromycin was correlated with the presence of the ermB resistance gene. Both resistant isolates belonged to clonal complex 1 (CC1), and analysis of these in the context of global CC1 isolates suggested that they were more similar to isolates from India rather than the other CC1 isolates included in this study. This study provides baseline AMR data for L. monocytogenes isolated in Australia, identifies key genetic markers underlying this resistance, and highlights the need for global molecular surveillance of resistance patterns to maintain control over the potential dissemination of AMR isolates.

Genomic and metabolic characterization of spoilage-associated Pseudomonas species.

Pseudomonas are common spoilage agents of aerobically stored fresh foods. Their ability to cause spoilage is species- and may be strain-specific. To improve our understanding of the meat and milk spoilage agents Pseudomonas fragi and Pseudomonas lundensis, we sequenced the genomes of 12 P. fragi and seven P. lundensis isolates. These genomes provided a dataset for genomic analyses. Key volatile organic compounds (VOCs) produced or metabolised by the isolates were determined during their growth on a beef paste and where possible, metabolic activity was associated with gene repertoire. Genome analyses showed that the isolates included in this work may belong to more than two Pseudomonas species with possible spoilage potential. Pan-genome analyses demonstrated a high degree of diversity among the P. fragi and genetic flexibility and diversity may be traits of both species. Growth of the P. lundensis isolates was characterised by the production of large amounts of 1-undecene, 5-methyl-2-hexanone and methyl-2-butenoic acid. P. fragi isolates produced extensive amounts of methyl and ethyl acetate and the production of methyl esters predominated over ethyl esters. Some of the P. fragi produced extremely low levels of VOCs, highlighting the importance of strain-specific studies in food matrices. Furthermore, although usually not considered to be denitrifiers, all isolates generated molecular nitrogen, indicating that at least some steps of this pathway are intact.

Vibrioferrin production by the food spoilage bacterium Pseudomonas fragi.

Pseudomonas fragi is a meat and milk spoilage bacterium with high iron requirements; however, mechanisms of iron acquisition remain largely unknown. The aim of this work was to investigate siderophore production as an iron acquisition system for P. fragi. A vibrioferrin siderophore gene cluster was identified in 13 P. fragi, and experiments were conducted with a representative strain of this group (F1801). Chromeazurol S assays showed that P. fragi F1801 produced siderophores under iron starvation at optimum growth and refrigeration temperature. Conversely, supplementation of low iron media with 50 µM FeCl3 repressed transcription of the vibrioferrin genes and siderophore production. Disruption of the siderophore receptor (pvuA) caused polar effects on downstream vibrioferrin genes, resulting in impaired siderophore production of the ΔpvuA mutant. Growth of this mutant was compared to growth of a control strain (Δlip) with wild-type vibrioferrin genes in low iron media supplemented with iron chelators 2,2΄-bipyridyl or apo-transferrin. While 25 µM 2,2΄-bipyridyl caused impaired growth of ΔpvuA, growth of the mutant was completely inhibited by 2.5 µM apo-transferrin, but could be restored by FeCl3 addition. In summary, this work identifies a vibrioferrin-mediated iron acquisition system of P. fragi, which is required for growth of this bacterium under iron starvation.

Porphyromonas gingivalis Uses Specific Domain Rearrangements and Allelic Exchange to Generate Diversity in Surface Virulence Factors.

Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis. The virulence of P. gingivalis is reported to be strain related and there are currently a number of strain typing schemes based on variation in capsular polysaccharide, the major and minor fimbriae and adhesin domains of Lys-gingipain (Kgp), amongst other surface proteins. P. gingivalis can exchange chromosomal DNA between strains by natural competence and conjugation. The aim of this study was to determine the genetic variability of P. gingivalis strains sourced from international locations over a 25-year period and to determine if variability in surface virulence factors has a phylogenetic basis. Whole genome sequencing was performed on 13 strains and comparison made to 10 previously sequenced strains. A single nucleotide polymorphism-based phylogenetic analysis demonstrated a shallow tri-lobed phylogeny. There was a high level of reticulation in the phylogenetic network, demonstrating extensive horizontal gene transfer between the strains. Two highly conserved variants of the catalytic domain of the major virulence factor the Kgp proteinase (KgpcatI and KgpcatII) were found. There were three variants of the fourth Kgp C-terminal cleaved adhesin domain. Specific variants of the cell surface proteins FimA, FimCDE, MfaI, RagAB, Tpr, and PrtT were also identified. The occurrence of all these variants in the P. gingivalis strains formed a mosaic that was not related to the SNP-based phylogeny. In conclusion P. gingivalis uses domain rearrangements and genetic exchange to generate diversity in specific surface virulence factors.

Baseline Practices for the Application of Genomic Data Supporting Regulatory Food Safety.

The application of new data streams generated from next-generation sequencing (NGS) has been demonstrated for food microbiology, pathogen identification, and illness outbreak detection. The establishment of best practices for data integrity, reproducibility, and traceability will ensure reliable, auditable, and transparent processes underlying food microbiology risk management decisions. We outline general principles to guide the use of NGS data in support of microbiological food safety. Regulatory authorities across intra- and international jurisdictions can leverage this effort to promote the reliability, consistency, and transparency of processes used in the derivation of genomic information for regulatory food safety purposes, and to facilitate interactions and the transfer of information in the interest of public health.

Insight into the Genome of Brochothrix thermosphacta, a Problematic Meat Spoilage Bacterium.

Brochothrix thermosphacta is a dominant but poorly studied meat spoilage organism. It is a close relative of the foodborne pathogen Listeria monocytogenes, and Brochothrix constitutes the second genus in the Listeriaceae family. Here, the genomes of 12 B. thermosphacta strains were sequenced, assembled into draft genomes, characterized, and compared with the genomes of Brochothrix campestris and L. monocytogenes Phenotypic properties including biogenic amine production and antibiotic and heavy metal susceptibilities were tested. Comparative genomic analyses revealed a high degree of similarity among the B. thermosphacta strains, with bacteriophage genes constituting a significant proportion of the accessory genome. Genes for the production of the malodorous compounds acetate, acetoin, butanediol, and fatty acids were found, as were stress response regulatory genes, which likely play important roles in the spoilage process. Amino acid decarboxylases were not identified in the genomes, and phenotypic testing confirmed their absence. Orthologs of Listeria virulence proteins involved in virulence regulation, intracellular survival, and surface protein anchoring were found; however, key virulence genes were absent. Analysis of antibiotic susceptibility showed that strains were sensitive to the four tested antibiotics, except for one tetracycline-resistant isolate with plasmid-mediated tetracycline resistance genes. Strains tolerated higher levels of copper and cobalt than of cadmium although not at concentrations high enough to categorize the strains as being resistant. This study provides insight into the Brochothrix genome, links previous phenotypic data and data provided here to the gene inventory, and identifies genes that may contribute to the persistence of this organism in the food chain.IMPORTANCE Despite increasing knowledge and advances in food preservation techniques, microbial spoilage of foods causes substantial losses, with negative social and economic consequences. To better control the contamination and microbial spoilage of foods, fundamental knowledge of the biology of key spoilage bacteria is crucial. As a common meat spoilage organism, B. thermosphacta contributes substantially to spoilage-associated losses. Nonetheless, this organism and particularly its genome remain largely unstudied. This study contributes to improving our knowledge of the Brochothrix genus. Spoilage-relevant pathways and genes that may play a role in the survival of this organism in a food processing environment were identified, linking previous phenotypic data and data provided here to the gene inventory of Brochothrix and establishing parallels to and differences from the closely related foodborne pathogen L. monocytogenes.

Draft Genome Sequences of 15 Isolates of Listeria monocytogenes Serotype 1/2a, Subgroup ST204.

Listeria monocytogenes sequence type 204 (ST204) strains have been isolated from a range of food, environmental, and clinical sources in Australia. This study describes the draft genome sequences of 15 isolates collected from meat and dairy associated sources.

Comparative Genomics of the Listeria monocytogenes ST204 Subgroup.

The ST204 subgroup of Listeria monocytogenes is among the most frequently isolated in Australia from a range of environmental niches. In this study we provide a comparative genomics analysis of food and food environment isolates from geographically diverse sources. Analysis of the ST204 genomes showed a highly conserved core genome with the majority of variation seen in mobile genetic elements such as plasmids, transposons and phage insertions. Most strains (13/15) harbored plasmids, which although varying in size contained highly conserved sequences. Interestingly 4 isolates contained a conserved plasmid of 91,396 bp. The strains examined were isolated over a period of 12 years and from different geographic locations suggesting plasmids are an important component of the genetic repertoire of this subgroup and may provide a range of stress tolerance mechanisms. In addition to this 4 phage insertion sites and 2 transposons were identified among isolates, including a novel transposon. These genetic elements were highly conserved across isolates that harbored them, and also contained a range of genetic markers linked to stress tolerance and virulence. The maintenance of conserved mobile genetic elements in the ST204 population suggests these elements may contribute to the diverse range of niches colonized by ST204 isolates. Environmental stress selection may contribute to maintaining these genetic features, which in turn may be co-selecting for virulence markers relevant to clinical infection with ST204 isolates.