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Neisseria meningitidis (meningococcus) colonizes the human nasopharynx, primarily as a commensal, but sporadically causing septicemia and meningitis. During colonization and invasion, it encounters different niches with specific nutrient compositions. Small noncoding RNAs (sRNAs) are used to fine-tune expression of genes, allowing adaptation to their physiological differences. We have previously characterized sRNAs ( Neisseria metabolic switch regulators [NmsRs]) controlling switches between cataplerotic and anaplerotic metabolism. Here, we extend the NmsR regulon by studying methylcitrate lyase (PrpF) and propionate kinase (AckA-1) involved in the methylcitrate cycle and serine hydroxymethyltransferase (GlyA) and 3-hydroxyacid dehydrogenase (MmsB) involved in protein degradation. These proteins were previously shown to be dysregulated in a Δ nmsRs strain. Levels of transcription of target genes and NmsRs were assessed by reverse transcriptase quantitative PCR (RT-qPCR). We also used a novel gene reporter system in which the 5' untranslated region (5' UTR) of the target gene is fused to mcherry to study NmsRs-target gene interaction in the meningococcus. Under nutrient-rich conditions, NmsRs downregulate expression of PrpF and AckA-1 by direct interaction with the 5' UTR of their mRNA. Overexpression of NmsRs impaired growth under nutrient-limiting growth conditions with pyruvate and propionic acid as the only carbon sources. Our data strongly suggest that NmsRs downregulate propionate metabolism by lowering methylcitrate enzyme activity under nutrient-rich conditions. Under nutrient-poor conditions, NmsRs are downregulated, increasing propionate metabolism, resulting in higher tricarboxylic acid (TCA) activities. IMPORTANCE Neisseria meningitidis colonizes the human nasopharynx, forming a reservoir for the sporadic occurrence of epidemic invasive meningococcal disease like septicemia and meningitis. Propionic acid generated by other bacteria that coinhabit the human nasopharynx can be utilized by meningococci for replication in this environment. Here, we showed that sibling small RNAs, designated NmsRs, riboregulate propionic acid utilization by meningococci and, thus, colonization. Under conditions mimicking the nasopharyngeal environment, NmsRs are downregulated. This leads to the conversion of propionic acid to pyruvate and succinate, resulting in higher tricarboxylic acid cycle activity, allowing colonization of the nasopharynx. NmsRs link metabolic state with colonization, which is a crucial step on the trajectory to invasive meningococcal disease.

Phase-variable Type I Restriction Modification (RM) systems are epigenetic regulatory systems that have been identified in numerous human bacterial pathogens. We previously showed that an emerging zoonotic lineage ofStreptococcus suisacquired a phase-variable Type I RM system named SsuCC20p. The SsuCC20p locus was present in the genome of disease-associated isolates from multiple streptococcal species. This indicates that it is not restricted toS. suisand can be acquired through horizontal gene transfer. We demonstrate that SsuCC20p phase-variability relies on a recombinase present within the locus.In vitro, only SsuCC20p is responsible for the genome methylation profiles that were detected in the representative zoonoticS. suisisolate 861160. In addition, we show that, contrary to previous observations,hsdSgenes located downstream of thehsdMgene and the recombinase gene, can contribute to the SsuCC20p genome methylation profile. SsuCC20p locked mutants expressing a singlehsdSeach showed unique genome methylation profiles. The differential genome methylation of the distinct locked mutants caused phase dependent differences in global gene expression in a growth condition dependent manner. We observed significant differences in virulence betweenhsdSlocked mutants in a zebrafish larvae infection model. These data indicate that the streptococcal phase-variable Type I RM system SsuCC20p can impact bacterial virulence via epigenetic regulation of gene expression and potentially contributes to the zoonotic potential ofS. suis.ImportancePhase-variation contributes to the virulence of bacterial pathogens as it allows a single strain to produce phenotypic diverse subpopulations. Phase-variable Restriction Modification (RM) systems are systems that allow for such phase-variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage ofStreptococcus suis. We show that the phase-variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and showed that the differential genome methylation within the phases causes differences in gene expression levels and virulence. Altogether, we show that the acquisition of a phase-variable RM system impacts virulence and can potentially contribute to the zoonotic potential ofS. suis. Bacterial pathogens can increase their virulence through acquisition of mobile elements containing epigenetic regulatory systems such as RM systems.

International audience; Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA-secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA, we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with enhanced activity of hGIIA on the cell membrane. Moreover, lspA deletion increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. MRSA wild type could be sensitized to hGIIA and daptomycin killing through exposure to LspA-specific inhibitors globomycin and myxovirescin A1. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting universal application of LspA inhibition. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological interference with LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically applied antibiotics.

Neisseria meningitidis(the meningococcus) colonizes the human nasopharynx, primarily as a commensal, but sporadically causing septicemia and meningitis. During colonization and invasion, it encounters different niches with specific nutrient compositions. Small non-coding RNAs (sRNAs) are used to fine-tune expression of genes allowing adaptation to their physiological differences. We have previously characterized sRNAs (Neisseria metabolic switch regulators [NmsRs]) controlling switches between cataplerotic and anaplerotic metabolism. Here we extend the NmsRs regulon by studying methylcitrate lyase (PrpF) and propionate kinase (AckA-1) involved in the methylcitrate cycle, and serine hydroxymethyltransferase (GlyA) and 3-hydroxyacid dehydrogenase (MmsB) involved in protein degradation. These proteins were previously shown to be dysregulated in a ΔnmsRsstrain. Levels of transcription of target genes and NmsRs were assessed by RT-qPCR. We also used a novel gene-reporter system, in which the 5’UTR of the target gene is fused to mcherry to study NmsRs-target gene interaction in the meningococcus.Under nutrient-rich conditions, NmsRs downregulate expression of PrpF and AckA-1, by direct interaction with the 5’ UTR of their mRNA. Overexpression of NmsRs impaired growth under nutrient-limiting growth conditions with pyruvate and propionic acid as the only carbon sources. Our data strongly suggest that NmsRs downregulate propionate metabolism by lowering methylcitrate enzyme activity under nutrient-rich conditions. Under nutrient-poor conditions, NmsRs are downregulated, increasing propionate metabolism, resulting in higher tricarboxylic acid (TCA) activities. This allows metabolism to support nasopharynx colonization with breakdown products of amino acids functioning as anaplerotic substrates, as highlighted by NmsRs regulation of GlyA and MmsB.SIGNIFICANCENeisseria meningitidiscolonizes the human adult nasopharynx, forming a reservoir for the sporadic occurrence of epidemic invasive meningococcal disease like septicemia and meningitis. Propionic acid generated by other bacteria that co-inhabit the human adolescent/adult nasopharynx can be utilized by meningococci for replication in this environment. Here we showed that the sibling small RNAs designated NmsRs (Neisseria metabolic switch regulators) riboregulate propionic acid utilization by meningococci, and thus, colonization. Under conditions mimicking the nasopharyngeal environment, NmsRs upregulated expression of enzymes of the methylcitrate cycle. This leads to the conversion of propionic acid to pyruvate and succinate, resulting in higher tricarboxylic acid cycle activity, allowing colonization of the nasopharynx. NmsRs link metabolic state with colonization, which is a crucial step on the trajectory to invasive meningococcal disease.

Chlamydia psittaci is an avian pathogen with zoonotic potential. In Australia, C. psittaci has been well reported as a cause of reproductive loss in mares which subsequently have been the source of infection and illness in some in-contact humans. To date, molecular typing studies describe the predominant and clonal C. psittaci sequence type (ST)24 strains in horse, psittacine, and human infections. We sought to assess the clonality between ST24 strains and the emergence of equine ST24 with a comprehensive genomics approach. We used culture-independent probe-based and metagenomic whole-genome sequencing to investigate 13 C . psittaci genomes from horses, psittacines, and a pigeon from Australia. Published genomes of 36 C . psittaci strains were also used to contextualise our Australian dataset and investigate lineage diversity. We utilised a single-nucleotide polymorphism (SNP) based clustering and multi-locus sequence typing (MLST) approach. C. psittaci has four major phylogenetic groups (PG1-4) based on core-genome SNP-based phylogeny. PG1 contained clonal global and Australian equine, psittacine, and human ST24 genomes, with a median pairwise SNP distance of 68 SNPs. PG2, PG3, and PG4 had greater genomic diversity, including diverse STs collected from birds, livestock, human, and horse hosts from Europe and North America and a racing pigeon from Australia. We show that the clustering of C. psittaci by MLST was congruent with SNP-based phylogeny. The monophyletic ST24 clade has four major sub-lineages. The genomes of 17 Australian human, equine, and psittacine strains collected between 2008 and 2021 formed the predominant ST24 sub-lineage 1 (emerged circa 1979). Despite a temporal distribution of 13 years, the genomes within sub-lineage 1 had a median pairwise SNP distance of 32 SNPs, suggesting a recent population expansion or potential cross-host transmission. However, two C. psittaci genomes collected in 2015 from Victorian parrots clustered into distinct ST24 sub-lineage 4 (emerged circa 1965) with ovine strain C19/98 from Germany. This work describes a comprehensive phylogenomic characterisation of ST24 and identifies a timeline of potential bird-to-equine spillover events.

Background Chlamydia abortus and Chlamydia psittaci are important pathogens of livestock and avian species, respectively. While C. abortus is recognized as descended from C. psittaci species, there is emerging evidence of strains that are intermediary, including C. psittaci strain 84/2334 that was isolated from a parrot. Methods Strain 84/2334 was analysed by multi-locus sequence typing of seven housekeeping genes and by analysis of five species-discriminant proteins. The strain was de novo sequenced for comparison with representative strains from chlamydial species. 16S rRNA and whole genome phylogenetic analyses, network and recombination analyses, and analyses of key virulence-associated genes were conducted to explore its evolutionary relationship with both C. psittaci and C. abortus. Results Typing, 16S rRNA and whole genome phylogenetic analyses, as well as network and recombination analyses showed that strain 84/2334 clusters closely with C. abortus. Analyses also suggested a closer evolutionary relationship with classical C. abortus strains, than to two other avian C. abortus or C. psittaci strains. Analyses of virulence-associated genes that exhibit greatest diversity within chlamydial species, reveal greater diversity than present in sequenced C. abortus genomes. The strain also possesses an extrachromosomal plasmid, as found in C. psittaci species but absent from all sequenced classical C. abortus strains. Conclusion The results are consistent with 84/2334 being a C. abortus ancestral strain, suggesting that it should be reclassified as C. abortus. The identification of a C. abortus strain bearing an extra-chromosomal plasmid has implications for plasmid-based transformation studies providing a potential route for future vaccine development.

Methicillin-resistantStaphylococcus aureus(MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA secreted phospholipase A2(hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, includingS. aureus. To determine hGIIA-resistance mechanisms of MRSA we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role oflspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in bothin vitroassays and an infection model in hGIIA-transgenic mice. Increased susceptibility of thelspAmutant was associated with faster and increased cell wall penetration of hGIIA. Moreover,lspAdeletion also increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. Exposure of MRSA wild-type to the LspA-specific inhibitors globomycin and myxovirescin A1 induced alspAmutant phenotype with regard to hGIIA and daptomycin killing. Analysis of >26,000S. aureusgenomes showed that LspA is highly sequence-conserved, suggesting that LspA inhibition could be applied universally. The role of LspA in hGIIA resistance was not restricted to MRSA sinceStreptococcus mutansandEnterococcus faecaliswere also more hGIIA-susceptible afterlspAdeletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological blocking of LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically-applied antibiotics.

Background Chlamydia abortus and Chlamydia psittaci are important pathogens of livestock and avian species, respectively. While C. abortus is recognized as descended from C. psittaci species, there is emerging evidence of strains that are intermediary between the two species, suggesting they are recent evolutionary ancestors of C. abortus. Such strains include C. psittaci strain 84/2334 that was isolated from a parrot. Our aim was to classify this strain by sequencing its genome and explore its evolutionary relationship to both C. abortus and C. psittaci. Results In this study, methods based on multi-locus sequence typing (MLST) of seven housekeeping genes and on typing of five species discriminant proteins showed that strain 84/2334 clustered with C. abortus species. Furthermore, whole genome de novo sequencing of the strain revealed greater similarity to C. abortus in terms of GC content, while 16S rRNA and whole genome phylogenetic analysis, as well as network and recombination analysis showed that the strain clusters more closely with C. abortus strains. The analysis also suggested a closer evolutionary relationship between this strain and the major C. abortus clade, than to two other intermediary avian C. abortus strains or C. psittaci strains. Molecular analyses of genes (polymorphic membrane protein and transmembrane head protein genes) and loci (plasticity zone), found in key virulence-associated regions that exhibit greatest diversity within and between chlamydial species, reveal greater diversity than present in sequenced C. abortus genomes as well as similar features to both C. abortus and C. psittaci species. The strain also possesses an extrachromosomal plasmid, as found in most C. psittaci species but absent from all sequenced classical C. abortus strains. Conclusion Overall, the results show that C. psittaci strain 84/2334 clusters very closely with C. abortus strains, and are consistent with the strain being a recent C. abortus ancestral species. This suggests that the strain should be reclassified as C. abortus. Furthermore, the identification of a C. abortus strain bearing an extra-chromosomal plasmid has implications for plasmid-based transformation studies to investigate gene function as well as providing a potential route for the development of a next generation vaccine to protect livestock from C. abortus infection.

Developed two decades ago as a molecular method to provide definite characterization of a bacterial isolate, Multilocus Sequence Typing (MLST) is today globally adopted as a universal fine-detailed molecular typing tool and has been applied to numerous pathogenic and nonpathogenic bacterial as well eukaryotic organisms. MLST utilizes DNA sequence of several conserved housekeeping (HK) genes which are assigned an allelic number, which then collectively constitute an allelic profile or sequence type (ST), a "molecular barcode" of the interrogated bacterial strain or a eukaryotic organism. Here, we describe the principles and molecular approaches for generating MLST data for an analysis of a bacteria in the order Chlamydiales, using a Chlamydia pecorum-specific MLST scheme as an example.

Chlamydia psittaci is traditionally regarded as a globally distributed avian pathogen that can cause zoonotic spill-over. Molecular research has identified an extended global host range and significant genetic diversity. However, Australia has reported a reduced host range (avian, horse, and human) with a dominance of clonal strains, denoted ST24. To better understand the widespread of this strain type in Australia, multilocus sequence typing (MLST) and ompA genotyping were applied on samples from a range of hosts (avian, equine, marsupial, and bovine) from Australia. MLST confirms that clonal ST24 strains dominate infections of Australian psittacine and equine hosts (82/88, 93.18%). However, this study also found novel hosts (Australian white ibis, King parrots, racing pigeon, bovine, and a wallaby) and demonstrated that strain diversity does exist in Australia. The discovery of a C. psittaci novel strain (ST306) in a novel host, the Western brush wallaby, is the first detection in a marsupial. Analysis of the results of this study applied a multidisciplinary approach regarding Chlamydia infections, equine infectious disease, ecology, and One Health. Recommendations include an update for the descriptive framework of C. psittaci disease and cell biology work to inform pathogenicity and complement molecular epidemiology.

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and showed 99.1% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight in the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is an ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs C. gallinacea induced mortality was observed, potentially strain dependent but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of (potential) virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as a (conditional) pathogen than an innocuous commensal.ImportanceChlamydiaceae are a family of bacteria comprising human and animal pathogens including the recently recognized Chlamydia gallinacea. C. gallinacea is widespread in poultry without causing clinical signs, which raises questions about its pathogenic potential. To assess this potential, two novel C. gallinacea strains were isolated, tested in infection experiments in embryonated chicken eggs and compared to C. psittaci. C. psittaci infection in poultry can result in severe systemic illness, depending on the conditions, and infections can be transmitted to humans. In the experiments C. gallinacea infection induced mortality of the embryo, but to a lower extent than infection with C. psittaci. Subsequent genome comparisons confirmed both C. gallinacea strains possess potential virulence genes typical for chlamydia, but fewer than C. psittaci. These results indicate C. gallinacea does have a pathogenic potential which warrants further research to elucidate its role as a poultry pathogen.

NrrF is a small regulatory RNA of the human pathogen Neisseria meningitidis. NrrF was previously shown to repress succinate dehydrogenase (sdhCDAB) under control of the ferric uptake regulator (Fur). Here, we provide evidence that cytochrome bc 1, encoded by the polycistronic mRNA petABC, is a NrrF target as well. We demonstrated differential expression of cytochrome bc 1 comparing wild‐type meningococci and meningococci expressing NrrF when sufficient iron is available. Using a gfp‐reporter system monitoring translational control and target recognition of sRNA in Escherichia coli, we show that interaction between NrrF and the 5′ untranslated region of the petABC mRNA results in its repression. The NrrF region essential for repression of petABC was identified by site‐directed mutagenesis and is fully conserved among meningococci. Our results provide further insights into the mechanism by which Fur controls essential components of the N. meningitidis respiratory chain. Adaptation of cytochrome bc 1 complex component levels upon iron limitation is post‐transcriptionally regulated via the small regulatory RNA NrrF.

The conserved RNA-binding protein, Hfq, has multiple regulatory roles within the prokaryotic cell, including promoting stable duplex formation between small RNAs and mRNAs, and thus hfq deletion mutants have pleiotropic phenotypes. Previous proteome and transcriptome studies of Neisseria meningitidis have generated limited insight into differential gene expression due to Hfq loss. In this study, reversed-phase liquid chromatography combined with data-independent alternate scanning mass spectrometry (LC-MSE) was utilized for rapid high-resolution quantitative proteomic analysis to further elucidate the differentially expressed proteome of a meningococcal hfq deletion mutant. Whole-cell lysates of N. meningitidis serogroup B H44/76 wild-type (wt) and H44/76 Delta hfq (Delta hfq) grown in liquid growth medium were subjected to tryptic digestion. The resulting peptide mixtures were separated by liquid chromatography (LC) prior to analysis by mass spectrometry (MSE). Differential expression was analyzed by Student's t-test with control for false discovery rate (FDR). Reliable quantitation of relative expression comparing wt and Delta hfq was achieved with 506 proteins (20%). Upon FDR control at q

We describe the incidence and patient characteristics of Streptococcus gallolyticus meningitis. We identified S. gallolyticus meningitis in a nationwide cohort of patients with community-acquired bacterial meningitis, and performed a systematic review and meta-analysis of all reported adult cases in the literature. Five cases were identified (0.3%) in a cohort of 1561 episodes of bacterial meningitis. In one patient, bowel disease (colon polyps) was identified as a predisposing condition for S. gallolyticus infection, whereas no patients were diagnosed with endocarditis. In a combined analysis of our patients and 37 reported in the literature, we found that the median age was 59 years. Predisposing factors were present in 21 of 42 patients (50%), and mainly consisted of immunosuppressive therapy (seven patients), cancer (four patients), and alcoholism (four patients). Colon disease was identified in 15 of 24 patients (63%) and endocarditis in five of 27 patients (18%). Co-infection with Strongyloides stercoralis was identified in 14 of 34 patients (41%), ten of whom were infected with human immunodeficiency virus or human T-lymphotropic virus. Outcomes were described for 37 patients; eight died (22%) and one (3%) had neurological sequelae. S. gallolyticus is an uncommon cause of bacterial meningitis, with specific predisposing conditions. When it is identified, consultation with a cardiologist and gastroenterologist is warranted to rule out underlying endocarditis or colon disease. Stool examinations for Strongyloides stercoralis should be performed in patients who have travelled to or originate from endemic areas.

Recent reports highlight a growing awareness of the potential for Chlamydia considered to be traditionally veterinary pathogens to cause cases of community-acquired pneumonia in humans. Cases of Chlamydia caviae, a guinea pig pathogen, were detected in individuals with serious pneumonia requiring hospitalisation. Source tracking linked these infections to pet guinea pigs. Studies of horses have found that the avian and zoonotic pathogen, Chlamydia psittaci, may be more common in these mammalian hosts, further highlighting the risk of exposure to chlamydial causes of CAP. The realisation that CAP can be caused by a broader range of chlamydial agents highlights the need for better collaboration between primary care physicians, public health officials and veterinarians. In a Dutch model for this approach, surveillance of veterinary chlamydiae as a cause of CAP has been improved through harmonisation of detection and molecular typing methods to facilitate an easier exchange of detection and molecular typing data to support source tracking efforts by public health and veterinary authorities.

Background Chlamydia trachomatis (Ct) plasmid has been shown to encode genes essential for infection. We evaluated the population structure of Ct using whole-genome sequence data (WGS). In particular, the relationship between the Ct genome, plasmid and disease was investigated. Results WGS data from 157 Ct isolates deposited in the Chlamydiales pubMLST database (http://pubMLST.org/chlamydiales/) were annotated with 902 genes including the core and accessory genome. Plasmid associated genes were annotated and a plasmid MLST scheme was defined allowing plasmid sequence types to be determined. Plasmid allelic variation was investigated. Phylogenetic relationships were examined using the Genome Comparator tool available in pubMLST. Phylogenetic analyses identified four distinct Ct core genome clusters and six plasmid clusters, with a strong association between the chromosomal genotype and plasmid. This in turn was linked to ompA genovars and disease phenotype. Horizontal genetic transfer of plasmids was observed for three urogenital-associated isolates, which possessed plasmids more commonly found in isolates resulting from ocular infections. The pgp3 gene was identified as the most polymorphic plasmid gene and pgp4 was the most conserved. Conclusion A strong association between chromosomal genome, plasmid type and disease was observed, consistent with previous studies. This suggests co-evolution of the Ct chromosome and their plasmids, but we confirmed that plasmid transfer can occur between isolates. These data provide a better understanding of the genetic diversity occurring across the Ct genome in association with the plasmid content. Electronic supplementary material The online version of this article (10.1186/s12864-018-4522-3) contains supplementary material, which is available to authorized users.

Streptococcus suis is an emerging zoonotic pathogen, which causes meningitis and septicaemia in humans and pigs [1, 2]. Ingestion of S. suis serotype 2 (SS2) contaminated food was identified as a risk factor for meningitis caused by SS2 in adult patients in Vietnam and Thailand [2], indicating that the gastrointestinal tract (GIT) might serve as an entry site of SS2 infection in humans [3, 4]. The upper respiratory tract (URT) is considered the main entry site of S. suis infection in pigs [5]. However, recent studies showed that the porcine GIT is rapidly colonized by S. suis after weaning [6] and that bacterial entry through the GIT occurs in piglets after mild stress induction [7], indicating that also the porcine GIT is an entry site of S. suis infection. The capsular polysaccharide (CPS), which determines the serotype, appears to play an essential role during the interaction with the URT epithelium [8]. Suilysin (Sly), a hemolytic pore-forming toxin, may also play a role facilitating translocation across the mucosal epithelium [9]. SS2 is responsible for most reported infections in humans and pigs worldwide [1, 10]. However, S. suis serotype 9 (SS9) is the most common serotype isolated from diseased pigs in Northern Europe [11] and is also prevalent in China [12] but has never been isolated from human patients [1, 10]. It is not known why isolates with serotypes that are highly prevalent among diseased pigs, such as SS9, have never been reported from human cases of SS infections. In addition to serotype, the genotype contributes to virulence of S. suis. Invasive S. suis strains are limited to certain sequence types (ST) and multi locus sequence typing (MLST) clonal complexes (CC) [13]. The majority of the SS2 strains isolated from human patients belong to CC1. However, we recently identified SS2 isolates from human patients in The Netherlands belonging to CC20, which is unrelated to CC1, indicating that multiple zoonotic clones are circulating worldwide [11]. In contrast, Dutch SS9 strains isolated from diseased pigs mainly belong to CC16, which is unrelated to both CC1 and CC20 [11, 14]. We aimed to provide experimental proof of concept that S. suis is capable of translocation in the GIT, in the absence of any invasive procedures. We then studied strain- and host-specific factors that may modulate S. suis infectivity at the intestinal mucosal surface, potentially providing clues to the zoonotic potential of SS2.

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae . Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease ( n = 101) and from carriage ( n = 103), and on non-typeable pneumococci from asymptomatic individuals ( n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae , targeting cpsA , lytA , piaB , ply , Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae , whereas assays targeting cpsA , ply , Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates.

Background Chlamydia suis is an important, globally distributed, highly prevalent and diverse obligate intracellular pathogen infecting pigs. To investigate the prevalence and genetic diversity of C. suis in China, 2,137 nasal, conjunctival, and rectal swabs as well as whole blood and lung samples of pigs were collected in 19 regions from ten provinces of China in this study. Results We report an overall positivity of 62.4% (1,334/2,137) of C. suis following screening by Chlamydia spp. 23S rRNA-based FRET-PCR and high-resolution melting curve analysis and confirmatory sequencing. For C. suis-positive samples, 33.3 % of whole blood and 62.5% of rectal swabs were found to be positive for the C. suis tetR(C) gene, while 13.3% of whole blood and 87.0% of rectal swabs were positive for the C. suis tet(C) gene. Phylogenetic comparison of partial C. suis ompA gene sequences revealed significant genetic diversity in the C. suis strains. This genetic diversity was confirmed by C. suis-specific multilocus sequence typing (MLST), which identified 26 novel sequence types among 27 examined strains. Tanglegrams based on MLST and ompA sequences provided evidence of C. suis recombination amongst the strains analyzed. Conclusions Genetically highly diverse C. suis strains are exceedingly prevalent in pigs. As it stands, the potential pathogenic effect of C. suis on pig health and production of C. suis remains unclear and will be the subject of further investigations. Further study is also required to address the transmission of C. suis between pigs and the risk of 'spill-over' and 'spill-back' of infections to wild animals and humans. Electronic supplementary material The online version of this article (10.1186/s12917-017-1295-x) contains supplementary material, which is available to authorized users.

Background Streptococcus suis is a zoonotic pathogen, causing meningitis and septicemia. We previously demonstrated that the gastrointestinal tract (GIT) is an entry site for zoonotic S. suis infection. Here we studied the contribution of (S) under bar treptococcal (a) under bar(d) under bar hesin (P) under bar rotein (SadP) to host-pathogen interaction at GIT level. Methods SadP expression in presence of Intestinal Epithelial Cells (IEC) was compared with expression of other virulence factors by measuring transcript levels using quantitative Real Time PCR (qRT-PCR). SadP variants were identified by phylogenetic analysis of complete DNA sequences. The interaction of SadP knockout and complementation mutants with IEC was tested in vitro. Results Expression of sadP was significantly increased in presence of IEC. Sequence analysis of 116 invasive strains revealed five SadP sequence variants, correlating with genotype. SadP1, present in zoonotic isolates of clonal complex 1, contributed to binding to both human and porcine IEC and translocation across human IEC. Antibodies against the globotriaosylceramide Gb3/CD77 receptor significantly inhibited adhesion to human IEC. Conclusion SadP is involved in the host-pathogen interaction in the GIT. Differences between SadP variants may determine different affinities to the Gb3/CD77 host-receptor, contributing to variation in adhesion capacity to host IEC and thus to S. suis zoonotic potential

Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. pneumoniae arginine synthesis genes promote growth and virulence in invasive pneumococcal disease.

Many studies have aimed to set up boundaries for the classification and definition of prokaryotic genus and species classification; however, studies that focused on genus-level genomic differences for existing taxonomy systems are limited. Recently, a novel method was described for prokaryotic genus delineation using the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance (Qin et al. A proposed genus boundary for the prokaryotes based on genomic insights. J Bacteriol 2014; 196 :2210–5). Here, we extended the POCP analysis of the order Chlamydiales and pairwise compared all currently recognized species and candidate species of the family Chlamydiaceae as well as some species from other families. Using the taxonomy advised by the International Committee on Systematics of Prokaryotes, subcommittee on the taxonomy of the Chlamydiae , POCP analysis revealed that all pairwise comparisons of species from different families resulted in values lower than 50%, the proposed threshold for genus boundary. In contrast, all interspecies pairwise comparisons of species from the single genus within the family Chlamydiaceae resulted in POCP values higher than 70%. We conclude that the recommended genus classification of the family Chlamydiaceae is rational and that POCP analyses can provide a robust genomic index for the taxonomy of members of the order Chlamydiales in terms of genus demarcation.

Streptococcus suis is a zoonotic swine pathogen and a major public health concern in Asia, where it emerged as an important cause of bacterial meningitis in adults. While associated with food-borne transmission in Asia, zoonotic S. suis infections are mainly occupational hazards elsewhere. To identify genomic differences that can explain zoonotic potential, we compared whole genomes of 98 S. suis isolates from human patients and pigs with invasive disease in the Netherlands, and validated our observations with 18 complete and publicly available sequences. Zoonotic isolates have smaller genomes than non-zoonotic isolates, but contain more virulence factors. We identified a zoonotic S. suis clone that diverged from a non-zoonotic clone by means of gene loss, a capsule switch, and acquisition of a two-component signalling system in the late 19th century, when foreign pig breeds were introduced. Our results indicate that zoonotic potential of S. suis results from gene loss, recombination and horizontal gene transfer events.

Hfq is a highly conserved pleiotropically acting prokaryotic RNA-binding protein involved in the post-transcriptional regulation of many stress-responsive genes by small RNAs. In this study, we show that Hfq of the strictly human pathogen Neisseria meningitidis is involved in the regulation of expression of components involved in general metabolic pathways, iron metabolism and virulence. A meningococcal hfq deletion strain (H44/76Deltahfq) is impaired in growth in nutrient-rich media and does not grow at all in nutrient-limiting medium. The growth defect was complemented by expression of hfq in trans. Using proteomics, the expression of 28 proteins was found to be significantly affected upon deletion of hfq. Of these, 20 proteins are involved in general metabolism, among them seven iron-responsive genes. Two proteins (PilE, TspA) are involved in adherence to human cells, a step crucial for the onset of disease. One of the differentially expressed proteins, GdhA, was identified as an essential virulence factor for establishment of sepsis in an animal model, studied earlier. These results show that in N. meningitidis Hfq is involved in the regulation of a variety of components contributing to the survival and establishment of meningococcal disease.

A real-time PCR assay with a DNA purification and inhibition control (internal control; IC) was developed to detect Chlamydophila psittaci DNA in human clinical samples. Novel C. psittaci-specific primers targeting the ompA gene were developed. The IC DNA contained the same primer-binding sites and had the same length and nucleotide content as the C. psittaci DNA amplicon, but had a shuffled probe-binding region. The lower limit of detection was 80 target copies/PCR, corresponding to 6250 copies /mL in a clinical sample. Specificity was tested using reference strains of 30 bacterial species. No amplification was observed from any of these samples. Respiratory samples from eight patients were positive with this PCR. Six of these patients were confirmed as positive for C. psittaci with serological testing. Two patients had increasing antibody titres, but did not fulfil criteria proposed previously for serologically proven Chlamydia spp. infection. The real-time PCR described in this paper is a sensitive, specific and rapid method to detect C. psittaci DNA in human clinical respiratory samples.

The gene prmC , encoding the putative S -adenosyl- l -methionine (AdoMet)-dependent methyltransferase (MTase) of release factors (RFs) of the obligate intracellular pathogen Chlamydia trachomatis , was functionally analyzed. Chlamydial PrmC expression suppresses the growth defect of a prmC knockout strain of Escherichia coli K-12, suggesting an interaction of chlamydial PrmC with E. coli RFs in vivo. In vivo methylation assays carried out with recombinant PrmC and RFs of chlamydial origin demonstrated that PrmC methylates RFs within the tryptic fragment containing the universally conserved sequence motif Gly-Gly-Gln. This is consistent with the enzymatic properties of PrmC of E. coli origin. We conclude that C. trachomatis PrmC functions as an N 5 -glutamine AdoMet-dependent MTase, involved in methylation of RFs.

Objectives The aetiology of primary sclerosing cholangitis (PSC) is unknown, and the role of micro-organisms has been studied only to a limited extent. We tested the hypothesis that past or persisting infection with common viruses or atypical bacteria might play a role in genetically susceptible hosts. Design Case-control study. Methods Serological screening for antibodies against 22 viruses as well as Chlamydia spp. and Mycoplasma pneumoniae was carried out in 41 well-established PSC patients. All 5110 sera tested in 1997 for these microorganisms at our laboratory served as a background reference group. Subsequently, Chlamydia antilipopolysaccharide (LPS) antibodies were determined by enzyme-linked immunosorbent assay (ELISA) in the PSC group and in three race-matched control groups (inflammatory bowel disease (IBD) group, n = 35; non-IBD patients group, n = 39; healthy blood donor group, n = 40). Subtyping in Chlamydia trachomatis and C. pneumoniae serotypes by specific anti-major outer membrane protein (MOMP) assays was carried out in the four groups. Immunohistochemical staining using specific markers for chlamydiae was carried out on liver biopsies of 14 PSC patients. Results There was a markedly elevated seroprevalence of Chlamydia-LPS antibodies compared with the 1997 reference group. The odds ratios (ORs) for the presence of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies for the PSC patients versus the control group were 2.4 (95% confidence interval (CI) 1.1 to 5.4),1.9 (95% CI 0.9 to 4.0) and 6.7 (95% CI 3.0 to 17.0), respectively. All other micro-organisms tested showed normal antibody profiles that did not differ from the 1997 reference group. The seroprevalence of Chlamydia-anti-LPS antibodies was elevated markedly in the PSC patients compared with the IBD, non-IBD and blood donor groups. The outcomes in the C, trachomatis and C. pneumoniae anti-MOMP assays did not correlate with the anti-LPS-positive PSC sera. The actual presence of Chlamydia bodies in liver tissue could not be demonstrated. Conclusion Our findings suggest an association between PSC and (previous) infection with Chlamydia. Eur J Gastroenterol Hepatol 14:641-648 (C) 2002 Lippincott Williams Wilkins

Chlamydia pecorum is globally associated with several ovine diseases including keratoconjunctivitis and polyarthritis. The exact relationship between the variety of C. pecorum strains reported and the diseases described in sheep remains unclear, challenging efforts to accurately diagnose and manage infected flocks. In the present study, we applied C. pecorum multi-locus sequence typing (MLST) to C. pecorum positive samples collected from sympatric flocks of Australian sheep presenting with conjunctivitis, conjunctivitis with polyarthritis, or polyarthritis only and with no clinical disease (NCD) in order to elucidate the exact relationships between the infecting strains and the range of diseases. Using Bayesian phylogenetic and cluster analyses on 62 C. pecorum positive ocular, vaginal and rectal swab samples from sheep presenting with a range of diseases and in a comparison to C. pecorum sequence types (STs) from other hosts, one ST (ST 23) was recognised as a globally distributed strain associated with ovine and bovine diseases such as polyarthritis and encephalomyelitis. A second ST (ST 69) presently only described in Australian animals, was detected in association with ovine as well as koala chlamydial infections. The majority of vaginal and rectal C. pecorum STs from animals with NCD and/or anatomical sites with no clinical signs of disease in diseased animals, clustered together in a separate group, by both analyses. Furthermore, 8/13 detected STs were novel. This study provides a platform for strain selection for further research into the pathogenic potential of C. pecorum in animals and highlights targets for potential strain-specific diagnostic test development.

Neisseria meningitidis is a gram-negative bacterium that may cause meningitis, sepsis, or both. The increase in the incidence of meningococcal disease in various countries in the past 2 decades is mainly due the genotypically related lineage III meningococci. The chromosomal DNA differences between lineage III strains and non-lineage III strains were identified using representational difference analysis. Thus, a 1.8-kb locus that is specific for lineage III meningococci was identified. The locus contains three open reading frames encoding the Nme SI restriction-modification system. The methyltransferase gene was cloned and expressed in Escherichia coli . Site AGTACT was found to be modified by the enzyme. In conclusion, lineage III strains differ from endemic strains by the presence of a specific restriction-modification system. This restriction-modification system may contribute to the clonal and hypervirulent character of lineage III strains by influencing horizontal gene transfer and transcription.

Reactive arthritis (ReA) after infections with various gram-negative bacteria is strongly associated with the MHC class I molecule HLA-B27. It is supposed that the B27 molecule itself plays a role in the pathogenesis of ReA by presenting antigenic peptides to cytotoxic T lymphocytes. The peptide repertoires presented by Salmonella - , Shigella - and non-infected cells were compared to identify such peptides. From the peptides isolated from the B27 molecules of these cells, profiles were generated by reversed-phase chromatography and peaks present in the profiles from infected cells but not in profiles from non-infected cells were studied for their peptide compositions. Some sequences with identity to those in human histone H3, human ribosomal protein S17 and the heavy chain of HLA-B27 itself were detected only in profiles from infected cells. All peptides identified from infected cells contained the B*2705 peptide-binding motif. The data suggest that HLA-B27-positive cells infected with ReA-inducing bacteria show an increased presentation of certain self-peptides. There was no evidence for altered peptide-binding specificity of B27 after infection. However, the interpretations were hampered by the variation in peptide presentation between different experiments.

The inflammatory response to the presence of Ureaplasma urealyticum or Mycoplasma hominis in the lower genital tract of subfertile men without any signs or symptoms of infection was investigated by measuring the concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in seminal plasma. Semen samples were collected from 30 culture-positive subfertile males and 23 culture-negative subfertile males. Enzyme-linked immunosorbent assays showed that IL-8 was present in relatively high concentrations (0.12-4.8 ng/ml) in all semen samples investigated. In contrast, the other cytokines were only detectable in 72% (IFN-gamma), 44% (IL-6) and 19% (TNF-gamma) of the samples and were present in relatively low concentrations (1-410 pg/ml). Seminal plasma cytokine concentrations were similar in samples from culture-positive and culture-negative males. These data strongly indicate that the presence of U. urealyticum or M. hominis in the lower genital tract of subfertile males reflects a silent colonisation rather than infection.