Phenotypic & molecular characterization of AmpC ß-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. from five Indian Medical Centers.

BACKGROUND & OBJECTIVES: AmpC ß-lactamases which are often plasmid mediated hydrolyze all ß-lactam antibiotics except cefepime and carbapenems. We evaluated the presence of AmpC ß-lactamases among Enterobacteriaceae strains recovered prospectively from patients at five Indian tertiary care centres. METHODS: The study included 909 consecutive Gram-negative isolates recovered from clinically significant specimens during June 2007 - May 2008 as part of an ICMR-ESBL study. Among the study isolates, 312 were found to be cefoxitin resistant by disc diffusion test (DDT). Minimum inhibitory concentration (MIC) determination by E test was done against amikacin, levofloxacin, impinem, meropenem, ertapenem, tigecycline and piperacillin-tazobactam. Combined DDT using phenyl boronic acid as inhibitor with cefoxitin was used for phenotypic confirmation of AmpC phenotype. The common Amp C genotypes ACC, FOX, MOX, DHA, CIT and EBC were detected by multiplex PCR. RESULTS: Plasmid mediated Amp C phenotype was confirmed in 114 of the 312 (36.5%) cefoxitin resistant isolates with 255 (81.7%) showing multidrug resistance. Susceptibility to tigecycline was highest (99%) followed by imipenem, meropenem (97%), ertapenem (89%), amikacin (85%), and piperacillin-tazobactam (74.6%). Levofloxacin resistance was 82 per cent. ESBL co carriage was observed among 92 per cent of Amp C producers. Among 114 Amp C producers, 48 could be assigned a genotype, this included CIT- FOX (n = 25), EBC (n = 10), FOX (n = 4), CIT (n = 3), EBC-ACC (n = 2) and one each of DHA, EBC-DHA, FOX -DHA and FOX-EBC-DHA. INTERPRETATION & CONCLUSIONS: Overall, AmpC phenotypes were found in 12.5 per cent isolates, multidrug resistance and ESBL co-carriage among them was high suggesting plasmid mediated spread. The study results have implications in rational antimicrobial therapy and continued surveillance of mechanisms of resistance among nosocomial pathogens.

Potential of mycobacterial excretory secretory protein antigens (sevatb ES-31, ES-43, EST-6 and ES-20) as biomarkers to detect Mycobacterium tuberculosis bacilli.

There is a need for a simple and reliable method to identify Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM). The utility of mycobacterial ES-31, ES-43, EST-6 or ES-20 antigen as a biomarker for differentiation of Mycobacterium tuberculosis bacilli from nontuberculous mycobacteria was explored using Fluorescein isothiocyanate conjugated antibodies against these antigens. Detection of these antigens was done from M.tb H(37)Ra and H(37)Rv DSS antigen. The presence of antigen in bacilli using FITC labelled antibody was indicated by green fluorescence on the cell surface while, its absence by no fluorescence under microscope. In M.tb H(37)Ra and H(37)Rv bacilli, fluorescence was observed on addition of FITC labelled anti ES-31 and anti ES-43 antibody; whereas no fluorescence was observed in case of EST-6 and ES-20 antibody conjugates. However all the antigens were detected in detergent soluble sonicate antigen of tubercle bacilli on addition of FITC conjugates. Fluorescence was not observed for ES-31, ES-43, EST-6 and ES-20 antigen in any of the tested NTM as well as in Escherichia coli. SEVA TB ES-31 and ES-43 may be used as biomarkers to distinguish M.tuberculosis bacilli from NTM.

Drug susceptibility of rapid and slow growing non-tuberculous mycobacteria isolated from symptomatics for pulmonary tuberculosis, Central India.

BACKGROUND: Non-tuberculous mycobacteria (NTM) are emerging as important pathogens. Their treatment also differs from that of Mycobacterium tuberculosis. In India, any datum on them is scarce as species identification and drug susceptibility are not performed in most laboratories. Susceptibility also differs from one geographic area to another, and in our country, there are no data even to guide the clinicians to start treatment empirically. METHODOLOGY: The present study endeavours to generate drug susceptibility data on NTM isolated from sputum samples collected and stored from 6445 symptomatics for pulmonary tuberculosis during a prevalence survey and from specimens received from the hospital. Isolates were not necessarily associated with the disease. Species were identified and antibiotic susceptibility was performed using micro-broth dilution technique as per the standard Clinical and Laboratory Standards Institute guidelines. RESULTS: A total of 65 NTM with 11 species were identified, of which 27 belonged to Mycobacterium fortuitum complex, 14 Mycobacterium gordonae, 9 Mycobacterium avium, 7 Mycobacterium flavescens, 4 Mycobacterium scrofulaceum and one each of others. Sensitivity to amikacin for M. fortuitum was 95.22% (20 out of 21), followed by ciprofloxacin (76.19%) and clarithromycin (71.42%). All the 9 M. avium isolates, 11 of M. gordonae (78.57%), 5 of M. flavescens and 2 of M. scrofulaceum were sensitive to clarithromycin. All NTM were resistant to first-line antitubercular drugs except 8, which were sensitive to streptomycin. CONCLUSIONS: Drug sensitivity of NTM varies from species to species. While amikacin was the best for rapidly growing mycobacteria, clarithromycin was the most active drug against M. avium and other slow growers.

Prevalence of tuberculous lymphadenitis in children in Wardha district, Maharashtra State, India.

SETTING: Rural area of Wardha district, Maharashtra State, Central India. OBJECTIVE: To determine the prevalence of tuberculous lymphadenitis in children aged 0-14 years in the study area and to assess factors that may contribute towards the prevalence. DESIGN: House to house survey of a population of 23,229 in 35 neighbouring villages with 7900 children aged 0-14 years from May 1993 to May 1994 and from March 1995 to February 1996. RESULTS: The prevalence of tuberculous lymphadenitis/1000 children was 4.43. The maximum prevalence was in the 5-9 years age group. The prevalence was 34 times higher in children with positive family history of tuberculosis than in those without a history. There was an association between prevalence and the living standards of the children, with a higher prevalence in families that belonged to an underprivileged social class living in thatched, improvised houses. Multiple cervical lymph nodes >2 cm and with matting and fluctuation were found to be characteristic clinical features. CONCLUSION: The prevalence of peripheral lymphadenopathy was 27.2/1000 children and that of tuberculous lymphadenitis was 4.43/1000. Positive history of contact in the family was a significant epidemiological indicator of tuberculous glands.

Mycotic keratitis due to Engyodontium album: first case report from India.

Engyodontium album is a rare and an unusual human pathogen. It is a common inhabitant of waste and moist material and frequently isolated from substrates such as paper, jute, linen and painted walls. This fungus grew within 3 days on SDA with chloramphenicol from corneal scrapping of a 70-year-old male farmer with a history of trauma by unknown vegetative matter. The fungus can be confused with Tritirachium sp and Beauveria sp.

Evaluation of variants of carbol fuchsin solution to stain acid-fast bacilli in-situ by the pot method.

BACKGROUND: Ziehl-Neelsen (ZN) staining requires heating, and pre-stained smears contain viable bacilli. OBJECTIVE: To evaluate four variants of carbol fuchsin solution by the pot method and compare the results with ZN staining, taking culture as gold standard. METHOD: Five hundred sputum samples from presumptive tuberculosis cases were homogenised and divided into two parts. One part was subjected to routine ZN staining and culture on solid medium, the other was equally distributed into four pots. Equal quantities of the basic fuchsin (BF) variant were added to each pot. Variant I contained 2% BF with 10% phenol and 4% ammonium sulphate (PhAS), while Variant II had 0.6% BF with PhAS; Variants III and IV contained respectively 2% and 0.6% BF with 10% phenol only. After 1 h, smears were made from each pot and culture was performed on Löwenstein-Jensen medium. Smear results were compared with the ZN results and evaluated against culture. RESULTS AND CONCLUSION: Variant III gave excellent results compared to ZN (κ = 0.97), with sensitivity, specificity, and positive and negative predictive values similar to those of ZN, taking culture as gold standard. Pot contents were negative for Mycobacterium tuberculosis culture.

Prevalence of sputum-positive pulmonary tuberculosis in tribal and non-tribal populations of the Ashti and Karanja tahsils in Wardha district, Maharashtra State, India.

SETTING: Ashti and Karanja tahsils, Wardha district, Maharashtra State, Central India. OBJECTIVE: To find and compare the prevalence of bacillary positive pulmonary tuberculosis amongst the different tribes and in the non-tribal population. DESIGN: Prevalence study of pulmonary tuberculosis by house-to-house survey of symptoms among tribal (n = 20596) and non-tribal (n = 93 670) populations aged 5 years and over, between September 1989 and November 1990. RESULTS: The prevalence of smear and/or culture-positive tuberculosis/100000 population was 133 in the tribal and 144 in the non-tribal population. The difference in prevalence of symptomatic individuals and sputum-positive cases among the tribal and the non-tribal populations was statistically significant only in the symptomatic individuals/100000 (P = 0.01). The prevalence of cases in both groups was higher in males than females; however this difference was significant only in the tribal group (P = 0.05). Only two of the 46 tribes encountered, the Mana and Pawara tribes, showed a high prevalence, of 730 and 612/100000, respectively. The three other tribes with positive cases (the Gond group) had prevalences comparable to that of the nontribal population. CONCLUSION: The prevalence of tuberculosis in tribal people was comparable to that of the non-tribal population.

Characteristics of Vibrio cholera 0139 strains isolated in Sevagram (Maharashtra) during April-August 1993.

A total of 44 strains of Vibrio 0139 serotype isolated between April and August 1993 at Sevagram (Wardha) were examined for expression of a number of biochemical and physiological characteristics. All strains fermented lactose within 24 h and belonged to Heiberg group III. Salt tolerance to 8 per cent NaCl was seen in 22.72 per cent strains. Haemolysis of sheep RBCs and haemagglutination of human 'O', chicken and rabbit RBCs was consistently positive. All the strains were sensitive to tetracycline and resistant to polymyxin B and cotrimoxazole.

Leptospirosis--a case report.

Outbreaks of Leptospirosis have been reported from various parts of India including Maharashtra. We report a case of leptospirosis from Wardha District, Maharashtra from where the disease has not been reported so far.

Evaluation of routine laboratory methods in the diagnosis of tubercular lymphadenitis.

Lymph node biopsies from 64 cases suspected of Tuberculosis were subjected to fluorescent smear microscopy, culture and histopathology. Fluorescent microscopy was found to be the most sensitive technique (32.8%) followed by culture (20.3%) and histopathology (14.06%). Thus, for the diagnosis of tubercular lymphadenitis requisition for bacteriological investigations must be sent along with histopathology, particularly in early cases.

Comparision of Three Laboratory Tests for Detection of AmpC ß Lactamases in Klebsiella Species and E. Coli.

BACKGROUND AND OBJECTIVE: AmpC ß lactamases are one of the important causes of drug resistance in gram negative bacteria. Failure to detect these enzymes in the laboratory has contributed to therapeutic failures but there are till date no standard guideline available. This study was therefore undertaken to evaluate three phenotypic laboratory tests and the inhibitors used in two of the tests to detect AmpC ß lactamases produced by E. coli and Klebsiella species as they are most commonly isolated organisms. METHODS: E. coli and Klebsiella isolates from different clinical samples were tested for ESBLs production as per CLSI guidelines and excluded from the study. The non-ESBLs isolates were then screened for AmpC ß lactamases production, by cefoxitin and then confirmed by three different methods, i.e., Disc Potentiation Test (DPT) , Double Disc Synergy Test (DDST) and Modified Three Dimensional Test (M3DT) which in the absence of molecular methods, was taken as the gold standard. Boronic acid and cloxacillin were used as inhibitory agents in the Disc Potentiation and Double Disc synergy Tests. RESULTS: A total of 2,933 isolates were tested out of which 165 isolates were detected as non ESBLs producers,135 (81.82%) when screened for AmpC ß lactamases based on resistance to cefoxitin were labelled as positive. 30 (18.18%) cefoxitin sensitive isolates were labelled as probably non AmpC producers . M3DT, in addition to detecting all the 135 (100%) cefoxitin resistant isolates, also detected 5 (16.67%) cefoxitin sensitive isolates as AmpC producers. Other phenotypic tests, DPT and DDST with different inhibitors like boronic acid and cloxacillin in different potencies were all found to be less sensitive. The best results among these two methods were obtained with DDST using cloxacillin 500µg. CONCLUSION: In the absence of recommended guidelines for AmpC detection, the study reports, among the tests performed, M3DT as the best phenotypic method for AmpC confirmation, as it is not only the most sensitive but also specific test for AmpC as it rules out the resistance due to other mechanisms like the porin channel.

Trends in the annual risk of tuberculous infection in India.

SETTING: Twenty-four districts in India. OBJECTIVES: To evaluate trends in annual risk of tuberculous infection (ARTI) in each of four geographically defined zones in the country. STUDY DESIGN: Two rounds of house-based tuberculin surveys were conducted 8-9 years apart among children aged 1-9 years in statistically selected clusters during 2000-2003 and 2009-2010 (Surveys I and II). Altogether, 184,992 children were tested with 1 tuberculin unit (TU) of purified protein derivative (PPD) RT23 with Tween 80 in Survey I and 69,496 children with 2TU dose of PPD in Survey II. The maximum transverse diameter of induration was measured about 72 h after test administration. ARTI was computed from the prevalence of infection estimated using the mirror-image method. RESULTS: Estimated ARTI rates in different zones varied between 1.1% and 1.9% in Survey I and 0.6% and 1.2% in Survey II. The ARTI declined by respectively 6.1% and 11.7% per year in the north and west zones; no decline was observed in the south and east zones. National level estimates were respectively 1.5% and 1.0%, with a decline of 4.5% per year in the intervening period. CONCLUSION: Although a decline in ARTI was observed in two of the four zones and at national level, the current ARTI of about 1% in three zones suggests that further intensification of TB control activities is required.

Comparison of disc and MIC reduction methods with polymerase chain reaction for the detection of metallo-ß-lactamase in Pseudomonas aeruginosa.

PURPOSE: The present study was undertaken to evaluate the screening antibiotic, confirmatory phenotypic test and agent against PCR as gold standard and to detect the prevalent MBL gene. MATERIALS AND METHODS: Three hundred and twenty-six Pseudomonas aeruginosa isolates were screened for resistance to Imipenem (IPM), Meropemem (MEM) and Ceftazidime (CAZ) by disc diffusion. Isolates resistant to any of these were considered screen test-positive for MBL and were subjected to Double disc synergy test (DDST) and Disc potentiation test (DPT: Using IPM, MEM and CAZ alone and with EDTA), Minimum inhibitory concentration (MIC) reduction [four-fold or more reduction in MIC of IPM and MEM in presence of chelators: EDTA and 1,10-phenanthroline (EPI/EPM: EDTA-phenanthroline- Imipenem/Meropenem Broth Microdilution method)] and polymerase chain reaction (PCR) for blaIMP and blaVIM . RESULTS: Screen test-positives by MEM and CAZ were 19.3% as against 17.8% by IPM. MEMDDST, DPT and EPM confirmed 100% screen-test positives as against 93.7% by CAZ DDST and DPT-2, 76.2% by CAZ DPT-1, 88.9% by IPM DDST, 85.7% by IPM DPT-1 and 92.1% by EPI. IPMand CAZ DDST together confirmed 100% while IPM and CAZ DPT-2 confirmed 96.8%. All 63 screen-test positives showed the presence of blaVIM . CONCLUSIONS: MEM was found to be the best screening and confirmatory agent for MBL detection and blaVIM was found to be the prevalent MBL gene in this part of the country.

Detection of metallo-ß-lactamases producing Acinetobacter baumannii using microbiological assay, disc synergy test and PCR.

BACKGROUND: One leading factor responsible for resistance in Acinetobacter baumannii, an important opportunist in health care institutions globally, is the production of carbapenamases like metallo-ß-lactamases (MBLs), which hydrolyze a variety of ß-lactams including penicillin, cephalosporins and carbapenems. However, neither any standard guidelines are available nor any method has been found to be perfect for their detection. Various methods have shown discordant results, depending upon the employed methodology, ß-lactamase substrate and MBL inhibitor used. This study aims to evaluate two phenotypic methods against PCR as gold standard among carbapenem resistant A. baumannii for identifying MBL producers. MATERIALS AND METHODS: A total of 130 A. baumannii were screened for imipenem and meropenem resistance by Kirby-Bauer disc diffusion method. Phenotypic expression of MBL was detected by EDTA-imipenem-microbiological (EIM) assay and extended EDTA disc synergy (eEDS) test and presence of bla-IMP and bla-VIM was detected by PCR in all the carbapenem resistant isolates. RESULTS: Of the 43 imipenem and/or meropenem resistant A. baumannii isolates, 4 (9.3%) were found to be MBL producers by EIM and 3 (6.97%) by eEDS. Only bla-VIM gene was detected in 7 (16.28%) by PCR. In addition EIM detected 14 (32.56%) carbapenem resistant non-metallo enzyme producers. CONCLUSION: Of the two MBL genes targeted, bla-VIM was only detected and that too in isolates resistant to both imipenem and meropenem. Further, EIM was useful in differentiating MBL from non-metalloenzymes producers.

Comparison of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide tube method with the conventional method and real-time polymerase chain reaction for the detection of rifampicin resistance in Mycobacterium tuberculosis.

Colorimetric methods are cheap, reproducible, and rapid methods of detecting drug resistance in Mycobacterium tuberculosis. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) method is one such technique that has been established in our laboratory to detect rifampicin resistance. The present study compared the results of the MTT method with those of the proportion method and real-time polymerase chain reaction (RTPCR) in order to establish sensitivity and specificity of MTT. The mutations for rifampicin resistance occur in rpoB gene, and the commonest reported are in codons 526 and 531. Therefore, RTPCR was targeted at these two codons. The concordance of MTT with the proportion method and RTPCR was 94 and 72.77%, respectively, and that of RTPCR with the proportion method was 77.77%. While the study confirmed that the MTT method is a good method for detecting rifampicin resistance, it also brought out the fact that RTPCR when targeted for limited mutations is not a good tool. Either the genotypic method used should target the total 81-bp rpoB genome or methods such as DNA sequencing should be used. For resource-constraint laboratories, the MTT method can be considered as a better choice.

Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples.

PURPOSE: Clindamycin is commonly used in the treatment of erythromycin resistant Staphylococcus aureus causing skin and soft tissue infections. In vitro routine tests for clindamycin susceptibility may fail to detect inducible clindamycin resistance due to erm genes resulting in treatment failure, thus necessitating the need to detect such resistance by a simple D test on routine basis. MATERIALS AND METHOD: 247 Staphylococcus aureus isolates were subjected to routine antibiotic susceptibility testing including oxacillin (1ìg) by Kirby Bauer disc diffusion method. Inducible clindamycin resistance was detected by D test, as per CLSI guidelines on erythromycin resistant isolates. RESULTS: 36 (14.5%) isolates showed inducible clindamycin resistance, nine (3.6%) showed constitutive resistance while remaining 35 (14.1%) showed MS phenotype. Inducible resistance and MS phenotype were found to be higher in MRSA as compared to MSSA (27.6%, 24.3% and 1.6%, 4% respectively). CONCLUSION: Study showed that D test should be used as a mandatory method in routine disc diffusion testing to detect inducible clindamycin resistance.

Disseminated disease caused by Mycobacterium simiae in AIDS patients: a report of three cases.

Disseminated disease caused by Mycobacterium simiae, a slowly growing nontuberculous mycobacterium, has been rarely reported in the literature. We report on three AIDS patients who were found to suffer from mycobacteraemia caused by M. simiae in a rural tertiary-care hospital in central India.