Rachna Singh

Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study.

The presence of persister cells and small-colony variants (SCVs) has been associated with enhanced antibiotic resistance of many organisms in biofilms. This study investigated whether persisters and/or SCVs contribute to the antibiotic resistance of Staphylococcus aureus biofilms. A detailed dose-dependent killing of biofilms and planktonic cells with five antibiotics (oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin) was analysed by treating them with each antibiotic at a concentration of 0-100 microg ml(-1) at 37 degrees C for 48 h. The killing of biofilm cells by all of the antibiotics showed the presence of persister cells - most cells in the population died, leaving a fraction that persisted, even at higher concentrations of the antibiotics. These persisters represented a transient resistant phenotype and reverted to a killing curve resembling that of the wild-type parent upon re-exposure to the antibiotics. SCVs were observed in biofilms only after treatment with ciprofloxacin, and these SCVs were of a transient nature. The treatment of planktonic cells with oxacillin, cefotaxime, ciprofloxacin and vancomycin killed the entire population and no persisters were detected. Transient SCVs, observed in planktonic cells following exposure to these antibiotics, were killed at higher antibiotic concentrations. The treatment of planktonic cells with amikacin yielded a small subpopulation of survivors that included persisters (at numbers significantly lower than for the biofilms) and highly resistant, stable SCVs with an increased biofilm-forming capacity in comparison with the wild-type parent. Thus the high resistance of S. aureus biofilms to multiple unrelated antibiotics is largely dependent on the presence of persister cells. Biofilms harbour a large number of persisters in comparison with planktonic cultures, which either do not harbour persisters or harbour only a small number. SCVs, although not specifically associated with S. aureus biofilms, have an increased biofilm-forming capacity and this may explain the frequent isolation of SCVs from biofilm-associated infections. The intrinsic resistance of these variants may in turn contribute to the enhanced antibiotic resistance of the biofilms thus formed.

Enhanced production of exopolysaccharide matrix and biofilm by a menadione-auxotrophic Staphylococcus aureus small-colony variant.

The role of Staphylococcus aureus small-colony variants (SCVs) in the pathogenesis of biofilm-associated infections remains unclear. This study investigated the mechanism behind increased biofilm-forming potential of a menadione-auxotrophic Staphylococcus aureus SCV compared with the wild-type parental strain, as recently reported by our laboratory. SCVs displayed an autoaggregative phenotype, with a greater amount of polysaccharide intercellular adhesin (PIA), significantly reduced tricarboxylic acid cycle activity and a decreased susceptibility to aminoglycosides and cell-wall inhibitors compared with wild-type. The biofilms formed by the SCV were highly structured, consisting of large microcolonies separated by channels, and contained more biomass as well as significantly more PIA than wild-type biofilms. The surface hydrophobicity of the two phenotypes was similar. Thus, the autoaggregation and increased biofilm-forming capacity of menadione-auxotrophic Staphylococcus aureus SCVs in this study was related to the enhanced production of PIA in these variants.

Quorum sensing-mediated regulation of staphylococcal virulence and antibiotic resistance.

Accessory gene regulator (agr)-mediated quorum sensing plays a central role in staphylococcal pathogenesis. It primarily upregulates secreted virulence factors and downregulates cell surface proteins, thereby governing invasiveness of staphylococci and cell dispersal from biofilms. Except for α- and β-PSMs, which are directly controlled by AgrA, the effector functions of agr are primarily mediated by RNAIII, a regulatory RNA encoded by this operon. agr phenotype and expression considerably influence the chronicity of an infection. It has also been linked with altered susceptibility of Staphylococcus aureus against antibiotics. Four classes of S. aureus and Staphylococcus epidermidis AIPs exist based on sequence variation, and lead to inter-strain and species cross-inhibition. Certain agr classes have been associated with specific clonal complexes, disease syndromes and intermediate-susceptibility to glycopeptides. It is also being investigated as a prophylactic and therapeutic target. This article describes the presently available literature regarding the role of agr in S. aureus and S. epidermidis infections.

Isolation and characterisation of an anticryptococcal protein in human cerebrospinal fluid.

An earlier study reported that human cerebrospinal fluid (CSF) has fungistatic activity for Cryptococcus neoformans. The present study reports that molecular sieve fractionation of concentrated CSF yielded three protein peaks, one of which (p2) had anticryptococcal activity. On a DEAE-Sephacel anion-exchange column the active molecular sieve peak (p2) gave two peaks that contained anticryptococcal activity. The first (DEAE-1) eluted with 0.1 M NaCl and the second (DEAE-2) eluted with 0.2 M NaCl in buffer. Fungistatic activity of DEAE-1 was reversed by FeCl3. Moreover, FeCl3 reversed inhibition of C. neoformans growth by CSF. In contrast, activity of DEAE-2 was not reversed by FeCl3, indicating that inhibition was produced by an iron-independent mechanism. Immunoblot assays showed that transferrin was present in DEAE-1 but not in DEAE-2, whereas albumin was present in DEAE-2 but not in DEAE-1. On NuPAGE, DEAE-1 protein migrated as a single band corresponding to transferrin and DEAE-2 protein gave a single band corresponding to albumin. In control experiments, human serum albumin subjected to the same isolation protocol acquired anticryptococcal activity similar to that of DEAE-2. Therefore, CSF albumin (DEAE-2) activity was associated with the isolation protocol. These data indicate that transferrin, present in or isolated from CSF, sequesters trace amounts of ferric iron, inhibits growth of C. neoformans and acts as an innate defence mechanism.

Sigma S-Dependent Antioxidant Defense Protects Stationary-Phase Escherichia coli against the Bactericidal Antibiotic Gentamicin

ABSTRACT Stationary-phase bacteria are important in disease. The σs-regulated general stress response helps them become resistant to disinfectants, but the role of σs in bacterial antibiotic resistance has not been elucidated. Loss of σs rendered stationary-phase Escherichia coli more sensitive to the bactericidal antibiotic gentamicin (Gm), and proteomic analysis suggested involvement of a weakened antioxidant defense. Use of the psfiA genetic reporter, 3′-(p-hydroxyphenyl) fluorescein (HPF) dye, and Amplex Red showed that Gm generated more reactive oxygen species (ROS) in the mutant. HPF measurements can be distorted by cell elongation, but Gm did not affect stationary-phase cell dimensions. Coadministration of the antioxidant N-acetyl cysteine (NAC) decreased drug lethality particularly in the mutant, as did Gm treatment under anaerobic conditions that prevent ROS formation. Greater oxidative stress, due to insufficient quenching of endogenous ROS and/or respiration-linked electron leakage, therefore contributed to the greater sensitivity of the mutant; infection by a uropathogenic strain in mice showed this to be the case also in vivo. Disruption of antioxidant defense by eliminating the quencher proteins, SodA/SodB and KatE/SodA, or the pentose phosphate pathway proteins, Zwf/Gnd and TalA, which provide NADPH for ROS decomposition, also generated greater oxidative stress and killing by Gm. Thus, besides its established mode of action, Gm also kills stationary-phase bacteria by generating oxidative stress, and targeting the antioxidant defense of E. coli can enhance its efficacy. Relevant aspects of the current controversy on the role of ROS in killing by bactericidal drugs of exponential-phase bacteria, which represent a different physiological state, are discussed.

Methicillin-resistant Staphylococcus aureus carriage screening in intensive care

Methicillin-resistant Staphylococcus aureus (MRSA) has been associated with considerable morbidity and mortality and is a major public health issue world-wide. It has established itself as a common nosocomial pathogen and is being increasingly implicated in both health-care and community associated infections in developing nations including India.[1] Recent studies indicate that MRSA is endemic in our country, with an isolation rate of nearly 27%, 49% and 47% amongst clinical isolates of S. aureus from out-patients, ward-in-patients and intensive care unit (ICU) patients respectively.[2]

Cellular Response of Escherichia coli to Microgravity and Microgravity Analogue Culture

Spaceflight and spaceflight-analogue studies reveal that microgravity or aspects of the microgravity environment impact the physiology, stress resistance, and molecular biology of microbes. Escherichia coli is an ideal model bacterium for microgravity and microgravity analogue investigations, as it is the most well-characterized organism, and has pathogenic strains that could cause infections during spaceflight. It is also a key player in ecosystems critical for sustaining life in space, such as renewal of resources like oxygen and water, as well as waste recycling. This chapter reviews the current literature on the effect of microgravity and modeled microgravity systems on E. coli physiology and molecular biology, with implications for risk assessment and astronaut health as well as beneficial applications for the general public.