New population of Solanum pimpinellifolium backcross inbred lines as a resource for heat stress tolerance in tomato.

The occurring temperature increase in crop production areas worldwide is generating conditions of heat stress that negatively affect crop productivity. Tomato (Solanum lycopersicum), a major vegetable crop, is highly susceptible to elevated temperatures. Under such conditions, fruit set is dramatically reduced, leading to significant yield losses. Solanum pimpinellifolium, a wild species closely related to the cultivated tomato, was shown to have beneficial attributes under various abiotic stress growth conditions. We have utilized a new population of backcross inbred lines originated from a cross between S. pimpinellifolium and S. lycopersicum, in order to evaluate its potential as a new genetic resource for improvement of reproductive performance of cultivated tomato under heat stress conditions. This population was screened for various heat stress-related traits, under controlled heat stress and non-stress conditions. Our results show that significant variation exists for all the heat stress related traits that were examined and point at individual lines with better reproductive performance under heat stress conditions that share a common introgression from the wild S. pimpinellifolium parent, suggesting several candidate genes as potential drivers of thermotolerance. Thus, our results place this population as a valuable new resource for the discovery of heat stress related genetic loci for the future development of heat stress tolerant tomato cultivars.

Gardnerella Species and Their Association With Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a condition marked by high vaginal bacterial diversity. Gardnerella vaginalis has been implicated in BV but is also detected in healthy women. The Gardnerella genus has been expanded to encompass 6 validly named species and several genomospecies. We hypothesized that particular Gardnerella species may be more associated with BV. METHODS: Quantitative polymerase chain reaction (PCR) assays were developed targeting the cpn60 gene of species groups including G. vaginalis, G. piotii/pickettii, G. swidsinskii/greenwoodii, and G. leopoldii. These assays were applied to vaginal swabs from individuals with (n = 101) and without BV (n = 150) attending a sexual health clinic in Seattle, Washington. Weekly swabs were collected from 42 participants for up to 12 weeks. RESULTS: Concentrations and prevalence of each Gardnerella species group were significantly higher in participants with BV; 91.1% of BV-positive participants had 3 or more Gardnerella species groups detected compared to 32.0% of BV-negative participants (P < .0001). BV-negative participants with 3 or more species groups detected were more likely to develop BV within 100 days versus those with fewer (60.5% vs 3.7%, P < .0001). CONCLUSIONS: These results suggest that BV reflects a state of high Gardnerella species diversity. No Gardnerella species group was a specific marker for BV.

Biofilm-Forming Capacity and Drug Resistance of Different Gardnerella Subgroups Associated with Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common infection of the lower reproductive tract among women of reproductive age. Recurrent infections and antibiotic resistance associated with biofilms remain significant challenges for BV treatment. Gardnerella species are commonly found in women with and without BV, indicating that genetic differences among Gardnerella isolates may distinguish pathogenic from commensal subgroups. This study isolated 11 Gardnerella strains from vaginal samples obtained from women with BV before or after treatment. The biofilm formation ability of each strain was examined by crystal violet staining. Eight strains were selected using phylogenetic analysis of the cpn60 sequences and classified as subgroups A (6/8), B (1/8), and D (1/8). The biofilm formation ability and antibiotic resistance profile of these strains was compared among the subgroups. Subgroup D had the strongest biofilm formation ability. Six of the planktonic strains exhibited resistance to the first-line BV drug, metronidazole, and one to clindamycin. Moreover, biofilm formation in vitro increased strain resistance to clindamycin. Two strains with strong biofilm ability, S20 and S23, and two with weak biofilm ability, S24 and S25, were selected for comparative genomic analysis. S20 and S23 were found to contain four key genes associated with biofilm formation and more genes involved in carbohydrate synthesis and metabolism than S24 and S25. Identifying differences in the expression of virulence factors between Gardnerella subgroups could inform the development of novel treatments for BV.

Maternal vaginal microbiome composition does not affect development of the infant gut microbiome in early life.

Birth mode has been implicated as a major factor influencing neonatal gut microbiome development, and it has been assumed that lack of exposure to the maternal vaginal microbiome is responsible for gut dysbiosis among caesarean-delivered infants. Consequently, practices to correct dysbiotic gut microbiomes, such as vaginal seeding, have arisen while the effect of the maternal vaginal microbiome on that of the infant gut remains unknown. We conducted a longitudinal, prospective cohort study of 621 Canadian pregnant women and their newborn infants and collected pre-delivery maternal vaginal swabs and infant stool samples at 10-days and 3-months of life. Using cpn60-based amplicon sequencing, we defined vaginal and stool microbiome profiles and evaluated the effect of maternal vaginal microbiome composition and various clinical variables on the development of the infant stool microbiome. Infant stool microbiomes showed significant differences in composition by delivery mode at 10-days postpartum; however, this effect could not be explained by maternal vaginal microbiome composition and was vastly reduced by 3 months. Vaginal microbiome clusters were distributed across infant stool clusters in proportion to their frequency in the overall maternal population, indicating independence of the two communities. Intrapartum antibiotic administration was identified as a confounder of infant stool microbiome differences and was associated with lower abundances of Escherichia coli, Bacteroides vulgatus, Bifidobacterium longum and Parabacteroides distasonis. Our findings demonstrate that maternal vaginal microbiome composition at delivery does not affect infant stool microbiome composition and development, suggesting that practices to amend infant stool microbiome composition focus factors other than maternal vaginal microbes.

Chaperonin: Co-chaperonin Interactions.

Co-chaperonins function together with chaperonins to mediate ATP-dependent protein folding in a variety of cellular compartments. Chaperonins are evolutionarily conserved and form two distinct classes, namely, group I and group II chaperonins. GroEL and its co-chaperonin GroES form part of group I and are the archetypal members of this family of protein folding machines. The unique mechanism used by GroEL and GroES to drive protein folding is embedded in the complex architecture of double-ringed complexes, forming two central chambers that undergo conformational rearrangements that enable protein folding to occur. GroES forms a lid over the chamber and in doing so dislodges bound substrate into the chamber, thereby allowing non-native proteins to fold in isolation. GroES also modulates allosteric transitions of GroEL. Group II chaperonins are functionally similar to group I chaperonins but differ in structure and do not require a co-chaperonin. A significant number of bacteria and eukaryotes house multiple chaperonin and co-chaperonin proteins, many of which have acquired additional intracellular and extracellular biological functions. In some instances, co-chaperonins display contrasting functions to those of chaperonins. Human HSP60 (HSPD) continues to play a key role in the pathogenesis of many human diseases, in particular autoimmune diseases and cancer. A greater understanding of the fascinating roles of both intracellular and extracellular Hsp10 on cellular processes will accelerate the development of techniques to treat diseases associated with the chaperonin family.

Research Progress on the Correlation Between Gardnerella Typing and Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common infectious disease of the reproductive tract in women of childbearing age. It often manifests as an imbalance in the vaginal microbiome, including a decrease in Lactobacillus and an increase in anaerobic bacteria. While Gardnerella spp. are considered a major cause of BV, they are also detected in the vaginal microbiome of healthy women. G. vaginalis was the only recognized species of Gardnerella until a recent study characterized three new species, G. leopoldii, G. piotii, and G. swidsinskii. This review describes the different types and genetic diversity of Gardnerella, as well as new findings on the correlation between different Gardnerella spp. and BV.

cpn60 barcode sequences accurately identify newly defined genera within the Lactobacillaceae.

The cpn60 barcode sequence has been established as an informative target for microbial species identification. Applications of cpn60 barcode sequencing are supported by the availability of "universal" PCR primers for amplification and a curated reference database of cpn60 sequences, cpnDB. A recent reclassification of lactobacilli involving the definition of 23 new genera provided an opportunity to update cpnDB and to determine if the cpn60 barcode could be used for accurate identification of species consistent with the new framework. Analysis of 275 cpn60 sequences representing 258/269 of the validly named species in Lactobacillus, Paralactobacillus, and the 23 newer genera showed that cpn60-based sequence relationships were generally consistent with whole-genome-based phylogeny. Aligning or mapping full-length barcode sequences or a 150 bp subsequence resulted in accurate and unambiguous species identification in almost all cases. Taken together, our results show that the combination of available reference sequence data, "universal" barcode amplification primers, and the inherent sequence diversity within the cpn60 barcode makes it a useful target for the detection and identification of lactobacilli, as defined by the latest taxonomic framework.

The Exoproteome of Staphylococcus pasteuri Isolated from Cervical Mucus during the Estrus Phase in Water Buffalo (Bubalus bubalis).

Bacterial extracellular proteins participate in the host cell communication by virtue of the modulation of pathogenicity, commensalism and mutualism. Studies on the microbiome of cervical mucus of the water buffalo (Bubalus bubalis) have shown the occurrence of Staphylococcus pasteuri and that the presence of this bacterium is indicative of various physiological and reproductive states in the host. Recently, S. pasteuri has been isolated from the cervical mucus of the buffalo during the different phases of estrous cycle, and has proved to be much more pronounced during the estrus phase. The basis underlying the availability of a significantly increased S. pasteuri population, specifically during the estrus phase, is not known. Consequently, it is important to determine the significance of the specific abundance of S. pasteuri during the estrus phase of the buffalo host, particularly from the perspective of whether this bacterial species is capable of contributing to sexual communication via its extracellular proteins and volatiles. Therefore, the relevance of S. pasteuri exoproteome in the buffalo cervical mucus during the estrus phase was analyzed using LC-MS/MS. As many as 219 proteins were identified, among which elongation factor Tu (EF-Tu), 60-kDa chaperonin (Cpn60), enolase, fructose-bisphosphate aldolase class 1 (FBP aldolase), enoyl-[acyl-carrier-protein] reductase [NADPH] (ENR) and lipoprotein (Lpp) were the functionally important candidates. Most of the proteins present in the exoproteome of S. pasteuri were those involved in cellular-metabolic functions, as well as catalytic- and binding activities. Moreover, computational studies of Lpp have shown enhanced interaction with volatiles such as acetic-, butanoic-, isovaleric- and valeric acids, which were identified in the cervical mucus S. pasteuri culture supernatant. The present findings suggest that S. pasteuri extracellular proteins may play an important role in buffalo sexual communication during the estrus phase.

Gardnerella subgroup dominant microbiomes are associated with divergent cervicovaginal immune responses in a longitudinal cohort of Kenyan women.

Most cervicovaginal microbiome-immunology studies to date have relied on 16S rDNA microbial profiling which does not resolve the molecular subgroups of Gardnerella, believed to be central to the pathogenesis of bacterial vaginosis (BV) and subsequent risk of HIV acquisition. Here we used the cpn60 universal target which in addition to other microbial taxa, resolves four Gardnerella subgroups, for cervicovaginal microbial profiling in a longitudinal cohort of Kenyan women to examine associations with cellular and soluble markers of inflammation and HIV susceptibility. Participants (N = 41) were sampled, contributing 362 samples for microbiome analysis. All non-Lactobacillus dominant microbial communities were associated with high pro-inflammatory cytokine levels. Divergent associations were observed among different Gardnerella subgroup dominated communities with respect to the chemokine IP-10. Specifically, Gardnerella subgroup A dominant and polymicrobial communities were associated with reduced concentrations of IP-10 in adjusted linear mixed models (p<0.0001), compared to microbial communities dominated by Lactobacillus (non-iners) species. However, these associations did not translate to significant differences in the proportion or absolute number of CCR5, HLA-DR and CD38 expressed on cervical CD4+ T- cells. These findings suggest that some associations between Gardnerella subgroup dominant microbiomes and mucosal immunity differ and are relevant for the study of BV-pathogenesis and understanding the mechanisms of BV-associated HIV risk.

Improved folding of recombinant protein via co-expression of exogenous chaperones.

Expression of heterologous genes in Escherichia coli is a routine technology for recombinant protein production, but the predictable recovery of properly folded and uniformly bioactive material remains a challenge. Misfolded proteins typically accumulate as insoluble inclusion bodies, and a variety of strategies have been employed in efforts to increase the yield of soluble product. One technique is the overexpression of E. coli protein chaperones during recombinant protein induction, in an effort to increase the folding capacity of the bacterial host. We have developed an alternative approach, by supplementing the host protein folding machinery with chaperones from other species. Extremophiles have evolved under conditions (extremes of temperature, salinity, pressure, and/or pH) that make them attractive candidates for possessing chaperones with novel folding activities. The green fluorescent protein (GFP) of Aequorea victoria, which is predominantly insoluble under typical recombinant expression culture conditions, was employed as an in vivo indicator of protein folding activity for chaperone homologs from a variety of extremophiles. For a subset of the chaperones tested, co-expression with GFP promoted an increase in both fluorescence signal intensity as well as the amount of GFP recovered in the soluble protein fraction. Several archaeal chaperones were also found to be able to refold soluble Lyt_Orn C40 peptidase from inclusion bodies in vitro. In particular, Pf Cpn(MA), a mutant chaperonin which exhibited significant refolding activity, is also shown to deconstruct the morphology and structure of inclusion bodies (Kurouski et al., 2012). Hence, the simple and rapid GFP assay provides a tool to screen for extremophilic chaperones that exhibit folding activity under E. coli growth conditions, and suggests that increasing the repertoire of heterologous chaperones might provide a partial but general solution to the problem of recombinant protein insolubility.

Early Neonatal Meconium Does Not Have a Demonstrable Microbiota Determined through Use of Robust Negative Controls with cpn60-Based Microbiome Profiling.

Detection of bacterial DNA within meconium is often cited as evidence supporting in utero colonization. However, many studies fail to adequately control for contamination. We aimed to define the microbial content of meconium under properly controlled conditions. DNA was extracted from 141 meconium samples and subjected to cpn60-based microbiome profiling, with controls to assess contamination throughout. Total bacterial loads of neonatal meconium, infant stool, and controls were compared by 16S rRNA quantitative PCR (qPCR). Viable bacteria within meconium were cultured, and isolate clonality was assessed by pulsed-field gel electrophoresis (PFGE). Meconium samples did not differ significantly from controls with respect to read numbers or taxonomic composition. Twenty (14%) outliers with markedly higher read numbers were collected significantly later after birth and appeared more like transitional stool than meconium. Total bacterial loads were significantly higher in stool than in meconium, which did not differ from that of sequencing controls, and correlated well with read numbers. Cultured isolates were most frequently identified as Staphylococcus epidermidis, Enterococcus faecalis, or Escherichia coli, with PFGE indicating high intraspecies diversity. Our findings highlight the importance of robust controls in studies of low microbial biomass samples and argue against meaningful bacterial colonization in utero. Given that meconium microbiome profiles could not be distinguished from sequencing controls, and that viable bacteria within meconium appeared uncommon and largely consistent with postnatal skin colonization, there does not appear to be a meconium microbiota. IMPORTANCE Much like the recent placental microbiome controversy, studies of neonatal meconium reporting bacterial communities within the fetal and neonatal gut imply that microbial colonization begins prior to birth. However, recent work has shown that placental microbiomes almost exclusively represent contamination from lab reagents and the environment. Here, we demonstrate that prior studies of neonatal meconium are impacted by the same issue, showing that the microbial content of meconium does not differ from negative controls that have never contained any biological material. Our culture findings similarly supported this notion and largely comprised bacteria normally associated with healthy skin. Overall, our work adds to the growing body of evidence against the in utero colonization hypothesis.

A peptide derived from chaperonin 60.1, IRL201104, inhibits LPS-induced acute lung inflammation.

Chaperonin 60.1 (Cpn60.1) is a protein derived from Mycobacterium tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of nonallergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5,000 ng/kg) or IRL201104 (0.00025-2.5 ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4 h after LPS administration. In some experiments, mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analyzed for inflammasome function. Human umbilical vein endothelial cells (HUVECs) were analyzed for adhesion molecule expression. Human neutrophils were analyzed for integrin expression, chemotaxis, and cell polarization. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and annexin A1 knockout mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVECs or integrin expression, chemotaxis, or polarization of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1ß and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent, the proresolving factor annexin A1.

Discrimination of Gardnerella Species by Combining MALDI-TOF Protein Profile, Chaperonin cpn60 Sequences, and Phenotypic Characteristics.

The description of Gardnerella vaginalis was recently updated and three new species, including nine genome species within Gardnerella, were defined using whole genome sequences and matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. A fast and simple method based on readily available techniques would be of immense use to identify Gardnerella species in research and clinical practice. Here we show that 34 previously characterized Gardnerella isolates were assigned to the species using partial chaperonin cpn60 sequences. The MALDI Biotyper from Bruker Daltonik GmbH demonstrated the capability to differentiate the phylogenetically diverse groups composed of G. vaginalis/G. piotii and G. leopoldii/G. swidsinskii. Among the phenotypic properties that characterize Gardnerella species are sialidase and ß-galactosidase activities. Our data confirmed that the NanH3 enzyme is responsible for sialidase activity in Gardnerella spp. isolates. Almost all G. piotii isolates displayed a sialidase positive phenotype, whereas the majority of G. vaginalis strains were sialidase negative. G. leopoldii and G. swidskinskii displayed a sialidase negative phenotype. ß-galactosidase is produced exclusively in G. vaginalis strains. Earlier determined phenotypic characteristics associated with virulence of Gardnerella isolates now assigned to the defined species may provide insights on how diverse species contribute to shaping the vaginal microbiome.

Different and diverse anaerobic microbiota were seen in women living with HIV with unsuppressed HIV viral load and in women with recurrent bacterial vaginosis: a cohort study.

OBJECTIVE: To compare the vaginal microbiota of women living with HIV (WLWH) with the vaginal microbiota of women with recurrent bacterial vaginosis (BV) and healthy women without HIV to determine if there are differences in the vaginal microbiome, what factors influence these differences, and to characterise HIV clinical parameters including viral load and CD4 count in relation to the vaginal microbiome. DESIGN: Observational cohort study. SETTING: Canada. POPULATION: Women aged 18-49 years who were premenopausal and not pregnant were recruited into three cohorts: healthy women, WLWH and women with recurrent BV. METHODS: Demographic and clinical data were collected via interviews and medical chart reviews. Vaginal swabs were collected for Gram-stain assessment and microbiome profiling using the cpn60 barcode sequence. MAIN OUTCOME MEASURES: To compare overall community composition differences, we used compositional data analysis methods, hierarchical clustering and Kruskal-Wallis tests where appropriate. RESULTS: Clinical markers such as odour and abnormal discharge, but not irritation, were associated with higher microbial diversity. WLWH with unsuppressed HIV viral loads were more likely than other groups to have non-Gardnerella-dominated microbiomes. HIV was associated with higher vaginal microbial diversity and this was related to HIV viral load, with unsuppressed women demonstrating significantly higher relative abundance of Megasphaera genomosp. 1, Atopobium vaginae and Clostridiales sp. (all P < 0.05) compared with all other groups. CONCLUSIONS: In WLWH, unsuppressed HIV viral loads were associated with a distinct dysbiotic profile consisting of very low levels of Lactobacillus and high levels of anaerobes. TWEETABLE ABSTRACT: Vaginal microbiomes in WLWH with viral load >50 copies/ml have distinct dysbiotic profiles with high levels of anaerobes.

Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing.

The pig colon is the habitat of diverse Brachyspira species, of which only a few are of clinical importance. Methods for identification have shifted from phenotypic to molecular testing over the last two decades. Following the emergence of B. hampsonii it became evident that relying on species-specific PCRs carries the risk of overlooking important new species. Consequently, sequencing was proposed as an unbiased alternative for identification of isolates. So far, the main target for identification across species has been the NADH oxidase gene (nox). However, multiple copies of this gene in the genome and potential lateral gene transfer reduce confidence when using this gene. This study compared identification and phylogentic relationship inferred from nox sequencing to that inferred from sequencing of the cpn60 universal target using a collection of 168 isolates from different Brachyspira species. The majority of isolates had an identical identification with both methods. There were a few outliers in the trees with uncertain assignment to a species by BLAST analysis. A few major discrepancies pertained to the pathogenic species B. hampsonii (2), B. pilosicoli (1) and B. suanatina (1). Weakly haemolytic variants of B. hyodysenteriae were assigned to the correct species by both methods. Some of the isolates identified as B. hampsonii also had a weakly haemolytic phenotype.

The Chloroplast Envelope Protease FTSH11 - Interaction With CPN60 and Identification of Potential Substrates.

FTSH proteases are membrane-bound, ATP-dependent metalloproteases found in bacteria, mitochondria and chloroplasts. The product of one of the 12 genes encoding FTSH proteases in Arabidopsis, FTSH11, has been previously shown to be essential for acquired thermotolerance. However, the substrates of this protease, as well as the mechanism linking it to thermotolerance are largely unknown. To get insight into these, the FTSH11 knockout mutant was complemented with proteolytically active or inactive variants of this protease, tagged with HA-tag, under the control of the native promoter. Using these plants in thermotolerance assay demonstrated that the proteolytic activity, and not only the ATPase one, is essential for conferring thermotolerance. Immunoblot analyses of leaf extracts, isolated organelles and sub-fractionated chloroplast membranes localized FTSH11 mostly to chloroplast envelopes. Affinity purification followed by mass spectrometry analysis revealed interaction between FTSH11 and different components of the CPN60 chaperonin. In affinity enrichment assays, CPN60s as well as a number of envelope, stroma and thylakoid proteins were found associated with proteolytically inactive FTSH11. Comparative proteomic analysis of WT and knockout plants, grown at 20°C or exposed to 30°C for 6 h, revealed a plethora of upregulated chloroplast proteins in the knockout, some of them might be candidate substrates. Among these stood out TIC40, which was stabilized in the knockout line after recovery from heat stress, and three proteins that were found trapped in the affinity enrichment assay: the nucleotide antiporter PAPST2, the fatty acid binding protein FAP1 and the chaperone HSP70. The consistent behavior of these four proteins in different assays suggest that they are potential FTSH11 substrates.

Hetero-oligomeric CPN60 resembles highly symmetric group-I chaperonin structure revealed by Cryo-EM.

The chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as a model system, we found that in vivo the chloroplast chaperonin consists of CPN60α, CPN60ß1 and CPN60ß2 and the co-chaperonin of the three subunits CPN20, CPN11 and CPN23. In Escherichia coli, CPN20 homo-oligomers and all possible other chloroplast co-chaperonin hetero-oligomers are functional, but only that consisting of CPN11/20/23-CPN60αß1ß2 can fully replace GroES/GroEL under stringent stress conditions. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60α:CPN60ß1:CPN60ß2. The cryo-EM structures of endogenous CPN60αß1ß2/ADP and CPN60αß1ß2/co-chaperonin/ADP were solved at resolutions of 4.06 and 3.82 Å, respectively. In both hetero-oligomeric complexes the chaperonin subunits within each ring are highly symmetric. Through hetero-oligomerization, the chloroplast co-chaperonin CPN11/20/23 forms seven GroES-like domains, which symmetrically interact with CPN60αß1ß2. Our structure also reveals an uneven distribution of roof-forming domains in the dome-shaped CPN11/20/23 co-chaperonin and potentially diversified surface properties in the folding cavity of the CPN60αß1ß2 chaperonin that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.

Evaluation of dual target-specific real-time PCR for the detection of Kingella kingae in a Danish paediatric population.

BACKGROUND: We aimed to evaluate the relevance of dual target real-time polymerase chain (PCR) assays targeting the rtxA and cpn60 genes of the paediatric pathogen Kingella kingae. We also studied for the first time the clinical and epidemiological features of K. kingae infections in a Danish population. METHOD: Children with K. kingae-positive cultures were identified from 11,477 children and 86 children younger than 16 years old from whom blood cultures and joint fluid cultures were obtained between January 2010 and November 2016. Results were then compared to microbiological results obtained from 29 joint fluids (28 children) tested by dual target K. kingae real-time PCR from September 2014 to November 2016. Epidemiological data of all children with microbiologically confirmed K. kingae infections were collected. RESULTS: From 2010 to 2016, we diagnosed 17 children with microbiological-proven K. kingae infections. During this period, blood cultures from five children and joint fluid cultures from a single child yielded K. kingae. Dual target K. kingae real-time PCR allowed us to increase the diagnostic yield of K. kingae infections by detecting the organism in 12 of 29 (41.4%) specimens. Notably, the 12 real-time PCR-positive specimens were rtxA-positive whereas only 10 (83.3%) were cpn60-positive. PCR-positive children were significantly younger than PCR-negative children (p-value: .01). A significant seasonal variation was found for patients with proven K. kingae infection (p-value: <.001), with a peak in autumn. CONCLUSION: Dual target-specific real-time PCR markedly improved the detection of K. kingae in clinical specimens when compared to culture methods.

Molecular characterization and phylogenetic analysis of Clostridium perfringens from animals and their environments by cpn60 UT sequencing analysis.

Clostridium perfringens an ubiquitous environmental bacterium causes major food borne illnesses, digestive diseases and several soft tissue infections in humans and animals. In the present study, toxin typing of 91 C. perfringens isolates from animals with enteric diseases and their environments revealed the presence of type A and C strains. Enterotoxin gene (cpe), responsible for majority of the food poisoning incidences in humans and enteric infections in animals was present in 60.43 % of the isolates of which 76.3% and 23. 36% were chromosomal and plasmid borne respectively. Neighbour-joining tree inferred from cpn60 UT nucleotide sequences could differentiate the cpe+ve isolates from the cpe-ve isolates, provide clear distinction between the cpe-IS1470 and cpe-IS1151 genotypes and segregate type A and C strains in separate clusters. The present study is the first report on the utilization of cpn60 UT region for C. perfringens phylogeny analysis and demonstrates that cpn60 UT analysis alone, to a greater extent can be a simple, rapid and efficient method for differentiating between cpe+ve and cpe-ve strains or toxin types. The cpb2 gene was observed among 30 isolates of which 16.6% were from porcine sources while the rest were of non-porcine and environmental origin. The cpb2 sequences obtained in the present study though similar among them were diverse both from the consensus and atypical cpb2 sequences reported globally and formed a separate cluster. The study thus reports of novel cpb2 gene variant and warrants its characterization through further studies.

Microbial profiling of cpn60 universal target sequences in artificial mixtures of vaginal bacteria sampled by nylon swabs or self-sampling devices under different storage conditions.

The vaginal microbiome is increasingly characterized by deep sequencing of universal genes. However, there are relatively few studies of how different specimen collection and sample storage and processing influence these molecular profiles. Here, we evaluate molecular microbial community profiles of samples collected using the HerSwab™ self-sampling device, compared to nylon swabs and under different storage conditions. In order to minimize technical variation, mixtures of 11 common vaginal bacteria in simulated vaginal fluid medium were sampled and DNA extracts prepared for massively parallel sequencing of the cpn60 universal target (UT). Three artificial mixtures imitating commonly observed vaginal microbiome profiles were easily distinguished and proportion of sequence reads correlated with the estimated proportion of the organism added to the artificial mixtures. Our results indicate that cpn60 UT amplicon sequencing quantifies the proportional abundance of member organisms in these artificial communities regardless of swab type or storage conditions, although some significant differences were observed between samples that were stored frozen and thawed prior to DNA extraction, compared to extractions from samples stored at room temperature for up to 7days. Our results indicate that an on-the-market device developed for infectious disease diagnostics may be appropriate for vaginal microbiome profiling, an approach that is increasingly facilitated by rapidly dropping deep sequencing costs.