Journal of Microbiology and Related Research

 Introduction: India carries about 25% burden of tuberculosis. It is estimated that about 15­20% of all TB cases are of extrapulmonary origin. Recently, molecular amplification methods such as PCR and RT PCR have attracted interest of laboratorians as the diagnosis can be made in a short time and treatment initiated at the proper time.The present study was undertaken to evaluate the role of Truelab Micro PCR (Molbio diagnostics) in diagnosis of EPTB. Materials and Methods: This was a retrospective study conducted between January 2014 to June 2016.Atotal of 447 patients suspected of having EPTB clinically were included in the study. Specimens were collected from patients, both male and female, from different sites like pus, body fluids, lymph node aspirates, CSF, semen, urine, menstrual blood and endometrial tissue etc.according to standard protocols. All the specimens were run on Truelab MicroPCR system(Molbio diagnostics) according to standard protocols. Results: A total of 447 patients data was used for this study. Maximum number of samples were endometrial tissue (59.9%) followed by lymph node aspirates (11.63%), pus (4.92%),urine (4.47%), pleural fluid & menstrual blood (3.57%) CSF (3.13%), Ascitic fluid (2.90%) Synovial fluid (1.1%), vitreous fluid and peritoneal fluid (0.89%) , BAL fluid (0.67%) . Out of these different extra pulmonary samples 19(4.25%) were positive by RT PCR and 95.75% were negative. Highest positivity rate was observed inpus (27.2%), followed by CSF (21.4%), menstrual blood & pleural fluid (6.25%), lymph node aspirates(5.76%), urine(5%) and 1.49% in endometrial tissue. Conclusion: Truelab Micro PCR is portable,easy to use and provides accurate diagnosis of disease like EPTB with a rapid turn around time. It does not require skilled manpower and elaborate infrastructure and can be easily installed in resource limited settings. A complete patient centric solution for patients suffering from EPTB is a necessity and rapid, accurate diagnosis with Truelab Micro PCR and prompt therapywill help control TB burden in India.

Keywords: EPTB; Real Time PCR; Molecular Methods; True Lab MicroPCR.

 Background: Burn injuries constitute a major health concern with respect to morbidity and mortality. Burns are a very common injury, serious burn injuries are ex­cruciatingly painful and require special care to prevent infection and reduce the severity of scarring. Objectives: To determine the predominant bacterial pathogens in the burn wound infection with age, sex and cause of injury, and to know the antibiotics sensitivity profiles of the isolates obtained. Material & Method: Burn Patients admitted within 24 hours were included. Whereas Patients admitted after 24 hours were excluded. Two burn swabs were aseptically collected on admission before the start of antibiotics. One swab was subjected for gram staining and the other for culture. All specimens were inoculated on 5% blood agar and MacConkey agar and incubated. Isolated organisms were subjected for Antibiotic Susceptibility. Result: Maximum patients (50%) belonged to 21­ 40 years age group. and males 39 (65%) were predominant over Female as 21 (35%), Cause of burn in Maximum number of patients was fire burn 25 (41.6 %) Among single isolates Pseudomonas aeruginosa was leading (20.8%) followed by Acinetobacter baumannii (15.4%), staphylococcus aureus (14.1%) and Klebsiella pneumoniae (7.5%). Antibiotics susceptibility pattern of gram positive bacteria showed good sensitivity to Gentamycin, Linezolid, Vancomycin, Ciprofloxacin and Clindamycin. Antibiotics susceptibility pattern of gram Negative bacteria showed good sensitivity to Imipenem, Meropenem, Tobramycin, Amikacin, Kenamycin, Cefoperazone, and Tetracycline. Conclusion: Microbial colonization was present right from the time of admission in the majority of swabs. The commonest organism isolated was Pseudomonas Aeruginosa followed by Acinetobacter baumannii, staphylococcus aureus, Klebsiella pneumoniae, Enterobacter, Enterococcus species Escherichia coli, proteus mirabilis, CONS and Citrobacter species.

Keywords: Burn wounds, Invasive Infection, Pseudomonas Aeruginosa, Antibiotic Susceptibility.

 Background: In recent years,Staph.aureus,both coagulase positive and negative,have shown resistance to commonly used antibiotics used to treat infections.Over the last two decades,methicillin resistant strains (MRSA) have also been on a rise specially in patients admitted to ICUs and immunocompromised patients. The drug resistance mechanism of MRSA has been studied extensively in the past decade.Our study aims to study the sensitivity pattern of Staph. aureus in different clinical specimens and to study thedifferent resistance mechanisms. Objective: The present retrospective study highlights the susceptibility pattern and resistance mechanism ofStaph.aureus in clinical specimens obtained in our Microbiology department from June 2015 to June 2016. Materials and Methods: This was a retrospective study of Staph.aureus isolates from different clinical specimens including urine, blood, pus, vaginal swab, semen, aural swab, BAL fluid, conjunctival swab etc obtained from out patients at our Diagnostic Clinic Microbiology department from June 2015 to June 2016 and reported in VItek II (Biomerieux) according to CLSI guidelines. A total of 278 samples were reviewed. Results: Out of278 Staph.aureus isolates grown in the lab from different clinical specimens the sensitivity pattern showed highest sensitivity for Tigecycline(91.7%),followed bypenicillin (91.3%), ,Gentamycin (80.9%),tetracycline (66%),levofloxacin(65.4%),Rifampicin(63.6%), Linezol id(63.3%), Daptomycin(50.3%), Vancomycin (46,7%), Teicoplanin (42%). 117 cases showed resistance to aminoglycosides by KAN (APH(3")­III) mechanism,Cefoxitin screen was positive in 166 patients,APH(3")­III in 117 patients,MecA gene in 113 cases,ANT(4’)(4"))In 106 patients,acquired penicillinase in 77 ,SGASGB in 49 cases,efflux mechanism in 30 cases and inducible clindamycin resistance in 24 cases. Conclusion: According to our study Tigecycline, followed by Penicillin, Gentamycin ,Tetracycline, Levofloxacin and Linezolid are the antibiotics of choice for treating Staph aureus infections in the present scenario. With the introduction of automated equipments like Vitek II our understanding of the resistance mechanism is increasing.A larger study population would be required for a better understanding of resistance mechanisms.

Keywords: Staph Aureus; Vitec II; Resistance Mechanism; MRSA; Meca Gene.

 Preventing infections in patients undergoing surgical procedures is a primary goal for all members of the healthcare team. This is especially important in today’s dynamic healthcare environment, in the face of newly recognized pathogens, well­known microorganisms that have become resistant to treatment modalities, and the economic pressures to reduce. Healthcare­associated infections. A key infection control practice for reducing the likelihood of a surgical site infection is proper reprocessing of surgical instruments. In its 2008 Guideline for Disinfection and Sterilization in Healthcare Facilities, the Centers for Disease Control and Prevention (CDC) notes that failure to properly disinfect and sterilize equipment carries not only the risk associated with breach of host barriers, but also the risk for person­to­person transmission as well as transmission of environmental pathogens; furthermore, thorough cleaning is required before disinfection and sterilization because inorganic and organic materials that remain on the surfaces of instruments interfere with the effectiveness of these processes. Proteases are the most important type of enzyme to look for when choosing an enzymatic detergent for medical use because there is a high content of protein in most body fluids (including blood, tissue and mucous) which cannot be easily removed with regular detergents/surfactants and water, proteases break down protein into individual amino acids or short string of amino acid. Amino acid and peptides are much more soluble in water and will float away the surface of the instrument. In the present study the slaughterhouse drainage sample collected was screened for presence of bacteria, which can utilize blood protein as their protein source. The samples were serially diluted on blood agar. From these plates, depending upon their morphological, microscopical and physiological characters, the isolates were suspected to be Bacillus cereus. The colonies were confirmed by cultivating on selective media. On PEMBA (Polymyxin pyruvate egg­yolk mannitol bromothymol blue agar) media the isolated colonies were peacock blue in colour. This confirms the presence of Bacillus cereus. The isolates have been designated as Bacillus cereus KLM1, KLM2, KLM3 and KLM4. Of these KLM4 isolate was chosen as potential producer of protease enzyme depending upon its zone of lysis on fibrin plates and studied further for production of protease enzyme and its compatibility for the use of enzymatic detergent preparation for the cleaning surgical instruments.

Keywords: Protease; Zymography; Bacillus Cereus KLM4

 The canine parvovirus which causes myocarditis and enteritis is considered to be a very serious pathogen causing mortality in pups. The original strain CPV2 is found to be continuously evolving and presently three antigenic strains CPV­2a, CPV­2b and CPV­2c found to be circulating worldwide. In the present study 52 fecal samples were collected from 1­2 month old pups showing clinical signs of CPV from Hisar (India) and screened using vp2 gene specific seminested polymerase chain reaction assay. Three fecal samples were found positive for CPV infection. The PCR products were allowed for nucleic acid sequencing and nucleotide sequences were deposited to GenBank with accession numbers KC012923, KC012928 and KC012932. The sequence analysis using various bioinformatics tools revealed that all the three isolates belong to CPV­2a genotype and are much closer to other CPV­2a genotype from India and China. The present study showed that the predominant genotype running in India is CPV2a. Therefore, CPV2a strain should be incorporated in vaccine formulation.

Keywords: CPV; CPV­2a; PCR; Genotype.

 The role of chemical agents in aggravation or amelioration of infertility is widely acknowledged, however, their impact in cases of microbial induced infertility is yet to be deciphered. Further, Escherichia coli is known to reduce the reproductive potential of male mice therefor in the light of present information, this work was carried out to study the effect of the chemical agents viz. carnitine, tamoxifen and nictotine on E. coli induced infertility. For this, in vitro effect of carnitine/tamoxifen/nicotine was assessed on human spermatozoa when coincubated with E. coli. The results showed that carnitine and a tamoxifen at lower concentrations enhanced the motility and viability of sperms but at higher concentrations they proved to be toxic. Nicotine on the other hand was found to negatively influence the motility and viability of spermatozoa at all concentrations. For in vivo relevance of these results, male Balb/c mice were intraperitoneally inoculated with these chemical compounds challenged intravasaly with E. coli. The results revealed that carnitine and tamoxifen were able to ameliorate the negative impact of E. coli on male reproductive potential but nicotine aggravated the same. Therefore, it could be speculated that a healthier lifestyle has the ability to avert the microbial induced decreased reproductive potential.

Keywords: Escherichia coli; Tamoxifen; Nicotine.

 A study was conducted to evaluate C­reactive protein(CRP) as a screening tool for neonatal sepsis. CRP is one of the most studied and most used laboratory tests for neonatal sepsis. As part of the acute phase reaction to infection, it plays a central role in the humoral response to bacterial invasion. CRP is useful for monitoring the response to treatment and guiding antibiotic therapy. 75 neonates with risk factors and clinical features suggestive of CRP sepsis were selected as per operational definition and fulfilling the inclusion and exclusion criteria. Detailed physical examination was carried out. Blood sample for culture and CRP was taken from all neonates. CRP performed by semi quantitative latex agglutination method. Positive culture were the ‘gold standard’ against which the performance of CRP, abnormal white blood cell count (WBC) and platelet count were compared. Among 75 septic screens, 40 (53.33%) neonates had positive cultures. The sensitivity, specificity and positive predictive value of CRP was 92.5%, 89.74%, 90.24% respectively. Abnormal platelet count had lowest specificity(62.86%) and sensitivity(37.5%) among them. CRP assay is a valuable adjuvent in screening for neonatal sepsis, complementing clinical decision­making.

Keywords: C­Reactive Protein; Acute Phase Reactant; Neonatal Sepsis.