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.

Necrotizing fasciitis caused by a variant of epidemic methicillin resistant Staphylococcus aureus -15.

We report a case of necrotizing fasciitis (NF), caused by community-acquired epidemic methicillin resistant Staphylococcus aureus 15 (EMRSA 15). The patient had a prolonged recovery period following treatment with antibiotics and surgical debridement of the infected part. Molecular characterization revealed that the isolate carried Staphylococcal Cassette Chromosome mec (SCC mec) type IV harboring Panton-Valentine Leucocidin (pvl) gene and having accessory gene regulator (agr) type I. The isolate was positive for enterotoxin gene cluster (egc). Pulsed field gel electrophoresis patterns revealed that the isolate belonged sequence type 22, which is an Indian variant of EMRSA 15, reported earlier.

A variant epidemic methicillin resistant Staphylococcus aureus-15 cavernous sinus thrombosis and meningitis: a rare occurrence with unusual presentation.

Septic cavernous sinus thrombosis (CST) is an uncommon clinical syndrome. Although Staphylococcus aureus (S aureus) is the most common bacterial pathogen causing CST, it is infrequent as a cause of meningitis. We report the first case of CST and meningitis from Bengaluru, Karnataka, caused by community-acquired epidemic methicillin resistant Staphylococcus aureus-15 (EMRSA-15), in a previously healthy individual without known risk factors; the patient recovered following treatment with vancomycin. The isolate was genotyped as belonging to staphylococcal cassette chromosome mec type IV and sequence type 22 and carried the panton-valentine leucocidin gene. It is the first Indian EMRSA-15 disease isolate from a case of meningitis. EMRSA-15 has been a major problem in hospitals in UK and it is a cause for great concern in Indian hospitals too.

Inhibition enzyme-linked immunosorbent assay for serotyping of group B streptococcal isolates.

Group B Streptococcus (GBS) is one of the most common organisms causing neonatal sepsis as well as serious infections in adults. Serotyping the organism is important in studying the epidemiology of the disease as well as deciding a course of treatment. There are several methods available for serotyping. Most of them need high-titered sera and are not quantitative. We are reporting a new inhibition enzyme-linked immunosorbent assay (ELISA) for serotyping which is sensitive and specific compared to the conventional methods but does not need high-titered serotype-specific antisera, as the specificity is controlled by the polysaccharide coating on the ELISA plates. The method can also be quantitative, and we have measured polysaccharide elaborated by different serotype V strains. Thus, the inhibition ELISA method will be useful in serotyping for epidemiological studies, assessing virulence, and performing strain selection for vaccine production.

Involvement of phospholipid end groups of group C Neisseria meningitidis and Haemophilus influenzae type b polysaccharides in association with isolated outer membranes and in immunoassays.

There are several bacterial polysaccharides (PSs) which contain a terminal lipid moiety. It has been postulated that these terminal lipid moieties anchor the PSs to the outer membrane of the bacteria. Our studies have shown that incubation of native PS from group C Neisseria meningitidis or Haemophilus influenzae type b with isolated outer membrane vesicles results in association of a portion of the PS with the vesicles. Removal of the terminal lipid from the PS by treatment with phospholipase A2 or phospholipase D eliminates this association. In other studies, it was shown that delipidated PSs are not suitable as solid-phase antigens in a currently used enzyme-linked immunosorbent assay (ELISA). Measurement of antibody units in the reference sera by using delipidated PSs as antigens in an ELISA yielded negligible absorbance compared with native PSs when methylated human serum albumin was used to coat the PSs to the plate. Nevertheless, phospholipase A2 and phospholipase D treatment did not noticeably affect antigenic epitopes, since soluble group C PS without the terminal lipid bound antibody as effectively as the native PS did, as measured by a competitive inhibition assay. Both hydrophobic and electrostatic interactions are important for the binding of group C N. meningitidis PS to the ELISA plate, while charge interactions seem to be sufficient for binding the more negatively charged H. influenzae type b PS.

Characterization of a stress protein from group B Neisseria meningitidis.

Increased levels of a 65-kDa stress protein (Msp65) were observed in group B Neisseria meningitidis grown under stationary-growth conditions. Electron microscopy showed two apposing rings of seven subunits, a structure typical of Escherichia coli GroEL. Msp65 was not found in either the periplasmic space or the outer membrane. Several important differences between the GroEL analogs of N. meningitidis and Neisseria gonorrhoeae are discussed.

Disseminated meningococcal infection in HIV-seropositive patients.

OBJECTIVE: Although increasing numbers of infections due to various bacterial pathogens have been described in HIV-infected individuals, there have been few reports to date of disseminated Neisseria meningitidis infections in such individuals. We describe here the presentation and clinical course of systemic meningococcal infection in two HIV-1-seropositive men and the response to meningococcal vaccine in one. DESIGN AND METHODS: Retrospective analysis of case reports of two patients identified in a municipal hospital in Denver, Colorado, USA, and evaluation by enzyme-linked immunosorbent assay of antibody response to quadrivalent (A, C, Y, W-135) meningococcal vaccine. RESULTS: A 27-year-old HIV-seropositive man with bacteremic group Y meningococcal pneumonia and a 45-year-old man with AIDS and group B meningococcal arthritis both responded to short-term antibiotic therapy without recurrence. The second patient responded to meningococcal vaccination with seroconversion to all four serogroups. CONCLUSIONS: Disseminated meningococcal infection, although rare in HIV-infected individuals, may present with a variety of clinical manifestations and responds well to antibiotic therapy. Meningococcal vaccine appears to be immunogenic in such individuals.

Quantitation of human IgG subclass antibodies to Haemophilus influenzae type b capsular polysaccharide. Results of an international collaborative study using enzyme immunoassay methodology.

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.

Specificity of antibodies to O-acetyl-positive and O-acetyl-negative group C meningococcal polysaccharides in sera from vaccinees and carriers.

Most group C Neisseria meningitidis strains produce an O-acetyl-positive polysaccharide, a homopolymer of alpha-2----9-linked N-acetylneuraminic acid with O-acetyl groups at the C-7 and C-8 of its sialic acid residues. The majority of disease isolates have been reported to contain this polysaccharide. Some strains produce group C polysaccharide lacking O-acetyl groups. The licensed vaccine contains the O-acetyl-positive polysaccharide. We have measured the antibody specificities to the two polysaccharides in sera from asymptomatic group C meningococcal carriers and vaccinated adults by a new enzyme-linked immunosorbent assay (ELISA) procedure using methylated human serum albumin for coating the group C polysaccharide onto microtiter plates. Inhibition of binding of serum antibodies to polysaccharide-coated plates was measured by ELISA after incubation with O-acetyl-positive and O-acetyl-negative group C polysaccharides. Greater inhibition of binding of carrier sera was observed with the homologous polysaccharide. There was substantial inhibition of binding of vaccinee sera to the O-acetyl-positive polysaccharide-coated plate following preincubation with O-acetyl-positive polysaccharide, but homologous inhibition on plates coated with the O-acetyl-negative polysaccharide required much higher concentrations of polysaccharide. Carrier sera may have a higher proportion of antibodies with greater specificity for the O-acetyl-negative polysaccharide, while vaccinee sera contain antibodies with greater affinity for the O-acetyl-positive polysaccharide. Studies with monoclonal antibodies specific for O-acetyl-positive and O-acetyl-negative polysaccharides reveal that the percentage of group C meningococcal disease caused by O-acetyl-negative strains remains about 15%, as found over 15 years ago.

Risk factors for carriage of meningococcus in the Los Angeles County men's jail system.

Ten cases of meningococcal meningitis in the Los Angeles County men's jail system in 1986 were the first known reported cases in this population. New cases have continued into 1990. Nineteen of 21 symptomatic cases identified by serogroup from the men's jail occurring through 1988 had serogroup C. The prevalence of meningococcal carriage and potential risk factors were studied in 1988 among 150 men booked to enter the jail, 350 inmates being released, and 100 jail staff. The prevalence of meningococcal carriage among releases, bookings, and staff were 25.4%, 18.7%, and 5.0%, respectively. Among releases, imprisonment longer than a threshold of 28 days increased carriage of serogroup C 10.0 times (95% confidence interval (CI) 4.6-21.6). Among bookings, household crowding increased serogroup C carriage 8.2 times (95% CI 1.5-45.3). Direct and passive smoking at home increased carriage of any serogroup 5.2 (95% CI 1.2-47.5) and 2.5 (95% CI 1.1-5.8) times, respectively. Feasible potential interventions include banning smoking in the jail and immunization with quadrivalent meningococcal vaccine of booked men sentenced for one month or more.

Comparison of a new, rapid enzyme-linked immunosorbent assay with latex particle agglutination for the detection of Haemophilus influenzae type b infections.

A new, rapid enzyme-linked immunosorbent assay (ELISA) for the detection of polyribosylribitol phosphate of Haemophilus influenzae type b was compared with a commercially available latex particle agglutination (LPA) system (Bactigen; Wampole Laboratories, Cranbury, N.J.). By adding specimens and the anti-polyribosylribitol phosphate immunoglobulin-enzyme conjugate to the solid phase in a single step, it was possible to complete the ELISA procedure in 30 min. The ELISA was capable of detecting 0.3 ng of polyribosylribitol phosphate per ml in cerebrospinal fluid, 0.6 ng/ml in urine, and 1.2 ng/ml in serum; the in vitro sensitivity of LPA in these body fluids was 0.6, 0.3, and 0.3 ng/ml, respectively. Both procedures detected polyribosylribitol phosphate in specimens from 25 patients with bacteriologically confirmed H. influenzae type b infections. The specificity of ELISA appeared to be superior to that of LPA. ELISA was positive in only one of seven patients who had a positive LPA test and a clinical illness that was not compatible with haemophilus infection. Moreover, five patients with bacteriologically confirmed infections due to other pathogens (Streptococcus pneumoniae type 14 [two patients], Neisseria meningitidis group C, Escherichia coli K100, and Staphylococcus aureus) had false-positive LPA tests; only two (E. coli and S. aureus) were positive by ELISA. A total of 108 samples from 61 patients who had no evidence of haemophilus infections were negative by both procedures. The ELISA is a rapid, sensitive, and specific alternative to LPA for the detection of haemophilus polyribosylribitol phosphate.

Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity.

Middle T antigen of polyoma virus is associated principally with the plasma membrane. Comparison of the trypsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [gamma-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32PO4 were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.