Antimicrobial susceptibility profiles of gram-negative bacteria causing infections collected across India during 2014–2016: Study for monitoring antimicrobial resistance trend report

Background: The emergence of antibiotic resistance among bacterial pathogens in the hospital and community has increased the concern to the health-care providers due to the limited treatment options. Surveillance of antimicrobial resistance (AMR) in frequently isolated bacterial pathogens causing severe infections is of great importance. The data generated will be useful for the clinicians to decide empiric therapy on the local epidemiological resistance profile of the antimicrobial agents. This study aims to monitor the distribution of bacterial pathogen and their susceptibility pattern to the commonly used antimicrobial agents. Materials and Methods: This study includes Gram-negative bacilli collected from intra-abdominal, urinary tract and respiratory tract infections during 2014–2016. Isolates were collected from seven hospitals across India. All the study isolates were characterised up to species level, and minimum inhibitory concentration was determined for a wide range of antimicrobials included in the study panel. The test results were interpreted as per standard Clinical Laboratory Standards Institute guidelines. Results: A total of 2731 isolates of gram-negative bacteria were tested during study period. The most frequently isolated pathogens were 44% of Escherichia coli (n = 1205) followed by 25% of Klebsiella pneumoniae (n = 676) and 11% of Pseudomonas aeruginosa (n = 308). Among the antimicrobials tested, carbapenems were the most active, followed by amikacin and piperacillin/tazobactam. The rate of extended-spectrum beta-lactamase (ESBL)-positive isolates were ranged from 66%–77% in E. coli to 61%–72% in K. pneumoniae, respectively. Overall, colistin retains its activity in > 90% of the isolates tested and appear promising. Conclusion: Increasing rates of ESBL producers have been noted, which is alarming. Further, carbapenem resistance was also gradually increasing, which needs much attention. Overall, this study data show that carbapenems, amikacin and colistin continue to be the best agents available to treat drug-resistant infections. Thus continuous monitoring of susceptibility profile of the clinically important Gram-negative pathogens is of great importance to guide effective antimicrobial therapy.

In silico and In vitro activity of ceftolozane/tazobactam against pseudomonas aeruginosa collected across Indian hospitals

Ceftolozane/tazobactam is a novel antimicrobial agent with activity against Pseudomonas aeruginosa and other common Gram-negative pathogens. In this study, we determined the antimicrobial susceptibility for a total of 149 clinical isolates of P. aeruginosa for the most commonly used antimicrobials including the new agent ceftolozane/tazobactam (C/T). Broth microdilution was performed to determine the minimum inhibitory concentration against various antimicrobials including C/T. Among the ?-lactam/?-lactamase inhibitor, overall susceptibility was 67%, 55% and 51% for C/T, Piperacillin/Tazobactam (P/T) and Cefoperazone/Sulbactam, respectively. The variations in the susceptibility rates were noted among the three different ?-lactam/?-lactamase inhibitors. Interestingly, 33% susceptibility was noted for C/T against isolates that were resistant to P/T, indicating the higher activity of C/T. This finding suggests about 33% of the P/T-resistant isolates can still be treated effectively with C/T. C/T could be a better alternative for the treatment of ESBL-producing organism, and thereby usage of higher antimicrobials can be minimised.

Methicillin resistant Staphylococcus aureus (MRSA) in India Prevalence & susceptibility pattern.

Background & objectives: Methicillin resistant Staphylococcus aureus (MRSA) is endemic in India and is a dangerous pathogen for hospital acquired infections. This study was conducted in 15 Indian tertiary care centres during a two year period from January 2008 to December 2009 to determine the prevalence of MRSA and susceptibility pattern of S. aureus isolates in India. Methods: All S. aureus isolates obtained during the study period in the participating centres were included in the study. Each centre compiled their data in a predefined template which included data of the antimicrobial susceptibility pattern, location of the patient and specimen type. The data in the submitted templates were collated and analysed. Results: A total of 26310 isolates were included in the study. The overall prevalence of methicillin resistance during the study period was 41 per cent. Isolation rates for MRSA from outpatients, ward inpatients and ICU were 28, 42 and 43 per cent, respectively in 2008 and 27, 49 and 47 per cent, respectively in 2009. The majority of S. aureus isolates was obtained from patients with skin and soft tissue infections followed by those suffering from blood stream infections and respiratory infections. Susceptibility to ciprofloxacin was low in both MSSA (53%) and MRSA (21%). MSSA isolates showed a higher susceptibility to gentamicin, co-trimoxazole, erythromycin and clindamycin as compared to MRSA isolates. No isolate was found resistant to vancomycin or linezolid. Interpretation & conclusions: The study showed a high level of MRSA in our country. There is a need to study epidemiology of such infections. Robust antimicrobial stewardship and strengthened infection control measures are required to prevent spread and reduce emergence of resistance.

In-house, simple & economical phage technique for rapid detection of rifampicin, isoniazid, ethambutol, streptomycin & ciprofloxacin drug resistance using Mycobacterium tuberculosis isolates.

Background & objectives: Multiple drug resistance (MDR) among Mycobacterium tuberculosis poses a serious therapeutic problem. Early detection of MDR can be valuable but the conventional drug susceptibility tests take 4-6 wk time after the laboratory isolation of M. tuberculosis. The bacterial phage assay has been reported as a rapid tool for rifampicin susceptibility testing of tubercle bacilli using the suspension of isolated cultures. The present study was aimed to set up a phage assay for testing drug susceptibility to isoniazid (INH), rifampicin, ethambutol, streptomycin and ciprofloxacin in M. tuberculosis isolates. Methods: Mueller-Hinton broth instead of Middle Brook 7H9 broth was used to make it more economical. The phage assay was compared with the proportion method using 100 M. tuberculosis isolates from pulmonery TB cases. Phage assay results were available in 48 h for rifampicin and streptomycin while 72 h required for INH, ethambutol and ciprofloxacin. The assay was compared with gold standard proportion method. Interpretation of the results was easy and clear. Results: In the present study, sensitivity and specificity of the phage assay when compared to proportion method were in the range of 97 to 100 per cent for all the drugs except for ciprofloxacin for which it was 93 and 96 per cent, respectively. Interpretation & conclusions: The phage assay was economic, easy to perform and rapid for the detection of drug resistance in M. tuberculosis isolates with no requirement of expensive equipment. It is within the reach of microbiology laboratories in developing countries having high loads of tuberculosis.

Surface disinfection by exposure to germicidal UV light.

The present study was aimed to design a simple model to check efficacy of germicidal UV tube, to standardise the position, distance and time for UV light and also to find out its efficacy against medically important bacteria, the bacterial spores and fungi. The microbial cultures tested included gram positive and gram negative bacteria, bacterial spores and fungal spores. The microbes streaked on solid media were exposed to UV light. The inactivation of the order of four logs was observed for bacteria. UV light can have efficient inactivation of bacteria up to a distance of eight feet on either side and exposure time of 30 minutes is adequate.

Chikungunya infection in neonates.

We describe two neonates in whom chikungunya infection was confirmed by RNA PCR. Important clinical features include apnea, fever, erythematous maculo-papular rash and generalized hyperpigmentation.

Chronic knee monoarthritis caused by Mycobacterium chelonae.

Extrapulmonary tuberculosis occurs in 20% of all patients with tuberculosis and tubercular arthritis occurs in 10% of those with extrapulmonary tuberculosis. Arthritis caused by Mycobacterium tuberculosis is not uncommon in India. However, arthritis caused by Mycobacterium chelonae has not been reported to the best of our knowledge. We report a patient with arthritis caused by Mycobacterium chelonae in whom the diagnosis was confirmed by smear and culture of acid-fast bacilli. Polymerase chain reaction of the synovial fluid using IS6110 was negative.

Role of cancer antigen-125 from pleural & ascitic fluid samples in non malignant conditions.

BACKGROUND & OBJECTIVE: CA-125, an ovarian tumor marker is known to increase in non malignant conditions such as tubercular and non tubercular pleuritis and ascites. We undertook this study to evaluate non-specific rise in CA-125 levels in conditions associated with pleural effusion and ascites and also to understand the mechanism of its secretion. METHODS: CA-125 levels in 38 pleural and 46 ascitic fluid samples from non malignant cases and 10 blood samples from pulmonary tuberculosis cases were estimated by ELISA. The ascitic fluid samples were collected from cases of bacterial peritonitis, tuberculosis, hepatitis, cirrhosis of other aetiology and pleural fluid samples were from cases of tubercular, pyogenic, cardiomegaly and other conditions. RESULTS: Both ascitic and pleural fluid samples (transudative and exudative) showed elevated CA- 125 levels. The CA-125 levels were significantly higher in ascitic fluid samples than in pleural fluid samples. INTERPRETATION & CONCLUSION: Our findings showed that elevated levels of CA-125 in pleural and ascitic fluid could be because of varied aetiologies which need to be ruled out before considering malignancy. Peritoneum has a greater capacity to secrete CA-125 than the pleural epithelium and the secretion occurs following inflammation or mechanical distress. Pulmonary tuberculosis as a closed lesion without involvement of pleural epithelium does not evoke high CA-125 release.

Elisa kit evaluation for IGG and IGM antibodies to A-60 tubercular protein antigen.

AIMS: The purpose of this study is to evaluate the A-60 antigen-based enzyme-linked immuno sorbent assay (ELISA) test for its sensitivity, specificity, and other related statistical parameters. SETTINGS AND DESIGN: Sera from 114 healthy volunteers, 105 bacteriologically confirmed cases of pulmonary tuberculosis (PTB), 59 sera from family contacts of PTB, and 40 sera from cases of lung infections other than tuberculosis collected from September to December 2003 were used for the kit evaluation. METHODS AND MATERIALS: Enzyme-linked immuno sorbent assay test using tuberculosis A-60 antigen-based kit manufactured by Anda Biologicals, France was used for the evaluation. STATISTICAL ANALYSIS: Differences in the optical density (OD) values for immunoglobulins G (IgG), and immunoglobulins M (IgM) antibodies in various groups were studied using t-test. RESULTS: On the basis of the findings the threshold value was setup as 400 U for IgG and mean OD for sera from healthy volunteers +2SD as the threshold for IgM. The sensitivity was 80% and specificity 95.8% for the IgG antibody test. The efficiency and predictive values were also high. The sensitivity for IgM was low (28.5%) but the specificity was high (95.7%). None of the 40 nontubercular lung infection cases were positive for the IgG and IgM antibody test for A-60, whereas five and three cases of 59 family contacts of PTB were positive for IgG and IgM antibody test. The test reproducibility was good for both IgG and IgM. CONCLUSION: IgG antibody test using A-60 antigen has good sensitivity and specificity, whereas IgM antibody test had high specificity but low sensitivity. Multicentric trials suggested evaluation of the diagnostic utility of the test for the extra-PTB.

Biomedical waste in laboratory medicine: audit and management.

Pathology, microbiology, blood bank and other diagnostic laboratories generate sizable amount of biomedical waste (BMW). The audit of the BMW is required for planning proper strategies. The audit in our laboratory revealed 8 kgs anatomical waste, 600 kgs microbiology waste, 220 kgs waste sharps, 15 kgs soiled waste, 111 kgs solid waste, 480 litres liquid waste along with 33,000 litres per month liquid waste generated from labware washing and laboratory cleaning and 162 litres of chemical waste per month. Section wise details are described in the text. Needle sharps are collected in puncture proof containers and the needles autoclaved before sending to needle pit. The glass forms the major sharp category and is disinfected with hypochlorite before washing/recycling. All microbiology waste along with containers/plates/tubes are autoclaved before recycling/disposal. The problem of formalin fixed anatomical waste as histology specimens is pointed out. The formalin containing tissues cannot be sent for incineration for the fear of toxic gas release and the guidelines by the Biomedical waste rule makers need to be amended for the issue. The discarded/infected blood units in blood bank need to be autoclaved before disposal since chemical treatments are difficult or inefficient. The liquid waste management needs more attention and effluent treatment facility needs to be viewed seriously for hospital in general. The segregation of waste at source is the key step and reduction, reuse and recycling should be considered in proper perspectives.

Modified blood supplemented Lowenstein Jensen medium improves recovery of mycobacteria from sputum samples.

Lowenstein Jensen medium containing 3% human blood in CPDA anticoagulant was compared with plain LJ medium for mycobacterial growth using 565 sputum samples. Mycobacterial growth appeared on both the media in case of 148 samples. However, growth was faster by one week and colony size larger over blood supplemented LJ in 53 of the 145 culture positives. Additional 12 samples which showed no growth on plain LJ could grow only on LJ supplemented with blood. While 3 samples revealed scanty growth on plain LJ alone. The experience suggests that two LJ slants; one plain and the other supplemented with blood be in inoculated routinely to increase speed of growth and recovery of mycobacteria from clinical samples.

Current status of drug resistance among gram-negative bacilli isolated from admitted cases in a tertiary care centre.

AIMS: To study the prevalence of drug resistance among gram-negative bacilli and susceptibility to the Cefoperazone + Sulbactam and Piperacillin + Tazobactam combination among the hospital isolates resistant to all other antimicrobial agents. METHODS: A total of 367 gram-negative bacilli isolated from various pathological samples were tested for susceptibility to antimicrobial agent by the disc diffusion technique and 300 isolates of gram-negative bacilli which were resistant to all the other antibiotics were checked for sensitivity to piperacillin + tazobactam and cefoperazone + sulbactam combination. RESULTS: Resistance to ampicillin and amoxicillin + clavulanic acid ranged from (72.1-83.8%) which was highest. Resistance to cotrimoxazole, gentamicin, netilmycin, quinolones and all the generation of cephalsporins was often above 50%. piperacillin + tazobactam combination had the lowest incidence of resistance (14 to 20.5%). CONCLUSION: Drug resistance for most of the antimicrobial agents was greater than 50% including the various generations of cephalosporins. Inefficiency of amoxicillin + clavulanic acid (resistance among 72-79.5% isolates) suggests the presence of extended spectrum beta-lactamases among the isolates. Addition of sulbactam to cefoperazone showed increased activity (resistance 27 to 34%) but piperacillin + tazobactam was the most effective combination in vitro.

Hypochlorite (1%) is inefficient in decontaminating blood containing hypodermic needles.

Infectious biomedical waste and sharps have a potential hazard of transmission of pathogens. Among sharps, used needles form a major share and disinfection by 1 % hypochlorite is recommended in biomedical waste management rules of India. The aim of the present study was to evaluate the efficacy of hypochlorite for the decontamination of needles. Needles (16 g) filled with suspensions of standard strains and clinical isolates of gram positive and gram negative bacteria in plain normal saline and in human blood containing anticoagulant, were exposed to 1% hypochlorite and the surviving bacteria were subjected to viable counts. The observations indicated that 85 - 90 % of the needles filled with bacterial suspensions in saline are disinfected to a level of> 5 log bacterial reduction (standard disinfection) on exposure to hypochlorite but only 15 to 30% needles contaminated with the challenge bacteria suspended in blood showed> 5 log reduction in viable counts. Thus, hypochlorite treatment is inadequate for disinfecting needles contaminated with pathogenic bacteria in presence of blood and should not be recommended as an option for disinfection of the needles.

Prevalence of fungal meningitis among HIV positive & negative subjects in Indore (MP state).

The aim of the study was to find out the prevalence of fungal meningitis among AIDS cases and to assess the prognosis of fungal meningitis among HIV positive & negative subjects. The study comprised of 15 & 10 cases of fungal meningitis among HIV positive & negative subjects respectively during the study period 1992-2001. India ink preparation and Gram's staining procedures were carried out on the centrifuged CSF deposits. The CSF deposits were also used for bacterial and fungal culture. In the present study the prevalence of fungal meningitis was noted among 15 (3.1%) of 483 AIDS cases. Twelve of them had cryptococcal meningitis while 3 were infected with Candida albicans. Four AIDS cases presented fungal meningitis as a primary opportunistic infection and HIV status was confirmed in 4 of them after the diagnosis of fungal meningitis. 13 of the 15 cases were in the age group 26-40 yrs while one was 55 yrs old and the other 16 month old child; these two cases had blood transfusion transmitted and vertically transmitted mode of HIV transmission respectively. Further, only two of 15 cases were females and both acquired HIV infection through blood transfusion. Overall prognosis of fungal meningitis was poor among HIV positives and 7 of the 15 cases died in hospital within 2-3 weeks after diagnosis of cryptococcal meningitis. Among HIV negative subjects, ten cases of fungal meningitis (3 with Candida albicans and 7 with Cryptococcous neoformans) were noted at our end and nine of them had immunocompromised status (3 cases of renal transplant on immunosuppressives, 3 cases neonates/infant and 2 diabetic subjects. The fungal meningitis is one of the important causes of morbidity & mortality among immunocompromised among HIV positive subjects.

Drug resistance among tubercle bacilli from pulmonary tuberculosis cases in central India.

An estimate of drug resistance is extremely important in the epidemiology and control of tuberculosis. Data on drug resistance among mycobacterial isolates from sputum samples analysed at Microbiology dept. of Choithram Hospital and Research Centre, Indore, M.P. is presented here. Drug sensitivity testing was carried out on 1426 Mycobacterial isolates by the method of proportion using critical concentration in Lowenstein Jensen medium. Resistance for Isoniazid, streptomycin, and pyrazinamide was found to be high (54.2%, 41.5% and 50% respectively) and was followed by resistance to rifampin (25%) and ethambutol (22%). Resistance for kanamycin, p-aminosalicylic acid, thiacetazone and ciprofloxacin was much lower (18%, 13%, 6.5% and 3.6% respectively). Only 12% of the isolates were sensitive to all the anti-TB drugs while resistance to two, three, and four or more drugs was in the range of 20-25%. Pattern wise, simultaneous resistance to INF and Rifampin with or without resistance to other drugs was observed in 8.1% while resistance for Isoniazid + pyrazinamide and Isoniazid + streptomycin was 11.9 and 11.5% respectively. Resistance for Isoniazid + ethambutol was the lowest (5.1%). Growing multiple drug resistance among tubercle bacilli warrant urgent attention in tuberculosis control programme.