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.
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
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
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
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
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
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.
Abstract
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.
High mortality and hospitalization rates have seen
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
into food production environments (FPEs) due to its ubiquitous nature. In addition to this, the capacity of
strains to colonize FPEs can lead to repeated identification of
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
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
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
within biofilms in food production facilities.
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.
•We sequenced 12 Pseudomonas fragi and seven Pseudomonas lundensis genomes.•We present a first insight into the genomes of these meat and milk spoilage species.•Metabolic activity and intra-/interspecies diversity of isolates was investigated.•Both species were associated with production of odour-active volatile compounds.•Some isolates produced notably low levels of volatile compounds.
is a dominant but poorly studied meat spoilage organism. It is a close relative of the foodborne pathogen
, and
constitutes the second genus in the
family. Here, the genomes of 12
strains were ...sequenced, assembled into draft genomes, characterized, and compared with the genomes of
and
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
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
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
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.
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,
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
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
and establishing parallels to and differences from the closely related foodborne pathogen
.
Microbial spoilage of meat during chilled aerobic storage causes significant financial losses to the industry. Even with modern day preservation techniques, spoilage remains an unsolved problem. ...Spoilage of meat is a complex process that involves the activity of endogenous enzymes and microorganisms. Psychrotrophic Pseudomonas species are the key microorganisms that cause spoilage in aerobically stored chilled meat. Spoilage pseudomonads are highly robust and able to withstand stressful environmental conditions that would otherwise inhibit the growth of other spoilage organisms. In order to implement efficient control measures, and to minimize spoilage, a thorough understanding of the characteristics of spoilage pseudomonads is essential. This review focuses on the spoilage process and the key metabolic attributes of the main psychrotrophic spoilage Pseudomonas species to explain their predominance on meat over other psychrotrophic bacteria. This review also highlights less studied, but important, characteristics of psychrotrophic pseudomonads such as biofilm formation and quorum sensing in the context of meat spoilage. The importance of the use of model systems that are closely applicable to the food industry is also discussed in detail.
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.
•A novel cadA cadmium resistance gene variant (cadA7) was identified.•Cadmium resistance was widespread among isolates (63%).•50% of the strains were resistance to disinfectants.•No resistance was observed to clinically important antimicrobials.
is a keystone pathogen of chronic periodontitis. The virulence of
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.
can exchange chromosomal DNA between strains by natural competence and conjugation. The aim of this study was to determine the genetic variability of
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 (Kgp
I and Kgp
II) 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
strains formed a mosaic that was not related to the SNP-based phylogeny. In conclusion
uses domain rearrangements and genetic exchange to generate diversity in specific surface virulence factors.
Psychrotrophic Pseudomonas species form biofilms on meat during refrigerated and temperature abuse conditions. Biofilm growth leads to slime formation on meat which is a key organoleptic degradation ...characteristic. Limited research has been undertaken characterising biofilms grown on meat during chilled aerobic storage. In this work, biofilms formed by two key meat spoilage organisms, Pseudomonas fragi and Pseudomonas lundensis were studied in situ using five strains from each species. Biofilm structures were studied using confocal microscope images, cellular arrangement, cell counts and biomass quantifications. This work demonstrated that highly dense, compact biofilms are a characteristic of P. fragi strains. P. lundensis formed biofilms with loosely arranged cells. The cells in P. fragi biofilm appear to be vertically oriented whereas this characteristic was absent in P. lundensis biofilms formed under identical conditions. Despite the continued access to nutrients, biofilms formed on meat by proteolytic Pseudomonas species dispersed after a population maximum was reached.
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