Phase Variation During Host Colonization and Invasion by Campylobacter jejuni and Other Campylobacter Species.

Phase variation (PV) is a phenomenon common to a variety of bacterial species for niche adaption and survival in challenging environments. Among Campylobacter species, PV depends on the presence of intergenic and intragenic hypermutable G/C homopolymeric tracts. The presence of phase-variable genes is of especial interest for species that cause foodborne or zoonotic infections in humans. PV influences the formation and the structure of the lipooligosaccharide, flagella, and capsule in Campylobacter species. PV of components of these molecules is potentially important during invasion of host tissues, spread within hosts and transmission between hosts. Motility is a critical phenotype that is potentially modulated by PV. Variation in the status of the phase-variable genes has been observed to occur during colonization in chickens and mouse infection models. Interestingly, PV is also involved in bacterial survival of attack by bacteriophages even during chicken colonization. This review aims to explore and discuss observations of PV during model and natural infections by Campylobacter species and how PV may affect strategies for fighting infections by this foodborne pathogen.

Assessment of fHbp Expression Level by Reverse Transcriptase Quantitative PCR and Promoter Sequence Analysis.

Reverse transcriptase quantitative PCR (RT qPCR) is widely used for assessing the levels of expression of specific genes in various organisms. Here we describe a RT qPCR assay designed to determine the level of expression of fHbp in multiple isolates of Neisseria meningitidis. The level of expression is measured by a two-step qPCR and is associated with a promoter region analysis.

Clustered intergenic region sequences as predictors of factor H Binding Protein expression patterns and for assessing Neisseria meningitidis strain coverage by meningococcal vaccines.

Factor H binding protein (fHbp) is a major protective antigen in 4C-MenB (Bexsero®) and Trumenba®, two serogroup B meningococcal vaccines, wherein expression level is a determinant of protection. Examination of promoter-containing intergenic region (IGR) sequences indicated that nine fHbp IGR alleles covered 92% of 1,032 invasive meningococcal strains with variant 1 fHbp alleles. Relative expression values for fHbp were determined for 79 meningococcal isolates covering ten IGR alleles by quantitative reverse transcriptase polymerase chain reaction (qRT PCR). Derivation of expression clusters of IGR sequences by linear regression identified five expression clusters with five nucleotides and one insertion showing statistically associations with differences in expression level. Sequence analysis of 273 isolates examined by the Meningococcal Antigen Typing Scheme, a sandwich ELISA, found that coverage depended on the IGR expression cluster and vaccine peptide homology combination. Specific fHbp peptide-IGR expression cluster combinations were designated as 'at risk' for coverage by 4C-MenB and were detected in multiple invasive meningococcal disease cases confirmed by PCR alone and occurring in partially-vaccinated infants. We conclude that sequence-based analysis of IGR sequences is informative for assessing protein expression and has utility for culture-independent assessments of strain coverage by protein-based vaccines.

Rise in Group W Meningococcal Carriage in University Students, United Kingdom.

MenACWY conjugate vaccination was recently introduced in the United Kingdom for adolescents and young adults to reduce disease from infection by Neisseria meningitidis group W. We conducted a cross-sectional meningococcal carriage study in first-year UK university students. Despite 71% MenACWY vaccine coverage, carriage of group W increased substantially.

Draft genome of Gemmata massiliana sp. nov, a water-borne Planctomycetes species exhibiting two variants.

Gemmata massiliana is a new Planctomycetes bacterium isolated from a hospital water network in France, using a new culture medium. It is an aerobic microorganism with optimal growth at pH 8, at 30 °C and salinity ≤ 1.25 % NaCl. G. massiliana is resistant to ß-lactam antibiotics, due to lack of peptidoglycan in its cell wall.G. massiliana shares a 97 % 16S rRNA gene sequence similarity with the nearest species, Gemmata obscuriglobus; and 99 % similarity with unnamed soil isolates. Its 9,249,437-bp genome consists in one chromosome and no detectable plasmid and has a 64.07 % G + C content, 32.94 % of genes encoding for hypothetical proteins. The genome contains an incomplete 19.6-kb phage sequence, 26 CRISPRs, 3 CAS and 15 clusters of secondary metabolites. G. massiliana genome increases knowledge of a poorly known world of bacteria.

Molecular diversity of the Planctomycetes in the human gut microbiota in France and Senegal.

Until now, Planctomycetes bacteria were considered as environmental organisms. Nevertheless, some studies detected Planctomycetes DNA from human gut. We therefore explored the human gut Planctomycetes content. Planctomycetes-specific PCR primers were designed to amplify a 240-bp 16S rRNA gene fragment in human stool specimens from individuals in France and in Senegal and from endocarditis patients receiving antibiotics in France. PCR products were then cloned and sequenced. PCR detection revealed a significantly higher prevalence (1.8% vs 0.4%, p = 0.05) and higher diversity (62 vs 6 phylotypes, p = 0.02) of Planctomycetes 16S rRNA gene in stool specimens collected in Senegal than in France. Also, stool specimens from endocarditis patients exhibited non-significantly higher prevalence (0.6% vs 0.4%) and the ratio of phylotypes by positive patient (3 vs 1.5) than those collected from untreated French individuals. Gemmata sp. related sequences were found in 6/12 individuals. Planctomycetes organisms are a part of the human digestive tract microbiota. Their diversity varied by environment including the geographical origin of the individual and antibiotics treatment.

Peptidoglycan: a post-genomic analysis.

BACKGROUND: To derive post-genomic, neutral insight into the peptidoglycan (PG) distribution among organisms, we mined 1,644 genomes listed in the Carbohydrate-Active Enzymes database for the presence of a minimal 3-gene set that is necessary for PG metabolism. This gene set consists of one gene from the glycosyltransferase family GT28, one from family GT51 and at least one gene belonging to one of five glycoside hydrolase families (GH23, GH73, GH102, GH103 and GH104). RESULTS: None of the 103 Viruses or 101 Archaea examined possessed the minimal 3-gene set, but this set was detected in 1/42 of the Eukarya members (Micromonas sp., coding for GT28, GT51 and GH103) and in 1,260/1,398 (90.1%) of Bacteria, with a 100% positive predictive value for the presence of PG. Pearson correlation test showed that GT51 family genes were significantly associated with PG with a value of 0.963 and a p value less than 10(-3). This result was confirmed by a phylogenetic comparative analysis showing that the GT51-encoding gene was significantly associated with PG with a Pagel's score of 60 and 51 (percentage of error close to 0%). Phylogenetic analysis indicated that the GT51 gene history comprised eight loss and one gain events, and suggested a dynamic on-going process. CONCLUSIONS: Genome analysis is a neutral approach to explore prospectively the presence of PG in uncultured, sequenced organisms with high predictive values.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of environmental organisms: the Planctomycetes paradigm.

We have developed a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based identification technique for Planctomycetes organisms, which are used here as bacteria of suitable diversity at genus and species level for testing resolution of the method. Planctomyces maris ATCC 29201, Planctomyces brasiliensis ATCC 49424(T) , P. brasiliensis ATCC 49425, Planctomyces limnophilus ATCC 43296(T) , Blastopirellula marina ATCC 49069(T) , Rhodopirellula baltica DSM 10527(T) and Gemmata obscuriglobus DSM 5831(T) were cultured on half-strength marine broth and agar, or alternatively on caulobacter broth and agar. The resulting pellets of organisms (liquid) or colonies (solid agar) were directly applied to a MALDI-TOF plate. This yielded a reproducible, unique protein profiles comprising 23-39 peaks ranging in size from 2403 to 12 091 Da. These peaks were unambiguously distinguished from any of the 3038 bacterial spectra in the Brüker database. Matrix-assisted laser desorption/ionization time-of-flight patterns were similar for isolates grown in solid and in liquid medium, albeit the patterns from solid growth were more easily interpretable. After the incorporation of the herein determined profiles into the Brüker database, Planctomycetes isolates were blindly identified within 10 min, with an identification score in the range of 1.8 to 2.3. Matrix-assisted laser desorption/ionization time-of-flight-based clustering of these Planctomycetes organisms was consistent with 16S rDNA-based phylogeny. However, the incorporation of additional non-Planctomycetes MALDI-TOF profiles in the analysis resulted in inconsequential clustering. In conclusion, MALDI-TOF protein profiling is a new approach for the rapid and accurate identification of cultured environmental organisms, as illustrated in this study through the analysis of Planctomycetes.

Genotyping of Mycobacterium avium complex organisms using multispacer sequence typing.

Mycobacterium avium complex (MAC) currently comprises eight species of environmental and animal-associated, slowly-growing mycobacteria: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense , Mycobacterium bouchedurhonense, Mycobacterium marseillense and Mycobacterium timonense. In humans, MAC organisms are responsible for opportunistic infections whose unique epidemiology remains poorly understood, in part due to the lack of a genotyping method applicable to all eight MAC species. In this study we developed multispacer sequence typing (MST), a sequencing-based method, for the genotyping of MAC organisms. An alignment of the genome sequence of M. avium subsp. hominissuis strain 104 and M. avium subsp. paratuberculosis strain K-10 revealed 621 intergenic spacers <1000 bp. From these, 16 spacers were selected that ranged from 300 to 800 bp and contained a number of variable bases, <50 within each of the 16 spacers. Four spacers were successfully PCR-amplified and sequenced in 11 reference strains. Combining the sequence of these four spacers in 106 MAC organisms, including 83 M. avium, 11 M. intracellulare , six M. chimaera, two M. colombiense and one each of M. arosiense, M. bouchedurhonense, M. marseillense and M. timonense, yielded a total of 45 spacer types, with an index of discrimination of 0.94. Each spacer type was specific for a species and certain spacer types were specific for subspecies of M. avium. MST is a new method for genotyping of organisms belonging to any one of the eight MAC species tested in this study.

Mycobacterium marseillense sp. nov., Mycobacterium timonense sp. nov. and Mycobacterium bouchedurhonense sp. nov., members of the Mycobacterium avium complex.

An rpoB sequence-based evaluation of 100 Mycobacterium avium complex (MAC) clinical isolates led to the identification of five respiratory tract isolates that were potential representatives of three novel MAC species. Distinctive phenotypic features of isolates 62863 and 5356591(T) included a pseudomycelium morphology and both esterase and acid phosphatase activities. These two isolates exhibited sequence similarities of 99.8 % for the 16S rRNA gene, 86.3 and 86.1 % for 16S-23S rRNA gene internal transcribed spacer (ITS-1) sequence, 96.7 and 97.8 % for rpoB and 97.6 and 97.4 % for hsp65, respectively, with the type strain of Mycobacterium chimaera, the most closely related species. Isolates 3256799 and 5351974(T) lacked alpha-mannosidase and beta-glucosidase activities. They exhibited sequence similarities of 99.6 % for the 16S rRNA gene, 90.1 and 90.4 % for ITS-1, 97.8 % for rpoB and 98.0 and 98.1 % for hsp65, respectively, with the type strain of M. chimaera, the most closely related species. Isolate 4355387(T) lacked urease and alpha-glucosidase activities, but it exhibited valine arylamidase, cystine arylamidase and acid phosphatase activities. It had sequence similarities of 99.3 % for the 16S rRNA gene, 51.8 % for ITS-1, 97.1 % for rpoB and 97.8 % for hsp65 with the type strain of Mycobacterium colombiense, the most closely related species. A phylogenetic tree based on concatenated 16S rRNA gene, ITS-1, rpoB and hsp65 sequences showed the uniqueness of these five isolates as representatives of three novel species, with bootstrap values >/=95 % in all nodes. On the basis of these phenotypic and genetic characteristics, these five isolates are proposed as representatives of three novel MAC species: Mycobacterium marseillense sp. nov., with strain 5356591(T) (=CCUG 56325(T) =CIP 109828(T) =CSUR P30(T)) as the type strain; Mycobacterium timonense sp. nov., with strain 5351974(T) (=CCUG 56329(T) =CIP 109830(T) =CSUR P32(T)) as the type strain; and Mycobacterium bouchedurhonense sp. nov., with strain 4355387(T) (=CCUG 56331(T) =CIP 109827(T) =CSUR P34(T)) as the type strain.