Lalitha Biswas

Genome Analysis of the Meat Starter Culture Bacterium Staphylococcus carnosus TM300

ABSTRACT The Staphylococcus carnosus genome has the highest GC content of all sequenced staphylococcal genomes, with 34.6%, and therefore represents a species that is set apart from S. aureus, S. epidermidis, S. saprophyticus, and S. haemolyticus. With only 2.56 Mbp, the genome belongs to a family of smaller staphylococcal genomes, and the ori and ter regions are asymmetrically arranged with the replichores I (1.05 Mbp) and II (1.5 Mbp). The events leading up to this asymmetry probably occurred not that long ago in evolution, as there was not enough time to approach the natural tendency of a physical balance. Unlike the genomes of pathogenic species, the TM300 genome does not contain mobile elements such as plasmids, insertion sequences, transposons, or STAR elements; also, the number of repeat sequences is markedly decreased, suggesting a comparatively high stability of the genome. While most S. aureus genomes contain several prophages and genomic islands, the TM300 genome contains only one prophage, ΦTM300, and one genomic island, νSCA1, which is characterized by a mosaic structure mainly composed of species-specific genes. Most of the metabolic core pathways are present in the genome. Some open reading frames are truncated, which reflects the nutrient-rich environment of the meat starter culture, making some functions dispensable. The genome is well equipped with all functions necessary for the starter culture, such as nitrate/nitrite reduction, various sugar degradation pathways, two catalases, and nine osmoprotection systems. The genome lacks most of the toxins typical of S. aureus as well as genes involved in biofilm formation, underscoring the nonpathogenic status.

Small-Colony Variant Selection as a Survival Strategy for Staphylococcus aureus in the Presence of Pseudomonas aeruginosa

ABSTRACT Previously it has been demonstrated that Staphylococcus aureus is sensitive toward Pseudomonas-secreted exotoxins, which preferentially target the electron transport chain in staphylococci. Here it is shown that a subpopulation of S. aureus survives these respiratory toxins of Pseudomonas aeruginosa by selection of the small-colony variant (SCV) phenotype. Purified pyocyanin alone causes the same effect. A hemB mutant of S. aureus survives cocultivation with P. aeruginosa without a decrease in CFU.

Lipoproteins in Staphylococcus aureus Mediate Inflammation by TLR2 and Iron-Dependent Growth In Vivo

Lipoproteins (Lpp) are ligands of TLR2 and signal by the adaptor MyD88. As part of the bacterial cell envelope, Lpp are mainly involved in nutrient acquisition for Staphylococcus aureus. The impact of Lpp on TLR2-MyD88 activation for S. aureus in systemic infection is unknown. S. aureus strain SA113 deficient in the enzyme encoded by the prolipoprotein diacylglyceryl transferase gene (Δlgt), which attaches the lipid anchor to pro-Lpp, was used to study benefits and costs of Lpp maturation. Lpp in S. aureus induced early and strong cytokines by TLR2-MyD88 signaling in murine peritoneal macrophages. Lpp contributed via TLR2 to pathogenesis of sepsis in C57BL/6 mice with IL-1β, chemokine-mediated inflammation, and high bacterial numbers. In the absence of MyD88-mediated inflammation, Lpp allowed bacterial clearing from liver devoid of infiltrating cells, but still conferred a strong growth advantage in mice, which was shown to rely on iron uptake and storage in vitro and in vivo. With iron-restricted bacteria, the Lpp-related growth advantage was evident in infection of MyD88−/−, but not of C57BL/6, mice. On the other hand, iron overload of the host restored the growth deficit of Δlgt in MyD88−/−, but not in immunocompetent C57BL/6 mice. These results indicate that iron acquisition is improved by Lpp of S. aureus but is counteracted by inflammation. Thus, lipid anchoring is an evolutionary advantage for S. aureus to retain essential proteins for better survival in infection.

Role of the Twin-Arginine Translocation Pathway in Staphylococcus

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (DeltatatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of DeltatatAC and Deltatat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.

Antibiotic resistance in Pseudomonas aeruginosa and alternative therapeutic options

Pseudomonas aeruginosa is a leading cause of nosocomial infections and is responsible for ∼10% of all hospital-acquired infections worldwide. It continues to pose a therapeutic challenge because of the high rate of morbidity and mortality associated with it and the possibility of development of drug resistance during therapy. Standard antibiotic regimes against P. aeruginosa are increasingly becoming ineffective due to the rise in drug resistance. With the scope for developing new antibiotics being limited, alternative treatment options are gaining more and more attention. A number of recent studies reported complementary and alternative treatment options to combat P. aeruginosa infections. Quorum sensing inhibitors, phages, probiotics, anti-microbial peptides, vaccine antigens and antimicrobial nanoparticles have the potential to act against drug resistant strains. Unfortunately, most studies considering alternative treatment options are still confined in the pre-clinical stages, although some of these findings have tremendous potential to be turned into valuable therapeutics. This review is intended to raise awareness of several novel approaches that can be considered further for combating drug resistant P. aeruginosa infections.

Impact of Staphylococcus aureus on pathogenesis in polymicrobial infections.

ABSTRACT Polymicrobial infections involving Staphylococcus aureus exhibit enhanced disease severity and morbidity. We reviewed the nature of polymicrobial interactions between S. aureus and other bacterial, fungal, and viral cocolonizers. Microbes that were frequently recovered from the infection site with S. aureus are Haemophilus influenzae, Enterococcus faecalis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Corynebacterium sp., Lactobacillus sp., Candida albicans, and influenza virus. Detailed analyses of several in vitro and in vivo observations demonstrate that S. aureus exhibits cooperative relations with C. albicans, E. faecalis, H. influenzae, and influenza virus and competitive relations with P. aeruginosa, Streptococcus pneumoniae, Lactobacillus sp., and Corynebacterium sp. Interactions of both types influence changes in S. aureus that alter its characteristics in terms of colony formation, protein expression, pathogenicity, and antibiotic susceptibility.

Amidase, a cell wall hydrolase, elicits protective immunity against Staphylococcus aureus and S. epidermidis

The morbidity and the mortality associated with Staphylococcus aureus and S. epidermidis infections have greatly increased due to the rapid emergence of highly virulent and antibiotic resistant strains. Development of a vaccine-based therapy is greatly desired. However, no staphylococcal vaccine is available till date. In this study, we have identified Major amidase (Atl-AM) as a prime candidate for future vaccine design against these pathogens. Atl-AM is a multi-functional non-covalently cell wall associated protein which is involved in staphylococcal cell separation after cell division, host extracellular matrix adhesion and biofilm formation. Atl-AM is present on the surface of diverse S. aureus and S. epidermidis strains. When used in combination with Freunds adjuvant, Atl-AM generated a mixed Th1 and Th2 mediated immune response which is skewed more toward Th1; and showed increased production of opsonophagocytic IgG2a and IgG2b antibodies. Significant protective immune response was observed when vaccinated mice were challenged with S. aureus or S. epidermidis. Vaccination prevented the systemic dissemination of both organisms. Our results demonstrate the remarkable efficacy of Atl-AM as a vaccine candidate against both of these pathogens.

Impact of cell wall peptidoglycan O-acetylation on the pathogenesis of Staphylococcus aureus in septic arthritis

Staphylococcus aureus (S. aureus) is one of the most common pathogen causing septic arthritis. To colonize the joints and establish septic arthritis this bacterium needs to resist the host innate immune responses. Lysozyme secreted by neutrophils and macrophages is an important defense protein present in the joint synovial fluids. S. aureus is known to be resistant to lysozyme due to its peptidoglycan modification by O-acetylation of N-acetyl muramic acid. In this study we have investigated the role of O-acetylated peptidoglycan in septic arthritis. Using mouse models for both local and hematogenous S. aureus arthritis we compared the onset and progress of the disease induced by O-acetyl transferase mutant and the parenteral wild type SA113 strain. The disease progression was assessed by observing the clinical parameters including body weight, arthritis, and functionality of the affected limbs. Further X-ray and histopathological examinations were performed to monitor the synovitis and bone damage. In local S. aureus arthritis model, mice inoculated with the ΔoatA strain developed milder disease (in terms of knee swelling, motor and movement functionality) compared to mice inoculated with the wild type SA113 strain. X-ray and histopathological data revealed that ΔoatA infected mice knee joints had significantly lesser joint destruction, which was accompanied by reduced bacterial load in knee joints. Similarly, in hematogenous S. aureus arthritis model, ΔoatA mutant strain induced significantly less severe clinical septic arthritis compared to its parental strain, which is in accordance with radiological findings. Our data indicate that peptidoglycan O-acetylation plays an important role in S. aureus mediated septic arthritis.

Autochthonous Blastomycosis of the Adrenal: First Case Report from Asia

Systemic endemic mycoses, such as blastomycosis, are rare in Asia and have been reported as health risks among travelers who visit or reside in an endemic area. Adrenal involvement is rarely seen in blastomycosis and has never been reported from Asia. We report the first case of blastomycosis with bilateral involvement of the adrenals in a diabetic patient residing in the state of Arunachal Pradesh, India.

Detection of occult hepatitis B and window period infection among blood donors by individual donation nucleic acid testing in a tertiary care center in South India

With the introduction of highly sensitive hepatitis B surface antigen immunoassay, transfusion associated HBV infection have reduced drastically but they still tend to occur due to blood donors with occult hepatitis B infection (OBI) and window period (WP) infection. Sera from, 24338 healthy voluntary blood donors were screened for HBsAg, HIV and HCV antibody using Vitros Enhanced Chemiluminescent Immunoassay. The median age of the donor population was 30 (range 18–54) with male preponderance (98%). All serologically negative samples were screened by nucleic acid testing (NAT) for viral DNA and RNA. NAT-positive samples were subjected to discriminatory NAT for HBV, HCV, and HIV and all samples positive for HBV DNA were tested for anti-HBc, anti-HBs, HBeAg. Viral load was determined using artus HBV RG PCR Kit. Of the 24,338 donors screened, 99.81% (24292/24338) were HBsAg negative of which NAT was positive for HBV DNA in 0.0205% (5/24292) donors. Four NAT positive donors had viral load of <200 IU/ml making them true cases of OBI. One NAT positive donor was negative for all antibodies making it a case of WP infection. Among OBI donors, 75% (3/4) were immune and all were negative for HBeAg. Precise HBV viral load could not be determined in all (5/5) NAT positive donors due to viral loads below the detection limit of the artus HBV RG PCR Kit. The overall incidence of OBI and WP infections was found to be low at 1 in 6503 and 1 in 24214 donations, respectively. More studies are needed to determine the actual burden of WP infections in Indian blood donors.