Leptospira interrogans biofilm transcriptome highlights adaption to starvation and general stress while maintaining virulence.

Life-threatening Leptospira interrogans navigate a dual existence: surviving in the environment and infecting mammalian hosts. Biofilm formation is presumably an important survival strategy to achieve this process. Understanding the relation between biofilm and virulence might improve our comprehension of leptospirosis epidemiology. Our study focused on elucidating Leptospira's adaptations and regulations involved in such complex microenvironments. To determine the transcriptional profile of Leptospira in biofilm, we compared the transcriptomes in late biofilms and in exponential planktonic cultures. While genes for motility, energy production, and metabolism were downregulated, those governing general stress response, defense against metal stress, and redox homeostasis showed a significant upsurge, hinting at a tailored defensive strategy against stress. Further, despite a reduced metabolic state, biofilm disruption swiftly restored metabolic activity. Crucially, bacteria in late biofilms or resulting from biofilm disruption retained virulence in an animal model. In summary, our study highlights Leptospira's adaptive equilibrium in biofilms: minimizing energy expenditure, potentially aiding in withstanding stresses while maintaining pathogenicity. These insights are important for explaining the survival strategies of Leptospira, revealing that a biofilm lifestyle may confer an advantage in maintaining virulence, an understanding essential for managing leptospirosis across both environmental and mammalian reservoirs.

Climate-driven models of leptospirosis dynamics in tropical islands from three oceanic basins.

BACKGROUND: Leptospirosis is a neglected zoonosis which remains poorly known despite its epidemic potential, especially in tropical islands where outdoor lifestyle, vulnerability to invasive reservoir species and hot and rainy climate constitute higher risks for infections. Burden remains poorly documented while outbreaks can easily overflow health systems of these isolated and poorly populated areas. Identification of generic patterns driving leptospirosis dynamics across tropical islands would help understand its epidemiology for better preparedness of communities. In this study, we aim to model leptospirosis seasonality and outbreaks in tropical islands based on precipitation and temperature indicators. METHODOLOGY/PRINCIPAL FINDINGS: We adjusted machine learning models on leptospirosis surveillance data from seven tropical islands (Guadeloupe, Reunion Island, Fiji, Futuna, New Caledonia, and Tahiti) to investigate 1) the effect of climate on the disease's seasonal dynamic, i.e., the centered seasonal profile and 2) inter-annual anomalies, i.e., the incidence deviations from the seasonal profile. The model was then used to estimate seasonal dynamics of leptospirosis in Vanuatu and Puerto Rico where disease incidence data were not available. A robust model, validated across different islands with leave-island-out cross-validation and based on current and 2-month lagged precipitation and current and 1-month lagged temperature, can be constructed to estimate the seasonal dynamic of leptospirosis. In opposition, climate determinants and their importance in estimating inter-annual anomalies highly differed across islands. CONCLUSIONS/SIGNIFICANCE: Climate appears as a strong determinant of leptospirosis seasonality in tropical islands regardless of the diversity of the considered environments and the different lifestyles across the islands. However, predictive and expandable abilities from climate indicators weaken when estimating inter-annual outbreaks and emphasize the importance of these local characteristics in the occurrence of outbreaks.

Ertapenem Supplemented Selective Media as a New Strategy to Distinguish ß-Lactam-Resistant Enterobacterales: Application to Clinical and Wastewater Samples.

The increase in carbapenem-resistant Enterobacterales (CRE) is mostly driven by the spread of carbapenemase-producing (CP) strains. In New Caledonia, the majority of carbapenemases found are IMP-type carbapenemases that are difficult to detect on routine selective media. In this study, a culture-based method with ertapenem selection is proposed to distinguish non-CRE, non-CP-CRE, and CP-CRE from samples with very high bacterial loads. Firstly, assays were carried out with phenotypically well-characterized ß-lactam-resistant Enterobacterales isolates. Then, this approach was applied to clinical and environmental samples. Presumptive CP-CRE isolates were finally identified, and the presence of a carbapenemase was assessed. In a collection of 27 phenotypically well-characterized ß-lactam-resistant Enterobacterales, an ertapenem concentration of 0.5 µg·mL-1 allowed distinguishing CRE from non-CRE. A concentration of 4 µg·mL-1 allowed distinguishing CP-CRE from non-CP-CRE after nine hours of incubation. These methods allowed isolating 18 CP-CRE from hospital effluents, including the first detection of a KPC in New Caledonia. All these elements show that this cost-effective strategy to distinguish ß-lactam-resistant Enterobacterales provides fast and reliable results. This could be applied in the Pacific islands or other resource-limited settings, where limited data are available.

Corrigendum: Escape of TLR5 recognition by Leptospira spp.: A rationale for atypical endoflagella.

[This corrects the article DOI: 10.3389/fimmu.2020.02007.].

A fusidic acid-resistant (PVL+) clone is associated with the increase in methicillin-resistant Staphylococcus aureus in New Caledonia.

OBJECTIVES: Since 2014, Staphylococcus aureus methicillin resistance has been rapidly increasing in New Caledonia and is associated with potential serious clinical repercussions. In the present study, we investigated the epidemiology of methicillin-resistant S. aureus (MRSA) in New Caledonia and the possible emergence of a particular clonal strain. METHODS: An overview of the distribution of MRSA in New Caledonia in 2019 is presented. We collected and analysed 171 clinical MRSA isolates from New Caledonia medical laboratories during August and September 2019. Among this collection, 49 representative isolates were analyzed by the French National Reference Center for Staphylococci using the StaphyType DNA microarray, allowing genetic characterization of the isolates. RESULTS: Among the 1144 S. aureus isolated over the year 2019, 442 isolates (39%) were resistant to methicillin, and 62% of these isolates were resistant to fusidic acid (FA). During the inclusion period, FA resistance rate was similar (60%). Genetic characterization evidenced CC6 as the predominant clonal complex (70%) with 26 isolates (53%) identified as CC6-MRSA-[IV+fus] (PVL+). CONCLUSIONS: These findings demonstrated a low diversity of MRSA in New Caledonia, with the dominance of a clonal complex not reported previously. The frequent fusidic acid (FA) resistance in MRSA was associated with a high prevalence of fusC gene, suggesting that FA misuse contributed to driving the selection of this clone. Our findings suggest the recommendation to stop the topical use of FA to control the emergence of this severe MRSA clone and decrease the rate of MRSA in New Caledonia.

Original Leptospira spp. in island's native terrestrial mammals: A case study in Pteropus spp. bats of New Caledonia.

Leptospirosis is a bacterial zoonosis that occurs in tropical and subtropical regions worldwide. Chiroptera are known to be a formidable reservoir of zoonotic pathogens, including leptospires. The epidemiology of leptospirosis in bats in the Pacific Islands is poorly known, both in terms of prevalence and in terms of the bacterial strains involved. A strong host specificity between leptospiral strains and their mammalian reservoir is recognized. This phenomenon has notably been studied recently in bat communities, providing strong evidence of co-evolution. In New Caledonia, a biodiversity hotspot where leptospirosis is endemic and enzootic, Chiroptera are the only indigenous terrestrial mammals. In this study, we aimed to investigate leptospires associated with three flying fox species in New Caledonia. Kidneys and urine samples of Pteropus spp. from captures and seizures were analysed. Among 254 flying foxes analysed, 24 harboured pathogenic leptospires corresponding to an observed prevalence of 9.45% with 15.8% on the Main Island and 4.3% on Loyalty Islands. The analysis of the rrs gene, lfb1, and MLST sequences evidenced four distinct clusters of undescribed strains, likely corresponding to undescribed species. All four strains belong to the Group I of pathogenic Leptospira spp., which includes Leptospira interrogans, Leptospira noguchii, and Leptospira kirschneri. We detected pathogenic leptospires in all three Pteropus spp. studied (including two endemic species) with no evidence of host specificity in two co-roosting species. For a better understanding of Leptospira-host co-evolution, notably to genetically characterize and evaluate the virulence of these original bat-associated leptospires, it is essential to improve isolation techniques. Flying foxes are traditionally hunted and eaten in New Caledonia, a massive cause of bat-human interactions. Our results should encourage vigilance during these contacts to limit the spillover risk of these pathogens to humans.

Unraveling the invisible leptospirosis in mainland Southeast Asia and its fate under climate change.

Leptospirosis is a neglected waterborne zoonosis of growing concern in tropical and low-income regions. Endemic in Southeast Asia, its distribution and environmental factors such as climate controlling its dynamics remain poorly documented. In this paper, we investigate for the first time the current and future leptospirosis burden at a local scale in mainland Southeast Asia. We adjusted machine-learning models on incidence reports from the Thai surveillance system to identify environmental determinants of leptospirosis. The explanatory variables tested in our models included climate, topographic, land cover and soil variables. The model performing the best in cross-validation was used to estimate the current incidence regionally in Thailand, Myanmar, Cambodia, Vietnam and Laos. It then allowed to predict the spatial distribution of leptospirosis future burden from 2021 to 2100 based on an ensemble of CMIP6 climate model projections and 4 Shared Socio-economics Pathways ranging from the most optimistic to the no-climate policy outcomes (SSP1-2.6, SSP2-4.5, SSP3-7.0 and SSP5-8.5). Leptospirosis incidence was best estimated by 10 environmental variables: four landscape-, four rainfall-, two temperature-related variables. Of all tested scenario, the worst-case scenario of climate change (SSP5-8.5) surprisingly appeared as the best-case scenario for the future of leptospirosis since it would induce a significant global decline in disease incidence in Southeast Asia mainly driven by the increasing temperatures. These global patterns are however contrasted regionally with some regions showing increased incidence in the future. Our work highlights climate and the environment as major drivers of leptospirosis incidence in Southeast Asia. Applying our model to regions where leptospirosis is not routinely monitored suggests an overlooked burden in the region. As our model focuses on leptospirosis responses to environmental drivers only, some other factors, such as poverty, lifestyle or behavioral changes, could further influence these estimated future patterns.

Clinical Evaluation of the Modified Faine Criteria in Patients Admitted with Suspected Leptospirosis to the Territorial Hospital, New Caledonia, 2018 to 2019.

Leptospirosis is endemic in New Caledonia. Clinical diagnosis is often difficult and its evolution can be fatal. Leptospirosis requires specific management before biological confirmation. Modified Faine criteria (Faine Score) have been suggested to diagnose leptospirosis on epidemiological (parts A and B) and biological (part C) criteria. The main objective of our study was to assess the relevance of the epidemiological-clinical modified Faine score, parts A and B (MF A + B), in patients with suspected leptospirosis in New Caledonia. A monocentric case-control study was conducted in suspect patients for whom a Leptospira polymerase chain reaction (PCR) test was done within the first 7 days of signs onset at the tertiary hospital from January 2018 to January 2019. Cases and control subjects were matched 1:2 in the gender and age categories. Bivariate, and then multivariable, analyses studied the association between the MF A + B score and a positive Leptospira PCR test, adjusted on the variables retained. In all, 35 cases and 70 control subjects matched for age and gender were analyzed. Multivariable analysis by logistic regression found a significant association between an MF A + B score taken from the categories "possible leptospirosis" (score, 20-25) and "presumed leptospirosis" (score, > 26), and the case or control subject status (P < 0.0001). Model performance was high, with an area under the curve value of 99.27%, 93.55% sensitivity, and 96.36% specificity, which classified subjects correctly in 95.35% of cases. Our study suggests using the MF A + B score to identify possible cases of leptospirosis and initiate antibiotic therapy before biological confirmation in New Caledonia. This score should be evaluated in areas where more differential diagnoses exist and where PCR is not widely available.

Prevention and control of highly antibiotic-resistant bacteria in a Pacific territory: Feedback from New Caledonia between 2004 and 2020.

OBJECTIVE: Carbapenemase-producing Enterobacteriaceae (CRE) and Enterococcus faecium resistant to vancomycin (VRE) constitute major threats to public health worldwide. The Pacific area is concerned and has implemented strategies to control antimicrobial resistance (AMR). However, accurate epidemiological data are rarely reported. Our study aimed to present the strategies applied to prevent and control the spread of highly resistant bacteria in the Pacific territory of New Caledonia. PATIENTS AND METHODS: Cohort prospective study of all cases of highly resistant bacteria (HRB) isolated in New Caledonia from September 2004 to December 2020. Evaluation of the impact of the infection control measures implemented in healthcare settings: screening strategy, cohorting unit, IT tools and control of antibiotic prescriptions. RESULTS: A total of 346 patients with HRB were identified. Most of them (63.0%) were infected or colonized by VRE (n=218) and 128 by CRE. While the number of CREs significantly increased from 2013 to 2020 (P<0.0001), control procedures have limited their dissemination. Most patients were colonized by IMP-4-CRE (n=124/128). The incidence density of VRE significantly decreased from 38.52 for 100,000 hospitalisation-days in 2015 to 4.19 for 100,000 hospitalisation-days in 2019 due to systematic screening of patients before sanitary repatriation from Australia and cohorting implementation. The risk of VRE diffusion is now well under control. CONCLUSIONS: Our study confirms that it is possible to control the spread of AMR in a circumscribed territory by means of a global control strategy involving screening, cohorting unit, IT tools and antibiotic prescription controls.

Leptospira ainlahdjerensis sp. nov., Leptospira ainazelensis sp. nov., Leptospira abararensis sp. nov. and Leptospira chreensis sp. nov., four new species isolated from water sources in Algeria.

Leptospira strains were isolated from freshwater sampled at four sites in Algeria and characterized by whole-genome sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The cells were spiral-shaped and motile. Phylogenetic and MALDI-TOF MS analyses showed that the strains can be clearly distinguished from the other described species in the genus Leptospira, therefore representing two novel species of the pathogen subclade P1 and two novel species of the saprophyte subclade S1. The names Leptospira ainlahdjerensis sp. nov. (type strain 201903070T=KIT0297T=CIP111912T), Leptospira ainazelensis sp. nov. (201903071T=KIT0298T=CIP111913T), Leptospira abararensis sp. nov. (201903074T=KIT0299T=CIP111914T) and Leptospira chreensis (201903075T=KIT0300T=CIP111915T) are proposed.

Eosinophilic meningitis in New Caledonia: The role of Angiostrongylus cantonensis?

INTRODUCTION: Eosinophilic meningitis is a rare form of meningitis with sequelae or death occurring in approximately 2-3% of cases. The most frequent etiological agent is the parasite Angiostrongylus cantonensis. The aim of this study was to characterize New Caledonian cases and to assess the extent to which of A. cantonensis was involved. MATERIAL AND METHODS: We performed a retrospective study of all cases of eosinophilic meningitis (EM) admitted to the Territorial Hospital of New Caledonia, from 2004 to 2019. We performed a descriptive and a multivariate analysis to identify association of variables with severe and fatal cases (or cases with sequelae). CONCLUSION: Angiostrongyliasis was confirmed as being responsible for 17 of the 92 reported EM cases in New Caledonia from 2004 to 2019 with most being young adults and non-walking infants, and with two peaks of incidence one during the dry season and one during the rainy season. Considering the high incidence and regularity of cases, the potential reservoirs should be identified to target prevention campaigns.

Ending the Neglect of Treatable Bacterial Zoonoses Responsible for Non-Malaria Fevers.

Bacterial zoonotic diseases such as leptospirosis, Q fever, melioidosis, spotted fever group rickettsioses, and brucellosis are increasingly recognized causes of non-malaria acute fevers. However, though readily treatable with antibiotics, these diseases are commonly misdiagnosed resulting in poor outcomes in patients. There is a considerable deficit in the understanding of basic aspects of the epidemiology of these neglected diseases and diagnostic tests for these zoonotic bacterial pathogens are not always available in resource-poor settings. Raising awareness about these emerging bacterial zoonoses is directly beneficial to the patients by allowing a test-and-treat approach and is essential to control these life-threatening diseases.

Age-specific epidemiology of human leptospirosis in New Caledonia, 2006-2016.

With over one million cases worldwide annually and a high fatality in symptomatic forms, human leptospirosis is a growing public health concern for the most vulnerable populations, especially in the context of global warming and unplanned urbanization. Although the Asia-Pacific region is particularly affected, accurate epidemiological data are often lacking. We conducted an eleven-year retrospective laboratory-based epidemiological survey of human leptospirosis in New Caledonia. From 2006 to 2016, 904 cases were laboratory-confirmed, including 29 fatalities, corresponding to an average annual incidence of 30.6/100,000 and a case fatality rate of 3.2%. Over the period, there was a major shift from indirect serological diagnosis by MAT to direct diagnosis by real-time PCR, a more specific and sensitive test when performed early in the course of the disease. The systematic implementation of genotyping informed on the variety of the infective strains involved, with a predominance of serogroups Icterohaemorrhagiae and Pyrogenes. The epidemiological pattern showed a marked seasonality with an annual peak in March-April. Interestingly, the seasonal peak in children of school age was significantly earlier and corresponded to school holidays, suggesting that attending school from February on could protect children from environment-borne leptospirosis.

Escape of TLR5 Recognition by Leptospira spp.: A Rationale for Atypical Endoflagella.

Leptospira (L.) interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagellae that allow their motility. L. interrogans are stealth pathogens that escape the innate immune recognition of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, which senses peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits. To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by qPCR. We did not identify any protective or inflammatory role of murine TLR5 for controlling pathogenic Leptospira. Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5 signaling. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, but did not, or barely induced stimulation via murine TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, after using antimicrobial peptides to destabilize live leptospires, we detected TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in the flagellin proteins, we further showed that neither FlaA nor Fcp participated in the recognition by TLR5, suggesting a role for the FlaB. FlaB have structural homology to Salmonella FliC, and possess conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and in mice downregulated the expression of the FlaB, but not the FlaA subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagellae, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape the TLR5 recognition and the induced immune response.

Leptospira interrogans Retains Direct Virulence After Long Starvation in Water.

Mostly studied as a zoonosis, leptospirosis is also an environment-borne infection and most human cases originate from soil or water contaminations. Yet, only few studies have been interested in the survival of pathogenic Leptospira in freshwater. In this study, water microcosms were designed to evaluate the survival and virulence of Leptospira spp. for 2 years. Four commercial bottled drinking waters and a non-ionized water, all previously filter-sterilized, were studied. Either one of two Leptospira interrogans strains, one Leptospira borgpetersenii strain, or a saprophytic Leptospira biflexa was inoculated in these waters under nutrient-deprived conditions. Molecular, microscopic and cultural approaches were used to study Leptospira survival. Direct virulence of the pathogens was assessed using animal challenge without re-culturing. Our results confirmed the capacity of pathogenic Leptospira to survive for more than a year in water. In addition, we showed the ability of L. interrogans in nutrient-deprived conditions to directly cause systemic infection in susceptible animals. To our knowledge, this is the first report of direct infection of a susceptible host with Leptospira following a long starvation and survival period in nutrient-deprived water. Our results also suggest that Leptospira turned into a physiological "survival" state in harsh freshwater conditions. These data are of prime importance considering that freshwater is a major source of Leptospira infections. Environmental survival and virulence of pathogenic Leptospira spp. are becoming a crucial challenge to determine the environmental risk and adopt relevant prevention and control strategies.

Isolation and Culture of Leptospira from Clinical and Environmental Samples.

Leptospires, the etiological agents of leptospirosis, are fastidious slow-growing organisms. Here we describe the isolation and routine maintenance of leptospires from clinical (blood, urine, or tissue) and environmental (water or soil) samples. Using combinations of filtration, agar plating, and selective agents, leptospires can be isolated in pure cultures even from complex contaminated sources in standard EMJH culture medium.

Use of MALDI-ToF Mass Spectrometry for Identification of Leptospira.

Medical microbiology has used phenotypical and metabolic criteria to identify bacterial pathogens for decades. However, no such criteria have been applied to identify leptospires at the species level. In the recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) has emerged as new tool for the identification of bacterial species in the medical microbiology laboratory. This technology has rapidly gained more and more popularity. Actually, this technique is sensitive and economic, saving both labor and bench costs, but also rapid, significantly reducing turnaround time from isolation to identification. MALDI-ToF MS provides an unprecedented tool for the rapid identification of Leptospira at the species level.

Biofilm Formation and Quantification Using the 96-Microtiter Plate.

Biofilm formation in microtiter plates is certainly the most commonly used method to grow and study biofilm. This simple design is very popular due to its high-throughput screening capacities, low cost, and easy handling. In the protocol described here, we focus on the use of 96-well optically clear, polystyrene flat-bottom plate to study biofilm formation by Leptospira spp. and quantify the biofilm formation by crystal violet (CV) staining. We also describe an alternative method, based on phase contrast image analysis that we believe is more suitable for accurately quantifying biofilm growth by reducing handling of this fragile structure.

The zoonotic pathogen Leptospira interrogans mitigates environmental stress through cyclic-di-GMP-controlled biofilm production.

The zoonotic bacterium Leptospira interrogans is the aetiological agent of leptospirosis, a re-emerging infectious disease that is a growing public health concern. Most human cases of leptospirosis result from environmental infection. Biofilm formation and its contribution to the persistence of virulent leptospires in the environment or in the host have scarcely been addressed. Here, we examined spatial and time-domain changes in biofilm production by L. interrogans. Our observations showed that biofilm formation in L. interrogans is a highly dynamic process and leads to a polarized architecture. We notably found that the biofilm matrix is composed of extracellular DNA, which enhances the biofilm's cohesiveness. By studying L. interrogans mutants with defective diguanylate cyclase and phosphodiesterase genes, we show that biofilm production is regulated by intracellular levels of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) and underpins the bacterium's ability to withstand a wide variety of simulated environmental stresses. Our present results show how the c-di-GMP pathway regulates biofilm formation by L. interrogans, provide insights into the environmental persistence of L. interrogans and, more generally, highlight leptospirosis as an environment-borne threat to human health.