Isolation by culture and PCR identification of LipL32 gene of pathogenic Leptospira spp. in wild rats of Kuala Lumpur.

BACKGROUND: A study was conducted to confirm the status of rats as the carrier of pathogenic leptospira in Kuala Lumpur, Malaysia. METHOD: A total of 140 urine samples were collected from trapped rats. These samples were cultured in EMJH enriched media and 18 of these samples (12.9%) were found to be positive when observed under x40 by dark field microscope. Genomic DNA was extracted from all the 18 native isolates for PCR. RESULT: All the 18 isolates generated the expected 786 base pair band when the set of primers known to amplify LipL32 gene were utilized. These results showed that the primers were suitable to be used for the identification of pathogenic leptospira from the 18 rat samples. CONCLUSION: The sequencing of the PCR products and BLAST analysis performed on each representative isolates confirmed the pathogenic status of all these native isolates as the LipL32 gene was detected in all the Leptospira isolates. This indicates that the rats are carriers of the pathogenic leptospira in the study area, and therefore are of public health importance.

Investigation of a leptospirosis outbreak in triathlon participants, Réunion Island, 2013.

We report herein the investigation of a leptospirosis outbreak occurring in triathlon competitors on Réunion Island, Indian Ocean. All participants were contacted by phone or email and answered a questionnaire. Detection and molecular characterization of pathogenic Leptospira was conducted in inpatients and in rodents trapped at the vicinity of the event. Of the 160 athletes competing, 101 (63·1%) agreed to participate in the study. Leptospirosis was biologically confirmed for 9/10 suspected cases either by real-time PCR or serological tests (MAT or ELISA). The total attack rate, children's attack rate, swimmers' attack rate, and the attack rate in adult swimmers were respectively estimated at 8·1% [95% confidence interval (CI) 4·3-14·7], 0%, 12·7% (95% CI 6·8-22·4) and 23·1% (95% CI 12·6-33·8). Leptospirosis cases reported significantly more wounds [risk ratio (RR) 4·5, 95% CI 1·6-13], wore complete neoprene suits less often (RR 4·3, 95% CI 1·3-14·5) and were most frequently unlicensed (RR 6·6, 95% CI 2·9-14·8). The epidemiological investigation supported that some measures such as the use of neoprene suits proved efficient in protecting swimmers against infection. PCR detection in rats revealed high Leptospira infection rates. Partial sequencing of the 16S gene and serology on both human and animal samples strongly suggests that rats were the main contaminators and were likely at the origin of the infection in humans.

Outbreak of leptospirosis among canyoning participants, Martinique, 2011.

Two gendarmes who participated in canyoning activities on 27 June 2011 on the Caribbean island of Martinique were diagnosed with leptospirosis using quantitative real-time polymerase chain reaction (qPCR), 9 and 12 days after the event. Among the 45 participants who were contacted, 41 returned a completed questionnaire, of whom eight met the outbreak case definition. The eight cases sought medical attention and were given antibiotics within the first week after fever onset. No severe manifestations of leptospirosis were reported. In seven of the eight cases, the infection was confirmed by qPCR. Three pathogenic Leptospira species, including L. kmetyi, were identified in four of the cases. None of the evaluated risk factors were statistically associated with having developed leptospirosis. Rapid diagnostic assays, such as qPCR, are particularly appropriate in this setting ­ sporting events with prolonged fresh-water exposure ­ for early diagnosis and to help formulate public health recommendations. Participants in such events should be made specifically aware of the risk of leptospirosis, particularly during periods of heavy rainfall and flooding.

Human leptospira isolates circulating in Mayotte (Indian Ocean) have unique serological and molecular features.

Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira.

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

[Leptospirosis in French Guiana and the Guiana shield: Current knowledge in 2016]. / La leptospirose en Guyane française et sur le bouclier des Guyanes. État des connaissances en 2016.

Leptospirosis is a cosmopolitan zoonosis caused by bacteria of the genus Leptospira. Whether the distribution is worldwide, the hot and humid climate of the tropics is particularly conducive to its expansion. In most French overseas departments and territories, leptospirosis is considered as a public health problem. In French Guiana, a French department located in the northeastern part of the Amazon rainforest, it is supposed to be rare. The objective of this review was to make an inventory of the knowledge on human and animal leptospirosis in French Guiana and neighboring countries. A comprehensive search was conducted through the indexed and informal medical literature in English, French, Spanish and Portuguese. Thus, respectively ten and four publications were identified on human and animal leptospirosis in French Guiana, published between 1940 and 1995 in the form of case reports or case series. The publications concerning this disease in the other countries of the Guiana Shield, eastern Venezuela, Guyana, Suriname, and Brazilian state of Amapá, also scarce or nonexistent. However recent data from the French National Centre of leptospirosis showed a recent and sudden increase in the number of cases in the department, probably partly due to the development of diagnostic tools such as Elisa IgM serology. It is likely that leptospirosis is a neglected disease in the region, due to the lack of diagnostic tools readily available, the lack of knowledge of the local clinicians on this disease and the existence of many other pathogens with similar clinical presentation such as malaria, arboviruses and Q fever and Amazonian toxoplasmosis. The establishment of more large-scale studies on animal and human leptospirosis is necessary and urgent to know the true burden of this disease in our region.

Development of a species-specific probe for Mycobacterium xenopi.

In Europe, Mycobacterium xenopi is a frequently isolated species among opportunist mycobacteria, and represents one of the main agents of pulmonary infection due to non-tuberculous mycobacteria. Conventional identification of mycobacteria is a time-consuming and laborious process. In this study, we propose a rapid and simple method for the identification of M. xenopi using polymerase chain reaction. The amplified product consists of a specific probe, present in all 38 M. xenopi strains tested, which could not be amplified and did not present cross-hybridization with a set of 110 strains belonging to 23 other mycobacterial species. The probe was cloned and sequenced. Comparison with data bases revealed no significant homologies with previously described sequences.

Identification and characterization of IS-like elements in Mycobacterium gordonae.

A new insertion sequence, IS1512, from Mycobacterium gordonae was cloned and sequenced. This element is present in up to 10 copies which provides a high diversity for restriction fragment length polymorphism analysis. We have also identified truncated IS1512-like elements, including a truncated IS1512 and another truncated insertion sequence which displays homology with IS1512 and was designated IS1511. Sequences homologous to the previously described transposon Tn554 are inserted into these truncated insertion sequences. Insertion loci of IS1511/IS1512 are shown to be highly related to those described for IS256-like elements in other species. Alignment of the putative transposases from Rhodococcus and Mycobacterium suggests these insertion sequences may form a distinct closely homologous subclass within the IS256 family. Analysis comparing phylogenetic divergence of these elements with that of 16S rRNA and superoxide dismutase genes suggests horizontal transfer of a IS1511/IS1512 precursor into M. gordonae, and the occurrence of horizontal transfer between Mycobacteriaceae and Rhodococcaceae.

Diagnosis and epidemiology of leptospirosis.

Leptospirosis is a zoonosis found worldwide, the main reservoir of which is the rat. Human infection generally results from exposure to contaminated river or lake water or animals. Around 600 cases are diagnosed per year in France. Half of these cases occur in French overseas territories, where the incidence can be more than 100 times higher than in mainland France. Leptospirosis has been under-diagnosed because of non-specific symptoms, inadequate surveillance system, and lack of readily available quick and simple diagnostic tests. Most cases of leptospirosis are currently detected by PCR amplification of bacterial DNA from the blood during the first week after the onset of symptoms, or by detection of antibodies during the second week of the disease. More than 300 serovars have been identified among leptospires, including serovar Icterohaemorrhagiae, the most frequent in human infections. Leptospirosis remains a major public health issue in many developing countries, one century after discovering the causative agent. Leptospirosis is expected to become more important due to a rapid urbanization in developing countries (slums), global warming, and extreme climatic events (floods).

Comparative and functional genomic analyses of iron transport and regulation in Leptospira spp.

The spirochetes of the Leptospira genus contain saprophytic and pathogenic members, the latter being responsible for leptospirosis. Despite the recent sequencing of the genome of the pathogen L. interrogans, the slow growth of these bacteria, their virulence in humans, and a lack of genetic tools make it difficult to work with these pathogens. In contrast, the development of numerous genetic tools for the saprophyte L. biflexa enables its use as a model bacterium. Leptospira spp. require iron for growth. In this work, we show that Leptospira spp. can acquire iron from different sources, including siderophores. A comparative genome analysis of iron uptake systems and their regulation in the saprophyte L. biflexa and the pathogen L. interrogans is presented in this study. Our data indicated that, for instance, L. biflexa and L. interrogans contain 8 and 12 genes, respectively, whose products share homology with proteins that have been shown to be TonB-dependent receptors. We show that some genes involved in iron uptake were differentially expressed in response to iron. In addition, we were able to disrupt several putative genes involved in iron acquisition systems or iron regulation in L. biflexa. Comparative genomics, in combination with gene inactivation, gives us significant functional information on iron homeostasis in Leptospira spp.

Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto.

Leptospira interrogans sensu stricto is responsible for the most frequent and severe cases of human leptospirosis. The epidemiology and clinical features of leptospirosis are usually associated with the serovars and serogroups of Leptospira. Because of the difficulties associated with serological identification of Leptospira strains, we evaluated a novel PCR-based method for typing L. interrogans serovars. Based upon the genome sequence of L. interrogans serovar Lai type strain 5660, 44 loci were analyzed by PCR for their variability in size due to the presence of variable-number tandem repeats (VNTR). Seven VNTR loci were found to be powerful markers for serovar identification, epidemiology, and phylogenetic studies of L. interrogans. This rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.

Characterization of large linear plasmids in mycobacteria.

Linear plasmids were found in Mycobacterium xenopi, M. branderi, and M. celatum. These elements represented a wide size range (from 20 to 320 kb), had Streptomyces-like terminal structures, and defined five hybridization groups. Cross-hybridization and common restriction fragment length polymorphism patterns of plasmids from the different species suggest either genetic exchange or a common ancestry of the species.

Typing of Mycobacterium avium isolates by PCR.

A Mycobacterium avium typing method based on PCR amplification of genomic sequences located between the recently described repetitive elements IS1245 and IS1311 was developed. This method was applied to a set of epidemiologically related and unrelated strains and compared with restriction fragment length polymorphism analysis with IS1245 as the probe. This PCR typing consists of a rapid and simple technique, providing a reproducible M. avium characterization as discriminant as restriction fragment length polymorphism analysis.

Killing effect and antitoxic activity of the Leptospira interrogans toxin-antitoxin system in Escherichia coli.

We report the first evidence of a chromosome-encoded toxin-antitoxin locus in spirochetes. This locus has been found in the pathogenic spirochete Leptospira interrogans and exhibits homologies with the pem/chp loci. The L. interrogans chp locus consists of two genes: chpK (for "killer protein") and its upstream partner chpI (for "inhibitory protein"). Expression of ChpK in Escherichia coli results in the inhibition of bacterial growth. The coexpression of ChpI neutralizes ChpK toxicity. By Southern blot analysis, chp homologs were found in all representative pathogenic strains of L. interrogans.

Analyzing DNA strand compositional asymmetry to identify candidate replication origins of Borrelia burgdorferi linear and circular plasmids.

The Lyme disease agent Borrelia burgdorferi has a genome composed of a linear chromosome and a series of linear and circular plasmids. We previously mapped the oriC of the linear chromosome to the center of the molecule, where a pronounced switch in CG skew occurs. In this study, we analyzed B. burgdorferi plasmid sequences for AT and CG skew in an effort to similarly identify plasmid replication origins. Cumulative skew diagrams of the plasmids suggested that they, like the linear chromosome, replicate bidirectionally from an internal origin. The B. burgdorferi linear chromosome contains homologs to partitioning protein genes soj and spoOJ, which are closely linked to oriC at the minimum cumulative skew point of the 1-Mb molecule. A soj/parA homolog also maps to cumulative skew minima of the B. burgdorferi linear and circular plasmids, further suggesting that these regions contain the replication origin. The heterogeneity in these genes and in the nucleotide sequences of the putative origin regions could account for the mutual compatibility of the multiple DNA elements in B. burgdorferi.

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non-motile mutants deficient in endoflagella.

Leptospira spp. offer many advantages as model bacteria for the study of spirochaetes. However, homologous recombination between introduced DNA and the corresponding chromosomal loci has never been demonstrated. A unique feature of spirochaetes is the presence of endoflagella between the outer membrane sheath and the cell cylinder. We chose the flaB flagellin gene, constituting the flagellar core, as a target for gene inactivation in the saprophyte Leptospira biflexa. The amino acid sequence of the FlaB protein of L. biflexa was most similar to those of spirochaetes Brachyspira hyodysenteriae (agent of swine dysentery), Leptospira interrogans (agent of leptospirosis) and Treponema pallidum (agent of syphilis). A suicide vector containing the L. biflexa flaB gene disrupted by a kanamycin marker was UV irradiated or alkali denatured before electroporation. This methodology allowed the selection of many kanamycin-resistant colonies resulting from single and double cross-over events at the flaB locus. The double recombinant mutants are non-motile, as visualized in both liquid and semi-solid media. In addition, a flaB mutant selected for further analysis was shown to be deficient in endoflagella by electron microscopy. However, most of the transformants had resulted from a single homologous recombination event, giving rise to the integration of the suicide vector. We evaluated the effect of the sacB and rpsL genes in L. biflexa as potential counterselectable markers for allelic exchange, and then used the rpsL system for the positive selection of flaB double recombinants in a streptomycin-resistant strain. Like the flaB mutant studied above, the Strr double cross-over mutant was non-motile and deficient in endoflagella. Our results demonstrate that FlaB is involved in flagella assembly and motility. They also show the feasibility of performing allelic replacement in Leptospira spp. by homologous recombination.

Physical mapping of an origin of bidirectional replication at the centre of the Borrelia burgdorferi linear chromosome.

The Borrelia burgdorferi chromosome is linear, with telomeres characterized by terminal inverted repeats and covalently closed single-stranded hairpin loops. The replication mechanism of these unusual molecules is unknown. Previous analyses of bacterial chromosomes for which the complete sequence has been determined, including that of B. burgdorferi, revealed an abrupt switch in polarity of CG skew at known or putative origins of replication. We used nascent DNA strand analysis to physically map the B. burgdorferi origin to within a 2 kb region at the centre of the linear chromosome, and to show that replication proceeds bidirectionally from this origin. The results are consistent with replication models in which termination occurs at the telomeres after bidirectional, symmetrical elongation from the central origin. Sequences typical of origins of other bacterial chromosomes were not found at the origin of this spirochete. The most likely location of the replication origin of the linear chromosome is the 240 bp sequence between dnaA and dnaN where the switch in CG skew occurs.

Genotypic characterization of five subspecies of Mycobacterium kansasii.

Different molecular typing methods including restriction fragment length polymorphism (RFLP) analysis with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe, pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and PCR restriction analysis of the hsp-65 gene (PRA) were applied to clinical and water isolates of Mycobacterium kansasii. RFLP with the MPTR probe, PRA, PFGE, and AFLP analysis revealed five homogeneous clusters which appeared to be subspecies. RFLP with the MPTR probe and PRA gave patterns specific for each cluster, whereas PFGE and AFLP analysis gave polymorphic patterns. IS1652 was present in two of the five clusters and provided polymorphic patterns for one cluster only. The two IS1652-positive clusters were Accuprobe negative (Accuprobe test; Gen-Probe Inc.), and only two other clusters were Accuprobe positive. A PCR test based on the detection of a species-specific fragment (M. Yang, B.C. Ross, and B. Dwyer, J. Clin. Microbiol. 31:2769-2772, 1993) was positive for all M. kansasii strains. This PCR test is an accurate, rapid, and specific M. kansasii identification test. No subspecies was particularly more virulent, because all clusters contained clinical strains, from AIDS patients and non-AIDS patients, and environmental strains.

Molecular characterization of Mycobacterium avium complex isolates giving discordant results in AccuProbe tests by PCR-restriction enzyme analysis, 16S rRNA gene sequencing, and DT1-DT6 PCR.

Based on cultural and biochemical tests, a total of 84 strains (72 clinical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobacterium avium complex (MAC). They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and this was followed by selective amplification of DT6 and DT1 sequences. Seventy isolates gave concordant results; 63 were identified as M. avium, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods. Fourteen isolates gave discrepant results, as they were DT1 positive but gave negative results by the M. intracellulare AccuProbe test. Consequently, a detailed molecular analysis of all DT1-positive isolates (14 discrepant strains plus 5 M. intracellulare strains) was performed by PCR-restriction analysis (PRA) of the hsp65 gene and 16S rRNA gene sequencing. The results confirmed the reported heterogeneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished profiles. 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88917), the rest being related to MCRO19 (EMBL accession no. X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have characterized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing. However, all of them were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 positive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the majority of such isolates (14 of 19) were from the Indian subcontinent, with 12 of 14 being environmental isolates. Our study confirms the marked heterogeneity of M. intracellulare isolates and shows the utility of in-house DT1 PCR to detect this group of isolates, which would otherwise have been missed by the AccuProbe system in a routine clinical microbiology laboratory.

Use of different molecular typing techniques for bacteriological follow-up in a clinical trial with AIDS patients with Mycobacterium avium bacteremia.

One hundred ninety-six Mycobacterium avium isolates from blood samples recovered from 93 AIDS patients for several months were typed by serotyping, by IS1245 restriction fragment length polymorphism (RFLP) analysis and in some cases RFLP analysis with plasmids pVT2 and pLR7 as probes, and by pulsed-field gel electrophoresis (PFGE). PCR typing of single colonies was also used to detect polyclonal infections. Strains belonged mainly to serotypes 1, 4, and 8. pVT2- and pLR7-related plasmids were detected in strains from 49% of the patients. The IS1245 RFLP and PFGE analyses showed a 96.8% diversity of the M. avium strains from the 93 patients. The vast majority (95.2%) of infections were monoclonal, indicating that recent infection is unlikely, even at an advanced stage of AIDS. For one patient, sequential isolates gave divergent patterns of sensitivity and resistance to clarithromycin, but all were identified as the initial clone. RFLP analysis and PCR typing of single colonies allowed for the detection of three polyclonal infections during the bacteriological follow-up. Among strains from patients whose samples were positive by culture after treatment for 2 to 15 months, 97.4% were the same as the initial strain. In conclusion, relapses and failures were mostly due to the initial strain. These relapses and failures resulted either from the selection of resistant mutants or the reappearance of sensitive strains, suggesting the persistence of nonsterilized tissue reservoirs.