Rombocin, a Short Stable Natural Nisin Variant, Displays Selective Antimicrobial Activity against Listeria monocytogenes and Employs a Dual Mode of Action to Kill Target Bacterial Strains.

Nisin, with its unique mode of action and potent antimicrobial activity, serves as a remarkable inspiration for the design of novel antibiotics. However, peptides possess inherent weaknesses, particularly their susceptibility to proteolytic degradation, such as by trypsin, which limits their broader applications. This led us to speculate that natural variants of nisin produced by underexplored bacterial species can potentially overcome these limitations. We carried out genome mining of two Romboutsia sedimentorum strains, RC001 and RC002, leading to the discovery of rombocin A, which is a 25 amino acid residue short nisin variant that is predicted to have only four macrocycles compared to the known 31-35 amino acids long nisin variants with five macrocycles. Using the nisin-controlled expression system, we heterologously expressed fully modified and functional rombocin A in Lactococcus lactis and demonstrated its selective antimicrobial activity against Listeria monocytogenes. Rombocin A uses a dual mode of action involving lipid II binding activity and dissipation of the membrane potential to kill target bacteria. Stability tests confirmed its high stability at different pH values, temperatures, and in particular, against enzymatic degradation. With its gene-encoded characteristic, rombocin A is amenable to bioengineering to generate novel derivatives. Further mutation studies led to the identification of rombocin K, a mutant with enhanced bioactivity against L. monocytogenes. Our findings suggest that rombocin A and its bioengineered variant, rombocin K, are promising candidates for development as food preservatives or antibiotics against L. monocytogenes.

Cesin, a short natural variant of nisin, displays potent antimicrobial activity against major pathogens despite lacking two C-terminal macrocycles.

Nisin is a widely used lantibiotic owing to its potent antimicrobial activity and its food-grade status. Its mode of action includes cell wall synthesis inhibition and pore formation, which are attributed to the lipid II binding and pore-forming domains, respectively. We discovered cesin, a short natural variant of nisin, produced by the psychrophilic anaerobe Clostridium estertheticum. Unlike other natural nisin variants, cesin lacks the two terminal macrocycles constituting the pore-forming domain. The current study aimed at heterologous expression and characterization of the antimicrobial activity and physicochemical properties of cesin. Following the successful heterologous expression of cesin in Lactococcus lactis, the lantibiotic demonstrated a broad and potent antimicrobial profile comparable to that of nisin. Determination of its mode of action using lipid II and lipoteichoic acid binding assays linked the potent antimicrobial activity to lipid II binding and electrostatic interactions with teichoic acids. Fluorescence microscopy showed that cesin lacks pore-forming ability in its natural form. Stability tests have shown the lantibiotic is highly stable at different pH values and temperature conditions, but that it can be degraded by trypsin. However, a bioengineered analog, cesin R15G, overcame the trypsin degradation, while keeping full antimicrobial activity. This study shows that cesin is a novel (small) nisin variant that efficiently kills target bacteria by inhibiting cell wall synthesis without pore formation. IMPORTANCE The current increase in antibiotic-resistant pathogens necessitates the discovery and application of novel antimicrobials. In this regard, we recently discovered cesin, which is a short natural variant of nisin produced by the psychrophilic Clostridium estertheticum. However, its suitability as an antimicrobial compound was in doubt due to its structural resemblance to nisin(1-22), a bioengineered short variant of nisin with low antimicrobial activity. Here, we show by heterologous expression, purification, and characterization that the potency of cesin is not only much higher than that of nisin(1-22), but that it is even comparable to the full-length nisin, despite lacking two C-terminal rings that are essential for nisin's activity. We show that cesin is a suitable scaffold for bioengineering to improve its applicability, such as resistance to trypsin. This study demonstrates the suitability of cesin for future application in food and/or for health as a potent and stable antimicrobial compound.

Interrogating the Diversity of Vaginal, Endometrial, and Fecal Microbiomes in Healthy and Metritis Dairy Cattle.

The bovine genital tract harbors a dynamic microbiome. Genital tract microbial communities in healthy animals have been characterized using next-generation sequencing methods showing that microbe compositions differ between the vagina and uterus, more so during the postpartum period. Pre-calving fecal and vaginal, and endometrial swabs at the different postpartum intervals were collected from dairy cows. Microbiomes in these samples were determined based on bacterial 16S amplicon sequencing and compared between healthy (H; n = 10) control animals and cows that developed metritis (M; n = 10) within 21 days postpartum (DPP). Compared to healthy animals the pre-calving fecal and vaginal microbiomes of metritis animals were more abundant in sequences from the phylum Fusobacteria and the bacterial genera such as Escherichia-Shigella and Histophilus. In addition, compared to healthy animals, metritis cows harboured low microbial species diversity in the endometrium, as well as decreasing Proteobacteria and increasing Fusobacteria, Firmicutes, Actinobacteria, and Bacteroidetes abundances. The greatest taxonomic compositional deviations in endometrial microbial communities between the metritis and health cows were detected between 7 and 10 DPP. There was high taxonomic similarity detected between postpartum endometrial microbiomes and the prepartum vaginal and fecal microbiomes suggesting that colonization through bacteria ascending from the rectum and vagina to the uterine cavity might play a major role in establishing the endometrial microbiome postpartum. A deeper understanding of the establishment and dynamics of postpartum endometrial microbial communities in cows will thus provide crucial basic knowledge to guide the development of genital microbiome manipulation strategies for preventing uterine disease and improving fertility in dairy cows.

Comparative genomics of dairy-associated Staphylococcus aureus from selected sub-Saharan African regions reveals milk as reservoir for human-and animal-derived strains and identifies a putative animal-related clade with presumptive novel siderophore.

Staphylococcus aureus infection is considered to be a neglected tropical disease with huge impact on human and animal health alike. Dairy production in sub-Saharan Africa (SSA) relies heavily on various animals such as cows, goats, and camels, depending on the region. S. aureus causes mastitis and exhibits high prevalence in raw milk. The population structure including genotypic and phenotypic traits of dairy S. aureus in relation to animal and human isolates is, however, unknown for SSA. In this work, 20 S. aureus dairy isolates from East and West Africa were selected for comparative genomics and phenotypic analysis. Comparing their population structure revealed a large diversity of different origins suggesting milk to be a reservoir for human and animal strains alike. Furthermore, a novel putative siderophore was detected in multiple strains in a distinct animal-clade with strains of global origin. This putative siderophore shares a high genetic identity with that from Streptococcus equi suggesting possible horizontal gene transfer. These findings combined with the virulence genes harbored by these dairy-derived strains such as pvl, human evasion factor scn, various enterotoxin, leucocidin and antibiotic resistance genes, stresses the need for an integrative One Health approach to tackle the problem of S. aureus infections in animals and humans in sub-Saharan Africa.

Unraveling the Genotypic and Phenotypic Diversity of the Psychrophilic Clostridium estertheticum Complex, a Meat Spoilage Agent.

The spoilage of vacuum-packed meat by Clostridium estertheticum complex (CEC), which is accompanied by or without production of copious amounts of gas, has been linked to the acetone-butyrate-ethanol fermentation, but the mechanism behind the variable gas production has not been fully elucidated. The reconstruction and comparison of intra- and interspecies metabolic pathways linked to meat spoilage at the genomic level can unravel the genetic basis for the variable phenotype. However, this is hindered by unavailability of CEC genomes, which in addition, has hampered the determination of genetic diversity and its drivers within CEC. Therefore, the current study aimed at determining the diversity of CEC through comprehensive comparative genomics. Fifty CEC genomes from 11 CEC species were compared. Recombination and gene gain/loss events were identified as important sources of natural variation within CEC, with the latter being pronounced in genomospecies2 that has lost genes related to flagellar assembly and signaling. Pan-genome analysis revealed variations in carbohydrate metabolic and hydrogenases genes within the complex. Variable inter- and intraspecies gas production in meat by C. estertheticum and Clostridium tagluense were associated with the distribution of the [NiFe]-hydrogenase hyp gene cluster whose absence or presence was associated with occurrence or lack of pack distention, respectively. Through comparative genomics, we have shown CEC species exhibit high genetic diversity that can be partly attributed to recombination and gene gain/loss events. We have also shown genetic basis for variable gas production in meat can be attributed to the presence/absence of the hyp gene cluster.

Systematic-review and meta-analysis on effect of decontamination interventions on prevalence and concentration of Campylobacter spp. during primary processing of broiler chickens.

Scientific advances in pathogen decontamination offer great potential to reduce Campylobacter spp. during primary processing. The aim of this study was to collate data from eligible studies using systematic review, meta-analysis followed by meta-regression. Random effect meta-analysis revealed heterogenous (τ2 = 0.6, I2 = 98 %) pooled reduction in Campylobacter concentration of 0.6 log10 CFU/carcass and a decrease in relative risk of Campylobacter spp. prevalence in broiler carcasses by 57.2 %. Decontamination interventions during Inside-Outside-Carcass-Wash were most effective on concentration (0.8 log10 CFU/carcass) while those during evisceration were most effective on prevalence (78.0 % decrease in relative risk). Physical decontamination was more effective on Campylobacter prevalence (68.7 % decrease in relative risk) compared chemical treatment (30.3 %). Application through immersion was superior on Campylobacter concentration (0.9 log10 CFU/carcass odds reduction) to spraying (0.5 log10 CFU/carcass odds reduction). Publication bias and small study effect were observed in trials on Campylobacter prevalence but not for concentration. The meta-regression revealed four and seven potential modifier variables for concentration and prevalence respectively. This meta-analysis provides an overview of the expected magnitude in Campylobacter spp. concentration and prevalence with application of decontamination interventions on broiler carcasses along the slaughter process and forms a basis of quantitative microbial risk assessment and derivation of intervention measures. Even though modest microbial concentration reduction is reported there was a large decrease in contamination prevalence during processing interventions.

Whole Genome Sequence-Based Identification of Clostridium estertheticum Complex Strains Supports the Need for Taxonomic Reclassification Within the Species Clostridium estertheticum.

Isolates within the Clostridium estertheticum complex (CEC) have routinely been identified through the 16S rRNA sequence, but the high interspecies sequence similarity reduces the resolution necessary for species level identification and often results in ambiguous taxonomic classification. The current study identified CEC isolates from meat juice (MJS) and bovine fecal samples (BFS) and determined the phylogeny of species within the CEC through whole genome sequence (WGS)-based analyses. About 1,054 MJS were screened for CEC using quantitative real-time PCR (qPCR). Strains were isolated from 33 MJS and 34 BFS qPCR-positive samples, respectively. Pan- and core-genome phylogenomics were used to determine the species identity of the isolates. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were used to validate the species identity. The phylogeny of species within the CEC was determined through a combination of these methods. Twenty-eight clostridia strains were isolated from MJS and BFS samples out of which 13 belonged to CEC. At 95% ANI and 70% dDDH thresholds for speciation, six CEC isolates were identified as genomospecies2 (n=3), Clostridium tagluense (n=2) and genomospecies3 (n=1). Lower thresholds of 94% ANI and 58% dDDH were required for the classification of seven CEC isolates into species C. estertheticum and prevent an overlap between species C. estertheticum and Clostridium frigoriphilum. Combination of the two species and abolishment of current subspecies classification within the species C. estertheticum are proposed. These data demonstrate the suitability of phylogenomics to identify CEC isolates and determine the phylogeny within CEC.

Potential Influence of Regulation of the Food Value Chain on Prevalence and Patterns of Antimicrobial Resistance: the Case of Tilapia (Oreochromis niloticus).

The current study was designed to evaluate the potential impact of the level of regulation on the prevalence and patterns of antimicrobial agent resistance in bacteria isolated from fish. The study sites included two large lakes and both semiregulated and unregulated fish value chains. A total of 328 bacterial isolates belonging to 11 genera were evaluated for antimicrobial susceptibility testing using the disk diffusion method. The bacterial species were tested against 12 different antibiotics (trimethoprim-sulfamethoxazole, tetracycline, ampicillin, cefotaxime, chloramphenicol, nalidixic acid, amoxicillin, meropenem, ciprofloxacin, nitrofurantoin, cefuroxime, and kanamycin). Data analysis was done to assess the heterogeneity in proportion of resistant bacterial species within and between the two value chains using a random-effects model proposed by DerSimonian and Laird (Control Clin Trials 7:177-188, 1986). Statistical heterogeneity within and between groups was estimated using the Cochran chi-square test and the Cochrane I2 index. The overall proportion of bacterial species resistant to antimicrobial agents in semiregulated and unregulated value chains ranged from 0.00 to 0.88 and 0.09 to 0.95, respectively. Shigella spp. had the highest proportion of bacteria that were resistant to most of the antimicrobial agents used. The bacterial species were highly resistant to ampicillin and amoxicillin, and the highest multidrug resistance capacity was observed in Shigella spp. (18.3%, n = 328), Vibrio spp. (18.3%), and Listeria monocytogenes (12.2%). We observed strong heterogeneity within and between the two value chains regarding proportion of resistant bacterial species. Sun-dried fish in both value chains had significantly high proportions of resistant bacterial species. Comparing the two value chains, the unregulated value chain had a significantly higher proportion of bacterial species that were resistant. In order to mitigate the risk of transmitting antimicrobial-resistant bacteria to consumers along the fish value chain, good manufacturing practices coupled with identification and management of possible sources of contamination are recommended for fish and potentially other foods distributed along the less regulated value chains. IMPORTANCE In order to mitigate the risk of transmitting antimicrobial-resistant bacteria to consumers along the fish value chain, good manufacturing practices coupled with identification and management of possible sources of contamination are recommended for fish and potentially other foods distributed along the less regulated value chains.

Cold Shock Proteins Promote Nisin Tolerance in Listeria monocytogenes Through Modulation of Cell Envelope Modification Responses.

Listeria monocytogenes continues to be a food safety challenge owing to its stress tolerance and virulence traits. Several listeriosis outbreaks have been linked to the consumption of contaminated ready-to-eat food products. Numerous interventions, including nisin application, are presently employed to mitigate against L. monocytogenes risk in food products. In response, L. monocytogenes deploys several defense mechanisms, reducing nisin efficacy, that are not yet fully understood. Cold shock proteins (Csps) are small, highly conserved nucleic acid-binding proteins involved in several gene regulatory processes to mediate various stress responses in bacteria. L. monocytogenes possesses three csp gene paralogs; cspA, cspB, and cspD. Using a panel of single, double, and triple csp gene deletion mutants, the role of Csps in L. monocytogenes nisin tolerance was examined, demonstrating their importance in nisin stress responses of this bacterium. Without csp genes, a L. monocytogenes ΔcspABD mutant displayed severely compromised growth under nisin stress. Characterizing single (ΔcspA, ΔcspB, and ΔcspD) and double (ΔcspBD, ΔcspAD, and ΔcspAB) csp gene deletion mutants revealed a hierarchy (cspD > cspB > cspA) of importance in csp gene contributions toward the L. monocytogenes nisin tolerance phenotype. Individual eliminations of either cspA or cspB improved the nisin stress tolerance phenotype, suggesting that their expression has a curbing effect on the expression of nisin resistance functions through CspD. Gene expression analysis revealed that Csp deficiency altered the expression of DltA, MprF, and penicillin-binding protein-encoding genes. Furthermore, the ΔcspABD mutation induced an overall more electronegative cell surface, enhancing sensitivity to nisin and other cationic antimicrobials as well as the quaternary ammonium compound disinfectant benzalkonium chloride. These observations demonstrate that the molecular functions of Csps regulate systems important for enabling the constitution and maintenance of an optimal composed cell envelope that protects against cell-envelope-targeting stressors, including nisin. Overall, our data show an important contribution of Csps for L. monocytogenes stress protection in food environments where antimicrobial peptides are used. Such knowledge can be harnessed in the development of better L. monocytogenes control strategies. Furthermore, the potential that Csps have in inducing cross-protection must be considered when combining hurdle techniques or using them in a series.

Targeted Genome Mining Reveals the Psychrophilic Clostridium estertheticum Complex as a Potential Source for Novel Bacteriocins, Including Cesin A and Estercticin A.

Antimicrobial resistance in pathogenic bacteria is considered a major public health issue necessitating the discovery of alternative antimicrobial compounds. In this regard, targeted genome mining in bacteria occupying under-explored ecological niches has the potential to reveal such compounds, including bacteriocins. In this study, we determined the bacteriocin biosynthetic potential of the psychrophilic Clostridium estertheticum complex (CEC) through a combination of genome mining and phenotypic screening assays. The genome mining was performed in 40 CEC genomes using antiSMASH. The production of bacteriocin-like compounds was phenotypically validated through agar well (primary screening) and disk diffusion (secondary screening) assays using cell free supernatants (CFS) and partially purified extracts, respectively. Stability of four selected CFS against proteolytic enzymes, temperature and pH was determined while one CFS was analyzed by HRMS and MS/MS to identify potential bacteriocins. Twenty novel bacteriocin biosynthetic gene clusters (BBGC), which were classified into eight (six lantibiotics and two sactipeptides) distinct groups, were discovered in 18 genomes belonging to C. estertheticum (n = 12), C. tagluense (n = 3) and genomospecies2 (n = 3). Primary screening linked six BBGC with narrow antimicrobial activity against closely related clostridia species. All four preselected CFS retained activity after exposure to different proteolytic, temperature and pH conditions. Secondary screening linked BBGC1 and BBGC7 encoding a lantibiotic and sactipeptide, respectively, with activity against Bacillus cereus while lantibiotic-encoding BBGC2 and BBGC3 were linked with activity against B. cereus, Staphylococcus aureus (methicillin-resistant), Escherichia coli and Pseudomonas aeruginosa. MS/MS analysis revealed that C. estertheticum CF004 produces cesin A, a short natural variant of nisin, and HRMS indicated the production of a novel sactipeptide named estercticin A. Therefore, we have shown the CEC, in particular C. estertheticum, is a source of novel and stable bacteriocins that have activities against clinically relevant pathogens.

Detection of Psychrophilic Clostridium spp. in Fecal Samples from Cattle of Different Ages Sampled at the Slaughterhouse Level.

ABSTRACT: Clostridium estertheticum and C. estertheticum-like spp. are obligate anaerobic psychrophiles causing "blown pack" spoilage of chilled vacuum-packed meat. The present study aimed at detecting and isolating these spoilage bacteria in fecal samples of cattle of different ages at the slaughterhouse level. One hundred two swab fecal samples were obtained and enriched anaerobically in prereduced peptone-yeast-glucose-starch (PYGS) medium for 3 weeks at 4°C and then screened for C. estertheticum and C. estertheticum-like spp. by using a 16S rRNA gene-based real-time PCR (RT-PCR) assay. The RT-PCR-positive samples were further enriched for 3 weeks in prereduced PYGS medium and then subjected to an ethanol (50%, v/v) and lysozyme (4 mg/mL) treatment. Isolation was carried out anaerobically on Columbia agar with 5% defibrinated sheep blood at 4°C for 3 weeks. Isolated strains were identified morphologically and by the 16S rRNA gene. Forty (39%) of 102 samples were RT-PCR positive. The frequency of positive samples was the following: 9 (45%) of 20 in calves (aged ≤160 days), 23 (43%) of 54 in young cattle (aged 161 to 1,000 days), and 8 (29%) of 28 in cows or bulls (aged >1,000 days). Six strains were isolated from 6 of 40 RT-PCR-positive samples. Of these, five were from the calves (n = 1) and young cattle (n = 4). The six isolates were identified as C. estertheticum (n = 1), Clostridium frigoriphilum (n = 1), and C. estertheticum-like spp. (n = 4). The present findings confirm that feces of cattle are an important source of psychrophilic Clostridium spp. The fecal carriage among livestock animals at slaughter is strongly correlated with the risk of carcass contamination. Therefore, the maintenance of slaughter hygiene is of central importance.

Draft Genome Sequences of Two Clostridium algidicarnis Strains Isolated from Meat Juice Samples of Chilled Vacuum-Packed Lamb Meat.

Clostridium algidicarnis causes blown-pack spoilage of vacuum-packed meat. Here, we report the draft genome sequences of C. algidicarnis strains CM002 and CM003, isolated from unspoiled chilled vacuum-packed lamb. The genome sequences of CM002 and CM003 comprise 2,950,326 and 2,870,995 bp, respectively, and each have a GC content of 30.1%.

The Analysis of Field Strains Isolated From Food, Animal and Clinical Sources Uncovers Natural Mutations in Listeria monocytogenes Nisin Resistance Genes.

Nisin is a commonly used bacteriocin for controlling spoilage and pathogenic bacteria in food products. Strains possessing high natural nisin resistance that reduce or increase the potency of this bacteriocin against Listeria monocytogenes have been described. Our study sought to gather more insights into nisin resistance mechanisms in natural L. monocytogenes populations by examining a collection of 356 field strains that were isolated from different foods, food production environments, animals and human infections. A growth curve analysis-based approach was used to access nisin inhibition levels and assign the L. monocytogenes strains into three nisin response phenotypic categories; resistant (66%), intermediate (26%), and sensitive (8%). Using this categorization isolation source, serotype, genetic lineage, clonal complex (CC) and strain-dependent natural variation in nisin phenotypic resistance among L. monocytogenes field strains was revealed. Whole genome sequence analysis and comparison of high nisin resistant and sensitive strains led to the identification of new naturally occurring mutations in nisin response genes associated with increased nisin resistance and sensitivity in this bacterium. Increased nisin resistance was detected in strains harboring RsbUG77S and PBPB3V240F amino acid substitution mutations, which also showed increased detergent stress resistance as well as increased virulence in a zebra fish infection model. On the other hand, increased natural nisin sensitivity was detected among strains with mutations in sigB, vir, and dlt operons that also showed increased lysozyme sensitivity and lower virulence. Overall, our study identified naturally selected mutations involving pbpB3 (lm0441) as well as sigB, vir, and dlt operon genes that are associated with intrinsic nisin resistance in L. monocytogenes field strains recovered from various food and human associated sources. Finally, we show that combining growth parameter-based phenotypic analysis and genome sequencing is an effective approach that can be useful for the identification of novel nisin response associated genetic variants among L. monocytogenes field strains.

Draft Genome Sequences of Two Phylogenetically Distinct Clostridium gasigenes Strains, CM001 and CM004, Isolated from Chilled Vacuum-Packed Meat.

We present the draft genome sequences of Clostridium gasigenes strains CM001 and CM004. The genomes are 4,147,089 and 4,191,074 bp with GC contents of 28.7% and 28.8%, respectively. Although both strains belong to the same species, whole-genome sequence-based analyses reveal that the strains are phylogenetically distinct.

Comparative Genome Analysis and Phenotypic Characterization of Clostridium gasigenes CGAS001 Isolated From Chilled Vacuum-Packed Lamb Meat.

Genomic data for psychrophilic bacteria causing blown pack spoilage (BPS) are limited. This study characterizes the genome of a novel Clostridium gasigenes strain CGAS001 isolated from meat juice sample (MJS) of vacuum-packed lamb meat by comparing it with the type strain C. gasigenes DSM 12272 and five strains representing four other BPS-causing Clostridium sensu stricto species. Phenotypic characteristics of the strain, which include biochemical characteristics, antimicrobial resistance and production of putative polyketide, have been determined. The size of its draft genome is 4.1 Mb with 3,845 coding sequences, 28.7% GC content and 95 RNA genes that include 75 tRNAs, 17 rRNAs, and 3 ncRNAs. Average Nucleotide Identity (ANI) and digital DNA-DNA Hybridization (dDDH) predict that C. gasigenes CGAS001 and DSM 12272 constitute a single species (ANI and dDDH = 98.3% for speciation) but two distinct subspecies (dDDH = 73.3% for subspeciation). The genome is characterized by saccharolytic, lipolytic and proteolytic genes as well as hemolysins and phospholipases, which are consistent with its phenotype. The genome also reveals the ability of C. gasigenes to synthesize polyketides which is demonstrated by the antimicrobial activity of a crude polyketide extract against Listeria monocytogenes and Enterococcus devriesei. The strain is resistant to polymyxin B and streptomycin. The genetic and phenotypic analyses suggest that CGAS001 constitutes a novel subspecies of C. gasigenes adapted to a saprophytic lifestyle and can synthesize narrow spectrum antimicrobial compounds.

Draft Genome Sequence of Clostridium estertheticum CEST001, Belonging to a Novel Subspecies of C. estertheticum, Isolated from Chilled Vacuum-Packed Lamb Meat Imported to Switzerland.

We present the draft genome sequence of Clostridium estertheticum strain CEST001. The genome is 4.8 Mbp long with a GC content of 30.6%. The digital DNA-DNA hybridization values against four C. estertheticum strains indicate that C. estertheticum CEST001 belongs to a novel subspecies of C. estertheticum.

Draft Genome Sequence of Psychrobacter okhotskensis Strain 5179-1A, Isolated from a Raw Cured Ham Storage Crate.

We present the draft genome sequence of Psychrobacter okhotskensis strain 5179-1A, which was isolated from a raw cured ham storage crate. Its size and GC content are 3.4 Mb and 43.4%, respectively. The 16S rRNA sequences of strain 5179-1A and P. okhotskensis MD17T are 100% identical.

Detection of Psychrophilic Clostridium spp. Causing "Blown Pack" Spoilage in Meat Juice Samples from Chilled Vacuum-Packed Beef and Lamb Meat Imported from Different Countries to Switzerland.

"Blown pack" spoilage (BPS) of chilled vacuum-packed meat is mainly caused by anaerobic and psychrophilic Clostridium spp., including C. estertheticum, C. gasigenes, C. frigoriphilum, and C. frigidicarnis. Recently, its occurrence has been reported in several countries, especially in internationally traded meat. Therefore, this study aimed at detecting the occurrence of psychrophilic Clostridium spp. causing BPS in meat juice samples (MJS) from chilled vacuum-packed beef and lamb meat imported from other countries to Switzerland. One hundred fifty-four MJS (n = 78 from beef; n = 76 from lamb meat) were screened for psychrophilic Clostridium spp. by quantitative PCR, whereby MJS with a crossing point PCR cycle value <35 and >35 were considered positive and negative, respectively. Psychrophilic Clostridium spp. were detected in 10 MJS, of which 2 were from beef and 8 were from lamb meat. The two beef MJS originated from Spain and Lithuania, whereas the lamb MJS originated from New Zealand (six) and Australia (two). This is the first report of psychrophilic Clostridium spp. in MJS from chilled vacuum-packed beef and lamb meat imported from other countries to Switzerland and provides further evidence that the risk of BPS in lamb meat is higher than in beef.

Meta-Analysis and Meta-Regression Indicate Dynamic Prevalence and Moderators of Foodborne Pathogens in African Indigenous Fermented Milk.

As more microbiological data for indigenous fermented milk (IFM) becomes available, concern about their microbial safety becomes eminent. Nonetheless, these data are highly fragmented, and a tool is required to integrate existing data and to provide a basis for data-driven decision making for IFM's safety. Therefore, meta-analysis and meta-regression were conducted to estimate the prevalence of foodborne pathogens in IFM and to determine factors influencing the estimated values. Using Africa as a case, searches were systematically made for published data and relevant grey literature. Data from 18 studies in 15 countries were analyzed. Staphylococcus aureus (37%), pathogenic Escherichia coli (16%), Listeria monocytogenes (6%), and Salmonella spp. (3%) were the most prevalent pathogens with a pooled prevalence estimate of 12%. Heterogeneity among prevalence estimates was attributed to sampling point and microbial group but could be moderated by publication year, country cluster, and methods for microbial confirmation. The pooled prevalence estimates increased over time as more studies became available, whereby the odds were higher in studies from 2010 onwards than studies before 2010. From the analyses, S. aureus presented the greatest safety concern in African IFM. Future microbiological studies should take into consideration different IFM sampling points and advanced analytical methods to identify pathogens.

Relevant Aspects of Clostridium estertheticum as a Specific Spoilage Organism of Vacuum-Packed Meat.

Clostridium estertheticum is a psychrotolerant, gram-positive, motile, anaerobic, spore-forming, rod-shaped bacteria that causes blown pack spoilage (BPS). Spoilage occurs in vacuum-packed meat without temperature abuse. Having been reported in the last 30 years in several countries, BPS by Cl. estertheticum is a major issue around the world and presents a huge economic impact on the meat industry. Despite being an important spoilage microorganism, studies on Cl. estertheticum are challenged by numerous aspects. These include, lack or poor growth in laboratory media, long culturing periods, and unpredictable isolation on the media. These factors hamper the detection of Cl. estertheticum before occurrence of BPS, which further undermines efforts to prevent the occurrence of BPS. Nevertheless, considerable developments have taken place with regard to culture-independent methods. Although information on Cl. estertheticum is available, it is limited and remains highly fragmented. Therefore, this review collates the available information and discusses relevant aspects of Cl. estertheticum as a specific spoilage organism of BPS in vacuum-packed meat.