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

Cellular aggregation and biofilm formation in Leptospira is a recently studied phenomenon in both in vitro and in vivo systems.^1,2 However, few studies on this phenomenon have been reported that may have strong implications on the pathogenesis of Leptospira in human host. Antibiotics at subminimal inhibitory concentrations (sub-MICs) were found to induce biofilm formation in numerous other bacteria in vitro. The process of antibiotic-induced biofilm formation may have clinical significance as bacteria are exposed to sub-MIC levels at different treatment periods, in the beginning and end of a dosing regimen, between doses, or continuously during a low-dose therapy. ³ Recently, Wu et al. ⁴ reported that during leptospiral infection in a hamster model, low dose of norfloxacin and ciprofloxacin increased the bacterial loads and aggravated the tissue injury of target organs. This may be due to the antibiotic-induced biofilm formation.

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 antibiotics tested here are the drugs of choice for leptospirosis. Stock solutions of 1 mg/ml concentration of antibiotics, including tetracycline and doxycycline (Sigma-Aldrich), were prepared with reagent grade powders in Milli Q water as solvents as per the National Committee for Clinical Laboratory Standards document, M100-S24-CLSI 2014 recommendation. The MIC values of two antibiotics against 14 leptospiral strains were measured using the broth microdilution method. To quantify the amount of biofilm induction, 200 μl of Ellinghausen–McCullough–Johnson–Harris (EMJH) liquid medium was inoculated with leptospiral strains (2 × 10² cells/ml) in U-bottomed 96-well (polyvinyl plate) microtiter plates containing serial twofold dilutions ranging from 0.015 to 16 μg/ml. Similarly, the strains were cultured in 5 ml EMJH, containing serial twofold dilutions of antibiotics in 10 ml screw cap tubes. Control tubes or wells were maintained without the antibiotic. The autoaggregation of leptospiral cells was observed under 20× objective of a dark field microscope. The biofilm in a 96-well plate was quantified using the crystal violet method. For the enumeration of Leptospira cell, biofilms were rinsed twice with sterile phosphate-buffered saline to remove loosely adherent cells. Attached cells were scraped and vortexed for 15 min. DNA was extracted from the sediment by using QIAamp DNA Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. The quantitative real-time PCR assay (SYBR Green method) was performed by targeting the lipL32 gene, as described earlier. ⁵

In the absence of antibiotics, the 14 strains exhibited a range of biofilm phenotypes in a 96-well microtiter plate using crystal violet binding assay (Table 1). The range of MIC values for these strains was 4–8 μg/ml for doxycycline and 2–4 μg/ml for tetracycline. The induction of biofilm by sub-MIC level of tetracycline and doxycycline was both strain and antibiotic dependent. The biofilm induction values for six strains were 0.89–0.99 for tetracycline and 0.86–0.99 for doxycycline (Table 1). This suggests that biofilm induction was not observed at subinhibitory concentrations of these antibiotics for the six strains. However, for the other eight strains, the induction values were 1.3–10.3 for tetracycline and 1.3–5.5 for doxycycline. The maximum biofilm induction was observed at sub-MIC, ranging from 0.062 to 0.125 μg/ml for tetracycline and from 0.062 to 0.250 μg/ml for doxycycline. These values represent 1/32–1/64 and 1/16–1/64 of the MICs for the respective antibiotics. In the absence of antibiotics, few leptospiral strains including MT-10, But-6, Poi, Paidjain, and Salinem demonstrated a higher ability to form biofilm, in vitro, whereas few other strains, such as RGA, Pomona, Ballico, and CH-31 showed a moderate ability. At sub-MIC levels of antibiotics, these strains did not show any significant effect on biofilm induction. However, the strains that had lower ability to form biofilm in vitro, including Moskva V, Hond utrech-IV, Bankinang 1, L-14, and 507, demonstrated an enhanced biofilm induction. Among these, L-14 and 507 had the maximum biofilm induction value of 5–10. It is evident from our previous report that strains such as MT-10 and But-6 that showed maximum biofilm formation are resistant to tetracycline/doxycycline. ²

Biofilm induction can be triggered by a stress condition caused by low level of antibiotics. Once formed, the amount of antibiotic required for killing the bacteria would be many times higher than the concentration needed to kill “Planktonic” cells. This has been reported earlier for Leptospira biofilm, where the minimum bactericidal concentrations of antibiotics was six- to sevenfold higher (>1,600 μg/ml) than the planktonic cells (25–100 μg/ml). ²

This is the first report demonstrating the effect of sub-MIC level of antibiotic 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. ³ 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. This may lead to better treatment and management of patients and help in appropriate antibiotic therapy. Such knowledge is valuable for the development of strategies to enhance the efficacy of antibiotics, especially for the treatment of leptospirosis.