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Rhizobium zaerense sp. nov., a novel member of the Rhizobium leguminosarum species complex with a broad geographic distribution and multiple legume hosts.

A novel nitrogen-fixing rhizobial strain, designated Z1P35ᵀ, was isolated from root nodules of Pisum sativum grown in the Zaër region of Morocco. Phylogenetic analysis of the 16S rRNA gene placed strain Z1P35ᵀ within the genus Rhizobium, showing 100% sequence identity with several undescribed genospecies of the Rhizobium leguminosarum species complex (Rlc). Strain Z1P35ᵀ exhibited low average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with all described Rhizobium species, but high ANI and dDDH values (97.62 and 78.8%, respectively) with Rhizobium sp. SRDI565, representing genospecies M (GsM) of the Rlc, suggesting that Z1P35ᵀ represents a novel species corresponding to GsM within this complex. FastANI screening against all Rhizobium genomes available in GenBank revealed that Z1P35ᵀ shares ANI values above the bacterial species delimitation threshold with 17 unclassified strains, which, together with Z1P35ᵀ and Rhizobium sp. SRDI565 (GsM), form a distinct lineage within the Rlc. These 17 strains originate from root nodules of diverse legume hosts and are distributed across the Mediterranean region and Australia, including representatives of the symbiovars viciae and trifolii. Phylogenomic analysis further confirms the clustering of Z1P35ᵀ with Rhizobium sp. SRDI565 (GsM) and several undescribed Rhizobium strains, forming a unique taxonomic unit clearly distinct from other members of the Rlc. Strain Z1P35ᵀ has a genome of 7.6 Mb with a G+C content of 61 mol% and carries numerous genes associated with chemotaxis, nodulation, nitrogen fixation, phosphate solubilization, iron acquisition and abiotic stress tolerance. Differentiation of Z1P35ᵀ from described Rhizobium species was further supported by phenotypic and chemotaxonomic analyses. Based on these results, we conclude that Z1P35T belongs to a novel species, corresponding to genospecies M within the Rlc, for which we propose the name Rhizobium zaerense sp. nov. The type strain is Z1P35ᵀ (DSM 120601ᵀ=CCMM B1365ᵀ).

Phylogeny↗

Bacteremia and chorioamnionitis due to cryptic genospecies of Haemophilus Influenzae biotype I.

Nontypable strains of Haemophilus influenzae are well-known causes of maternal and neonatal infections. Using DNA-DNA hybridization techniques, some of these strains have been shown to belong to a cryptic genospecies of Haemophilus, which is distantly related to Haemophilus influenzae and Haemophilus hemolyticus. This report describes the first case of sepsis and chorioamnionitis due to Haemophilus influenzae biotype I, which was identified using the RapIDNH system and then confirmed by multilocus enzyme electrophoresis to belong to this cryptic genospecies of Haemophilus. The electromorph type 92 of the isolate was consistent with that of biotype I of the cryptic genospecies.

Adult↗

Sequence diversity of a gene encoding a putative primary sigma factor among Borrelia burgdorferi sensu lato strains.

Twenty-six strains of Borrelia burgdorferi sensu lato were subjected to polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis for assessing the sequence divergence of rpoD gene encoding the primary sigma factor. Four and five RFLP patterns were observed from two fragments of rpoD gene. Sequence analysis of a subgenic fragment covering region 1 through 4 from 13 strains of Borrelia burgdorferi s. 1. revealed that 21 of 450 deduced amino acid residues were diverged. These results indicate that the sequence heterogeneity of rpoD is present in different strains of Borrelia burgdorferi s. 1., and agreed well with the current classification of genospecies.

Amino Acid Sequence↗

Effect of chlorhexidine varnish on Actinomyces naeslundii genospecies in plaque from dental fissures.

This study describes the effects of varnish containing 40% chlorhexidine diacetate on Actinomyces naeslundii populations in plaque from human molar fissures. In each of 15 subjects two dental fissures with high levels of mutants streptococci were selected. The experimental treatment consisted of the single application of a small amount of chlorhexidine varnish onto the selected fissures. The varnish was removed 15 min after application. One month after varnish application a significant increase was observed in A. naeslundii counts while the number of mutans streptococci had decreased significantly compared with preexperimental levels. From 85 randomly selected Actinomyces isolates taken from blood agar plates before varnish application, 44% belonged to A. naeslundii genospecies 1 and 56% to A. naeslundii genospecies 2. From 106 isolates taken 1 month after chlorhexidine varnish application, 42% belonged to A. naeslundii genospecies 1 and 58% to A. naeslundii genospecies 2. At baseline 28% of A. naeslundii genospecies 1 strains were catalase-positive, but 1 month after varnish application 4% of the strains were catalase-positive (p < 0.05). It is concluded that chlorhexidine varnish application caused an increase of A. naeslundii in dental plaque, but induced no significant changes in the distribution of the two A. naeslundii genospecies.

Actinomyces↗

Extension of Ixodes ricinus ticks and agents of tick-borne diseases to mountain areas in the Czech Republic.

Along with the shift of the hard tick Ixodes ricinus to higher altitudes observed in the Czech Republic a corresponding shift of tick-borne infections to higher altitudes has been expected. Therefore, I. ricinus ticks, mainly nymphs, were investigated for the presence of tick-borne viruses, tick-borne encephalitis (TBE), Tribec and Eyach, and the spirochaete Borrelia burgdorferi sensu lato in the Sumava and Krkonose Mountains (Czech Republic). The TBE virus and different genospecies of B. burgdorferi s.l. were detected by RT-PCR and PCR, respectively. TBE virus was detected in ticks at 620 and 720 m above sea level (a.s.l.), B. burgdorferi s.l. was detected in ticks up to 1065 m a.s.l. Four genospecies of B. burgdorferi s.l. were identified, B. afzelii, B. garinii, B. burgdorferi sensu stricto, and B. valaisiana. Some nymphs carried multiple Borrelia infections. The conditions of tick-borne agents' distribution and potential epidemiological consequences are discussed.

Altitude↗

Metal resistance in Acinetobacter and its relation to beta-lactamase production.

Thirty nine clinical isolates of Acinetobacter belonging to six species were tested for resistance to 20 metal ions and their ability to produce beta-lactamase. Fifty two percent of the strains produced beta-lactamase. beta-Lactamase producers and non-producers were almost equally distributed in the different species. A. baumannii was the predominant biotype and was found to be most resistant to metals. Resistance to mercury was prevalent in beta-lactamase-producing A. baumannii only. Silver resistant strains of A. baumannii produced beta-lactamase. Sensitivity and resistance to copper and cadium was equally distributed between beta-lactamase producers and non-producers. beta-Lactamase-producer and -non-producer strains were uniformly sensitive to cadmium except Acinetobacter genospecies 1.

Acinetobacter↗

Characterization of Borrelia species isolated from ixodid ticks, Ixodes ovatus.

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.

Animals↗

Genetic characterization of a cryptic genospecies of Haemophilus causing urogenital and neonatal infections.

In recent years, reports originating from several areas of the world have identified biotype IV strains of Haemophilus influenzae as a cause of serious urogenital, neonatal, and mother-infant infections. Preliminary analysis of a sample of biotype IV isolates found evidence for a cryptic genospecies of Haemophilus (R. Quentin, A. Goudeau, R. J. Wallace, Jr., A. L. Smith, R. K. Selander, and J. M. Musser. J. Gen. Microbiol. 136:1203-1209, 1990). Eighteen biotype IV strains assigned to the cryptic genospecies were further characterized by their rDNA restriction fragment length polymorphism patterns and genomic DNA-DNA hybridization. Isolates of the cryptic genospecies have distinctive rDNA restriction fragment length polymorphisms that differ from those of Haemophilus haemolyticus and H. influenzae. Genomic hybridization studies show that these organisms are allied with H. influenzae and H. haemolyticus and suggest a distant trifurcation of H. influenzae, H. haemolyticus, and the cryptic genospecies.

Adult↗

Cell-mediated immune response to high-passage Borrelia spirochetes in C57bl/6 mice is strictly dependent on antigen specificity.

Inbred C57bl/6 mice were challenged with high-passage Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen specific T-cell response in vitro. Sonicated preparations of washed spirochetes were potent cell activators, capable of stimulating polyclonal proliferation after 72h of culture while increasing the incubation time up to 120h provoked specific cell-mediated response. Isolated murine spleocytes previously sensitized to B. burgdorferi sensu lato but not those from control mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation assay, Moreover, in mice presensitized to B. burgdorferi sensu lato, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies. The current study emphasises that the B. burgdorferi antigen-specific response may also be expected in different genospecies infections in men.

Animals↗

Diarrhea associated with Aeromonas species in children in day care centers.

Outbreaks of diarrhea caused by enteropathogens have been reported in day care centers (DCC), but Aeromonas species have not been implicated. This study evaluated 381 children involved in 51 outbreaks in four DCC to determine the association of Aeromonas species with diarrhea and to characterize the isolates. The organism was identified in two outbreaks of diarrhea. In one, Aeromonas species were isolated from 6 (24%) of 25 children and in the other from 5 (21%) of 24 children. Seven other Aeromonas strains from children in DCC were studied. Fourteen (78%) of 18 were Aeromonas caviae and 15 were from children with diarrhea. Of the isolates, 75% did not have plasmids detected; all others had unique plasmid patterns. All strains had different DNA content. Twenty-two control isolates of Aeromonas from children with diarrhea in Mexico and Dallas had different chromosomal DNA patterns. Most Aeromonas infections were associated with symptoms. Chromosomal DNA patterns differentiated Aeromonas strains better than did plasmid DNA patterns. The outbreaks of diarrhea were unusual in that several different Aeromonas genospecies were involved in each outbreak.

Aeromonas↗

[Advances in molecular biology of dermatophytes].

During the 44th meeting of The Japanese Society for Medical Mycology in Nagasaki, 2000, a forum was held entitled Advances in Molecular Biology of Dermatophytes. Based on the subject, target molecules and kind of approach, we selected seven presentations from over 100 of the poster abstracts. Six of them concerned identification and one concerned viability. Summaries of the 7 presentations are given in this article. Of presentations on the identification methods, 5 demonstrated their usefulness: 1) A sequence analysis of ITS 1 region in ribosomal DNA of several Microsporum species showed ITS 1 genospecies Arthroderma otae to be composed of A. otae, M. canis, M. equinum and M. audouinii. 2) RAPD may be useful for identifying isolates which are not clearly identifiable by conventional biological techniques. 3) Sequence analysis of CHS 1 was shown to be a rapid tool for species level identification of M. gypseum. 4) PCR-SSCP analysis was also useful for discrimination of dermatophytes with high reproducibility and sensitivity. 5) Strain identification of A. benhamiae isolates may be possible using RFLP analysis of NTS regions in ribosomal DNA. The other presentation concerning identification pointed out some important problems: RFLP of mitochondrial DNA and ITS sequencing of A. benhamiae showed that the results are sometimes in conflict with those obtained from biological techniques, or in some cases, between other molecular techniques. This implies that our concept of fungal species needs to be re-examined and perhaps amended. The presentation on viability introduced quantitative analysis of mRNA of ACT gene, a new application of a molecular technique. Since the mRNA expresses only in living cells, the method is highly useful as an indicator of fungal viability.

Actins↗

Fourth report of the cooperative, open-ended study of slowly growing mycobacteria by the International Working Group on Mycobacterial Taxonomy.

The open-ended study of the International Working Group on Mycobacterial Taxonomy is an ongoing project to characterize slowly growing strains of mycobacteria that do not belong to well-established or thoroughly characterized species. In this fourth report we describe two numerical taxonomic clusters that represent subspecies or biovars of Mycobacterium simiae, one cluster that encompasses the erstwhile type strain of the presently invalid species "Mycobacterium paraffinicum," one cluster that is phenotypically very similar to Mycobacterium avium and Mycobacterium intracellulare but may be a separate genospecies, one cluster that appears to be phenotypically distinct from M. avium but reacts with a nucleic acid probe specific for M. avium, and three tentatively defined clusters in proximity to a cluster that encompasses the type strain of Mycobacterium malmoense. Of special practical interest is the fact that one of the latter three clusters is composed of clinically significant scotochromogenic bacteria that can be misidentified as the nonpathogenic organism Mycobacterium gordonae if insufficient biochemical tests are performed.

Agglutination Tests↗

Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene.

To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recA gene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from B. burgdorferi reference strains and 16 were isolated from B. burgdorferi strains cultured from Ixodes ricinus ticks; 13 were directly isolated from nine human biopsy specimens and four I. ricinus tick midguts. The melting temperature of B. garinii was 2 degrees C lower than that of B. burgdorferi sensu stricto and B. afzelii. Melting curve analysis offers a rapid alternative for identification and detection of B. burgdorferi sensu lato genospecies.

Animals↗

Real-time homogeneous assay of rapid cycle polymerase chain reaction product for identification of Leptonema illini.

Partial 16S rDNA sequences of eight Leptospira-like field isolates that reacted weakly or not at all to microscope agglutination test were found to be similar to the 16S rDNA sequence of the nonpathogen Leptonema illini-type strain 3055. Comparison of these sequences with those of Leptospira 16S rDNA sequences revealed a Leptonema species signature sequence for which a forward amplification primer was designed. This primer was used in conjunction with a bacterial-specific 16S rDNA universal reverse primer for developing a LightCycler-based rapid PCR protocol in which fluorescence emission due to the binding of SYBR green I dye to the amplified products was continuously monitored. A melting temperature (T(m)) determined from the melting curve of the amplified product immediately after PCR confirmed that the product was of Leptonema. The protocol for 24 samples consisting of 30 PCR cycles and melting curve acquisitions required 30 min to complete and agarose gel electrophoresis of the PCR products was not necessary. The method was specific as PCR products were detected from the seven Leptonema reference strains and the eight field isolates that had been previously verified as Leptonema by 16S rDNA sequencing, but not from the two representative strains from each of the eight Leptospira genospecies tested.

Base Sequence↗

Application of chemotaxonomic techniques to the taxonomy of anaerobic bacteria.

The use of chemical characters in bacterial classification and identification has proved to be an essential component of modern systematics. Several clinically important anaerobic genera, such as Bacteroides, Clostridium, Eubacterium, Fusobacterium and Peptostreptococcus, are known to be heterogeneous of the basis of chemotaxonomic and genetic data and are in need of further examination. Recent work on bacteriodes has led to the genus being redefined and restricted to species within the former Bacteroides fragilis group, and a number of new genera have been proposed. It is important that suitable phenotypic characters are identified so that newly-defined genospecies can be differentiated in diagnostic laboratories.

Bacteria, Anaerobic↗

Comparison of erythema migrans caused by Borrelia afzelii and Borrelia garinii.

BACKGROUND: We compared epidemiological and clinical characteristics of patients with erythema migrans (EM) caused by Borrelia afzelii and Borrelia garinii. PATIENTS AND METHODS: 200 consecutive adult patients with B. afzelii isolated from the skin lesion and 53 consecutive adult patients with EM caused by B. garinii qualified for the present study. RESULTS: Comparison of the two groups revealed several distinctions. Patients with EM caused by B. garinii were older, had their skin lesions more often located on the trunk but less often on extremities, had shorter incubation and faster evolution of EM, more often reported associated local and certain systemic symptoms, had abnormal liver function test results more often and were more frequently seropositive. CONCLUSION: Early localized Lyme borreliosis caused by B. afzelii and B. garinii has distinct epidemiological and clinical characteristics. Clinical features of EM depend upon the genospecies of Borrelia burgdorferi sensu lato causing the illness.

Adolescent↗

Occurrence of Aeromonas spp. in samples of ground meat and chicken.

Aeromonas spp. was commonly isolated from ground meat and chicken samples at the retail level. The dominant species in ground meat were A. hydrophila and A. caviae. In chicken, A. sobria was common while A. caviae was isolated infrequently. Although A. hydrophila was isolated from 75% of ground meat samples and 62% of chicken samples, DL-lactate-positive A. hydrophila (genospecies 1) was isolated from only 25 or 37% or respective samples. Sorbitol-positive A. hydrophila (genospecies 3) was common in both ground meat and chicken.

Aeromonas↗