Genomic insights into the c-di-GMP signaling and biofilm development in the saprophytic spirochete Leptospira biflexa.

C-di-GMP is a bacterial second messenger with central role in biofilm formation. Spirochete bacteria from Leptospira genus present a wide diversity, with species of medical importance and environmental species, named as saprophytic. Leptospira form biofilms in the rat's reservoir kidneys and in the environment. Here, we performed genomic analyses to identify enzymatic and effector c-di-GMP proteins in the saprophytic biofilm-forming species Leptospira biflexa serovar Patoc. We identified 40 proteins through local alignments. Amongst them, 16 proteins are potentially functional diguanylate cyclases, phosphodiesterases, or hybrid proteins. We also identified nine effectors, including PilZ proteins. Enrichment analyses suggested that c-di-GMP interacts with cAMP signaling system, CsrA system, and flagella assembly regulation during biofilm development of L. biflexa. Finally, we identified eight proteins in the pathogen Leptospira interrogans serovar Copenhageni that share high similarity with L. biflexa c-di-GMP-related proteins. This work revealed proteins related to c-di-GMP turnover and cellular response in Leptospira and their potential roles during biofilm development.

Environmental Biofilms from an Urban Community in Salvador, Brazil, Shelter Previously Uncharacterized Saprophytic Leptospira.

Biofilms are complex microecosystems with valuable ecological roles that can shelter a variety of microorganisms. Spirochetes from the genus Leptospira have been observed to form biofilms in vitro, in rural environments, and in the kidneys of reservoir rats. The genus Leptospira is composed of pathogenic and non-pathogenic species, and the description of new species is ongoing due to the advent of whole genome sequencing. Leptospires have increasingly been isolated from water and soil samples. To investigate the presence of Leptospira in environmental biofilms, we collected three distinct samples of biofilms formed in an urban setting with poor sanitation: Pau da Lima, in Salvador, Bahia, Brazil. All biofilm samples were negative for the presence of pathogenic leptospires via conventional PCR, but cultures containing saprophytic Leptospira were identified. Whole genomes were generated and analyzed for twenty isolates obtained from these biofilms. For species identification, we used digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analysis. The obtained isolates were classified into seven presumptive species from the saprophytic S1 clade. ANI and dDDH analysis suggest that three of those seven species were new. Classical phenotypic tests confirmed the novel isolated bacteria as saprophytic Leptospira. The isolates presented typical morphology and ultrastructure according to scanning electron microscopy and formed biofilms under in vitro conditions. Our data indicate that a diversity of saprophytic Leptospira species survive in the Brazilian poorly sanitized urban environment, in a biofilm lifestyle. We believe our results contribute to a better understanding of Leptospira biology and ecology, considering biofilms as natural environmental reservoirs for leptospires.

Structural and functional characterization of an orphan ATP-binding cassette ATPase involved in manganese utilization and tolerance in Leptospira spp.

Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn(2+), we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn(2+), suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn(2+) toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg(2+)-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/ß subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase.

Standardization of chronic uterine infection of hamsters by Leptospira santarosai serovar Guaricura, from serogroup Sejroe.

Leptospirosis is an important zoonosis that in cattle is characterized as a reproductive disease. It is well reported that the main agent of bovine leptospirosis worldwide is Sejroe serogroup serovar Hardjo. Reproductive disease in cattle has several gaps in its knowledge and studies with Golden Syrian hamsters, experimentally infected, are limited. Therefore, a protocol that could reproduce the chronic genital disease in hamsters would be extremely valuable for the advance of the knowledge of that syndrome. The aim of this study was to establish an experimental protocol for chronic non-lethal genital infection of female hamsters by L. santarosai serovar Guaricura (Sejroe serogroup), strain 2013_VF52. For this, two concentrations (1.0 × 108 leptospires/mL and 1.0 × 104 leptospires/mL) were used intraperitoneally in female hamsters of 06-08 weeks of age. Hamsters that survived for up to forty days after inoculation were euthanized. Uterine and renal tissues were collected to evaluate leptospires' presence by PCR and culture. The protocol demonstrated that 1.0 × 104 leptospires/mL of the strain determined chronic genital leptospirosis in the hamster model. The standardization of a protocol for chronic genital leptospirosis in hamsters can be extremely useful for the understanding of the physiopathology of the infection, as the distribution of leptospires in the uterus and the agent-host interactions.

Simplified sewerage to prevent urban leptospirosis transmission: a cluster non-randomised controlled trial protocol in disadvantaged urban communities of Salvador, Brazil.

INTRODUCTION: Leptospirosis is a globally distributed zoonotic and environmentally mediated disease that has emerged as a major health problem in urban slums in developing countries. Its aetiological agent is bacteria of the genus Leptospira, which are mainly spread in the urine of infected rodents, especially in an environment where adequate sanitation facilities are lacking, and it is known that open sewers are key transmission sources of the disease. Therefore, we aim to evaluate the effectiveness of a simplified sewerage intervention in reducing the risk of exposure to contaminated environments and Leptospira infection and to characterise the transmission mechanisms involved. METHODS AND ANALYSIS: This matched quasi-experimental study design using non-randomised intervention and control clusters was designed to assess the effectiveness of an urban simplified sewerage intervention in the low-income communities of Salvador, Brazil. The intervention consists of household-level piped sewerage connections and community engagement and public involvement activities. A cohort of 1400 adult participants will be recruited and grouped into eight clusters consisting of four matched intervention-control pairs with approximately 175 individuals in each cluster in baseline. The primary outcome is the seroincidence of Leptospira infection assessed through five serological measurements: one preintervention (baseline) and four postintervention. As a secondary outcome, we will assess Leptospira load in soil, before and after the intervention. We will also assess Leptospira exposures before and after the intervention, through transmission modelling, accounting for residents' movement, contact with flooding, contaminated soil and water, and rat infestation, to examine whether and how routes of exposure for Leptospira change following the introduction of sanitation. ETHICS AND DISSEMINATION: This study protocol has been reviewed and approved by the ethics boards at the Federal University of Bahia and the Brazilian National Research Ethics Committee. Results will be disseminated through peer-reviewed publications and presentations to implementers, researchers and participating communities. TRIAL REGISTRATION NUMBER: Brazilian Clinical Trials Registry (RBR-8cjjpgm).

Leptospira interrogans biofilm formation in Rattus norvegicus (Norway rats) natural reservoirs.

Rattus norvegicus (Norway rat) is the main reservoir host of pathogenic Leptospira, the causative agent of leptospirosis, in urban environments. Pathogenic Leptospira forms biofilms in the environment, possibly contributing for bacterial survival and maintenance. Nonetheless, biofilms have not yet been studied in natural animal reservoirs presenting leptospiral renal carriage. Here, we described biofilm formation by pathogenic Leptospira inside the renal tubules of R. norvegicus naturally infected and captured in an urban slum endemic for leptospirosis. From the 65 rats carrying Leptospira in their kidneys, 24 (37%) presented biofilms inside the renal tubules. The intensity of leptospiral colonization in the renal tubules (OR: 1.00; 95% CI 1.05-1.1) and the type of occlusion pattern of the colonized renal tubules (OR: 3.46; 95% CI 1.20-9.98) were independently associated with the presence of Leptospira biofilm. Our data showed that Leptospira interrogans produce biofilms during renal chronic colonization in rat reservoirs, suggesting a possible role for leptospiral biofilms in the pathogenesis of leptospirosis and bacterial carriage in host reservoirs.

The OmpA-like protein Loa22 is essential for leptospiral virulence.

Pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetic manipulations of pathogenic species. In this study, we characterized a mutant obtained by insertion of the transposon Himar1 into a gene encoding a putative lipoprotein, Loa22, which has a predicted OmpA domain based on sequence identity. The resulting mutant did not express Loa22 and was attenuated in virulence in the guinea pig and hamster models of leptospirosis, whereas the genetically complemented strain was restored in Loa22 expression and virulence. Our results show that Loa22 was expressed during host infection and exposed on the cell surface. Loa22 is therefore necessary for virulence of L. interrogans in the animal model and represents, to our knowledge, the first genetically defined virulence factor in Leptospira species.

Investigation of chronic infection by Leptospira spp. in asymptomatic sheep slaughtered in slaughterhouse.

The study aimed to evaluate the histopathological characteristics of renal lesions in chronically infected sheep and with low titers of anti-Leptospira antibodies from a slaughterhouse. In the serological analysis, 24.74% (48/194) presented seroreactivity with a titer equal to or greater than 100. Among these seroreactive sheep, titers of 100 were predominant (58.33%, 28/48), with the highest titer being 1,600 (2.08%, 1/48). Serogroup Sejroe (sv. Hardjo) was the most frequent at 35.42% (17/48). Leptospiral DNA was verified in 4.12% (8/194) of the kidney samples tested, and no urine sample was positive. All the samples corresponded to the pathogenic species L. interrogans. The eight amplicons with 202-nucleotides were identical with two mismatches (presented 100% of identity) using the PCR targeting to secY gene. Histological sections of PCR-positive kidneys were submitted to direct detection by the anti-LipL32 immunohistochemistry (IHC) technique. The Leptospira spp. antigen was evident in 62.5% (5/8) of the kidneys. Positive staining was observed in the cytoplasm of tubular cells and in the form of brownish aggregates that adhered to tubular epithelial cells and projected into the lumen. Inflammatory lymphoplasmacytic infiltrate, ranging from mild to moderate, with multifocal distribution, was the predominant finding in seroreactive animals (33.33%, 16/48). The demonstration of the leptospiral antigen lining the renal tubules through IHC of naturally infected sheep confirmed by PCR characterizes renal colonization in a species with the presence of histological changes compatible with leptospirosis.

An alternative for the preadsorption step in the paratuberculosis serodiagnosis: Mycobacterium fortuitum.

ELISAs for paratuberculosis employ a preadsorption step with Mycobacterium phlei to diminish unspecific reactions As M. fortuitum is one of the most frequent environmental mycobacteria, the purpose of this pilot study was to evaluate its use as an alternative for the preadsorption in ELISAs for paratuberculosis. Results suggest that M. fortuitum can be an alternative instead of or associated to M. phlei with comparable results (κ > 0.8) to conventional ELISAs using M. phlei as a preadsorption antigen.

A serological study on Brucella abortus, caprine arthritis-encephalitis virus and Leptospira in dairy goats in Rio de Janeiro, Brazil.

In spite of the large number of goats found in several developing tropical countries, milk production remains unsatisfactory. The occurrence of infectious diseases, such as leptospirosis, brucellosis and caprine arthritis-encephalitis (CAE) may in part be responsible for sub-optimal production. In this study, 1000 serum samples were tested for leptospirosis, 953 for brucellosis and 562 for CAE. All tested flocks presented at least one seroreactive animal for leptospirosis and for CAE. Reactivity to leptospirosis was 11.1%, and serovar hardjo was the most frequently found. Anti-B. abortus agglutinins were found in 0.5% of the samples presented and 14.1% were seroreactive to CAE. Leptospirosis was considered to represent the major infectious problem in the studied goat flocks. The occurrence of infectious diseases in the tested flocks may represent an important factor contributing to the decreased productivity of the animals. These findings may be similar to those observed in other developing countries and require further study to define the relationship between seropositivity and reduced production.

Diagnosis of paratuberculosis in a dairy herd native to Brazil.

Paratuberculosis (PTB) in Brazil has previously only been reported in imported animals and is officially considered as an exotic disease. A dairy herd, which had no imported animals, presented clinically suspect animals and was investigated for paratuberculosis using faecal culture, histopathology, indirect ELISA and the agar gel immunodiffusion test. Infection with Mycobacterium avium subsp. paratuberculosis (Map) was confirmed by culture of faeces from five cows with clinical symptoms of PTB and in 7/24 randomly selected asymptomatic cows from the same herd. Two cows with clinical symptoms were necropsied and their tissues were positive for Map by culture and histopathology. Twelve asymptomatic, randomly selected cows were positive on ELISA. The results confirmed the presence of PTB in this dairy herd and for the first time demonstrated the disease in a herd of native-bred cattle in Brazil.

Transcriptome Sequencing Reveals Wide Expression Reprogramming of Basal and Unknown Genes in Leptospira biflexa Biofilms.

The genus Leptospira is composed of pathogenic and saprophytic spirochetes. Pathogenic Leptospira is the etiological agent of leptospirosis, a globally spread neglected disease. A key ecological feature of some pathogenic species is their ability to survive both within and outside the host. For most leptospires, the ability to persist outside the host is associated with biofilm formation, a most important bacterial strategy to face and overcome hostile environmental conditions. The architecture and biochemistry of leptospiral biofilms are rather well understood; however, the genetic program underpinning biofilm formation remains mostly unknown. In this work, we used the saprophyte Leptospira biflexa as a model organism to assess over- and underrepresented transcripts during the biofilm state, using transcriptome sequencing (RNA-seq) technology. Our results showed that some basal biological processes like DNA replication and cell division are downregulated in the mature biofilm. Additionally, we identified significant expression reprogramming for genes involved in motility, sugar/lipid metabolism, and iron scavenging, as well as for outer membrane-encoding genes. A careful manual annotation process allowed us to assign molecular functions to many previously uncharacterized genes that are probably involved in biofilm metabolism. We also provided evidence for the presence of small regulatory RNAs in this species. Finally, coexpression networks were reconstructed to pinpoint functionally related gene clusters that may explain how biofilm maintenance is regulated. Beyond elucidating some genetic aspects of biofilm formation, this work reveals a number of pathways whose functional dissection may impact our understanding of leptospiral biology, in particular how these organisms adapt to environmental changes. IMPORTANCE In this work, we describe the first transcriptome based on RNA-seq technology focused on studying transcriptional changes associated with biofilm growth in a member of the genus Leptospira. As many pathogenic species of this genus can survive inside the host but also persist in environmental water, mostly forming biofilms, identifying the molecular basis of this capacity can impact the understanding of how leptospires are able to fulfill a complete life cycle that alternates between adaptation to the host and adaptation to hostile external environmental conditions. We identified several genes and regulatory networks that can be the kickoff for deepening understanding of the molecular mechanisms involving bacterial persistence via biofilm formation; understanding this is important for the future development of tools for controlling leptospirosis.

Heterogenic colonization patterns by Leptospira interrogans in Rattus norvegicus from urban slums.

We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.

Molecular characterization of the first leptospires isolated from goats in Brazil.

Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.

Serological trail of Brucella infection in an urban slum population in Brazil.

INTRODUCTION: Brucellosis is a re-emerging zoonosis with new cases reported each year in many Latin American countries, but it is mostly under-recognized. This study presents a serological investigation of infection with Brucella abortus and Brucella canis in a poor urban community in the city of Salvador, Brazil. METHODOLOGY: Human sera (n = 180) were randomly selected from 3,171 samples taken from healthy individuals during 2003-2004 and tested with C-ELISA for B. abortus and I-ELISA for B. canis. RESULTS: Thirteen percent (24/180) of the individuals were positive for B. abortus and 4.6 % (8/174) were positive for B. canis. Among the variables studied only age (older than 45 years) appeared to be a risk factor for the detection of Brucella antibodies. CONCLUSION: These results indicate the presence of Brucella infection in this settlement and highlight the need to understand the epidemiology of infection under these circumstances to establish the necessary measures for surveillance and control.

The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis.

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p<0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.

Heterogenic colonization patterns by Leptospira interrogans in Rattus norvegicus from urban slums

Abstract We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.

Molecular characterization of the first leptospires isolated from goats in Brazil

Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.

Biofilm formation by saprophytic and pathogenic leptospires.

Leptospires exist as saprophytic organisms that are aquatic or as pathogens that are able to survive in water. Leptospirosis is transmitted to humans through environmental surface waters contaminated by the urine of mammals, usually rodents, which are chronically infected by pathogenic strains. The ecology of Leptospira spp. prompted us to evaluate if these spirochaetes were able to form biofilms. This study investigated the characteristics of biofilm development by both saprophytic and pathogenic Leptospira species using microscopic examinations and a polystyrene plate model. Biofilms were formed preferentially on glass and polystyrene surfaces. Electron microscopic images showed cells embedded in an extracellular matrix. The formation of such a biofilm is consistent with the life of saprophytic strains in water and may help pathogenic strains to survive in environmental habitats and to colonize the host.

Evaluation of a rapid slide agglutination test for the diagnosis of acute canine leptospirosis.

The standard method for the diagnostic of leptospirosis is the microscopic agglutination test (MAT). Nevertheless, it is time-consuming and presents risk of infection for laboratory personal. Several attempts to simplify and reduce the time, skill and expense required for serological testing were made. Rapid slide agglutination tests (RSAT) have been used nowadays as a reliable screening test for the detection of acute and recent infections by many laboratories. The purpose of that study was to evaluate a new antigen preparation for the rapid diagnoses of acute canine leptospirosis and to compare it with MAT results in clinically suspect serum samples. Two hundred and thirteen serum samples from dogs of both sexes and different ages were tested and 141 (66.2%) animals were considered as positive by MAT. The most frequent serovar was icterohaemorrhagiae, followed by canicola. RSAT results were very similar to those observed on MAT, with 139 (65.3%) reactive animals. Correlation between RSAT and MAT was positive (0.82) and significant (p < 0.01). Concordance of results was of 93.4% (199/ 213) of the animals correctly diagnosed by RSAT. Considering MAT as the standard test, sensitivity of RSAT was calculated on 94.3% (133/141) and specificity on 91.7% (66/72).