Coaggregation and biofilm formation of Leptospira with Staphylococcus aureus.

It is not known how Leptospira react to wound or a cut infected with microbes, such as pathogenic Staphylococcus, or their common habitat on oral or nasal mucosal membranes. In the present study, Staphylococcus aureus MTCC-737 showed strong co-aggregation with leptospiral strains (>75%, visual score of + 4) in vitro. All tested strains of Leptospira were able to form biofilm with S. aureus. Scanning electron microscopy analysis revealed intertwined networks of attached cells of L. interrogans and S. aureus, thus providing evidence of a matrix-like structure. This phenomenon may have implications in Leptospira infection, which occurs via cuts and wounds of the skin.

Correlation Between Physicochemical Properties of Soil and Presence of Leptospira.

Leptospirosis is an important global public health problem. Favourable environmental factors are influencing the survival of leptospires in soil, which is an important link in the transmission cycle. The present study was designed to understand the correlation between various soil nutrients and presence of Leptospira in soil samples of different regions of Andaman and Nicobar Islands. The study revealed a significant positive relationship between presence of Leptospira and concentration of iron, manganese and copper in soil. Presence of iron, manganese and copper in the soil may influence the survival and transmission of leptospirosis.

Changing trend in the seroprevalence and risk factors of human leptospirosis in the South Andaman Island, India.

Seroprevalence of leptospirosis among a healthy population of the South Andaman Island was assessed through random sampling. Previous studies have high seroprevalences of up to 55% in general population and 65% in agricultural labourers. The study subjects (1,181 in total, 781 rural and 400 urban) were interviewed and tested for antibodies against Leptospira. Multivariate models were developed to determine the risk factors in the rural and the urban population. The overall seroprevalence was 10.9%, with rural (12.9%) being higher than the urban subjects (7.0%). The commonest infecting serogroup was Icterohaemorrhagiae (53.5%), followed by Grippotyphosa (13.2%). Compared to the earlier observation, seroprevalence was lower and an apparent shift in the infecting serogroup was found. This shift was in concordance with the changing trend in animal population. Significant difference in risk factors, both in rural and urban areas, was also observed. Similar trends in seroprevalence are being observed around the world. Therefore, time to time prevalence studies are needed for the development of effective control measure.

Can Subminimal Inhibitory Concentrations of Antibiotics Induce the Formation of Biofilm in Leptospira?

Antibiotics at subminimal inhibitory concentrations (sub-MICs) are known to induce biofilm formation in numerous bacteria in vitro. In this report, the effect of sub-MIC levels of antibiotics (doxycycline and tetracycline) on biofilm formation by leptospiral reference strains and isolates was investigated. The sub-MIC levels of both tetracycline and doxycycline were able to induce biofilm in some of the leptospiral strains. This is the first report demonstrating the effect of sub-MIC level of antibiotics in inducing biofilm formation in Leptospira. The induction of biofilm may solely be a response to the amount of threshold stress enforced by low levels of antibiotics. The mechanism of biofilm induction by subinhibitory antibiotic concentrations needs to be explored further. Studies are required to understand the clinical relevance of the phenomenon and its contribution to biofilm formation in the host, resulting in the failure of antimicrobial therapy during the treatment of chronic leptospirosis.

Overexpression of heat shock GroEL stress protein in leptospiral biofilm.

Leptospira is the causative agent of leptospirosis, which is an emerging zoonotic disease. Recent studies on Leptospira have demonstrated biofilm formation on abiotic surfaces. The protein expressed in the biofilm was investigated by using SDS-PAGE and immunoblotting in combination with MALDI-TOF mass spectrometry. The proteins expressed in Leptospira biofilm and planktonic cells was analyzed and compared. Among these proteins, one (60 kDa) was found to overexpress in biofilm as compared to the planktonic cells. MALDI-TOF analysis identified this protein as stress and heat shock chaperone GroEL. Our findings demonstrate that GroEL is associated with Leptospira biofilm. GroEL is conserved, highly immunogenic and a prominent stress response protein in pathogenic Leptospira spp., which may have clinical relevance.

Prevalence and Diversity of Leptospires in Different Ecological Niches of Urban and Rural Areas of South Andaman Island.

Leptospirosis is an emerging disease around the globe. South Andaman Island is an endemic region for leptospirosis. We herein compared the prevalence of leptospires in urban and rural areas of South Andaman Island. The PCR detection and isolation of Leptospira revealed that pathogenic leptospires were prevalent in sewage water and household drainage water in urban areas and in paddy fields, vegetable field water, and stream water in rural areas. These results demonstrate that intermediates are ubiquitously present in the environment and may be responsible for asymptomatic infections, and also provide an insight into disease ecology.

In Vitro Antimicrobial Susceptibility of Pathogenic Leptospira Biofilm.

Pathogenic Leptospira spp. are the causative agent of leptospirosis. Biofilm formation in leptospires is a new area of study, and its role in pathogenesis is not fully explored. As in other biofilm-forming bacteria, Leptospira biofilm may play a significant role in antibiotic resistance. In this study, the antimicrobial susceptibility of Leptospira biofilm was investigated by 96-well plate assay using Alamar Blue. Leptospira biofilm showed five to sixfold increase in resistance in all the strains used. The range of minimal bactericidal concentrations for penicillin G, ampicillin, tetracycline, and doxycycline was 1,600 U/ml, 800-1,600 µg/ml, 800-1,600 µg/ml, and 800-1,600 µg/ml, respectively. In agar substrate, the biofilm showed six- to sevenfold increase in resistance to antibiotics compared to planktonic cell. The present study emphasizes the importance of biofilm formation and its antibiotic susceptibility patterns. This could pave the way for devising appropriate strategy to prevent the occurrence of potential chronic leptospirosis in endemic areas and also during an outbreak situation.

GC/MS profiling, in vitro anti-leptospiral and haemolytic activities of Boesenbergia rotunda (L.) Mansf. used as a medicinal plant by Nicobarese of Andaman and Nicobar Islands.

Leaves of the plant Boesenbergia rotunda are used by the Nicobarese tribe of Andaman and Nicobar Islands, India, to prepare traditional medicine for treating fever, headache and body ache. In the present investigation, methanol fraction of these leaves were analysed by GC/MS that revealed the presence of 25 compounds. The anti-leptospiral activity of methanol crude extract was determined by both microdilution and macrodilution methods. The MICs of the extract were tested against 24 pathogenic leptospiral strains and ranged between 62.5-125 µg/mL in both microdilution and macrodilution. The range of MBCs was 250 and 500 µg/mL in macrodilution and microdilution respectively. The crude extract was subjected to cytotoxic studies and found to have negligible or no haemolytic activity, exhibiting IC50 values of greater than 4 mg/mL. Further in vivo studies are needed to investigate the pharmacological and toxicological properties of Boesenbergia rotunda, before it can be considered as a new anti-leptospiral agent.

Coexistence and survival of pathogenic leptospires by formation of biofilm with Azospirillum.

Pathogenic Leptospira spp. represent one cause of leptospirosis worldwide and have long been regarded as solitary organisms in soil and aquatic environments. However, in the present study, Leptospira interrogans was observed to be associated with environmental biofilms with 21 bacterial isolates belonging to 10 genera. All 21 isolates were examined for their coaggregation and biofilm-forming ability with leptospires in vitro. Among these, Azospirillum brasilense RMRCPB showed maximum interspecies coaggregation with leptospiral strains (>75%, visual score of +4). Other significant coaggregating isolates belonged to the genera Sphingomonas, Micrococcus, Brevundimonas, Acinetobacter and Paracoccus. Biofilms of leptospires in combination with A. brasilense RMRCPB showed high resistance to penicillin G, ampicillin and tetracycline (minimum bactericidal concentration ≥800 µg/mL) and tolerance to UV radiation and high temperature (up to 49°C). This study hypothesized that biofilm formation with A. brasilense protects the pathogenic Leptospira from adverse environmental conditions/stress. This coexistence of pathogenic Leptospira with other bacteria may be the key factor for its persistence and survival. However, the mechanism of biofilm formation by leptospires needs to be explored to help devise an appropriate control strategy and reduce transmission of leptospires.