Indian Journal of Microbiology Research

Page No. 1-6

Authors: Lakshmi K., Santhanam R., Chitralekha S.

Tuberculous Meningitis (TBM) is one of the leading causes of deaths and disabilities in the developing nations like India. Rapid diagnosis of the cases is essential to minimise the mortality and morbidity. Although clinical diagnosis is made based on the presenting symptoms, signs and radiological imaging studies, confirmation of the diagnosis is still difficult and there are significant diagnostic and treatment challenges. Cerebro spinal fluid (CSF) examination shows a lymphocytic-predominant pleiocytosis, elevated protein, and normal or low glucose. Multiple and repeated Culture and CSF examination by acid fast staining and PCR usually may show good results. Empirical treatment with at least four first-line drugs, preferably isoniazid, rifampin, pyrazinamide, and streptomycin or ethambutol is usually started in clinical suspected cases with supportive initial CSF findings. Corticosteroid can be added as adjunctive treatment, but its benefits remains doubtful. Drug interactions, possibility of drug resistant tuberculosis and development of immune reconstitution inflammatory syndrome needs to be kept in mind in initiating the TBM treatment in patients who are HIV positive. This review article throws light into the current practice in the laboratory diagnosis and management of Tuberculous meningitis

Page No. 14-19

Authors: Jyotismita Khasnabis, Paulomi Adhikari, Deblina Chowdhury, Chandan Rai, Arindam Roy

Background: Snacks and sweets from road side eateries are cheap sources of nutrition but occasionally cause numerous public health hazards most importantly gastroenteritis. Various factors including poor infrastructure to prepare foods, post-preparation contamination during storage and handling as well as unhygienic surroundings may contaminate these foods with pathogenic bacteria. Bacillus cereus is one of the common and important food borne pathogens causing spoilage to the foods and producing gastroenteritis to the consumers. This study was carried out to enumerate B. cereus and assess their spoilage potentiality and antibiotic resistance pattern. Methods: Standard plate count was performed using plate count agar while presumptive B. cereus was isolated in Bacillus cereus agar base supplemented with polymyxin B selective supplement and egg yolk emulsion. Confirmation as B. cereus was done by morphological, biochemical and physiological methods. Confirmed isolates were tested for extracellular enzyme production and antibiotic resistance pattern. Result: Standard plate count exceeded 1010 cfu g-1 and B. cereus count was positive in 57% of the samples. All the 43 B. cereus isolates produced amylase and caseinase and were multiple drug resistant. None produced lipase. Conclusion: Presence of B. cereus with spoilage potential and multiple drug resistance require good manufacturing practices (GMPs) and good hygienic practices (GHPs) during food production and storage.

Page No. 26-30

Authors: Gomathi Maniyan, Dillirani Vedachalam, Nithya Chinnusamy

of life threatening nosocomial infections and as multidrug resistant pathogens. Acinetobacter species are the opportunistic pathogens with increasing prevalence in the nosocomial infections. Community acquired infections are also common in Acinetobacter. It accounts for 10% of all community-acquired bacteraemic pneumonias. Aim: To isolate, identify and detect Carbapenem resistance producing Acinetobacter spp., and confirm Oxacillanase (OXA) and Metallo Beta Lactamase (MBL) production by phenotypic& genotypic methods. Materials and Methods: This cross sectional study conducted in a tertiary care hospital for a period of one year and samples collected like pus, urine, endotracheal aspirates, blood, sputum and body fluids were identified using standard protocol, which includes Gram staining, test for motility, catalase test, oxidase test, OF test and various biochemical reactions. The resistant strains of Acinetobacter species as identified by phenotyping were subjected to molecular analysis of OXA-51, VIM and IMP genes. Results: Out of 110 isolates of non-fermenters, Acinetobacter baumannii accounted for 36 (81.8%) and Acinetobacter lwoffi 8 (18.2%). The antimicrobial susceptibility pattern revealed maximum resistance to Gentamycin (50%), Cotrimoxazole (47%), followed by Ciprofloxacin (50%) and Cefotaxime (32.2%). Sensitivity to Polymyxin B (100%) followed by Imipenem and Meropenem (75.5%). MBL production was 20.5%. Molecular characterization of MBL of Acinetobacter species revealed, OXA- 51 (33.3%), 3 (33.3%) for bla IMP. and 2(22.2%) isolates blaVIM positive. Conclusion: Acinetobacter baumannii were the most common isolate in this study. Differences in antimicrobial susceptibility pose a great problem in treating these infections. MBL production by these organisms leads to high morbidity and mortality and left with the only option of treating them by potentially toxic drugs like Colistin and Polymyxin B.

Page No. 36-39

Authors: Rajeswari Jayakumar

Background & Objectives: Decompensated liver disease (DCLD) is defined as irreversible chronic injury of the hepatic parenchyma. Bacterial infections are more common in decompensated liver disease and causes 30%-50% of deaths. Therefore, this study was done to determine the various bacterial agents causing infections in decompensated liver disease patients and to determine the drug susceptibility and resistance pattern. Methods: A prospective study was conducted over a period of one year in a tertiary care hospital. Ascitic fluid, urine, sputum, blood and wound swab were collected. All the samples were processed through Gram’s stain and culture. The organisms were identified by standard protocols and antibiotic susceptibility testing. Results &Conclusion: Out of 150 samples, culture positivity seen in 81(54%). In 81 culture positive isolates, 63(78%) were Gram Negative bacilli (GNB) and 18 (22%) were Gram Positive cocci (GPC). Among Gram negative bacilli, Escherichia coli and in Gram positive cocci, Staphylococcus aureus was the most common isolates. The most common infections were spontaneous bacterial peritonitis (27%) followed by urinary tract infections (26%), Spontaneous bacteraemia (19%), pneumonia (16%), and skin and soft-tissue infections (12%).

Page No. 44-47

Authors: Sathish J.V., Ram Mohan Reddy K., Sujith Pavan

Background: Due to large number of infections caused by biofilm producing organism, a reliable and reproducible method for its detection is necessary. Specific methods like Polymerase Chain Reaction (PCR) are available for detection of biofilm, but these are beyond the scope majority of laboratories in developing nations like India. Objective: To test reliability of Tube Adherence test as a screening tool for detection of biofilm formation among Staphylococcus. Materials and Methods: Various clinical samples were collected with universal precautions from patients with indwelling medical device for more than 48 hours. Screening for biofilm detection was done using Tube adherence method. Results: Tube method was 97.5% sensitive and 100% specific for detection of biofilm formation. Conclusion: Tube method can be used as a general screening method for detection of biofilm formation by Staphylococci

Page No. 52-58

Authors: Soumya Kaup, Sunitha B. U., Jaya Sankarankutty

Introduction: Emergence of multi-drug resistant (MDR) organisms is an important public health concern especially in developing countries. The present study was carried out to assess the current antimicrobial pattern of multi-drug resistant Escherichia coli. Materials and Methods: 346 consecutive, non-duplicate isolates of E. coli isolated during a period of two years were included in the study. The numbers of multi-drug resistant isolates were determined. Their antimicrobial resistance phenotypes and Multiple Antibiotic Resistance (MAR) index were determined. Results: Among the 346 isolates of E. coli, 297 (85.84%) were multi-drug resistant with 61.3% of the isolates being resistant to drugs in more than five antimicrobial categories. The Multiple Antibiotic Resistance index of 82.7% of isolates was found to be greater than 0.2. Carbapenems, aminoglycosides, Chloramphenicol and Nitrofurantoin were found to be the most effective agents against MDR E.coli. Conclusion: Antimicrobial resistance profiles of bacteria are ever-changing and periodic evaluation of resistance phenotypes of isolates is essential for the formulation of appropriate antibiotic policy and initiation of pertinent empirical therapy.

Page No. 64-67

Authors: Vijaya Shivanna, Achut Rao

Background: The extended spectrum β-lactamases(ESBLs), AmpCβ-lactamases and metalloβ-lactamases (MBLs), have emerged worldwide as a cause of antimicrobial resistance in Gram negative bacteria (GNB). So the present study was conducted to detect all three β-lactamases in GNB using disc potentiation tests. Materials and Methods: A total of 200 Gram negative clinical isolates from a tertiary care centre were identified and antibiotic susceptibility testing was done according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The screening and phenotypic confirmatory test recommended by CLSI were carried out for ESBL detection. Detection of AmpCβ-lactamase was done by phenyl boronic acid test. MBL were detected using EDTA disc potentiation test. Result: Of a total of 200 Gram negative isolates, pure ESBL were seen in 50(25%) isolates and pure AmpC were seen in 35(17.5%) isolates. ESBL and AmpC co-existed in 38(19%) isolates. AmpC and MBL co-occurred in a single isolate. Conclusion: we recommend the phenotypic tests using various inhibitors for the detection of β-lactamases.

Page No. 74-78

Authors: Alice Peace Selvabai, A. S. Shameem Banu, M. Jeya, Priyadarshini Shanmugam

Staphylococcus aureus remains as one of the most potent bacterial human pathogen because of its expression of various virulence factors and also due to its property of multidrug resistance. A total of 468 non –duplicate S.aureus strains obtained from various clinical specimens were included in the study. Methicillin Resistant S.aureus (MRSA) strains were screened for DNase production using DNase agar, hemolytic property of the isolates were detected in 5% sheep blood agar plates, hemagglutination property of the isolates were demonstrated using 1% O group RBC’s,slime production was detected using congo red agar medium and biofilm production was quantitatively assayed by microtitre plate method. Antimicrobial susceptibility profile was studied for the MRSA isolates. Out of 468 S.aureus strains, 114(24%) strains were detected as MRSA. Among the MRSA strains 99(86%) were positive for DNase, 77(67%) were showed beta hemolysis, 49 (42%) strains were positive for hemagglutination, 70 (61%) were slime producers, most of the strains were biofilm producers, 4(3.5%) were non-adherent, 85(74%) were weakly adherent, 23(20%) were moderately adherent and 2(1.7%) were strongly adherent. About 56%, 60% and 63% of the isolates were resistant to cotrimoxazole, erythromycin and ciprofloxacin respectively and about 22% and 33% of the isolates showed resistance towards clindamycin and gentamicin respectively. All the isolates were sensitive to vancomycin, teicoplanin and linezolid. About 10(8.7%) strains showed high level mupirocin resistance (HLMR) and 1(0.8%) strain showed low level mupirocin resistance (LLMR). Both HLMR and LLMR (100%) strains showed susceptibility to fusidic acid. MRSA infections remain a major threat in both community and nosocomial settings. Therefore a thorough understanding of its virulence mechanisms and regular surveillance of antimicrobial susceptibility pattern will help the clinician to choose appropriate treatment options and to control the emergence of multidrug resistant strains.

Page No. 83-86

Authors: Asha Pai KB, Manjunath R Kamath, Rekha Rai, Gopalakrishnan M

Background: Mupirocin has been recognised as the best and the most effective topical antimicrobial agent for decolonisation. With widespread use of mupirocin for decolonisation, increasing number of MRSA have developed resistance to mupirocin. The society of thoracic surgeons recommends a glycopeptide (vancomycin) for cardiac surgical prophylaxis in patients known to be colonised with MRSA. Development of resistance to vancomycin among MRSA is a cause of concern. Currently, there is very limited data regarding prevalence of mupirocin and vancomycin resistance in MRSA colonising the anterior nares of patients undergoing high risk surgeries. Aims: To assess the prevalence of mupirocin and vancomycin resistance in MRSA colonising anterior nares of patients undergoing cardiac surgical procedures in our tertiary care hospital. Material and Methods: Screening for MRSA was done by disc diffusion method using Cefoxitin disc. Screening for MuL and MuH was done by disc diffusion method using mupirocin disc of strength 5 µg and 200 µg respectively. Screening for detection of reduced susceptibility to vancomycin was done by Agar Dilution method. The MIC of mupirocin and vancomycin determined by Agar dilution method. Results: Twenty two percent of MRSA isolates had MuL and eleven percent had MuH. One of the mupirocin sensitive MRSA, had a presumptively reduced susceptibility to vancomycin by BHI agar screen method. Conclusion: Screening of patient populations for MRSA, MuH and VISA/VRSA colonisation, especially those undergoing high risk surgeries should be considered, to prevent self infection and nosocomial transmission of resistant strains.

Page No. 90-95

Authors: Garima, Deepti Chaurasia

Introduction: The multidrug resistant Enterobacteriaceae isolates pose not only therapeutic problems but also serious concerns for infection control management. Early detection of lactamase producers is crucial to establish appropriate antimicrobial therapy and to prevent their interhospital and intrahospital dissemination. Objective: To study the prevalence of ESBL producing bacteria among all the members of Enterobacteriaceae isolated from various clinical specimens collected from all wards across a tertiary care teaching hospital. Methodology: ESBL production was detected among all the Enterobacteriaceae using, Phenotypic Combined Disk Diffusion Test (PCDDT), as recommended by the Clinical Laboratory Standards Institute (CLSI). Results: There is a high prevalence (25.67%) of ESBL producers in our hospital and so, it is essential to report the ESBL production along with the routine sensitivity reports, which will help the clinician in prescribing proper antibiotics. Pediatric ward had the highest number of ESBL positive Blood cultures. The pus isolates culture sensitivity testing from Surgery ward, Orthopedics and Burn and Plastic Surgery ward showed maximum number of ESBL strains. ESBL isolates show coresistance to many other classes of antimicrobials. Carbapenems (Imipenem, Meropenem), BL/BLI combination viz. Cefoperazone-sulbactum and aminoglycoside (amikacin) came out to be effective against these. Conclusion: A high degree of ESBL producers and carbapenem resistant Enterobacteriaceae is concerning; with emerging resistance to colistin, raising the fear of a return to the preantibiotic era. An urgent intervention including creating awareness and establishment of robust infection control and antibiotic stewardship program is the most important need of the hour.

Page No. 99-103

Authors: Mangala Ghatole, Virendra Kashetty, Apeksha Ghule, Avinash Wuike

Background: Control of tuberculosis is hampered by emergence of drug resistance such as MDR, XDR & TDR. Treatment of this disease will be facilitated if early reliable drug susceptibility profile is available. Various commercial systems are available but in resource poor country like India cheaper colorimetric methods are evaluated. One such method is using 2,3,5- Triphenyl tetrazolium chloride (TTC), which is an oxidation–reduction dye indicating cellular viability and growth. Colorimetric methods used in liquid media are shown to give better results than using them in Lowenstein–Jensen medium (LJ). Material and Method: A total of 82 strains isolated from patients suffering pulmonary tuberculosis and identified as M. tuberculosis were subjected to DST for Isoniazid and Rifampicin by Indirect drug susceptibility testing using absolute concentration method. 1% TTC was incorporated in Middlebrook 7H9 broth along with the drugs and compared with drug containing LJ. Results: Among the 82 strains, 9 (10.9%) strains were resistant to INH by both methods. Whereas 06 (7.3%) were resistant only in TTC. (sensitivity-100%, specificity -60%, positive predictive value- 91.8% and negative predictive value of 100%, P≤0.0001). Similarly 11(13.4%) strains showed resistance to RMP by both methods, 08 (9.8%) were resistant by only TTC and 01 (1.2%) only by conventional method. (Sensitivity- 98.4%, specificity- 57.9%, PPV-88.6%, NPV-91.7%, P≤0.0001). Conclusion: Results were available for 60 (73.2%) and 70 (85.4%) of strains by TTC method for INH and RMP respectively in 07 days as against 28 days in conventional method.

Page No. 110-113

Authors: Meenakshi Aggarwal, Sonal Saxena

Urinary tract infections constitute a large share of human systemic infections and are one of the commonest bacterial infections that lead patients to seek medical care. Positive culture results are a prerequisite for timely and effective treatment of UTIs. In resource limited settings, conventional media and biochemicals are used for isolation of pathogens and a positive urine culture sometimes may require more than 48 hours for identification. To overcome this time lag, chromogenic media have been developed which help in early presumptive identification of the uropathogens and timely initiation of therapy. We have evaluated one such media Urichrom–II™ (International Microbio, France) in comparison to in house conventional media for growth of common urinary pathogens.

Page No. 118-125

Authors: Kulal B., Bhat K.S., Meundi M., Kotigadde S.

Aim of the study: To determine the fungi causing otomycosis and the associated bacterial pathogens in a rural set up in clinically suspected cases of otomycosis. Methodology: Ear swabs were collected from 100 clinically suspected cases of otomycosis and from 50 persons who were apparently healthy. All the samples were processed by direct microscopy of KOH mount and Gram staining. Cultures were carried out for both fungal and bacterial isolates, which were identified by standard procedures. The data were statistically analysed. Results: Among 150 samples, 51.3% yielded only fungal growth, 23.3% grew bacteria only and 19.3% showed mixed growth of fungi and bacteria. Major fungal isolates were Aspergillus spp. and Candida spp. One sample grew A. sydowii which is an uncommon agent causing otomycosis. Major bacterial isolates were Staphylococcus aureus and Pseudomonas spp. All pathogenic bacteria were sensitive to routinely used antibiotics. Samples taken from healthy persons did not yield any fungal or bacterial pathogens.