Gayathri Arakere

Genotyping of methicillin-resistant Staphylococcus aureus strains from two hospitals in Bangalore, South India.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen in India, and up to 70% methicillin resistance has been reported from hospitals in various parts of India. Hospitals use phenotyping for the most part, and molecular genotyping is not done. Here we report on the genotyping of 82 single-patient isolates from two hospitals in Bangalore, South India, for the first time. Most of the strains possessed type III or IIIA staphylococcal cassette chromosome (SCCmec) cassettes, and we did not detect strains with type I, IA, or II cassettes. Most isolates also contained the type III cassette chromosome recombinase (ccr) AB region. Multilocus sequence typing (MLST) and staphylococcal protein A (spa) typing of a selected number of isolates have been carried out. Although most isolates that were chosen for MLST and spa typing had the same patterns, they were quite diverse in their pulsed-field gel electrophoresis (PFGE) patterns. PFGE, MLST, and spa typing of the Indian strains revealed that they are related to the previously described Hungarian and Brazilian clones.

Clonal complexes and virulence factors of Staphylococcus aureus from several cities in India

BackgroundDiseases from Staphylococcus aureus are a major problem in Indian hospitals and recent studies point to infiltration of community associated methicillin resistant S. aureus (CA-MRSA) into hospitals. Although CA-MRSA are genetically different from nosocomial MRSA, the distinction between the two groups is blurring as CA-MRSA are showing multidrug resistance and are endemic in many hospitals. Our survey of samples collected from Indian hospitals between 2004 and 2006 had shown mainly hospital associated methicillin resistant Staphylococcus aureus (HA-MRSA) carrying staphylococcal cassette chromosome mec (SCCmec) type III and IIIA. But S. aureus isolates collected from 2007 onwards from community and hospital settings in India have shown SCCmec type IV and V cassettes while several variations of type IV SCCmec cassettes from IVa to IVj have been found in other parts of the world. In the present study, we have collected nasal swabs from rural and urban healthy carriers and pus, blood etc from in patients from hospitals to study the distribution of SCCmec elements and sequence types (STs) in the community and hospital environment. We performed molecular characterization of all the isolates to determine their lineage and microarray of select isolates from each sequence type to analyze their toxins, virulence and immune-evasion factors.ResultsMolecular analyses of 68 S. aureus isolates from in and around Bengaluru and three other Indian cities have been carried out. The chosen isolates fall into fifteen STs with all major clonal complexes (CC) present along with some minor ones. The dominant MRSA clones are ST22 and ST772 among healthy carriers and patients. We are reporting three novel clones, two methicillin sensitive S. aureus (MSSA) isolates belonging to ST291 (related to ST398 which is live stock associated), and two MRSA clones, ST1208 (CC8), and ST672 as emerging clones in this study for the first time. Sixty nine percent of isolates carry Panton- Valentine Leucocidin genes (PVL) along with many other toxins. There is more diversity of STs among methicillin sensitive S. aureus than resistant ones. Microarray analysis of isolates belonging to different STs gives an insight into major toxins, virulence factors, adhesion and immune evasion factors present among the isolates in various parts of India.ConclusionsS. aureus isolates reported in this study belong to a highly diverse group of STs and CC and we are reporting several new STs which have not been reported earlier along with factors influencing virulence and host pathogen interactions.

Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. Beta-lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.

Staphylococcus aureus eye infections in two Indian hospitals: emergence of ST772 as a major clone

Purpose The purpose of this study was to perform molecular characterization of Staphylococcus aureus isolates causing a variety of eye infections from two major eye care hospitals in India. Methods Twenty-four isolates from Aravind Eye Hospital, Madurai, India, and nine isolates from LV Prasad Eye Institute, Bhubaneswar, India, representing severe to nonsevere eye infections like microbial keratitis to lacrimal sac abscess, were characterized. Staphylococcal cassette chromosome mec typing, multilocus sequence typing, accessory gene regulator typing, staphylococcal protein A typing, and pulsed field gel electrophoresis were used, along with determination of the presence of Panton–Valentine leucocidin toxin and endotoxin gene cluster among each sequence type. Results The majority of eye infections, both severe and nonsevere, were caused by sequence type (ST)772, positive for the Panton–Valentine leucocidin gene, and carrying methicillin-resistant staphylococcal cassette chromosome mec type V cassette (22/33, 67%). Some of the other sequence types that caused severe eye infections were ST1 (9%), 5 (3%), 72 (6%), 88 (3%), 121 (3%), and 672 (3%). This is the first report of the presence of ST1 and 88 in India. Conclusion Although the number of isolates included in this study was small, most of the eye infections were caused by community-associated S. aureus where patients had no history of hospitalization or treatment in the past year. In the case of six severe infections, patients were admitted for surgeries and there is probability of hospital infection. In addition, only methicillin-resistant S. aureus isolates carrying staphylococcal cassette chromosome mec type V were detected. Epidemic methicillin-resistant Staphylococcus aureus 15 (ST22) is a major ST found in health care as well as community settings in non-eye infections in India, but only one methicillin-sensitive S. aureus isolate belonging to ST22 was detected. Predominantly ST772, along with a few other STs, caused the 33 eye infections studied.

Epidemic meticillin-resistant Staphylococcus aureus (EMRSA-15) variants detected in healthy and diseased individuals in India

This study provides what we believe to be the first report of the presence of EMRSA-15 and its variants isolated from nasal swabs from 13 healthy and diseased individuals in India. The majority of the isolates belonged to staphylococcal cassette chromosome mec (SCCmec) type IV and spa type t852, whilst four isolates were non-typable and heterotypic for the presence of the mecA gene. All non-typable isolates were positive for the orfX gene by PCR and belonged to spa types t005 and t2986. They may have variant SCCmec cassettes indicating genetic changes occurring in the Indian EMRSA-15. All isolates were positive for Panton-Valentine leukocidin and toxic shock syndrome toxin, which is a cause for concern. In addition to soft-tissue infections, the EMRSA-15 isolates from patients were also responsible for meningitis and brain abscesses, which is quite rare.

Characterization and Acceptor Preference of a Soluble Meningococcal Group C Polysialyltransferase

Vaccines against Neisseria meningitidis group C are based on its α-2,9-linked polysialic acid capsular polysaccharide. This polysialic acid expressed on the surface of N. meningitidis and in the absence of specific antibody serves to evade host defense mechanisms. The polysialyltransferase (PST) that forms the group C polysialic acid (NmC PST) is located in the cytoplasmic membrane. Until recently, detailed characterization of bacterial polysialyltransferases has been hampered by a lack of availability of soluble enzyme preparations. We have constructed chimeras of the group C polysialyltransferase that catalyzes the formation α-2,9-polysialic acid as a soluble enzyme. We used site-directed mutagenesis to determine the region of the enzyme necessary for synthesis of the α-2,9 linkage. A chimera of NmB and NmC PSTs containing only amino acids 1 to 107 of the NmB polysialyltransferase catalyzed the synthesis of α-2,8-polysialic acid. The NmC polysialyltransferase requires an exogenous acceptor for catalytic activity. While it requires a minimum of a disialylated oligosaccharide to catalyze transfer, it can form high-molecular-weight α-2,9-polysialic acid in a nonprocessive fashion when initiated with an α-2,8-polysialic acid acceptor. De novo synthesis in vivo requires an endogenous acceptor. We attempted to reconstitute de novo activity of the soluble group C polysialyltransferase with membrane components. We found that an acapsular mutant with a defect in the polysialyltransferase produces outer membrane vesicles containing an acceptor for the α-2,9-polysialyltransferase. This acceptor is an amphipathic molecule and can be elongated to produce polysialic acid that is reactive with group C-specific antibody.

Genome Sequencing Unveils a Novel Sea Enterotoxin-Carrying PVL Phage in Staphylococcus aureus ST772 from India

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772 suggests that widespread mobility of the sea enterotoxin might be a selective force behind its ‘transfer’ to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside the genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.

A novel type-III staphylococcal cassette chromosome mec (SCCmec) variant among Indian isolates of methicillin-resistant Staphylococcus aureus.

We identified a novel type-III staphylococcal cassette chromosome mec (SCCmec) element carried by eight methicillin-resistant Staphylococcus aureus (MRSA) strains from different wards and patients in an Indian hospital. Although the pulsed-field gel electrophoresis pattern and spa types of eight strains were identical and clonally related to other nosocomial Indian isolates that belonged to sequence type (ST) 239 and spa type t037, the minimum inhibitory concentration (MIC) of these eight variants was noticeably low compared with the typical type-III isolates from the same hospital, and we were unable to identify ccrC and hsdR by multiplex PCR, although mer operon and transposases A, B, and C of Tn554 were amplified. By amplifying the entire SCCmec region by long-range PCR and determining parts of the nucleotide sequences of one isolate (V14), we found that the strain carried a novel SCCmec element containing a 422 bp sequence, which is highly homologous to that identified in strain CCR1-9583, mer operon and plasmid pT181 integrated in tandem via IS431 in the J3 region. It also carried a cassette chromosome, previously reported to be an SCC-like element, downstream of type-III SCCmec. Because PCR amplification of representative genes showed that these eight strains carried the same genetic elements, they belong to a novel MRSA clone that differs from most nosocomial clones carrying type-III SCCmec and SCCmercury, despite belonging to the ST239 genotype.

Epidemiology of Staphylococcus aureus in Bangalore, India: emergence of the ST217 clone and high rate of resistance to erythromycin and ciprofloxacin in the community

This study aimed to determine the antibiotic susceptibility pattern of Staphylococcus aureus (SA) and the circulating clones in Bangalore, India. Susceptibility testing was performed for all cases of SA infections in a tertiary-care hospital. Panton-Valentine leucocidin (PVL) encoding genes were detected, and sequence type and spa type were determined. Out of the 92 collected strains, 52.2% were methicillin-resistant SA (MRSA), isolated from community-acquired (CA) infections in 60.4% and hospital-acquired (HA) infections in 39.6%. S. aureus isolates were also highly resistant to erythromycin (54.3%) and ciprofloxacin (70.6%) in methicillin-susceptible SA (MSSA) and MRSA, as well as in CA and HA infections. MRSA were found to be significantly more resistant to gentamicin (p <0.001), cotrimoxazole (p <0.001) and ciprofloxacin (p 0.001) than MSSA, but no significant difference was observed between CA- and HA-SA. ST217 appeared as a new emerging and prevalent clone, but ST772 remained the predominant clone, all being PVL-positive isolates. Our study points out the high prevalence of MRSA, even in the community, and the worrying increase of resistance to ciprofloxacin and erythromycin among CA-MSSA. Emergence of clone ST217 is reported for the first time in India.

Community-acquired methicillin-resistant Staphylococcus aureus pyomyositis with myelitis: A rare occurrence with diverse presentation

Staphylococcus aureus is the most common bacterial pathogen implicated in pyomyositis. There are increasing reports of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections. The present case report brings out the diverse clinical manifestations of MRSA infection in the form of paraspinal pyomyositis, myelitis, spinal osteomyelitis, and pneumonia. Molecular typing of the organism confirmed the diagnosis. Patient was successfully treated with vancomycin and surgical drainage. Consideration of the possibility of methicillin-resistance and appropriate antibiotic selection is vital in the treatment of serious community-acquired staphylococcal infections.