Campylobacter prevalence from food, animals, human and environmental samples in Iran: a systematic review and meta-analysis.

BACKGROUND: Campylobacter regarded as a major cause of foodborne gastroenteritis in humans. The present study aimed to determine the prevalence of campylobacter in food, animal and human samples of Iran. RESULTS: Quantitative synthesis was performed from 119 articles. White meat had the highest pooled prevalence of Campylobacter spp. (43.9%). Pooled prevalence of 7.9% and 5.5% for Campylobacter, respectively, were determined for red meat and eggs from Iran. Campylobacter was seen in 14.9% of environmental samples and 8.4% of human samples. In most of the samples C. jejuni had higher frequency than C. coli. Most of the isolated Campylobacter harbored several of the known virulence related genes of this pathogen. CONCLUSION: Chicken was identified as the Campylobacter reservoir. As such preventive strategies in all stages of poultry production until consumption are necessary to control foodborne human infection with Campylobacter in Iran.

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages.

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.

Strain Variability of Listeria monocytogenes under NaCl Stress Elucidated by a High-Throughput Microbial Growth Data Assembly and Analysis Protocol.

Listeria monocytogenes causes the severe foodborne illness listeriosis and survives in food-associated environments due to its high stress tolerance. A data assembly and analysis protocol for microbial growth experiments was compiled to elucidate the strain variability of L. monocytogenes stress tolerance. The protocol includes measurement of growth ability under stress (step 1), selection of a suitable method for growth parameter calculation (step 2), comparison of growth patterns between strains (step 3), and biological interpretation of the discovered differences (step 4). In step 1, L. monocytogenes strains (n = 388) of various serovars and origins grown on media with 9.0% NaCl were measured using a Bioscreen C microbiology reader. Technical variability of the growth measurements was assessed and eliminated. In step 2, the growth parameters determined by Gompertz, modified-Gompertz, logistic, and Richards models and model-free splines were compared, illustrating differences in the suitability of these methods to describe the experimental data. In step 3, hierarchical clustering was used to describe the NaCl tolerance of L. monocytogenes measured by strain-specific variation in growth ability; tolerant strains had higher growth rates and maximum optical densities and shorter lag phases than susceptible strains. The spline parameter area under the curve best classified "poor," "average," and "good" growers. In step 4, the tested L. monocytogenes lineage I strains (serovars 4b and 1/2b) proved to be significantly more tolerant toward 9.0% NaCl than lineage II strains (serovars 1/2a, 1/2c, and 3a). Our protocol provides systematic tools to gain comparable data for investigating strain-specific variation of bacterial growth under stress.IMPORTANCE The pathogen Listeria monocytogenes causes the foodborne disease listeriosis, which can be fatal in immunocompromised individuals. L. monocytogenes tolerates several environmental stressors and can persist in food-processing environments and grow in foodstuffs despite traditional control measures such as high salt content. Nonetheless, L. monocytogenes strains differ in their ability to withstand stressors. Elucidating the intraspecies strain variability of L. monocytogenes stress tolerance is crucial for the identification of particularly tolerant strains. To enhance reliable identification of variability in bacterial stress tolerance phenotypes, we compiled a large-scale protocol for the entire data assembly and analysis of microbial growth experiments, providing a systematic approach and checklist for experiments on strain-specific growth ability. Our study illustrated the diversity and strain-specific variation of L. monocytogenes stress tolerance with an unprecedented scope and discovered biologically relevant serovar- and lineage-dependent phenotypes of NaCl tolerance.

East and West African milk products are reservoirs for human and livestock-associated Staphylococcus aureus.

Staphylococcus aureus frequently isolated from milk products in sub-Saharan Africa (SSA) is a major pathogen responsible for food intoxication, human and animal diseases. SSA hospital-derived strains are well studied but data on the population structure of foodborne S. aureus required to identify possible staphylococcal food poisoning sources is lacking. Therefore, the aim was to assess the population genetic structure, virulence and antibiotic resistance genes associated with milk-derived S. aureus isolates from Côte d'Ivoire, Kenya and Somalia through spa-typing, MLST, and DNA microarray analysis. Seventy milk S. aureus isolates from the three countries were assigned to 27 spa (7 new) and 23 (12 new) MLST sequence types. Milk-associated S. aureus of the three countries is genetically diverse comprising human and livestock-associated clonal complexes (CCs) predominated by the CC5 (n = 10) and CC30 (n = 9) isolates. Panton-Valentine leukocidin, toxic shock syndrome toxin and enterotoxin encoding genes were predominantly observed among human-associated CCs. Penicillin, fosfomycin and tetracycline, but not methicillin resistance genes were frequently detected. Our findings indicate that milk-associated S. aureus in SSA originates from human and animal sources alike highlighting the need for an overarching One Health approach to reduce S. aureus disease burdens through improving production processes, animal care and hygienic measures.

Prevalence of subtilase cytotoxin-encoding subAB variants among Shiga toxin-producing Escherichia coli strains isolated from wild ruminants and sheep differs from that of cattle and pigs and is predominated by the new allelic variant subAB2-2.

Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Three allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, subAB2-1, located on the pathogenicity island SE-PAI and subAB2-2 located in an outer membrane efflux protein (OEP) region. SubAB is becoming increasingly recognized as a toxin potentially involved in human pathogenesis. Ruminants and cattle have been identified as reservoirs of subAB-positive STEC. The presence of the three subAB allelic variants was investigated by PCR for 152 STEC strains originating from chamois, ibex, red deer, roe deer, cattle, sheep and pigs. Overall, subAB genes were detected in 45.5% of the strains. Prevalence was highest for STEC originating from ibex (100%), chamois (92%) and sheep (65%). None of the STEC of bovine or of porcine origin tested positive for subAB. None of the strains tested positive for subAB1. The allelic variant subAB2-2 was detected the most commonly, with 51.4% possessing subAb2-1 together with subAB2-2. STEC of ovine origin, serotypes O91:H- and O128:H2, the saa gene, which encodes for the autoagglutinating adhesin and stx2b were significantly associated with subAB-positive STEC. Our results suggest that subAB2-1 and subAB2-2 is widespread among STEC from wild ruminants and sheep and may be important as virulence markers in STEC pathogenic to humans.

Cold growth behaviour and genetic comparison of Canadian and Swiss Listeria monocytogenes strains associated with the food supply chain and human listeriosis cases.

Sixty-two strains of Listeria monocytogenes isolated in Canada and Switzerland were investigated. Comparison based on molecular genotypes confirmed that strains in these two countries are genetically diverse. Interestingly strains from both countries displayed similar range of cold growth phenotypic profiles. Based on cold growth lag phase duration periods displayed in BHI at 4 °C, the strains were similarly divided into groups of fast, intermediate and slow cold adaptors. Overall Swiss strains had faster exponential cold growth rates compared to Canadian strains. However gene expression analysis revealed no significant differences between fast and slow cold adapting strains in the ability to induce nine cold adaptation genes (lmo0501, cspA, cspD, gbuA, lmo0688, pgpH, sigB, sigH and sigL) in response to cold stress exposure. Neither was the presence of Stress survival islet 1 (SSI-1) analysed by PCR associated with enhanced cold adaptation. Phylogeny based on the sigL gene subdivided strains from these two countries into two major and one minor cluster. Fast cold adaptors were more frequently in one of the major clusters (cluster A), whereas slow cold adaptors were mainly in the other (cluster B). Genetic differences between these two major clusters are associated with various amino acid substitutions in the predicted SigL proteins. Compared to the EGDe type strain and most slow cold adaptors, most fast cold adaptors exhibited five identical amino acid substitutions (M90L, S203A/S203T, S304N, S315N, and I383T) in their SigL proteins. We hypothesize that these amino acid changes might be associated with SigL protein structural and functional changes that may promote differences in cold growth behaviour between L. monocytogenes strains.

Characterization of Listeria monocytogenes strains isolated during 2011-2013 from human infections in Switzerland.

Listeria monocytogenes, an emerging foodborne pathogen, can cause in the population at risk severe infections that are associated with high case fatality rates. A total of 93 L. monocytogenes strains isolated from different patients in Switzerland from July 2011 to September 2013 were further characterized. Septicemia was reported for 74.2% of the patients, meningitis for 10.8%, and abortion for 3.2%. The majority of the strains belonged to serotype 1/2a (n=58) followed by serotype 4b (n=28), 1/2b (n=5), and 1/2c (n=2). The strains represented 35 multilocus sequence typing sequence types, 8 of which were designated for the first time. Sequence analysis of the inlA gene in the 35 sequence types showed that most of the strains encoded full-length proteins. Screening for Listeriolysin S showed the presence of this virulence factor in 29 of the 33 genetic lineage I strains. By using ApaI and AscI for pulsed-field gel electrophoresis, most strains showed distinguishable patterns.

Harnessing Vaginal Probiotics for Enhanced Management of Uterine Disease and Reproductive Performance in Dairy Cows: A Conceptual Review.

Uterine disease in cattle impairs reproductive performance and profitability and increases antibiotic use and antimicrobial resistance. Thus, probiotics offer a promising alternative therapy. This review presents conceptual findings on the efficacy of probiotics in managing uterine diseases and fertility in cows. Probiotics containing Lactobacillus spp. and Bifidobacterium spp. individually or as composite formulations are known to improve fertility. Strategic intravaginal administration of these formulations would likely enhance uterine immunity, particularly during the postpartum period. While current findings on the benefits to uterine health are encouraging, there is still significant knowledge missing, including a lack of empirical information from large-scale field trials. This review underscores the need for evidence-based guidelines for probiotics, such as genomic selection of formulations, targeted delivery, or potential synergy with other interventions. Future research should address these gaps to maximize the potential of probiotics in managing uterine diseases and enhancing the reproductive health of dairy cattle.

A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Examination of food chain-derived Listeria monocytogenes strains of different serotypes reveals considerable diversity in inlA genotypes, mutability, and adaptation to cold temperatures.

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.

Identification of genes involved in serum tolerance in the clinical strain Cronobacter sakazakii ES5.

BACKGROUND: Cronobacter spp. are opportunistic pathogens that can cause septicemia and infections of the central nervous system primarily in premature, low-birth weight and/or immune-compromised neonates. Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteremia. It was the aim of the current study to identify genes involved in serum tolerance in a selected Cronobacter sakazakii strain of clinical origin. RESULTS: Screening of 2749 random transposon knock out mutants of a C. sakazakii ES 5 library for modified serum tolerance (compared to wild type) revealed 10 mutants showing significantly increased/reduced resistance to serum killing. Identification of the affected sites in mutants displaying reduced serum resistance revealed genes encoding for surface and membrane proteins as well as regulatory elements or chaperones. By this approach, the involvement of the yet undescribed Wzy_C superfamily domain containing coding region in serum tolerance was observed and experimentally confirmed. Additionally, knock out mutants with enhanced serum tolerance were observed. Examination of respective transposon insertion loci revealed regulatory (repressor) elements, coding regions for chaperones and efflux systems as well as the coding region for the protein YbaJ. Real time expression analysis experiments revealed, that knock out of the gene for this protein negatively affects the expression of the fimA gene, which is a key structural component of the formation of fimbriae. Fimbriae are structures of high immunogenic potential and it is likely that absence/truncation of the ybaJ gene resulted in a non-fimbriated phenotype accounting for the enhanced survival of this mutant in human serum. CONCLUSION: By using a transposon knock out approach we were able to identify genes involved in both increased and reduced serum tolerance in Cronobacter sakazakii ES5. This study reveals first insights in the complex nature of serum tolerance of Cronobacter spp.

Cold shock proteins contribute to the regulation of listeriolysin O production in Listeria monocytogenes.

Cold shock proteins (Csps) are multifunctional nucleic acid binding proteins used to regulate a wide range of gene expression responses in bacteria. We report here that Csps regulate the production of the pore-forming cytolysin listeriolysin (LLO) and hemolysis phenotypes in Listeria monocytogenes. A triple csp gene deletion mutant incapable of producing any Csps, as well as double csp gene deletion mutants only producing either CspA or CspD, caused less hemolysis and produced lower LLO concentration. On the other hand, another double csp gene deletion mutant that produces CspB retained hemolysis and LLO production levels that are similar to the parental wild-type strain. Transcription analysis showed that in absence of all three csp genes or cspB alone, L. monocytogenes cells have decreased levels of hly gene transcripts, which code for the synthesis of LLO proteins. A comparative examination of mRNA stability showed that hly transcripts were more rapidly degraded in L. monocytogenes triple csp gene deletion mutant cells that are not capable of producing Csps. Overall, our results indicate that Csps, in particular CspB, are important components of gene expression regulatory mechanisms that promote efficient LLO production and hence virulence responses of L. monocytogenes.

Stress Lowers Staphylococcal Enterotoxin C Production Independently of Agr, SarA, and SigB.

Staphylococcal enterotoxin C (SEC) can cause staphylococcal food poisoning, one of the most prevalent foodborne intoxications. It is produced by Staphylococcus aureus during growth in the food matrix. While the surrounding bacteria in food matrices usually repress the growth of S.aureus, the organism possesses a remarkable growth advantage under stressful conditions encountered in many foods. Examples for such food matrices are pastry and bakery products with their high sugar content that lowers water availability. While S. aureus can still grow in these challenging environments, it remains unclear how these conditions affect SEC expression. Here, the influence of 30% glucose on sec mRNA in a qPCR assay and SEC protein expression was investigated for the first time in an ELISA. In addition, regulatory knockout mutants Δagr, ΔsarA, and ΔsigB were generated to investigate regulatory gene elements in glucose stress. In five out of seven strains, glucose stress led to a pronounced decrease in sec mRNA transcription and SEC protein levels were substantially lower under glucose stress. It could be shown that key regulatory elements Δagr, ΔsarA, and ΔsigB in strain SAI48 did not contribute to the pronounced downregulation under glucose stress. Based on these findings, glucose effectively lowers SEC synthesis in the food matrix. However, the mechanism by which it acts on toxin expression and regulatory elements in S. aureus remains unclear. Future studies on other regulatory elements and transcriptomics may shed light on the mechanisms.

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.

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.

Phenotypic and transcriptomic analyses of Sigma L-dependent characteristics in Listeria monocytogenes EGD-e.

In this study the phenotypic and transcriptomic traits associated with the alternative sigma factor protein Sigma L in Listeria monocytogenes EGD-e were investigated. It was demonstrated that Sigma L is required for efficient growth in presence of stress associated with food preservative measures such as low temperature and organic acids. Furthermore, besides attenuation of swarming motility, the disruption of Sigma L in this bacterium also reduces resistance to a diverse range of toxic compounds, including some of the antibiotics used in listeriosis treatment. Genes under Sigma L-dependent transcriptional regulation were identified based on comparison of transcriptomes between exponentially growing cells of the EGD-e sigL null mutant and its parental strain cultivated under cold stress (3 °C) and optimized (37 °C) temperature conditions. Four hundred and forty genes under positive Sigma L-dependent transcriptional regulation were identified. The Sigma L regulon as revealed under these conditions comprises genes that code for proteins with diverse cellular functions including protein synthesis, nutrient transport, energy metabolism, cell envelope synthesis, and motility. The diverse range of transcriptome alterations induced by a sigL null mutation is thus consistent with the multiple phenotypic defects observed in the EGD-e ΔsigL mutant. These results demonstrate that Sigma L provides important global transcription regulatory functions in L. monocytogenes EGD-e. These promote execution of various cellular processes and stress adaptation responses thereby enabling this bacterium to overcome various food preservation measures as well as antibiotics and other toxic chemicals.

Mild NaCl Stress Influences Staphylococcal Enterotoxin C Transcription in a Time-Dependent Manner and Reduces Protein Expression.

Enterotoxins (SEs) produced by Staphylococcus aureus are the cause of serious food intoxications. Staphylococcal enterotoxin C (SEC) is one of the main contributors, as it is often highly expressed. S. aureus possesses a competitive growth advantage over accompanying bacterial flora under stress conditions encountered in foods, such as high NaCl concentrations. However, the influence of NaCl as an external stressor on SEC expression is still unclear. We investigated the influence of 4.5% NaCl on sec mRNA and SEC protein levels. A qRT-PCR assay revealed that NaCl stress leads to time-dependently decreased or elevated sec mRNA levels for most strains. SEC protein levels were generally decreased under NaCl stress. Our findings suggest that NaCl stress lowers overall SEC concentration and time-dependently affects sec mRNA levels.

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.

Deciphering the global roles of Cold shock proteins in Listeria monocytogenes nutrient metabolism and stress tolerance.

Listeria monocytogenes (Lm) accounts for serious public health and food safety problems owing to its stress resilience and pathogenicity. Based on their regulatory involvement in global gene expression events, cold-shock domain family proteins (Csps) are crucial in expression of various stress fitness and virulence phenotypes in bacteria. Lm possesses three Csps (CspA, CspB, and CspD) whose regulatory roles in the context of the genetic diversity of this bacterium are not yet fully understood. We examined the impacts of Csps deficiency on Lm nutrient metabolism and stress tolerance using a set of csp deletion mutants generated in different genetic backgrounds. Phenotype microarrays (PM) analysis showed that the absence of Csps in ∆cspABD reduces carbon (C-) source utilization capacity and increases Lm sensitivity to osmotic, pH, various chemical, and antimicrobial stress conditions. Single and double csp deletion mutants in different Lm genetic backgrounds were used to further dissect the roles of individual Csps in these phenotypes. Selected PM-based observations were further corroborated through targeted phenotypic assays, confirming that Csps are crucial in Lm for optimal utilization of various C-sources including rhamnose and glucose as well as tolerance against NaCl, ß-phenyethylamine (PEA), and food relevant detergent stress conditions. Strain and genetic lineage background-based differences, division of labour, epistasis, and functional redundancies among the Csps were uncovered with respect to their roles in various processes including C-source utilization, cold, and PEA stress resistance. Finally, targeted transcriptome analysis was performed, revealing the activation of csp gene expression under defined stress conditions and the impact of Csps on expression regulation of selected rhamnose utilization genes. Overall, our study shows that Csps play important roles in nutrient utilization and stress responses in Lm strains, contributing to traits that are central to the public health and food safety impacts of this pathogen.

Nitrite stress increases staphylococcal enterotoxin C transcription and triggers the SigB regulon.

Staphylococcal food poisoning is a common food intoxication caused by staphylococcal enterotoxins. While growth of Staphylococcus aureus is not inhibited by the meat-curing agent nitrite, we hypothesize that nitrite has an influence on enterotoxin C (SEC) expression. We investigated the influence of 150 mg/l nitrite on SEC expression at mRNA and protein level in seven strains expressing different SEC variants. Additionally, regulatory knockout mutants (Δagr, ΔsarA, and ΔsigB) of high SEC producing strain SAI48 were investigated at mRNA level. Our findings suggest that nitrite effectively increases sec mRNA transcription, but the effects on SEC protein expression are less pronounced. While Δagr mutants exhibited lower sec mRNA transcription levels than wildtype strains, this response was not stress specific. ΔsigB mutants displayed a nitrite stress-specific response. Whole genome sequencing of the strains revealed a defective agr element in one strain (SAI3). In this strain, sec transcription and SEC protein synthesis was not affected by the mutation. Consequently, additional regulatory networks must be at play in SEC expression. Comparison of our findings about SEC with previous experiments on SEB and SED suggest that each SE can respond differently, and that the same stressor can trigger opposing responses in strains that express multiple toxins.