rpoB Sequences Research Articles

Mycobacterium setenseInfection in Humans

<i>Mycobacterium setense</i>Infection in Humans

Lack of Evidence for “ Acinetobacter septicus ” as a Species Different from Acinetobacter ursingii ?

In their recent paper, Kilic et al. (2) reported on a hospital outbreak of bloodstream infections caused by a non-glucose-acidifying Acinetobacter strain. The authors showed that this strain (AK001) was phenotypically most similar to Acinetobacter ursingii but had two properties atypical of this species: i.e., the ability to hemolyse sheep blood and the inability to grow on citrate. Additional taxonomic methods to clarify the taxonomic status of AK001 included 16S rRNA and rpoB gene sequence analysis and DNA-DNA reassociation. Although the results of the sequence analyses supported the close relationship of the strain to A. ursingii, the value of DNA-DNA reassociation between AK001 and the A. ursingii type strain was found to be 64.7 to 68.7%, which is slightly less than 70%, the recommended threshold for species delineation (6). The authors concluded that the strain represents a new species, for which the name “Acinetobacter septicus” was proposed. As, in our opinion, the data provided by Kilic et al. (2) do not convincingly substantiate the proposal of a novel species, we analyzed strain AK001 using methods that had previously been used to resolve Acinetobacter taxonomy (3). AK001 was obtained by the Culture Collection, University of Goteborg (CCUG) from Y.-W. Tang and was designated CCUG 56015. The strain was shown to have properties consistent with the original description of A. ursingii, except for the inability to utilize citrate (4). It is of note, however, that there are other citrate-negative A. ursingii strains in our current collection, which indicates the intraspecies variation of this property. Importantly, the hemolytic activity of AK001 reported by Kilic et al. (2) could not be confirmed, although sheep blood agar plates obtained from different suppliers were used at different cultivation temperatures and with several control hemolytic Acinetobacter strains. In the study by Kilic et al. (2), the 16S rRNA gene sequences of AK001 were 99.5% similar to that of the A. ursingii type strain, which is the value that can be found in strains within one species (4). To obtain more comprehensive information on the rpoB-based position of AK001 within the genus Acinetobacter, we determined the rpoB sequence of AK001 and compared it to that of eight A. ursingii strains and all hitherto-described species. The similarity values between AK001 (GenBank no. {type:entrez-nucleotide,attrs:{text:EU742582,term_id:190688713,term_text:EU742582}}EU742582) and the A. ursingii strains ({type:entrez-nucleotide,attrs:{text:EU477105,term_id:526150767,term_text:EU477105}}EU477105 and {type:entrez-nucleotide-range,attrs:{text:EU742575 to EU742581,start_term:EU742575,end_term:EU742581,start_term_id:190688699,end_term_id:190688711}}EU742575 to EU742581) ranged from 96.9% to 98.0%, which partially overlaps with the range of intraspecies values found for Acinetobacter beijerinckii (3). In contrast, the rpoB similarity values between AK001 and the other species were between 78.8% and 84.5%. The proposal of “A. septicus” was based mainly on the results of DNA-DNA reassociation. However, DNA-DNA reassociation values close to the proposed value of 70% have to be treated with caution as incongruent results for the same strains can be obtained in different laboratories and/or by different methods, which may have taxonomic implications (5). Concerning also other limitations of DNA-DNA reassociation (1), no conclusions based on these intermediate values should be made without further evidence of genetic isolation between strains. To address this problem, the relatedness of AK001 to hitherto-described species was investigated by AFLP (Keygene NV) analysis, a whole-genome fingerprinting method. Previous studies using this method have shown that strains of the same (genomic) species cluster together at a ≥50% similarity level (4). AFLP analysis showed that AK001 and 25 reference A. ursingii strains were linked in a distinct cluster at a 59% similarity level, while AK001 was separated from other species at 34% similarity, which strongly supports the assignment of AK001 to A. ursingii. Based on the above arguments, strain AK2001 is considered as belonging to A. ursingii and not representing a novel species.

Complete rpoB gene sequencing as a suitable supplement to DNA-DNA hybridization for bacterial species and genus delineation

DNA-DNA hybridization (DDH), the gold standard for bacterial species delineation, is a laborious method and the alternative, average nucleotide identity (ANI), a genomic sequence-derived parameter, is not applicable to non-sequenced species. A universal cut-off value to delineate bacterial species does not exist, yet a DDH value <70 % and ANI <95+/-0.5 % have proved useful in selected examples. We herein compare published values for DDH and ANI with sequence similarity of rpoB gene sequences retrieved from GenBank for strains of 230 bacterial species representative of 45 genera. Intraspecific rpoB sequence similarity was 98.2-100 %. We observed that an rpoB gene sequence similarity <or=97.7 % significantly correlated with a DDH value <70 % and an ANI value <94.3 %. An rpoB gene sequence similarity <85.5 % correlated with membership of different genera. When applied to fastidious and as-yet-uncultivated organisms lacking experimental DDH values, these cut-off values suggested that 'Candidatus Blochmannia pennsylvanicus' and 'Candidatus Blochmannia floridarius' may belong to different genera, that the different endosymbiotic Buchnera aphidicola organisms may belong to different genera and that, while the tsetse fly enteric symbiont Sodalis glossinidius may belong to the Enterobacteriaceae, the endocellular obligate symbiont Wigglesworthia glossinidia from the same host may belong to the group of as-yet-uncultivated gammaproteobacteria. rpoB gene sequence similarity provides an efficient supplement to DDH and ANI measurements to delineate bacterial species and genera, including delineation of as-yet-uncultivated, non-sequenced organisms.

How to identify Raoultella spp. including R. ornithinolytica isolates negative for ornithine decarboxylase? The reliability of the chromosomal bla gene

Although Raoultella planticola and Raoultella ornithinolytica were described more than 20 years ago, identifying them remains difficult. The reliability of the chromosomal bla gene for this identification was evaluated in comparison with that of the 16S rDNA and rpoB genes in 35 Raoultella strains from different origins. Of the 26 strains previously identified as R. planticola by biochemical tests alone or in association with molecular methods, 21 harboured a bla gene with 99.8% identity with the bla gene of two reference R. ornithinolytica strains ( bla ORN gene) and 5 harboured a bla gene with 99.2% identity with the bla gene of two reference R. planticola strains ( bla PLA gene). The 9 isolates previously identified as R. ornithinolytica harboured a bla ORN gene. The bla gene-based identification was confirmed by 16S rDNA and rpoB sequencing. The 21 isolates newly identified as R. ornithinolytica had a test negative for ornithine decarboxylase (ODC). Molecular experiments suggested one copy of ODC-encoding gene in both ODC-negative R. ornithinolytica and R. planticola strains and two copies in ODC-positive R. orninthinolytica strains. Analysis of the 35 bla genes allowed us (i) to confirm an identity of only 94% between the bla genes of the two Raoultella species while this identity was > 98% for rpoB and > 99% for 16S rDNA genes and (ii) to develop and successfully apply a bla PCR RFLP assay for Raoultella spp. identification. Overall, this study allowed us to discover ODC-negative R. ornithinolytica and to provide a reliable Raoultella identification method widely available as not requiring sequencing equipment.

A traditional Sudanese fermented camel's milk product, Gariss, as a habitat of Streptococcus infantarius subsp. infantarius

Samples of the traditional Sudanese fermented camel's milk product Gariss representing 9 different regions in Sudan were microbiologically characterized using an integrated approach including phenotypic and genotypic methods. Lactic acid bacteria [log(CFU/g) = 7.76–8.66] and yeasts [log(CFU/g) = 6.05–7.79] were detected in high numbers. A total of 180 LAB isolates were identified of which 123 isolates were found to belong to the Streptococcus bovis group. Thirteen representative isolates were characterized by PCR amplification and sequencing of the housekeeping genes rpoB and sodA and the streptococcal glucosyltransferase gene ( gtf). All thirteen isolates were identified as Streptococcus infantarius subsp. infantarius, a potential human pathogen. The gene encoding the virulence determinant gtf was detected in 10 of the 13 tested strains. The same isolates were able to survive exposure to 0.3% (w/v) oxgall for 4 h and pH=2.7 for 1--4 h. Also Lactobacillus fermentum were detected in high numbers, whereas Enterococcus faecium and Lactobacillus helveticus were detected more occasionally. The yeast microflora in all Gariss samples consisted of Kluyveromyces marxianus and Issatchenkia orientalis with the former being predominant in 7 out of 9 samples.

The predominant cultivable Veillonella spp. of the tongue of healthy adults identified using rpoB sequencing

The predominant Veillonella spp. were isolated from the dorsum surface of the tongues of 11 healthy adults and identified to species level using rpoB sequencing because 16S ribosomal RNA sequence analysis does not reliably differentiate between all members of this genus. In all, 253 isolates were identified and the mean proportion (+/- SE) of Veillonella spp. per sample was 16.2 (+/- 3.6) with a range of 3.0% to 36.3% of the total anaerobic colony count. The predominant species were Veillonella atypica (10/11), Veillonella dispar (9/11) and Veillonella rogosae (8/11) because they were isolated from the majority of subjects. Veillonella parvula was isolated from only one subject while Veillonella dentocariosi and Veillonella montpelleriensis were not isolated from any subject.

LethalMycobacterium massilienseSepsis, Italy

To the Editor: A strain of Mycobacterium massiliense was isolated from the blood of a kidney transplant patient in Italy at the same time she was diagnosed as having pulmonary tuberculosis. M. massiliense bacteremia appears to have played a role in her sudden death. The patient, a 63-year-old woman who had had a kidney transplant 10 years earlier and was receiving immunosuppressive treatment with cyclosporine, azathioprine, and prednisolone, was hospitalized in an intensive care unit because of septic shock, a stuporous condition, hypotension, respiratory insufficiency, and acute renal failure. Results of initial microbiologic investigations (cultures of blood, urine, and bronchial aspirate, and nasal and pharyngeal swabs; tests for antigens of Legionella pneumophila, pneumococcus, and Cryptococcus neoformans; and serologic analysis for pneumotropic viruses and bacteria) were negative. Hematologic analysis showed leukopenia with high neutrophil counts. Septic shock–specific therapy was given along with wide-spectrum antimicrobial drug therapy (levofloxacin, trimethoprim-sulfamethoxazole, piperacillin-tazobactam, and fluconazole). After moderate improvement, the patient’s general condition worsened and prompted a new round of microbiologic tests, including those for mycobacteria. After acid-fast bacilli were detected in a bronchial aspirate and results of nucleic acid amplification (Amplicor; Roche, Basel, Switzerland) were positive for M. tuberculosis complex, she was given standard antituberculosis treatment. She died the next day. Three blood cultures obtained 7 days before the death of the patient showed positive results in aerobic bottles but not in anaerobic bottles (BacT/ALERT SA; bioMerieux, Marcy l’Etoile, France). Subcultures spread onto blood agar yielded small white colonies of gram-positive bacilli, including branched forms, within 24 hours. One month earlier, the patient had been seen as an outpatient with intermittent fever, and unidentified gram-positive bacilli were observed in her blood culture. Genetic sequencing of the first one third of the 16S rDNA gene (1) of the strain (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU370523,term_id:169264479}}EU370523) showed 99.8% identity with M. abscessus, M. bolletii, M. chelonae, and M. massiliense. To discriminate between these 4 species, a 723-bp fragment of the RNA polymerase β subunit (rpoB) gene (2) also was sequenced (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EU370524,term_id:169264486}}EU370524). Sequencing showed 100% similarity with M. massiliense; the next most closely related species was M. bolletii (98.6% similarity). An isolate of M. tuberculosis that was susceptible to all first-line antituberculosis drugs was recovered from this patient’s bronchial aspirate in both solid and liquid media. Immunocompromised patients, including those with organ transplants, are known to be prone to mycobacterial infections (3). Mixed mycobacterial infections also have been reported (4). This newly described species has been isolated from pulmonary fluids (5), blood (6), intramuscular injection sites (7), and surgical wounds (8). Because sequencing 16S rDNA does not differentiate M. abscessus, M. bolletii, M. chelonae, and M. massiliense (5), use of the rpoB sequence is crucial. As reported for isolates of M. massiliense (5–8), our isolate was characterized by high MICs (9) (broth microdilution by using the Sensititer RGMYCO; Trek Diagnostic Systems Inc., Cleveland, OH, USA) to most of the antimicrobial drugs tested. The isolate was sensitive only to clarithromycin and amikacin and showed borderline sensitivity to linezolid (Table). Table Antimicrobial drug resistance pattern of the strain of Mycobacterium massiliense isolated from the patient, Italy* Despite co-infection with M. tuberculosis, the patient’s death was likely caused by M. massiliense bacteremia. The patient died before the isolate was identified as a mycobacterium, and unfortunately, none of the drugs used empirically was active against this organism. Only speculations can be made about how this patient acquired the M. massiliense infection. However, 5 months before her hospital admission, the patient had received a coxofemoral arthroprosthesis as a result of a fall and had since complained of generalized bone pain and had remained bedridden. Although in this case no proof exists, infection caused by rapidly growing mycobacteria after surgical intervention (10) is well known and should be considered. M. massiliense has been distinguished from M. abscessus by sequencing of the rpoB gene (2). Because this technology is available in relatively few clinical laboratories, cases of infection with M. massiliense may be mistakenly attributed to M. abscessus. Although infections with M. massiliense may be underrecognized, reports of these infections are raising concern. The capacity of this bacteria to infect different body sites is further evidence for the pathogenic potential of a rapidly growing mycobacteria in human infections (10).

세포벽 분해 관련 효소의 생산균주 분리 및 CMCase의 특성

CMCase를 생산하는 Klebsiella sp. Sc를 부산 인근의 토양에서 분리하였다. 이 균주는 16S rDNA와 RNA polymerase B subunit (rpoB) 유전자의 유전적 계통 분석을 통하여 동정을 시행하였다. 그 결과 Klebsiella sp. 속들의 균주들과 99% 이상의 상동성을 나타내었다. CMCase 유전자는 Sc의 게놈 라이브러리를 구축하여 찾았다. CMCase 유전자는 37,666 Da의 분자량을 가지는 333개의 아미노산을 암호화하고 있는 1,002개의 뉴클레오티드들로 구성된 것으로 밝혀졌다. 추정된 CMCase의 단백질 서열은 Cellulomonas uda와 단백질과 95%의 identity와 97%의 positive를 가지는 것으로 나타났으며, Erwinia chrysanthemi와는 각각 58%와 72%를 가지는 것으로 나타났다. 그리고 CMCase은 ASDGDTLIAWALLRAQKQW와 같은 보존 영역을 가지고 있었는데, 이는 glycosyl hydrolase family 8의 보존영역(A-[ST]-D-[AG]-D-X(2)-[IM]-A-[SA]-[LIVM]-[LIVMG]-A-X(3)-[FW])와 일치하는 것으로 나타났다. 이는 klebsiells sp. Sc의 CMCase는 glycosyl hydrolase family 8에 속한다는 것을 나타낸다. A CMCase-producing bacterium, Klebsiella sp. Sc, was isolated from Busan area's soil sample. This strain was identified on the basis of phylogenetic analysis of the 165 rDNA and rpoB (RNA polymerase B subunit) sequences. The gene encoding CMCase was cloned by genome library. The CMCase gene consisted of an open reading frame of 1,002 nucleotides and encodes 333 amino acids with a deduced molecular weight of 37,666 Da. The deduced amino acid sequence of the CMCase from Klebsiella sp. exhibits 95% identities and 97% positives to those from Cellulomonas uda and 58% identities and 72% positives to those from Erwinia chrysanthemi. The CMCase contain a conserved motif with the consensus sequence, ASDGDTLIAWALLRAQKQW. This result shows CMCase protein from Klebsiella sp. Sc belongs to glycoside hydrolase family 8, A-[ST]-D-[AG]-D-X(2)-[IM]-A-[SA]-[LIVM]-[LIVMG]-A-X(3)-[FW].

Molecular and phenotypic comparison of phaeochromycin-producing strains of Streptomyces phaeochromogenes and Streptomyces ederensis

Streptomyces strain LL-P018 produces the phaeochromycins, novel anti-inflammatory polyketides. This organism was identified as a strain of Streptomyces phaeochromogenes by physiological and genetic taxonomic analysis. In order to gain greater taxonomic perspective, LL-P018 was compared to related strains from major culture collections by 16S rRNA gene sequence, ribotype, HPLC-MS metabolite profile, and rpoB sequence. Using BioNumerics software, genetic and chemical fingerprint data were integrated via multivariate cluster analysis into a single, robust comparison. Based upon this analysis, strain LL-P018 is very closely related to the type strains of both S. phaeochromogenes and Streptomyces ederensis, indicating that these two types may in fact represent a single species. This novel comparative multi-cluster analysis is most useful for clarifying relationships between closely related species.

Phylogenetic relationships amongst the saltwater members of the genus Bacteriovorax using rpoB sequences and reclassification of Bacteriovorax stolpii as Bacteriolyticum stolpii gen. nov., comb. nov.

Members of the saltwater genus Bacteriovorax, formerly known as the marine Bdellovibrio, are obligate predatory bacteria that prey selectively on other Gram-negative bacteria. Previous phylogenetic analysis based on the 16S rRNA genes of saltwater Bacteriovorax isolates from environmental samples revealed 11 distinct phylogenetic clusters based on > or =96.5 % gene sequence similarity. In other micro-organisms, the gene coding for the beta-subunit of RNA polymerase (rpoB) has been shown to be more discriminating than 16S rRNA genes. In this study, rpoB sequences from Bacteriovorax isolates were analysed to determine whether the results would be consistent with those based on 16S rRNA gene sequences. A 1242 bp region of the rpoB gene from 74 saltwater Bacteriovorax strains and two freshwater isolates, Bacteriovorax stolpii Uki2T and Peredibacter starrii A3.12T, was amplified by PCR and analysed. The sequences were aligned and phylogenetic trees were constructed using a neighbour-joining algorithm. The resulting tree showed that the rpoB sequences produced smaller subdivisions of isolates, but were nevertheless consistent with the clusters determined using 16S rRNA gene sequences. Thus, the highly conserved 16S rRNA gene sequences provided good phylogenetic information and the rpoB gene sequences permitted greater differentiation in order to further subdivide phylogenetically distinct groups within the genus Bacteriovorax. Also, on the basis of the extensive diversity and large distance between the saltwater members of the genus Bacteriovorax and the freshwater/soil Bacteriovorax, a reclassification of Bacteriovorax stolpii as Bacteriolyticum stolpii gen. nov., comb. nov. is proposed. A new family, Peredibacteraceae fam. nov., is also described.

Yersinia massiliensis sp. nov., isolated from fresh water

Two bacterial organisms, 50640T and 823, were isolated from fresh water in Marseilles, France, and were further identified as members of the genus Yersinia on the basis of their phenotypic characteristics and 16S rRNA gene sequencing. Their unique phenotypic profile differed from that of closely related species of Yersinia bercovieri and Yersinia mollaretii by exhibiting positive indole and inositol tests, and from that of Yersinia frederiksenii by lacking the ability to ferment l-rhamnose. A polyphasic approach, including almost complete 16S rRNA gene sequencing (1461 bp) and partial sequencing of hsp60 (683 bp), gyrB (662 bp), sodA (624 bp) and rpoB (1049 bp) showed that isolates 50640T and 823 exhibited 98.5, 93.5, 90.4, 92.4 and 96.6 % similarity with Y. mollaretii, 98.7, 93.0, 90.1, 89.1 and 96.2 % with Y. bercovieri, and 98.4, 93.2, 89.8, 88.9 and 95.2 % with Y. frederiksenii, respectively. Both isolates exhibited an identical 16S rRNA gene sequence and differed by one to five point mutations in housekeeping gene sequences. Phylogenetic reconstructions based on the combination of these four housekeeping genes indicated that the two isolates formed a unique branch supported by a bootstrap value of 93 %. Their unique phenotypic traits, 16S rRNA gene sequence, together with housekeeping gene sequences exhibiting <97 % similarity with closely related species, and phylogenetic analyses suggested that the two isolates represent a so far undescribed Yersinia species. The name Yersinia massiliensis sp. nov. is proposed for this new taxon (type strain 50640T=CIP 109351T=CCUG 53443T; isolate 823=CIP 109352=CCUG 53444).

Diversity of Veillonella spp. from Sound and Carious Sites in Children

Detailed data on the distribution of Veillonella in caries-free and caries-active subjects are scarce. We hypothesized that the diversity of the genus would be lower in caries lesions than in plaque from caries-free individuals. The proportions of Veillonella were not significantly different in the two groups. All isolates (n = 1308) were genotyped by REP-PCR, and different genotypes (n = 170) were identified by 16S rRNA, dnaK, and rpoB sequencing. V. parvula, V. dispar, and V. atypica were in both groups, V. denticariosi only in caries lesions, and V. rogosae only from the caries-free individuals (p < 0.009). Lesions were more likely to harbor a single predominant species (p = 0.0018). The mean number of genotypes in the lesions was less than in the fissure (p < 0.001) or buccal (p = 0.011) sites. The Veillonella from caries-free sites were more diverse than those from caries lesions, and may be related to the acidic environment of caries lesions.

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

To investigate the applicability of rpoB gene, which encodes the beta subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP- and BOX-polymerase chain reaction) were also used. rpoB DNA (458 bp) were amplified from 21 Geobacillus- and Bacillus type strains, producing different BOX- and REP-PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90-100% (16S rRNA) and 74-100% (rpoB) similarity among examined bacteria. BOX- and REP-PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.

Characterization ofBartonellaStrains Isolated from Black-Tailed Prairie Dogs (Cynomys ludovicianus)

Thirty bartonella strains were isolated from the blood of black-tailed prairie dogs (Cynomys ludovicianus) from Boulder County, Colorado, USA. The bacteria appeared as small, fastidious, aerobic, Gram-negative rods. The partial sequences of the citrate synthase gene (gltA) demonstrated five unique genetic variants. Phylogenetic analysis based on sequences of gltA, 16S rRNA, rpoB, ftsZ, and ribC showed that the black-tailed prairie dog-related Bartonella variants comprise a distinct monophyletic clade that is closely related to Bartonella washoensis, a species isolated from a human patient and subsequently from ground squirrels. These variants, however, are grouped together in 100% of the bootstrapped trees. These variants were not found in other small mammals trapped during the same study, showing some evidence of host specificity. We believe that the group being described here is typical of the black-tailed prairie dog. We propose to name the bacteria Candidatus Bartonella washoensis subsp. cynomysii. The type strain is CL8606co(T)(=ATCC BAA-1342(T) = CCUG 53213(T)), which is the representative isolate of the dominant variant of the characterized group.

Molecular characterization of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures.

An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belém, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak, all of which were originally attributed to a single strain of M. abscessus.

Phylogeny of γ-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.

Bartonella australissp. nov. from Kangaroos, Australia

To the Editor: During April–May 1999, 3 Bartonella isolates (AUST/NH1, AUST/NH2, AUST/NH3) were cultivated and established from the blood of 5 Macropus giganteus gray kangaroos from central coastal Queensland, Australia. We used multigene sequencing to evaluate whether these Bartonella isolates fulfill the minimum requirements for classification as a new species. DNA from each Bartonella isolate was extracted by using the QIAamp tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Partial PCR amplification and sequencing of the genes encoding the 16S rDNA (rrs), citrate synthase (gltA), β-subunit of the RNA polymerase (rpoB), and cell division protein (ftsZ), as well as for the 16S–23S rDNA intergenic spacer (ITS) were attempted by using previously described primers and conditions (1). Bartonella sp. isolates AUST/NH1 to AUST/NH3 exhibited identical sequences for all 4 genes and the spacer studied, and isolate AUST/NH1 was selected as type strain among kangaroo isolates. Similarity rates between strain Aust/NH1 and validated Bartonella species (Appendix Table) ranged from 84.7% to 91.6%, from 97.5% to 98.5%, from 79.6% to 87.2%, from 85.4% to 95.0%, and from 83.5% to 87.1% for the ITS and rrs, gltA, rpoB, and ftsZ genes, respectively. Therefore, for each of these 4 genes or the spacer, strain AUST/NH1 exhibited similarity rates with all other species lower than the cutoffs published to classify Bartonella isolates within a validated species (1). It may thus be regarded as a new species. To estimate the genomic G+C content of strain AUST/NH1, we amplified and sequenced its ftsY gene as described (2) by using the BartftsyF (5′-ATGACAAAAYCYTTTATMAA-3′) and BartftsyR (5′-TCATGAGTGTCTTCCTGC-3′) primers. The ftsY G+C content was 37.7%; the calculated genomic G+C content was 39.51%. The ftsY sequence was deposited in GenBank under accession no. {type:entrez-nucleotide,attrs:{text:DQ538398,term_id:109649511,term_text:DQ538398}}DQ538398. The phylogenetic relationships among the studied bartonellae were inferred from sequence alignments of each gene and from concatenated gene sequences by using the maximum parsimony and neighbor-joining methods within the MEGA version 2.1 software package (3) and the maximum-likelihood method within the PHYLIP software package (4). Using rrs, gltA, and rpoB sequences, the phylogenetic position of strain AUST/NH1 was supported by bootstrap values <70%. In contrast, by using the ITS, ftsZ, and concatenated sequences, strain AUST/NH1 clustered with a group of B. tribocorum, B. grahamii, and B. elizabethae, with elevated bootstrap values according to the 3 analysis methods (Figure). Figure Unrooted dendrogram showing the phylogenetic position of Bartonella sp. strain AUST/NH1 among Bartonella species inferred from the comparison of concatenated sequences from the rrs, gltA, intergenic spacer, rpoB, and ftsZ genes by the neighbor-joining ... The Bartonella strains we describe are the first, to our knowledge, obtained from kangaroos and, more generally, from marsupials. Before this study, the only 2 Bartonella species found in Australia were B. henselae (5) and B. quintana (6). We demonstrated that strain AUST/NH1 was reliably associated with a well-established cluster, including the rodent-associated B. elizabethae, B. grahamii, and B. tribocorum (7). Therefore, we are confident that the phylogenic position of the new Bartonella, which was similar according to 3 analysis methods and supported by high bootstrap values, is reliable. Although B. grahamii (8) and B. elizabethae (9), members of the same phylogenetic cluster as strain AUST/NH1, cause human infections, the pathogenicity of B. tribocorum is as yet unknown. Its pathogenicity should therefore be investigated, especially for persons who come in contact with kangaroos. B. australis is a facultative intracellular gram-negative bacterium. It grows on Columbia agar with 5% sheep blood at 32°C to 37°C in a moist atmosphere containing 5% CO2. A primary culture was obtained after 7 days, and subculture was obtained after 4 days under the same conditions. Colonies are homogeneous, smooth, round, and gray-white. The 3 strains tested were oxidase negative, catalase negative, and nonmotile. Pathogenicity for humans is, as yet, unknown. The type strain is strain AUST/NH1. The new species is distinguished from other Bartonella species by its 16S rRNA, gltA, rpoB, ftsZ gene sequences, as well as its 16S–23S rRNA ITS sequence. The estimated G+C content is 38%. The type strain exhibits a specific serotype (10) and was susceptible to amoxicillin, ceftriaxone, imipenem, erythromycin, clarithromycin, ofloxacin, ciprofloxacin, rifampin, and tetracycline (unpub. data). The type strain AUST/NH1 has been deposited in the Collection of the World Health Organization Collaborative Center for Rickettsioses, Borrelioses and Tick-borne Infections (CSUR), Marseille, France, under reference CSUR B1; in the Collection de l’Institut Pasteur (CIP) under reference CIP 108978T; and in the Culture Collection of the University of Goteborg (CCUG), Sweden, under reference CCUG 51999. The strains AUST/NH2 and AUST/NH3 have been deposited in CSUR under references CSUR B2 and CSUR B3, in the CIP under references CIP 108980 and CIP 108979, and in CCUG under references CCUG 52000 and CCUG 52001, respectively.

Expanding the Diagnostic Capabilities of Molecular Microbiology by Genomic Methods

Expanding the Diagnostic Capabilities of Molecular Microbiology by Genomic Methods

A New Method to Extract Dental Pulp DNA: Application to Universal Detection of Bacteria

BackgroundDental pulp is used for PCR-based detection of DNA derived from host and bacteremic microorganims. Current protocols require odontology expertise for proper recovery of the dental pulp. Dental pulp specimen exposed to laboratory environment yields contaminants detected using universal 16S rDNA-based detection of bacteria.Methodology/Principal FindingsWe developed a new protocol by encasing decontaminated tooth into sterile resin, extracting DNA into the dental pulp chamber itself and decontaminating PCR reagents by filtration and double restriction enzyme digestion. Application to 16S rDNA-based detection of bacteria in 144 teeth collected in 86 healthy people yielded a unique sequence in only 14 teeth (9.7%) from 12 individuals (14%). Each individual yielded a unique 16S rDNA sequence in 1–2 teeth per individual. Negative controls remained negative. Bacterial identifications were all confirmed by amplification and sequencing of specific rpoB sequence.Conclusions/SignificanceThe new protocol prevented laboratory contamination of the dental pulp. It allowed the detection of bacteria responsible for dental pulp colonization from blood and periodontal tissue. Only 10% such samples contained 16S rDNA. It provides a new tool for the retrospective diagnostic of bacteremia by allowing the universal detection of bacterial DNA in animal and human, contemporary or ancient tooth. It could be further applied to identification of host DNA in forensic medicine and anthropology.

Transmission of Mycobacterium tuberculosis in a Family Proved by Genotyping

Molecular genetic methods have been applied in various epidemiologic studies including investigations of disease acquisition by contact. This report describes the use of various molecular genetic methods in tracing possible household transmission of tuberculosis by contact. Four Mycobacterium tuberculosis strains, each from four members of a family, were first isolated and identified in the clinical laboratory of the Chest Hospital and were submitted to the National Reference Laboratory of Mycobacteriology for further confirmation and genotyping. In this study, IS6110 restriction fragment length polymorphism (RFLP), spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR), and rpoB gene sequencing were used for genotyping. All four strains were found to have identical spoligotypes, MIRU-VNTR patterns, and similar IS6110 RFLP profiles. The results of the drug susceptibility test and of rpoB sequencing showed that all four strains were rifampicin resistant. Household transmission through close contact was thus proved by genotyping. We conclude that all four family members were infected with the same lineage of M. tuberculosis.