Campylobacter portucalensis sp. nov., a new species of Campylobacter isolated from the preputial mucosa of bulls.

A new species of the Campylobacter genus is described, isolated from the preputial mucosa of bulls (Bos taurus). The five isolates obtained exhibit characteristics of Campylobacter, being Gram-negative non-motile straight rods, oxidase positive, catalase negative and microaerophilic. Phenotypic characteristics and nucleotide sequence analysis of 16S rRNA and hsp60 genes demonstrated that these isolates belong to a novel species within the genus Campylobacter. Based on hsp60 gene phylogenetic analysis, the most related species are C. ureolyticus, C. blaseri and C. corcagiensis. The whole genome sequence analysis of isolate FMV-PI01 revealed that the average nucleotide identity with other Campylobacter species was less than 75%, which is far below the cut-off for isolates of the same species. However, whole genome sequence analysis identified coding sequences highly homologous with other Campylobacter spp. These included several virulence factor coding genes related with host cell adhesion and invasion, transporters involved in resistance to antimicrobials, and a type IV secretion system (T4SS), containing virB2-virB11/virD4 genes, highly homologous to the C. fetus subsp. venerealis. The genomic G+C content of isolate FMV-PI01 was 28.3%, which is one of the lowest values reported for species of the genus Campylobacter. For this species the name Campylobacter portucalensis sp. nov. is proposed, with FMV-PI01 (= LMG 31504, = CCUG 73856) as the type strain.

Mycobacterium abscessus infection in the stomach of patients with various gastric symptoms.

Development of gastric diseases such as gastritis, peptic ulcer and gastric cancer is often associated with several biotic and abiotic factors. Helicobacter pylori infection is such a well-known biotic factor. However, not all H. pylori-infected individuals develop gastric diseases and not all individuals with gastric diseases are infected with H. pylori. Therefore, it is possible that other gastric bacteria may contribute to the formation and progression of gastric disease. The aim of this study was to isolate prevalent gastric bacteria under microaerobic condition and identify them by 16S rRNA gene sequence analysis. Analysis of gastric biopsies showed infection of Mycobacterium abscessus (phylum Actinobacteria) to be highly prevalent in the stomachs of subjects included. Our data show that of 129 (67 male and 62 female) patients with gastric symptoms, 96 (51 male and 45 female) showed the presence of M. abscessus in stomach tissues. Infection of M. abscessus in gastric epithelium was further confirmed by imaging with acid fast staining, immunohistochemistry and immunofluorescence. Our imaging data strongly suggested that M. abscessus is an intracellular colonizer residing inside the gastric epithelial cells rather than in macrophages. Additionally, phylogenetic analysis of the mycobacterial hsp65 gene showed that the nearest match to the M. abscessus strains isolated from our study subjects is the M. abscessus strain ATCC 19977. Surprisingly, the subjects studied, the prevalence of M. abscessus infection in stomach is even higher than the prevalence of H. pylori infection. This, to the best of our knowledge, is the first study showing the colonization of M. abscessus in human gastric mucosa among patients with various gastric symptoms. This study could provide usher in a new opportunity to understand the role of less studied gastric bacteria in the development of gastric diseases.

Manifestation of anaplasmosis as cerebral infarction: a case report.

BACKGROUND: Human granulocytic anaplasmosis is a tick-borne zoonotic disease caused by Anaplasma phagocytophilum, an obligate intracellular granulocytotropic bacterium. CASE PRESENTATION: A 70-year-old female patient was admitted with the clinical signs of fever and an altered state of consciousness 1 week after experiencing a tick bite while planting lawn grass. Magnetic resonance imaging, performed at the time of admission, indicated cerebral infarction in the left basal ganglia, whereas increasing immunofluorescence assay antibody titers for A. phagocytophilum were also documented. A. phagocytophilum was identified using groEL and ankA targeted polymerase chain reaction and sequencing. Because of severe thrombocytopenia, only doxycycline was administered, without any antiplatelet agents. Subsequently, the symptoms improved without any focal neurologic sequela. CONCLUSION: This is the first reported case of cerebral infarction occurrence in an anaplasmosis patient.

GroEL PCR- RFLP - An efficient tool to discriminate closely related pathogenic Vibrio species.

Vibrio sp. are autochthonous to marine and estuarine waters. Several species of Vibrio are pathogens. It is of utmost importance to detect and discriminate the Vibrio sp. that are often involved in food and water borne infections. Since 16S rRNA based identification has limited utility in differentiating the closely related pathogenic species from non pathogenic species, we have evaluated the discriminatory power of groEL PCR-RFLP for identification of closely related Vibrio sp. Accordingly, in the current study, the efficiency of groEL PCR- RFLP for detection and accurate differentiation of known pathogens among Vibrio sp. such as V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. fluvialis, V. alginolyticus, V. anguillarum was evaluated. PCR amplified groEL gene fragment of each Vibrio sp. was digested separately using 5 restriction enzymes viz. Hha1, Rsa1, Alu1, Dde1 and Mbo1. The accuracy of the method was further validated by insilico restriction analysis of multiple strains of each species using NEBcutter. The method proved to be efficient for detection and differentiation of Vibrio species under study. Phylogenetic analysis also revealed groEL gene to be a better phylogenetic marker for Vibrio compared to 16S rRNA. Hence, the method can be employed for accurate detection of Vibrio sp. including the closely related species.

Characterization of the Toxoplasma gondii hsp60 gene sequences from different hosts and geographical locations.

The intracellular protozoan Toxoplasma gondii is one of the most successful parasites, with the ability to invade all warm-blooded animals, including humans. T. gondii heat shock protein 60 (TgHSP60) plays an important role in intracellular survival and in the differentiation of the parasite, and is also recognized as being associated with its virulence. In the present study, we examined sequence variation in the hsp60 coding region among five T. gondii isolates from different hosts and geographical regions, which were compared with the corresponding sequences of strains ME49, 76K, and GT1 available in the ToxoDB databases. The length of the T. gondii hsp60 sequence was 1728 bp for all strains, and the A+T content ranged from 41.96 to 42.13%. The sequence alignment of the 8 T. gondii strains identified 20 variable positions (0-1.44%) and showed 1.16% overall sequence variation, suggesting a relatively considerable sequence diversity. Phylogenetic analysis of hsp60 sequences using Bayesian inference and maximum parsimony differentiated the two major clonal lineage types into their respective clusters, and thus separated atypical strains from classical genotypes. The results of the present study suggested that the coding region of the hsp60 gene may represent a novel genetic marker for intraspecies phylogenetic analyses of T. gondii.

Frequency and magnitude of Chlamydia trachomatis elementary body- and heat shock protein 60-stimulated interferon gamma responses in peripheral blood mononuclear cells and endometrial biopsy samples from women with high exposure to infection.

BACKGROUND: Cellular immune responses characterized by interferon gamma (IFN-gamma) production enhance clearance and confer protective immunity against Chlamydia trachomatis infection but have not been simultaneously investigated in systemic and mucosal compartments. METHODS: With use of the IFN-gamma enzyme-linked immunosorbent spot assay, we investigated immune responses to Chlamydia elementary body (EB) and 3 genotypically variant heat shock protein 60 (CHSP60) antigens using peripheral blood mononuclear cells and endometrial mononuclear cells obtained from a female sex worker cohort with high levels of exposure to C. trachomatis. RESULTS: Although we observed a marginally higher frequency of IFN-gamma responses to EB in peripheral blood mononuclear cells, compared with the frequency in endometrial mononuclear cells, the magnitudes of systemic and mucosal responses were similar except for preferential targeting of CHSP60 type 2 by endometrial mononuclear cells. Systemic and mucosal responses were highly correlated for EB and CHSP60 types 1 and 2 but not type 3. The frequency and magnitude of systemic responses specific for EB and CHSP60 type 1 were greater for CD4+ T cells than they were for CD8+ T cells, whereas preferential targeting by CHSP60 types 2 and 3 was undetectable. IFN-gamma response to CHSP60 type 1 by peripheral blood mononuclear cells was inversely correlated with systemic antibody titers to CHSP60 type 1. CONCLUSION: Systemic and mucosal IFN-gamma responses are correlated, with preferential systemic targeting of CD4+ T cells. Furthermore, CHSP60 type 1 response is largely CD4+ T cell mediated and follows discrete T helper 1 and T helper 2 pathways.

Species-level identification of clinical staphylococcal isolates based on polymerase chain reaction--restriction fragment length polymorphism analysis of a partial groEL gene sequence.

A pair of degenerate primers that amplified, by polymerase chain reaction (PCR), a partial groEL gene sequence (550 bp) was used for the identification of the 12 most common human staphylococcal pathogens. The amplified products were digested by AluI endonuclease, and distinctive PCR restriction fragment length polymorphism (RFLP) patterns for reference strains were obtained. This protocol was validated by the identification of 89 clinical staphylococcal isolates, and the results were compared with those obtained by the reference biochemical identification, showing 100% concordant results. Two species, Staphylococcus aureus and Staphylococcus lugdunensis, showed intraspecies polymorphisms on their PCR-RFLP patterns. All strains were also identified using the API Staph ID test (bioMérieux, Durham, NC) and the MicroScan WalkAway automated system (Dade Behring, West Sacramento, CA). When 17 Staphylococcus isolates were tested in a blind experiment by the PCR-RFLP of the groEL gene method, all strains were also correctly identified. We propose the PCR-RFLP of the groEL gene with AluI as a reliable and reproducible method for identification of Staphylococcus spp.

Evolution of assisted protein folding: the distribution of the main chaperoning systems within the phylogenetic domain archaea.

Newly made proteins must achieve a functional shape, the native configuration, before they can play their physiological roles in the cell. Proteins must also travel to the locale (e.g., the mitochondrion) in the cell where their functions are required. In these processes of folding into the native configuration and translocation to the place of work, proteins may be assisted by molecules called molecular chaperones. Stressors can unfold (denature) proteins, and genetic defects can cause misfolding and, in addition, both abnormalities can lead to polypeptide aggregation. Chaperones play a role in assisting refolding of partially denatured or misfolded proteins, thus preventing aggregation. Clearly, molecular chaperones are key cell components under normal, physiological circumstances, as well as in potentially harmful situations resulting from environmental or inherited factors. Hence, molecular chaperones constitute attractive targets for a variety of efforts aiming at improving the cell's performance, particularly under stress, to prevent disease, or at least to slow down its progression and to contain the deleterious effects of stress. In our efforts in this direction, we have undertaken to investigate the chaperoning systems of cells belonging to the phylogenetic domain Archaea. The findings reported here pertain to the distribution of the molecular chaperone machine, the chaperonins, and the prefoldins, among archaea. The genes hsp70(dnaK), hsp40(dnaJ), and grpE encoding the components of the molecular chaperone machine were present only in some archeaeal species: this contrasts with bacteria and eucarya, which do have the genes with no known exception. The group I, or bacterial, chaperonin-genes groEL and groES occured in the genomes of Methanosarcina species but were not found in any of the other archaea whose genomes have been sequenced. While all the archaea studied had between one and three chaperonins of group II (thermosome subunits), Methanosarcina acetivorans was exceptional since it had five of these chaperonins. This is the largest number of group II chaperonins ever found in a prokaryote. Furthermore, two of the M. acetivorans chaperonins were different from, albeit related to, the other known archaeal and eucaryal chaperonins of group II. Prefoldins were found in all archaea examined. Overall, the results provide clues to the evolution of the chaperoning systems, which must have played a critical role in survival since life started. Also, the data suggest new avenues of research for elucidating the evolution of assisted protein folding and for uncovering roles and interactions not yet described for these molecules.

Extensive profiling of a complex microbial community by high-throughput sequencing.

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.

Heat shock response and groEL sequence of Bartonella henselae and Bartonella quintana.

Transmission of Bartonella species from ectoparasites to the mammalian host involves adaptation to thermal and other forms of stress. In order to better understand this process, the heat shock response of Bartonella henselae and Bartonella quintana was studied. Cellular proteins synthesized after shift to higher temperatures were intrinsically labelled with [25S]methionine and analysed by gel electrophoresis and fluorography. The apparent molecular masses of three of the major heat shock proteins produced by the two Bartonella species were virtually identical, migrating at 70, 60 and 10 kDa. A fourth major heat shock protein was larger in B. quintana (20 kDa) than in B. henselae (17 kDa). The maximum heat shock response in B. quintana and B. henselae was observed at 39 degrees C and 42 degrees C, respectively. The groEL genes of both Bartonella species were amplified, sequenced and compared to other known groEL genes. The phylogenetic tree based on the groEL alignment places B. quintana and B. henselae in a monophyletic group with Bartonella bacilliformis. The deduced amino acid sequences of Bartonella GroEL homologues contain signature sequences that are uniquely shared by members of the Gram-negative alpha-purple subdivision of bacteria, which live within eukaryotic cells. Recombinant His6-GroEL fusion proteins were expressed in Escherichia coli to generate specific rabbit antisera. The GroEL antisera were used to confirm the identity of the 60 kDa Bartonella heat shock protein. These studies provide a foundation for evaluating the role of the heat shock response in the pathogenesis of Bartonella infection.

Ehrlichia sennetsu groE operon and antigenic properties of the GroEL homolog.

A clone expressing an immunoreactive 55-kilodalton (kDa) protein of Ehrlichia sennetsu, the causative agent of human Sennetsu ehrlichiosis, was isolated from a gene library of this organism. Sequence analysis of the DNA insert revealed two open reading frames, encoding proteins of 10,620 and 58,225 kDa, respectively. These deduced amino acid sequences were homologous to those of the GroES and GroEL heat shock proteins (HSP) of other bacteria, respectively. Phylogenetic trees based on GroES and GroEL homologs of several bacteria including E. sennetsu showed a relationship similar to that based on 16S rRNA gene sequences. The recombinant and native 55-kDa proteins of E. sennetsu, GroEL homolog, reacted with a monoclonal antibody (SPA807) which recognizes a homologous sequence between human and mycobacterial HSP60 and a polyclonal antibody (SPA804) to cyanobacteria HSP60, but not with antibodies to HSP60 of several other organisms used. Furthermore, anti-recombinant E. sennetsu 55-kDa protein antibody prepared in a rabbit was reactive to HSP60 antigens of other Ehrlichia and Rickettsia species, but not GroEL of E. coli. The recombinant 55-kDa protein would be a useful tool for studying the role of this antigen in the immune response to E. sennetsu infection.