Genomic evolution of ST228 SCCmec-I MRSA 10 years after a major nosocomial outbreak.

In this study, we investigated the genomic changes in a major methicillin-resistant Staphylococcus aureus (MRSA) clone following a significant outbreak at a hospital. Whole-genome sequencing of MRSA isolates was utilized to explore the genomic evolution of post-outbreak MRSA strains. The epidemicity of the clone declined over time, coinciding with the introduction of multimodal infection control measures. A genome-wide association study (GWAS) identified multiple genes significantly associated with either high or low epidemic success, indicating alterations in mobilome, virulence, and defense mechanisms. Random Forest models pinpointed a gene related to fibrinogen binding as the most influential predictor of epidemicity. The decline of the MRSA clone may be attributed to various factors, including the implementation of new infection control measures, single nucleotide polymorphisms accumulation, and the genetic drift of a given clone. This research underscores the complex dynamics of MRSA clones, emphasizing the multifactorial nature of their evolution. The decline in epidemicity seems linked to alterations in the clone's genetic profile, with a probable shift towards decreased virulence and adaptation to long-term carriage. Understanding the genomic basis for the decline of epidemic clones is crucial to develop effective strategies for their surveillance and management, as well as to gain insights into the evolutionary dynamics of pathogen genomes.

Taxon-rich transcriptomics supports higher-level phylogeny and major evolutionary trends in Foraminifera.

Foraminifera, classified in the supergroup Rhizaria, are a common and highly diverse group of mainly marine protists. Despite their evolutionary and ecological importance, only limited genomic data (one partial genome and nine transcriptomic datasets) have been published for this group. Foraminiferal molecular phylogeny is largely based on 18S rRNA gene sequence analysis. However, due to highly variable evolutionary rates of substitution in ribosomal genes plus the existence of intragenomic variation at this locus, the relationships between and within foraminiferal classes remain uncertain. We analyze transcriptomic data from 28 species, adding 19 new species to the previously published dataset, including members of the strongly under-represented class Monothalamea. A phylogenomic reconstruction of Rhizaria, rooted with alveolates and stramenopiles, based on 199 genes and 68 species supports the monophyly of Foraminifera and their sister relationship to Polycystinea. The phylogenomic tree of Foraminifera is very similar to the 18S rRNA tree, with the paraphyletic single-chambered monothalamids giving rise to the multi-chambered Tubothalamea and Globothalamea. Within the Monothalamea, our analyses confirm the monophyly of the giant, deep-sea xenophyophores that branch within clade C and indicate the basal position of monothalamous clades D and E. The multi-chambered Globothalamea are monophyletic and comprise the paraphyletic Textulariida and monophyletic Rotaliida. Our phylogenomic analyses support major evolutionary trends of Foraminifera revealed by ribosomal phylogenies and reinforce their current higher-level classification.

Benthic monitoring of oil and gas offshore platforms in the North Sea using environmental DNA metabarcoding.

Since 2010, considerable efforts have been undertaken to monitor the environmental status of European marine waters and ensuring the development of methodological standards for the evaluation of this status. However, the current routine biomonitoring implicates time-consuming and costly manual sorting and morphological identification of benthic macrofauna. Environmental DNA (eDNA) metabarcoding represents an alternative to the traditional monitoring method with very promising results. Here, we tested it further by performing eDNA metabarcoding of benthic eukaryotic communities in the vicinity of two offshore oil and gas platforms in the North Sea. Three different genetic markers (18S V1V2, 18S V9 and COI) were used to assess the environmental pressures induced by the platforms. All markers showed patterns of alpha and beta diversity consistent with morphology-based macrofauna analyses. In particular, the communities' structure inferred from metabarcoding and morphological data significantly changed along distance gradients from the platforms. The impact of the operational discharges was also detected by the variation of biotic index values, AMBI index showing the best correlation between morphological and eDNA data sets. Finally, the sediment physicochemical parameters were used to build a local de novo pressure index that served as benchmark to test the potential of a taxonomy-free approach. Our study demonstrates that metabarcoding approach outperforms morphology-based approach and can be used as a cost and time-saving alternative solution to the traditional morphology-based monitoring in order to monitor more efficiently the impact of industrial activities on marine biodiversity.

ScmR, a Global Regulator of Gene Expression, Quorum Sensing, pH Homeostasis, and Virulence in Burkholderia thailandensis.

The nonpathogenic soil saprophyte Burkholderia thailandensis is a member of the Burkholderia pseudomallei/B. thailandensis/B. mallei group, which also comprises the closely related human pathogens B. pseudomallei and Burkholderia mallei responsible for the melioidosis and glanders diseases, respectively. ScmR, a recently identified LysR-type transcriptional regulator in B. thailandensis, acts as a global transcriptional regulator throughout the stationary phase and modulates the production of a wide range of secondary metabolites, including N-acyl-l-homoserine lactones and 4-hydroxy-3-methyl-2-alkylquinolines and virulence in the Caenorhabditis elegans nematode worm host model, as well as several quorum sensing (QS)-dependent phenotypes. We have investigated the role of ScmR in B. thailandensis strain E264 during the exponential phase. We used RNA sequencing transcriptomic analyses to identify the ScmR regulon, which was compared to the QS-controlled regulon, showing a considerable overlap between the ScmR-regulated genes and those controlled by QS. We characterized several genes modulated by ScmR using quantitative reverse transcription-PCR or mini-CTX-lux transcriptional reporters, including the oxalate biosynthetic gene obc1 required for pH homeostasis, the orphan LuxR-type transcriptional regulator BtaR5-encoding gene, and the bsa (Burkholderia secretion apparatus) type III secretion system genes essential for both B. pseudomallei and B. mallei pathogenicity, as well as the scmR gene itself. We confirmed that the transcription of scmR is under QS control, presumably ensuring fine-tuned modulation of gene expression. Finally, we demonstrated that ScmR influences virulence using the fruit fly model host Drosophila melanogaster We conclude that ScmR represents a central component of theB. thailandensis QS regulatory network.IMPORTANCE Coordination of the expression of genes associated with bacterial virulence and environmental adaptation is often dependent on quorum sensing (QS). The QS circuitry of the nonpathogenic bacterium Burkholderia thailandensis, widely used as a model system for the study of the human pathogen Burkholderia pseudomallei, is complex. We found that the LysR-type transcriptional regulator, ScmR, which is highly conserved and involved in the control of virulence/survival factors in the Burkholderia genus, is a global regulator mediating gene expression through the multiple QS systems coexisting in B. thailandensis, as well as QS independently. We conclude that ScmR represents a key QS modulatory network element, ensuring tight regulation of the transcription of QS-controlled genes, particularly those required for acclimatization to the environment.

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans, Hyphomicrobium denitrificans, and Hyphomicrobium zavarzinii.

Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.

Methylophaga nitratireducenticrescens sp. nov. and Methylophaga frappieri sp. nov., isolated from the biofilm of the methanol-fed denitrification system treating the seawater at the Montreal Biodome.

Two bacterial strains, designated JAM1(T) and JAM7(T), were isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome, Canada. They were affiliated within the genus Methylophaga of the Gammaproteobacteria by analysis of the 16S rRNA gene sequences. Strain JAM1(T) had the capacity to grow under denitrifying conditions by reducing nitrate into nitrite which is unique among the species of the genus Methylophaga. Major fatty acids were C16:1ω7c or ω6c, C16:0 and C18:1ω7c or ω6c. The major ubiquinone was Q8. Both strains required vitamin B12 and Na(+) ions for growth. The genomes of strains JAM1(T) and JAM7(T) have been completely sequenced and showed a DNA G+C content of 44.7 mol% and 47.8 mol%, respectively. Growth occurred at pH 6-11 and at 0.5-8% NaCl. Both genomes contained predicted ORFs encoding the key enzymes of the ribulose monophosphate pathway. Also, operons encoding two nitrate reductases (Nar), two nitric oxide reductases (Nor), one nitrous oxide reductase (Nos) and one truncated nitrite reductase (NirK) were clustered in a 67 kb chromosomal region in strain JAM1(T). No such operons were found in strain JAM7(T). These results supported the affiliation of the two strains as novel species within the genus Methylophaga. The names Methylophaga nitratireducenticrescens sp. nov. for type strain JAM1(T) (=DSM 25689(T)=ATCC BAA-2433(T)) and Methylophaga frappieri sp. nov. for type strain JAM7(T) (=DSM 25690(T)=ATCC BAA-2434(T)) are proposed.

Hyphomicrobium nitrativorans sp. nov., isolated from the biofilm of a methanol-fed denitrification system treating seawater at the Montreal Biodome.

A budding prosthecate bacterial strain, designated NL23(T), was isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome, Canada. Phylogenetic analysis based on 16S rRNA (rRNA) gene sequences showed that the strain was affiliated with the genus Hyphomicrobium of the Alphaproteobacteria and was most closely related to Hyphomicrobium zavarzinii with 99.4 % sequence similarity. Despite this high level of 16S rRNA gene sequence similarity, DNA-DNA hybridization assays showed that strain NL23(T) was only distantly related to H. zavarzinii ZV-622(T) (12 %). Strain NL23(T) grew aerobically, but also had the capacity to grow under denitrifying conditions in the presence of nitrate without nitrite accumulation. Growth occurred at pH 7.0-9.5, with 0-1 % NaCl and at temperatures of 15-35 °C. Major fatty acids were C18 : 1ω7c or ω6c (84.6 %) and C18 : 0 (8.5 %), and major quinones were Q8 (5 %) and Q9 (95 %). The complete genome of the strain was sequenced and showed a DNA G+C content of 63.8 mol%. Genome analysis predicted open reading frames (ORF) encoding the key enzymes of the serine pathway as well as enzymes involved in methylotrophy. Also, ORF encoding a periplasmic nitrate reductase (Nap), a nitrite reductase (Nir), a nitric oxide reductase (Nor) and a nitrous oxide reductase (Nos) were identified. Our results support that strain NL23(T) represents a novel species within the genus Hyphomicrobium, for which the name Hyphomicrobium nitrativorans sp. nov. is proposed. The type strain is NL23(T) ( = ATCC BAA-2476(T) = LMG 27277(T)).

Identification and characterization of novel DIP-STRs from whole-genome sequencing data.

In an attempt to enhance forensic DNA mixture deconvolution several alternative DNA typing approaches have been developed. Among these, DIP-STR compound markers can resolve extremely unbalanced two-source DNA mixtures of same-or-opposite sex donors, up to a 1:1000 minor:major DNA ratio. A forensic set of 10 markers was validated for casework and a larger set of 23 DIP-STRs has proven suitable to biogeographic ancestry inference and for prenatal paternity testing. Yet, to promote the widespread use of this original approach, more markers and multiplex panels need to be developed. To this end, here we describe an extended set of forensic DIP-STRs identified using currently available whole-genome sequencing datasets. Complete lists of Indels and STRs were obtained from reported frequencies of genetic variants of 76,156 genomes. About 3000 identified DIP-STRs candidates were shorter than 200 bp and 500 showed high haplotype variability estimated using the genotypes of individuals homozygous for the DIP or the STR. Here, we present 23 additional DIP-STRs validated for sensitivity, specificity and Swiss population variability. Finally, a set of 30 markers comprising seven previously validated ones is proposed for the prospective development of a forensic DIP-STR multiplex panel.

Draft Genome Sequence of a Mixed-Serogroup W/Y Invasive Neisseria meningitidis Strain.

We report the genome of a Neisseria meningitidis strain (GE-156) that was isolated in Switzerland from a patient diagnosed with bacteremia. The strain belongs to a rare mixed serogroup W/Y and sequence type 11847 (clonal complex 167), as revealed by both routine laboratory examination and genomic sequencing.

Complete genome sequences of Methylophaga sp. strain JAM1 and Methylophaga sp. strain JAM7.

Methylophaga sp. strains JAM1 and JAM7 have been isolated from a denitrification system. Strain JAM1 was the first Methylophaga strain reported to be able to grow under denitrifying conditions. Here, we report the complete genome sequences of the two strains, which allowed prediction of gene clusters involved in denitrification in strain JAM1.

Effect of bacterial DNA enrichment on detection and quantification of bacteria in an infected tissue model by metagenomic next-generation sequencing.

Before implementing metagenomic next-generation sequencing (mNGS) in the routine diagnostic laboratory, several challenges need to be resolved. To address strengths and limitations of mNGS in bacterial detection and quantification in samples with overwhelming host DNA abundance, we used the pig muscle tissue spiked with a home-made bacterial mock community, consisting of four species from different phyla. From the spiked tissue, we extracted DNA using: (i) a procedure based on mechanical/chemical lysis (no bacterial DNA enrichment); (ii) the Ultra-Deep Microbiome Prep (Molzym) kit for bacterial DNA enrichment; and (iii) the same enrichment kit but replacing the original proteinase K treatment for tissue solubilization by a collagenases/thermolysin digestion and cell filtration. Following mNGS, we determined bacterial: 'host' read ratios and taxonomic abundance profiles. We calculated the load of each mock-community member by combining its read counts with read counts and microscopically-determined cell counts of other co-spiked bacteria. In unenriched samples, bacterial quantification and taxonomic profiling were fairly accurate but at the expense of the sensitivity of detection. The removal of 'host' DNA by the modified enrichment protocol substantially improved bacterial detection in comparison to the other two extraction procedures and generated less distorted taxonomic profiles as compared to the original enrichment protocol.

Gradient diffusion susceptibility testing for Neisseria gonorrhoeae: an accurate alternative to agar dilution in high-MIC strains?

INTRODUCTION: The correlation of antimicrobial susceptibility testing (AST) between agar dilution and gradient diffusion for Neisseria gonorrhoeae is not well established, especially in strains with high MICs. AIM: The objective of this study was to evaluate the accuracy of gradient diffusion for N. gonorrhoeae . METHODS: Fifty strains of N. gonorrhoeae , all tested by the agar dilution method according to CLSI methods and confirmed to be genetically distinct using molecular typing (NG-MAST), were selected. Isolates with high MICs were targeted. Gradient diffusion was performed for ceftriaxone (CRO), cefixime (CFX), azithromycin (AZT), tetracycline (TET) and fosfomycin (FOS) using two different commercial antimicrobial strips on different culture media (a non-commercial GC agar base with 1 % defined growth supplement and two commercial media). The performance of agar gradient diffusion was assessed based on accuracy, using essential and category agreements (EA and CA). RESULTS: Essential and categorical agreement were over 90 % for CRO, CFX and AZT on the two commercial agar media tested. Category disagreements were seen for CFX and AZT, mostly just very major errors. For TET, EA ranged from 80 to 96 % and CA ranged from 38 to 76 %, most of the misclassifications being minor errors. Finally, EA for FOS ranged between 80 and 98 %. CONCLUSION: Gradient diffusion is an accurate and acceptable alternative for CRO, CFX and AZT. Caution is advised when MICs are reported by gradient diffusion approach breakpoints because of the possibility of very major errors. The use of gradient diffusion is limited for TET because of the high rate of minor errors.

Dynamics of a methanol-fed marine denitrifying biofilm: 2-impact of environmental changes on the microbial community.

BACKGROUND: The biofilm of a methanol-fed, marine denitrification system is composed of a multi-species microbial community, among which Hyphomicrobium nitrativorans and Methylophaga nitratireducenticrescens are the principal bacteria involved in the denitrifying activities. To assess its resilience to environmental changes, the biofilm was cultivated in artificial seawater (ASW) under anoxic conditions and exposed to a range of specific environmental conditions. We previously reported the impact of these changes on the denitrifying activities and the co-occurrence of H. nitrativorans strain NL23 and M. nitratireducenticrescens in the biofilm cultures. Here, we report the impact of these changes on the dynamics of the overall microbial community of the denitrifying biofilm. METHODS: The original biofilm (OB) taken from the denitrification system was cultivated in ASW under anoxic conditions with a range of NaCl concentrations, and with four combinations of nitrate/methanol concentrations and temperatures. The OB was also cultivated in the commercial Instant Ocean seawater (IO). The bacterial diversity of the biofilm cultures and the OB was determined by 16S ribosomal RNA gene sequences. Culture approach was used to isolate other denitrifying bacteria from the biofilm cultures. The metatranscriptomes of selected biofilm cultures were derived, along with the transcriptomes of planktonic pure cultures of H. nitrativorans strain NL23 and M. nitratireducenticrescens strain GP59. RESULTS: High proportions of M. nitratireducenticrescens occurred in the biofilm cultures. H. nitrativorans strain NL23 was found in high proportion in the OB, but was absent in the biofilm cultures cultivated in the ASW medium at 2.75% NaCl. It was found however in low proportions in the biofilm cultures cultivated in the ASW medium at 0-1% NaCl and in the IO biofilm cultures. Denitrifying bacterial isolates affiliated to Marinobacter spp. and Paracoccus spp. were isolated. Up regulation of the denitrification genes of strains GP59 and NL23 occurred in the biofilm cultures compared to the planktonic pure cultures. Denitrifying bacteria affiliated to the Stappia spp. were metabolically active in the biofilm cultures. CONCLUSIONS: These results illustrate the dynamics of the microbial community in the denitrifying biofilm cultures in adapting to different environmental conditions. The NaCl concentration is an important factor affecting the microbial community in the biofilm cultures. Up regulation of the denitrification genes of M. nitratireducenticrescens strain GP59 and H. nitrativorans strain NL23 in the biofilm cultures suggests different mechanisms of regulation of the denitrification pathway in the biofilm. Other denitrifying heterotrophic bacteria are present in low proportions, suggesting that the biofilm has the potential to adapt to heterotrophic, non-methylotrophic environments.

Strain-level genetic diversity of Methylophaga nitratireducenticrescens confers plasticity to denitrification capacity in a methylotrophic marine denitrifying biofilm.

BACKGROUND: The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO[Formula: see text]) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59. METHODS: The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO[Formula: see text], nitrite (NO[Formula: see text]) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures. RESULTS: During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2-3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO[Formula: see text] under oxic culture conditions, and has a 24-h lag time before growth and NO[Formula: see text] reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO[Formula: see text] as it carries a gene encoding a NirK-type NO[Formula: see text] reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process. DISCUSSION: These results reinforce the unique trait of the species M. nitratireducenticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor.

Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1.

BACKGROUND: Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate ([Formula: see text]) to nitrite ([Formula: see text]). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. Here we characterized further the denitrifying activities of M. nitratireducenticrescens JAM1. METHODS: Series of oxic and anoxic cultures of strain JAM1 were performed with N2O, [Formula: see text] or sodium nitroprusside, and growth and N2O, [Formula: see text], [Formula: see text] and N2 concentrations were measured. Ammonium ([Formula: see text])-free cultures were also tested to assess the dynamics of N2O, [Formula: see text] and [Formula: see text]. Isotopic labeling of N2O was performed in 15NH4+-amended cultures. Cultures with the JAM1ΔnarG1narG2 double mutant were performed to assess the involvement of the Nar systems on N2O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochrome bc-type nitric oxide reductase (cnorB1 and cnorB2) and nitrous oxide reductase (nosZ), and also nnrS and norR that encode NO-sensitive regulators. RESULTS: Strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory [Formula: see text] reductase, [Formula: see text]-amended cultures produce N2O, representing up to 6% of the N-input. [Formula: see text] was shown to be the key intermediate of this production process. Upregulation in the expression of cnorB1, cnorB2, nnrS and norR during the growth and the N2O accumulation phases suggests NO production in strain JAM1 cultures. DISCUSSION: By showing that all the three denitrification reductases are active, this demonstrates that M. nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such an incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of the denitrification reactor from which it originates. The production of N2O in strain JAM1 did not involve Nar, contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using [Formula: see text] and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.

Comparison of sequential multiplex PCR, sequetyping and whole genome sequencing for serotyping of Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the major causes of pneumonia, meningitis and other pneumococcal infections in young children and elders. Determination of circulating S. pneumoniae serotypes is an essential service by public health laboratories for the monitoring of putative serotype replacement following the introduction of pneumococcal conjugate vaccines (PCVs) and of the efficacy of the immunization program. The Quellung method remains the gold standard for typing S. pneumoniae. Although this method is very effective, it is also costly, time consuming and not totally reliable due to its subjective nature. The objectives of this study were to test and evaluate the efficiency of 3 different molecular methods compared to the Quellung method. Sequential multiplex PCR, sequetyping and whole genome sequencing (WGS) were chosen and tested using a set of diverse S. pneumoniae. One-hundred and eighteen isolates covering 83 serotypes were subjected to multiplex PCR and sequetyping while 88 isolates covering 53 serotypes were subjected to WGS. Sequential multiplex PCR allowed the identification of a significant proportion (49%) of serotypes at the serogroup or subset level but only 27% were identified at the serotype level. Using WGS, 55% to 60% of isolates were identified at the serotype level depending on the analysis strategy used. Finally, sequetyping demonstrated the lowest performance, with 17% of misidentified serotypes. The use of Jin cpsB database instead of the GenBank database slightly improved results but did not significantly impact the efficiency of sequetyping. Although none of these molecular methods may currently replace the Quellung method, WGS remains the most promising molecular pneumococcal serotyping method.

Importance of the Two Dissimilatory (Nar) Nitrate Reductases in the Growth and Nitrate Reduction of the Methylotrophic Marine Bacterium Methylophaga nitratireducenticrescens JAM1.

Methylophaga nitratireducenticrescens JAM1 is the only reported Methylophaga species capable of growing under anaerobic conditions with nitrate as electron acceptor. Its genome encodes a truncated denitrification pathway, which includes two nitrate reductases, Nar1 and Nar2; two nitric oxide reductases, Nor1 and Nor2; and one nitrous oxide reductase, Nos; but no nitrite reductase (NirK or NirS). The transcriptome of strain JAM1 cultivated with nitrate and methanol under anaerobic conditions showed the genes for these enzymes were all expressed. We investigated the importance of Nar1 and Nar2 by knocking out narG1, narG2 or both genes. Measurement of the specific growth rate and the specific nitrate reduction rate of the knockout mutants JAM1ΔnarG1 (Nar1) and JAM1ΔnarG2 (Nar2) clearly demonstrated that both Nar systems contributed to the growth of strain JAM1 under anaerobic conditions, but at different levels. The JAM1ΔnarG1 mutant exhibited an important decrease in the nitrate reduction rate that consequently impaired its growth under anaerobic conditions. In JAM1ΔnarG2, the mutation induced a 20-h lag period before nitrate reduction occurred at specific rate similar to that of strain JAM1. The disruption of narG1 did not affect the expression of narG2. However, the expression of the Nar1 system was highly downregulated in the presence of oxygen with the JAM1ΔnarG2 mutant. These results indicated that Nar1 is the major nitrate reductase in strain JAM1 but Nar2 appears to regulate the expression of Nar1.

Complete Genome Sequence of Hyphomicrobium nitrativorans Strain NL23, a Denitrifying Bacterium Isolated from Biofilm of a Methanol-Fed Denitrification System Treating Seawater at the Montreal Biodome.

Hyphomicrobium nitrativorans strain NL23 has been isolated from the biofilm of a denitrification system treating seawater. This strain has the capacity to denitrify using methanol as a carbon source. Here, we report the complete genome sequence of this strain in an effort to increase understanding of the function of this bacterium within the biofilm.