Role of Mycoplasma pneumoniae infection in acute exacerbations of chronic obstructive pulmonary disease.

Eighty per cent of the cases of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) have an infective aetiology, atypical bacteria including Mycoplasma pneumoniae accounting for 5-10 % of these. However, the importance of association of M. pneumoniae with episodes of AECOPD still remains doubtful. The present study was therefore undertaken to delineate the extent of involvement of M. pneumoniae in patients with AECOPD at a referral hospital in Delhi, India. Sputum samples and throat swabs from a total of 100 AECOPD patients attending the Clinical Research Center of Vallabhbhai Patel Chest Institute, Delhi, were collected during a 2-year period (January 2004-June 2006). The samples were investigated for the presence of aerobic bacterial pathogens and M. pneumoniae. Diagnosis of infection with M. pneumoniae was based on culture, serology, direct detection of M. pneumoniae specific antigen and PCR. Bacterial aetiology could be established in 16 of the 100 samples studied. Pseudomonas spp. were recovered from eight cases, Streptococcus pneumoniae from four and Klebsiella spp. from two cases. Acinetobacter sp. and Moraxella catarrhalis were isolated from one case each. Serological evidence of M. pneumoniae infection and/or detection of M. pneumoniae specific antigen were seen in 16 % of the cases. One case with definite evidence of M. pneumoniae infection also had coinfection with Pseudomonas spp. However, no direct evidence of M. pneumoniae infection was found in our study population as defined by culture isolation or PCR. In conclusion, although the serological prevalence of M. pneumoniae infection in our study population was significantly higher than in the control group, there was no direct evidence of it playing a role in AECOPD.

Molecular typing of Salmonella enterica serotype Worthington isolates from infantile diarrhoea.

BACKGROUND AND OBJECTIVES: Salmonella Worthington has been known to be a causative agent for childhood diarrhoea. There is a paucity of information on the molecular relatedness of the strains isolated in various hospitals in India. The present study was carried out to attempt molecular typing of a cluster of Salmonella Worthington isolates obtained from cases of infantile diarrhoea during a six month period, from a tertiary care paediatric hospital in Delhi, India. METHODS: Nine isolates of S. Worthington obtained from faecal samples of infants suffering from diarrhoea during October 2001 to March 2002, were identified by the conventional biochemical methods and by serotyping. The antimicrobial susceptibility was determined by the disk diffusion method. Molecular typing was done by ribotyping. RESULTS: Eight patients were admitted to 3 different wards of the hospital and one was an outpatient. Four patients including the first patient visited the hospital with diarrhoea as the presenting symptom while five developed diarrhoea after admission. Stool microscopy showed no specific findings. Salmonella Worthington was isolated from stool cultures of these patients. Repeated cultures of the common drinking water source of the hospital and the milk supplied to children from central kitchen were negative for known pathogens. All S. Worthington isolates were resistant to all the beta-lactams tested including third generation cephalosporins. Eight isolates were sensitive to furazolidone and 6 to ciprofloxacin. Molecular characterization by ribotyping revealed four different clones. INTERPRETATION AND CONCLUSION: As four different ribotypes of the isolated Salmonella Worthington isolates were identified, it was clear that there was no single source of infection.

VEB-1 extended-spectrum ß-lactamase-producing multidrug-resistant Proteus mirabilis sepsis outbreak in a neonatal intensive care unit in India: clinical and diagnostic implications.

INTRODUCTION: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, multidrug-resistant (MDR) pathogens, are increasingly implicated in nosocomial outbreaksworldwide, particularly in neonatal intensive care units (NICUs). Proteus mirabilis is an uncommon nosocomial pathogen causing sepsis in neonates. CASE PRESENTATION: We report an outbreak of ESBL-positive MDR P. mirabilis sepsis involving five babies within 10 days in a NICU, which was promptly detected and managed. The aim of this study was to characterize the molecular mechanism of resistance to third-generation cephalosporins (3GCs) in the bacteria. Surveillance cultures were collected from health-care personnel (hand swabs, urine) and the surrounding patient-care environment. Ribotyping was performed to determine the clonality of the strain. Thirteen P. mirabilis were recovered from the blood cultures of the five babies and surveillance cultures. Twelve isolates were positive for the VEB-1 ESBL type, and were susceptible only to ciprofloxacin and carbapenems. There was an unusual phenotypic synergy observed between the 3GCs and imipenem/cefoxitin. The source of infection was traced to a contaminated multidose vial. The outbreak was associated with a high mortality (80 %). A change of empirical antibiotic policy to ciprofloxacin, with strict infection control measures, brought the outbreak to an abrupt end. CONCLUSION: This is believed to be the first report of a nosocomial outbreak of VEB-1 ESBL-producing P. mirabilis sepsis in neonates from India. The present report of infection due to VEB-1-producing P. mirabilis, an uncommon pathogen for an epidemic in a neonatal unit, highlights the growing significance of such Gram-negative bacteria as a cause of infections in newborns. Epidemic spread in a neonatal unit of an ESBL-producing Proteus species, which also had an intrinsically reduced susceptibility to imipenem, and resistance to colistin and tigecycline, can be a threatening situation and can result in high neonatal mortality unless recognized and controlled in a timely manner.

Synthesis of 4-aryl-7,7-dimethyl-1,2,3,4,5,6,7,8-octahydroquinazoline-2-one/thione-5-one derivatives and evaluation as antibacterials.

A novel, ecofriendly, one pot solvent free method for the synthesis of 4-aryl-7,7-dimethyl-1,2,3,4,5,6,7,8-octahydroquinazoline-2-one/thione-5-one derivatives is described which devoids the use of any organic solvents and auxiliaries. All the synthesized compounds were screened for their in vitro antibacterial activity against standard strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

Phenotypic characteristics of clinical isolates of Klebsiella pneumoniae & evaluation of available phenotypic techniques for detection of extended spectrum beta-lactamases.

BACKGROUND & OBJECTIVES: Identifying organisms that harbour extended spectrum beta lactamases (ESBLs) is a major challenge for a diagnostic clinical microbiology laboratory. Wide variety of ESBLs produced and lack of a sensitive phenotypic method for their detection make the detection of ESBLs difficult and is responsible for under-recognition. The present study was undertaken to evaluate phenotypic characteristics, initial screening tests and established confirmatory phenotypic methods for detection of ESBLs Klebsiella pneumoniae isolates prevalent in a hospital in north India. METHODS: One hundred, non-repeat clinical isolates of K. pneumoniae collected over a period of six months were included in the study. Susceptibilities of the isolates to 20 different antimicrobial agents were determined. Agar dilution and broth dilution methods were used to determine minimum inhibitory concentrations (MICs) of ceftazidime (CAZ) and cefotaxime (CTX). CAZ and CTX were used with and without clavulanic acid to detect ESBL harbouring isolates. Using agar dilution and broth dilution, MIC reduction of two and three doubling dilutions were evaluated as a criterion for ESBL harbouring isolates. Standard double disk synergy test (DDST) with disks placed at 30 mm and modified DDST with disks placed at 16 mm center-to-center distance, using at least two different third generation cephalosporins and combined disk method were also performed to detect ESBL harbouring isolates. RESULTS: Multi-drug resistance (resistance to three or more antimicrobials of different classes) was found among 94 per cent of the isolates. Pooling the results of all the three confirmatory techniques MIC reduction of >3 doubling dilutions using broth dilution method (using CTX and CAZ), combined disk method [(using CTX, ceftriaxone [(CRO), CAZ and aztreonam)] and standard DDST (using CTX, CRO, CAZ and aztreonam), revealed as many as 87 per cent of the isolates as ESBL producers. CTX had greater sensitivity in identifying isolates that harboured ESBLs. Modified DDST using CTX was as sensitive method as broth dilution method and combined disk method in detecting ESBL harbouring isolates. MIC reduction technique using agar dilution method and standard DDST had lowest overall sensitivity in detecting ESBLs. INTERPRETATION & CONCLUSION: Modified DDST using at least two different third generation cephalosporins was considered to be the best technique for detection of ESBL producing K. pneumoniae at our hospital. MIC reduction test with >2 doubling dilution reduction in MICs was found to be a better criterion than the presently recommended >3 doubling dilution reduction. For screening of potential ESBL producers, MIC determination using agar dilution was as good as that using broth dilution method. However, while performing MIC reduction test agar dilution method was found highly unreliable for detection of ESBL harbouring isolates.

A simple and rapid serum bactericidal assay and its evaluation in clinical isolates of Klebsiella pneumoniae.

A simple and rapid assay for the determination of serum bactericidal activity was developed and evaluated in 125 clinical isolates of Klebsiella pneumoniae. The serum reactivity against these isolates was concomitantly determined by the conventional viable count technique in order to compare the efficacy of the two techniques. The rapid assay could be completed within 5-8 h and the results were recorded in terms of visible change of colour of the culture medium. Of the 125 strains tested, more than 50% were found to be resistant to 20% normal human serum. There was an excellent agreement between the two methods. The degree of discordance observed in the results obtained by the two methods was statistically not significant (P>0.05). The conventional technique is labor intensive, cumbersome and time-consuming. The assay described here, on the other hand, is simple, easy and rapid enough to allow testing of a large number of bacterial isolates or recombinant clones in a single day. Thus, the assay can serve as an excellent alternative to the conventional technique for determining the bacterial serum susceptibility.

Chronic anaerobic pneumonitis in a seven-year-old girl.

A 7-year-old girl was referred for evaluation of chronic pulmonary disease associated with nasal symptoms of 4 years duration for which she had received frequent courses of antibiotics. Serial chest roentgenograms over a period of 2 years revealed a nonhomogeneous opacity in the right lower lung zone for which she had received 18 months of antituberculous therapy without relief. Evaluation of the patient led to the diagnosis of chronic anaerobic pneumonitis, a rare clinical entity in children. In addition, the patient also had bronchial asthma and chronic rhinitis. Therapy with oral phenoxymethylpenicillin and metronidazole for 6 weeks along with appropriate antiasthma medications abolished her symptoms and resulted in roentgenologic clearance.

Clindamycin in the treatment of anaerobic lung abscess.

A case of anaerobic lung abscess who had treatment failure after 4 weeks of supervised parenteral penicillin and oral metronidazole is described. Anaerobic pathogens resistant to one or the other of the above drugs were isolated. The patient had a striking clinical response to subsequent therapy with oral clindamycin. Failure of therapy should alert physicians to the possibility of infection with resistant anaerobic pathogens and in such situations, clindamycin is considered as an effective alternative.

Chronic anaerobic pneumonitis.

A case of chronic anaerobic pneumonitis, without any predisposing factors, nor the classical features often associated with it, which masqueraded as pulmonary tuberculosis is described. Therapy with metronidazole resulted in striking improvement.

Development of a computer software for analysis of SDS-PAGE protein fingerprints of bacterial isolates.

Protein fingerprinting is a widely used technique in epidemiological studies for typing bacterial strains. This study reports the development of a computer based gel analysis system. The system has the capability to analyse SDS-PAGE whole-cell protein profiles using digital image processing techniques. The software incorporates spatial and frequency domain operators for image enhancement, support for geometric correction of images and new algorithms for identification of strain tracks and protein bands. The system also provides facilities for correcting imaging defects for inter-gel comparison, similarity analysis, clustering and pictorial representation of results as a dendrogram. The software is highly interactive, user-friendly and can produce accurate results for differentiation of bacterial strains with minimal overhead of time.

Genotyping of methicillin-resistant Staphylococcus aureus in the Sultan Qaboos University Hospital, Oman reveals the dominance of Panton-Valentine leucocidin-negative ST6-IV/t304 clone.

The objective of this study was to determine the prevalence and distribution of methicillin-resistant Staphylococcus aureus (MRSA) genotypes circulating at a tertiary hospital in the Sultanate of Oman. A total of 79 MRSA isolates were obtained from different clinical samples and investigated using antibiogram, pulsed-field gel electrophoresis (PFGE), staphylococcal chromosome cassette mec (SCCmec), Spa typing and multilocus sequence typing (MLST). The isolates were susceptible to linezolid, vancomycin, teicoplanin, tigecycline and mupirocin but were resistant to tetracycline (30.4%), erythromycin (26.6%), clindamycin (24.1%), trimethoprim (19.0%), ciprofloxacin (17.7%), fusidic acid (15.2%) and gentamicin (12.7%). Molecular typing revealed 19 PFGE patterns, 26 Spa types and 21 sequence types. SCCmec-IV (86.0%) was the dominant SCCmec type, followed by SCCmec-V (10.1%). SCCmec-III (2.5%) and SCCmec-II (1.3%) were less common. ST6-IV/t304 (n = 30) and ST1295-IV/t690 (n = 12) were the dominant genotypes followed by ST772-V/t657 (n = 5), ST30-IV/t019/t021 (n = 5), ST22-IV/t852 (n = 4), ST80-IV/t044 (n = 3) and 18 single genotypes that were isolated sporadically. On the basis of SCCmec typing and MLST, 91.2% of the isolates were classified as community-associated MRSA and 8.8% of the isolates (consisting of four ST22-IV/t852, one ST239-III/t632, one ST5-III/t311 and one ST5-II/t003) were classified as healthcare-associated MRSA. The study has revealed the dominance of a Panton-Valentine leucocidin-negative ST6-IV/t304 clone and provided insights into the distribution of antibiotic resistance in MRSA at the tertiary hospital in Oman. It also highlights the importance of surveillance in detecting the emergence of new MRSA clones in a healthcare facility.

Evaluation of a modified double-disc synergy test for detection of extended spectrum beta-lactamases in AMPC beta-lactamase-producing proteus mirabilis.

The detection of extended-spectrum beta-lactamases (ESBLs) in gram-negative bacteria that produce AmpC beta-lactamases is problematic. In the present study, the performance of modified double-disc synergy test (MDDST) that employs a combination of cefepime and piperacillin-tazobactam for the detection of Proteus mirabilis producing extended spectrum and AmpC beta-lactamases was evaluated and compared with double-disc synergy test (DDST) and NCCLS phenotypic disc confirmatory test (NCCLS-PDCT). A total of 90 clinical isolates of P. mirabilis , which met the CLSI (Clinical and Laboratory Standards Institute) screening criteria that these had broth microdilution (BMD) MIC of > or =2 mg/mL for at least one extended spectrum cephalosporin [ceftazidime (CAZ), cefotaxime (CTX) and cefpodoxime], were selected for the study. MDDST detected ESBLs in 40/90 of the isolates, whereas DDST detected ESBLs in only 25 isolates. NCCLS-PDCT could detect ESBLs in 39 isolates using CAZ and CAZ + clavulanic acid (CLA) combination, whereas CTX and CTX + CLA combination could detect only 37 isolates as ESBL positive. As many as 34/40 ESBL positive isolates were confirmed to be AmpC beta-lactamase positive by the modified three-dimensional test (MTDT). MDDST and NCCLS-PDCT could detect ESBLs in all the 34 AmpC positive isolates, whereas DDST could detect ESBLs in only 19 isolates. The study demonstrated that MDDST is superior to DDST and as sensitive as NCCLS-PDCT. However, MDDST seems to have enhanced potential for the detection of ESBLs in AmpC beta-lactamase-producing P. mirabilis .

Veillonella as a cause of chronic anaerobic pneumonitis.

Anaerobes are not well recognized as a cause of chronic respiratory infections. A 44-year-old man was referred for evaluation of a progressive pulmonary disease of 7-month duration characterized by hemoptysis and fever. For these complaints, based on the radiological picture, he had already received antituberculous therapy without any relief. He was also subjected to bronchial artery embolization prior to referral. Evaluation of the patient led to a diagnosis of chronic anaerobic pneumonitis. Anaerobic culture of the computed tomography-guided transthoracic aspirate grew Fusobacterium and Veillonella species. Within 2 weeks of therapy with oral clindamycin, there was a dramatic relief in hemoptysis. This was accompanied by remarkable radiological clearance. This report underscores the importance of Veillonella species as a potential respiratory pathogen. A high index of suspicion is required to diagnose chronic anaerobic pneumonitis, which can mimic pulmonary tuberculosis, especially in tuberculosis endemic regions.

Molecular epidemiology of clinical isolates of ampc producing Klebsiella pneumoniae.

PURPOSE: AmpC producing K. pneumoniae have been increasingly reported from India but epidemiological studies are lacking. In the present study, molecular epidemiology of extended-spectrum AmpC beta-lactamases (ESACs) producing clinical isolates of K. pneumoniae prevalent in our hospital was studied. METHODS: Fifty-one non-repeat, consecutive, clinical isolates of K. pneumoniae producing AmpC enzymes, were subjected to whole cell protein profile analysis (SDS-PAGE) and ribotyping. The antimicrobial susceptibility was determined using standard disk diffusion technique. The isolates showing decreased susceptibility to cefoxitin (< 18 mm) or cefotetan (< 16 mm) were subjected to modified three- dimensional test for detection of AmpC enzyme. RESULTS: Six different types of protein profiles were observed. Ribotyping could further discriminate between the strains that were clustered by protein fingerprinting. Twelve different ribo-patterns were identified. Ribotyping was found to have a better Discriminatory Index (0.98) than that of SDS-PAGE (0.78). Of the 26 isolates that showed decreased susceptibility to cefoxitin and/or cefotetan 13 isolates were found to harbour AmpC enzyme. CONCLUSIONS: The study demonstrated the usefulness of SDS-PAGE whole cell protein profile analysis and ribotyping to identify the clonality of the ESACs isolates, the latter having a higher discriminatory power. The presence of ESACs isolates in the community as well as in hospital settings emphasizes the need for regular monitoring of antimicrobial resistance.

Liver abscess caused by Edwardsiella tarda biogroup 1 and identification of its epidemiological triad by ribotyping.

Two clinical isolates and an environmental isolate of Edwardsiella tarda biogroup 1 (ETB1), recovered from liver pus, the stool specimen and from the pond water of the village of the patient, diagnosed to have liver abscess, were found to be identical by protein fingerprinting and ribotyping. It can be construed that the pond water served as the source of infection. The epidemiological triad of the agent (ETB1), host (the patient) and environment (pond water) was thus established. This is the first report in which the triad for extraintestinal Edwardsiellosis caused by ETB1 has been identified. This also constitutes the first report of typing of ETB1 strains by SDS-PAGE and ribotyping.