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

Human infections with Shiga toxin-producing Escherichia coli, Switzerland, 2010-2014

Objectives: The aim of this study was to characterize a collection of 95 Shigatoxin-producing E.coli (STEC) isolated from human patients in Switzerland during 2010-2014. Methods: We performed O and H serotyping and molecular subtyping. Results: The five most common serogroups were O157, O145, O26, O103, and O146. Of the 95 strains, 35 (36.8%) carried stx1 genes only, 43 strains (45.2%) carried stx2 and 17 (17.9%) harbored combinations of stx1 and stx2 genes. Stx1a (42 strains) and stx2a (32 strains) were the most frequently detected stx subtypes. Genes for intimin (eae), hemolysin (hly), iron-regulated adhesion (iha), and the subtilase cytotoxin subtypes subAB1, subAB2-1, subAB2-2, or subAB2-3 were detected in 70.5, 83.2, 74.7, and 20% of the strains, respectively. Multilocus sequence typing assigned the majority (58.9%) of the isolates to five different clonal complexes (CC), 11, 32, 29, 20, and 165, respectively. CC11 included all O157:[H7] and O55:[H7] isolates. CC32 comprised O145:[H28] isolates, and O145:[H25] belonged to sequence type (ST) 342. CC29 contained isolates of the O26:[H11], O111:[H8] and O118:[Hnt] serogroups, and CC20 encompassed isolates of O51:H49/[Hnt] and O103:[H2]. CC165 included isolates typed O80:[H2]-ST301, all harboring stx2d, eae-ξ, hly, and 66.7% additionally harboring iha. All O80:[H2]-ST301 strains harbored at least 7 genes carried by pS88, a plasmid associated with extraintestinal virulence. Compared to data from Switzerland from the years 2000-2009, an increase of the proportion of non-O157 STEC infections was observed as well as an increase of infections due to STEC O146. By contrast, the prevalence of the highly virulent German clone STEC O26:[H11]-ST29 decreased from 11.3% during 2000-2009 to 1.1% for the time span 2010-2014. The detection of O80:[H2]-ST301 harboring stx2d, eae-ξ, hly, iha, and pS88 related genes suggests an ongoing emergence in Switzerland of an unusual, highly pathogenic STEC serotype. Conclusions: Serotyping and molecular subtyping of clinical STEC demonstrate that although STEC O157 predominates among STEC isolated from diseased humans, non-O157 STEC infections are increasing in Switzerland, including those due to STEC O146:[H2/H21/H28]-ST442/ST738 harboring subAB variants, and the recently emerged STEC O80:[H2]-ST301 harboring eae-ξ and pS88 associated extraintestinal pathogenic virulence genes

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

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

Listeriosis Caused by Persistence of Listeria monocytogenes Serotype 4b Sequence Type 6 in Cheese Production Environment

A nationwide outbreak of human listeriosis in Switzerland was traced to persisting environmental contamination of a cheese dairy with Listeria monocytogenes serotype 4b, sequence type 6, cluster type 7488. Whole-genome sequencing was used to match clinical isolates to a cheese sample and to samples from numerous sites within the production environment. Listeriosis is a potentially lethal infection, and the elderly population, pregnant women, and immunocompromised persons at particular risk (1). Foods, in particular ready-to-eat foodstuffs, including meat, fish, dairy products, fruits, and vegetables, represent the major vehicle for sporadic cases and outbreaks of listeriosis (2). Listeria monocytogenes serotype 4b sequence type 6 (ST6) has emerged since 1990 as a hypervirulent clone that is associated with particularly worse outcome for case-patients who have Listeria meningitis and therefore poses a particular threat to consumer health (3,4). L. monocytogenes ST6 is increasingly associated with outbreaks, including an outbreak linked to frozen vegetables in 5 countries in Europe during 2015–2018 (5), an outbreak associated with contaminated meat pâté in Switzerland during 2016 (6), and the largest listeriosis outbreak globally, which occurred in South Africa during 2017–2018 (7,8). More recently, the largest outbreak of listeriosis in Europe in the past 25 years was reported in Germany and was traced back to blood sausages contaminated with L. monocytogenes ST6 belonging to a particular clone referred to as Epsilon1a (9). Human listeriosis is a reportable disease in Switzerland. All cases of culture- or PCR-confirmed human listeriosis are reported to the Swiss Federal Office of Public Health (SFOPH). Diagnostic laboratories and regional (cantonal) laboratories forward isolates to the Swiss National Reference Centre for Enteropathogenic Bacteria and Listeria for strain characterization, ensuring early recognition of Listeria clusters among food isolates or human cases. We report an outbreak of listeriosis associated with cheese contaminated with L. monocytogenes 4b ST6 in Switzerland

Salmonella enterica serotype Virchow associated with human infections in Switzerland: 2004-2009

BACKGROUND: Salmonellosis is one of the most important foodborne diseases and a major threat to public health. Salmonella serotype Virchow ranks among the top five serovars in Europe. METHOD: A total of 153 strains isolated from different patients from 2004 through 2009 in Switzerland were further characterized by (i) assessing phenotypic antibiotic resistance profiles using the disk diffusion method and (ii) by genotyping using pulsed-field gel electrophoresis (PFGE) after macrorestriction with XbaI in order to evaluate strain relationship. RESULTS: The relative frequency of S. Virchow among other Salmonella serovars varied between 4th to 8th rank. The annual incidence ranged from 0.45/100'000 in 2004 to 0.40/100'000 in 2009. A total of 48 strains (32%) were resistant to one to 3 antimicrobials, 54 strains (36%) displayed resistance patterns to more than three antibiotics. No trend was identifiable over the years 2004 to 2009. We found a high prevalence (62%) of nalidixic acid resistant strains, suggesting an equally high rate of decreased fluoroqionolone susceptibility, whereas intermediate resistance to ciprofloxacin was negligible. Two strains were extended spectrum β-lactamase (ESBL) producers. Analysis of PFGE patterns uncovered a predominant cluster (similarity coefficient above 80%) consisting of 104 of the 153 strains. CONCLUSION: The worldwide increase of antibiotic resistances in Salmonella is an emerging public health problem. For Switzerland, no clear trend is identifiable over the years 2004 to 2009 for S. Virchow. Antimicrobial susceptibility and resistance profiles varied considerably within this period. Nevertheless, the situation in Switzerland coincided with findings in other European countries. Genotyping results of this strain collection revealed no evidence for an undetected outbreak within this time period

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. estertheticu