A non-canonical mismatch repair pathway in prokaryotes.

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS-MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS-MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.

Microbial genome dynamics in CNS pathogenesis.

The balancing act between microbes and their host in commensal and disease states needs to be deciphered in order to fully treat and combat infectious diseases. The elucidation of microbial genome dynamics in each instance is therefore required. In this context, the major bacterial meningitis pathogens are Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae. In prokaryotic CNS pathogenesis both the intact organism as well as its released components can elicit disease, often resulting in neurological sequelae, neurodegeneration or fatal outcome. The study of microbial virulence in CNS disease is expected to generate findings that yield new information on the general mechanisms of brain edema and excitatory neuronal disturbances due to meningitis, with significant potential for discoveries that can directly influence and inspire new strategies for prevention and treatment of this serious disease.

Antimutator role of DNA glycosylase MutY in pathogenic Neisseria species.

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2'-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC-->AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.

Analysis of the PilQ secretin from Neisseria meningitidis by transmission electron microscopy reveals a dodecameric quaternary structure.

PilQ is a member of the secretin family of outer membrane proteins and is specifically involved in secretion of type IV pili in Neisseria meningitidis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The quaternary structure of PilQ from N. meningitidis was analyzed by transmission electron microscopy by using a negative stain. Single particle averaging was carried out with a total data set of 650 individual particles, which produced a projection map generated from 296 particles at an estimated resolution of 2.6 nm. Oligomeric PilQ adopts a donut-like structure with an external ring that is 16.5 nm in diameter surrounding a central cavity that is 6.5 nm in diameter. Self-rotation and power spectrum analysis demonstrated the presence of 12-fold rotational symmetry, showing that PilQ is organized as a ring of 12 identical subunits. A model of the type IV meningococcal pilus fiber, based on the X-ray crystal structure of the N. gonorrhoeae pilin subunit, fitted neatly into the cavity, demonstrating how PilQ could serve as a channel for the growing pilus fiber.

Diagnostics of neisseriaceae and moraxellaceae by ribosomal DNA sequencing: ribosomal differentiation of medical microorganisms.

Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes.

Intracerebral abscess caused by Nocardia otitidiscaviarum in a renal transplant patient--cured by evacuation plus antibiotic therapy.

We present a 50-year-old female who experienced generalized convulsion 3 months after a successful cadaveric renal transplantation. The first cerebral CT scan indicated cerebral frontal infarction. Repeat CT some days later revealed progressive lesions, and a highly malignant tumor or abscess was suspected. Antifungal and broad-spectrum antibacterial therapy was initiated. Cerebral MRI could not differentiate between these conditions, but a neutrophil granulocyte scan strongly suggested an infectious process. A stereotactic puncture of the frontal lobe was followed by temporary improvement. A severe progressive left-sided hemiparalysis gave indication for a craniotomy with evacuation of the abscess 9 days later. Culture of aspirated pus yielded growth of a gram-positive, rod-shaped bacterium, later identified as Nocardia otitidiscaviarum by sequencing the 16S rRNA. The patient was treated with meropenem plus rifampicin intravenously for 6 weeks followed by oral ciprofloxacin and rifampicin for 2 months. Due to pharmacokinetic interaction with rifampicin, the prednisolone dose was doubled, and the dose of tacrolimus had to be tripled for maintenance of adequate trough concentrations. Five months following cessation of antibiotic treatment, the patient has regained normal strength and function in her left-sided extremities and has a serum creatinine level of about 160 micromol/l (1.8 mg/dl).

Structure and function of repetitive sequence elements associated with a highly polymorphic domain of the Neisseria meningitidis PilQ protein.

Secretins are a large family of proteins associated with membrane translocation of macromolecular complexes, and a subset of this family, termed PilQ proteins, is required for type IV pilus biogenesis. We analysed the status of PlIQ expression in Neisseria meningitidis (Mc) and found that PlIQ mutants were non-piliated and deficient in the expression of pilus-associated phenotypes. Sequence analysis of the 5' portion of the pilQ ORF of the serogroup B Mc strain 44/76 showed the presence of seven copies of a repetitive sequence element, in contrast to the situation in N. gonorrhoeae (Gc) strains, which carry either two or three copies of the repeat. The derived amino acid sequence of the consensus nucleotide repeat was an octapeptide PAKQQAAA, designated as the small basic repeat (SBR). This gene segment was studied in more detail in a collection of 52 Mc strains of diverse origin by screening for variability in the size of the PCR-generated DNA fragments spanning the SBRs. These strains were found to harbour from four to seven copies of the repetitive element. No association between the number of copies and the serogroup, geographic origin or multilocus genotype of the strains was evident. The presence of polymorphic repeat elements in Mc PilQ is unprecedented within the secretin family. To address the potential function of the repeat containing domain, Mc strains were constructed so as to express chimeric PilQ molecules in which the number of SBR repeats was increased or in which the repeat containing domain was replaced in toto by the corresponding region of the Pseudomonas aeruginosa (Pa) PilQ protein. Although the strain expressing PilQ with an increased number of SBRs was identical to the parent strain in pilus phenotypes, a strain expressing PilQ with the equivalent Pa domain had an eightfold reduction in pilus expression level. The findings suggest that the repeat containing domain of PilQ influences Mc pilus expression quantitatively but not qualitatively.

Antibiotic resistance of pneumococci in Norway.

A collection of 178 pneumococcal isolates found in Norway during the period 1987-1994 were tested for their susceptibility to benzylpenicillin, macrolides (azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin), fluoroquinolones (ciprofloxacin, sparfloxacin), imipenem, chloramphenicol, and vancomycin by a standard agar dilution procedure. To benzylpenicillin, two strains (1%) showed resistance and 14 strains (8%) intermediate susceptibility. Towards erythromycin, eight strains (4%) showed resistance and four strains (2%) intermediate susceptibility. Cross-resistance was demonstrated among the macrolides. Among the fluoroquinolones, intermediate susceptibility occurred with 42% of the isolates for sparfioxacin and 90% for ciprofloxacin; to the latter 5.1% proved resistant. The sum of intermediate and highly resistant isolates was 53% for chloramphenicol. Both penicillin-resistant strains were isolated during the last 2 years of collection and came from patients of non-Norwegian ethnic background. Imported strains appeared over represented among the strains resistant to penicillin and macrolides. Only imipenem and vancomycin showed full susceptibility for all pneumococci tested. An over representation of serogroup 6 strains was apparent among the strains with intermediate susceptibility and high resistance to benzylpenicillin. It is apparent that high-level resistance has, not so far, become a difficult problem in Norway. Nevertheless, the situation requires monitoring of the resistance level, particularly in meningitis and septic patients, and certainly in patients who cntail a higher than usual possibility of acquiring pneumococci from pools of resistant strains outside Norway (visitors, immigrants and recent returness from abroad).

Phylogeny of the family Moraxellaceae by 16S rDNA sequence analysis, with special emphasis on differentiation of Moraxella species.

Thirty-three strains previously classified into 11 species in the bacterial family Moraxellaceae were subjected to phylogenetic analysis based on 16S rRNA sequences. The family Moraxellaceae formed a distinct clade consisting of four phylogenetic groups as judged from branch lengths, bootstrap values and signature nucleotides. Group I contained the classical moraxellae and strains of the coccal moraxellae, previously known as Branhamella, with 16S rRNA similarity of > or = 95%. A further division of group I into five tentative clusters is discussed. Group II consisted of two strains representing Moraxella atlantae and Moraxella osloensis. These strains were only distantly related to each other (93.4%) and also to the other members of the Moraxellaceae (< or = 93%). Therefore, reasons for reclassification of these species into separate and new genera are discussed. Group III harboured strains of the genus Psychrobacter and strain 752/52 of [Moraxella] phenylpyruvica. This strain of [M.] phenylpyruvica formed an early branch from the group III line of descent. Interestingly, a distant relationship was found between Psychrobacter phenylpyruvicus strain ATCC 23333T (formerly classified as [M.] phenylpyruvica) and [M.] phenylpyruvica strain 752/52, exhibiting less than 96% nucleotide similarity between their 16S rRNA sequences. The establishment of a new genus for [M.] phenylpyruvica strain 752/52 is therefore suggested. Group IV contained only two strains of the genus Acinetobacter. Strategies for the development of diagnostic probes and distinctive sequences for 16S rRNA-based species-specific assays within group I are suggested. Although these findings add to the classificatory placements within the Moraxellaceae, analysis of a more comprehensive selection of strains is still needed to obtain a complete classification system within this family.

Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization, and 16S rRNA gene sequence analysis.

Although Mycobacterium ulcerans, M. marinum, and M. haemophilum are closely related, their exact taxonomic placements have not been determined. We performed gas chromatography of fatty acids and alcohols, as well as DNA-DNA hybridization and 16S rRNA gene sequence analysis, to clarify their relationships to each other and to M. tuberculosis. M. ulcerans and M. marinum were most closely related to one another, and each displayed very strong genetic affinities to M. tuberculosis; they are actually the two mycobacterial species outside the M. tuberculosis complex most closely related to M. tuberculosis. M. haemophilum was more distinct from M. ulcerans and M. marinum, and it appeared to be as related to these two species as to M. tuberculosis. These results are important with regard to the development of diagnostic and epidemiological tools such as species-specific DNA probes and PCR assays for M. ulcerans, M. marinum, and M. haemophilum. In addition, the finding that M. ulcerans and M. marinum are more closely related to M. tuberculosis than are other pathogenic mycobacterial species suggests that they may be evaluated as useful models for studying the pathogenesis of M. tuberculosis. M. marinum may be particularly useful in this regard since strains of this species grow much more rapidly than M. tuberculosis and yet can cause systemic disease in immunocompromised hosts.

Direct detection of Mycobacterium tuberculosis complex in clinical samples from patients in Norway by ligase chain reaction.

Our aim was to investigate the use of DNA amplification with the ligase chain reaction (LCR) for detection of the Mycobacterium tuberculosis complex directly in human clinical specimens. The LCR assay employed was the Abbott LCx MTB Assay, which uses the gene encoding protein antigen b as the target template. Four hundred eighty-two samples from 457 patients in one clinical microbiology laboratory in Norway were processed by routine culture analysis (BACTEC culture), direct microscopy (Ziehl-Neelsen staining) and LCR. Of the 118 specimens containing cultivable M. tuberculosis, 106 (90.6%) were detected by LCR. Among the 364 culture-negative specimens, 356 samples were negative also by LCR and 8 (1.6%) were positive by LCR. In five of the eight LCR-positive and culture-negative samples, another sample from the same patient was M. tuberculosis culture positive and/or the patient had symptoms of tuberculosis. In comparison with culture, the sensitivity of LCR was 96.7% for smear-positive samples and 72.0% for smear-negative samples, respectively. For all samples combined, the sensitivity, specificity, and positive and negative predictive values were 90.2, 99.2, 97.4, and 96.7%, respectively. Challenging the M. tuberculosis LCR test with DNAs and cultures from strains of Mycobacterium ulcerans and Mycobacterium marinum, which are the mycobacterial species most closely related to the M. tuberculosis complex, resulted in all-negative test results. The sensitivity, specificity, and positive and negative predictive values of BACTEC culture in comparison with the LCR test and clinical criteria were 95.9, 100, 100, and 98.6%, respectively. A certain prioritization of samples subjected to the LCR assay should be based on clinical indications and risks with regard to infection transmission and patient isolation policy. More automation and lower expenses are generally desired for nucleic acid amplification kits. However, this M. tuberculosis LCR assay represents a valuable tool in routine mycobacterial diagnostics.

The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships--a review.

Type-IV pilus expression plays a critical role in the interactions between Neisseria gonorrhoeae, Neisseria meningitidis and their human host. We have focused on experiments designed to elucidate the mechanisms of organelle biogenesis as one means of understanding the complexities of pilus biology in these species. Employing a variety of approaches, genes and gene products essential to pilus biogenesis have been identified and characterized. The findings indicate that the neisserial type-IV pilus biogenesis machinery is most closely related to that operating in Pseudomonas aeruginosa and other pseudomonad species. This interrelatedness is documented at the levels of gene organization, DNA homologies and identities between the primary structures of the components. Despite these similarities, the biological correlates of pilus expression in the pathogenic Neisseria are quite unique. The current status of our embryonic understanding of the factors influencing organelle biogenesis is presented. In the context of this workshop, emphasis has been placed on specific contributions made through studies of gonococci and meningococci to the field as a whole..

Moraxella boevrei sp. nov., a new Moraxella species found in goats.

Six Moraxella-like strains that formed a phenotypically homogeneous group were isolated from the nasal flora of healthy goats. Total genomic DNA-DNA hybridization, rRNA gene restriction pattern, DNA base composition, and genetic transformation studies were performed to determine the relationships of these bacteria to species belonging to the genus Moraxella and other fastidious gram-negative species. The new group of isolates was very homogeneous, as shown by rRNA gene restriction fragment length patterns (ribotyping), and these organisms displayed high relative binding ratios (RBRs) to each other in DNA-DNA hybridization experiments (RBRs, > or = 58%) but distinctly lower levels of DNA homology with all other species investigated. However, the RBRs obtained with species of the genus Moraxella were higher than the RBRs obtained with all other gram-negative strains examined. Although the new strains had most of the Moraxella bovis phenotypic characteristics except nitrate reduction, quantitative and qualitative genetic transformation data led to the conclusion that they belong to a distinct new cluster in the genus Moraxella. The results of this study, combined with the general morphological and phenotypic profiles of the new strains, are consistent with the creation of a new Moraxella species, for which the name Moraxella boevrei is proposed. Strain 88365 (= ATCG 700022 = CCUG 35435 = NCTC 12925 = CIP 104716) is the type strain of M. boevrei.

Fastidious gram-negative bacteria: meeting the diagnostic challenge with nucleic acid analysis.

The extended panorama of fastidious Gram-negative bacteria (FGNB) as opportunistic etiological agents of infectious diseases in immunocompromised patients is largely due to improved medical expertise and technology. The heightened awareness of infectious diseases due to FGNB species mandates comprehensive classification and identification systems as a basis for rapid and reliable diagnostics. The most useful approaches are combinations of nucleic acid techniques such as hybridization, genetic transformation, amplification and base sequence analysis with selected conventional criteria. Among these approaches, the widely distributed feature of natural competence in these organisms facilitates the use of the biological method of genetic transformation as a valuable addition to the more common nucleic acid techniques. We describe the development of the taxonomy of FGNB through the last four decades, with particular emphasis on the families Neisseriaceae, Moraxellaceae, and Pasteurellaceae.

Moraxella caprae sp. nov., a new member of the classical Moraxellae with very close affinity to Moraxella bovis.

Eight phenotypically homogeneous Moraxella-like strains were isolated from the nasal flora of healthy goats. Total genomic DNA-DNA hybridization, DNA base composition determination, and genetic transformation studies were performed to determine the relationships of these bacteria to the classical moraxellae. The eight new isolates exhibited very high levels of genetic affinity to Moraxella bovis, as shown by quantitative and qualitative genetic transformation data, and exhibited high DNA-DNA relative binding ratios to each other (63% or more) but lower levels of DNA homology with all of the other species investigated, including the closely related classical moraxellae. Our results, combined with the general morphologic and phenotypic profiles of these organisms, indicate that they should be classified with the classical moraxellae, and we propose the name Moraxella caprae for them. Strain 8897 (= CCUG 33296 [corrected] = NCTC 12877) is the type strain of M. caprae.

Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria.

Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. The assembly of these colonization factors is a complex process. This report describes a new pilus-assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre-pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus-assembly protein PilC of Pseudomonas aeruginosa and other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide of M(r) 38 kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group B polysaccharide-producing strain of N. meningitidis (Mc). The results indicate that the pilG genes and genetic organization at these loci in Gc and Mc are extremely conserved. Hybridization studies strongly suggest that pilG-related genes exist in commensal Neisseria species and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Mc chromosomes by allelic replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin-gene alterations or to polarity exerted on pilD expression. The results indicated that PilG exerts its own independent role in neisserial pilus biogenesis.

Detection of rifampin resistance among isolates of Mycobacterium tuberculosis from Mozambique.

Rifampin resistance in respiratory isolates of Mycobacterium tuberculosis from Mozambique was detected by screening for point mutations using polymerase chain reaction (PCR) and DNA sequence analysis. The target template was a 350-bp fragment of rpoB encoding the beta-subunit of the RNA polymerase. Of the 66 strains studied, 38 were rifampin resistant by susceptibility testing with the radiometric method, 3 were intermediately resistant, and 25 were susceptible to rifampin. In 39 of the 41 rifampin-resistant strains, base-substitutions in the rpoB fragment were detected, and a total of 13 distinct mutations affecting 6 amino acids were observed. One of these mutations (His-->Thr in amino acid 526) was not previously described. The isolates were also investigated by restriction fragment length polymorphism (RFLP) analysis using the insertion element IS6110 as a hybridization probe. A total of 47 RFLP patterns were identified, with up to 9 isolates having the same RFLP pattern. Strains with the same RFLP pattern harbored different mutations in rpoB, suggesting that acquisition of rifampin resistance followed the spread of a rifampin-susceptible clone. The data showed that rifampin resistance can be detected with a high sensitivity by DNA sequence analysis of this fragment of rpoB. However, a few strains with rifampin resistance due to factors other than base substitutions in rpoB could be missed.