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Introduction: Orthopaedic Implant associated infection is the major problem which leads to implant failure and economic burden to the patient. Implant-associated infections are due to the formation of biofilm at the implantation site leading to multidrug resistant organism. Aim: This study was done to evaluate the causative organisms, their antibiotic sensitivity pattern and their ability to form biofilm over the Implants used in Orthopaedic surgeries. Material and methods: This study was carried out in the department of Microbiology, Adichunchanagiri institute of Medical sciences from September 2019 to August 2022. Swabs from 100 patients who had undergone orthopaedic implant or prosthetic surgery and presented with signs and symptoms of infection were collected and processed as per standard procedures. All the isolates were subjected to detect biofilm production by Congo red agar method, MRSA detection by Cefoxitin disc diffusion test, vancomycin resistant Enterococcus by using Vancomycin (Minimum inhibitory concentration) MIC strips and Extended spectrum β lactamases (ESBL) production by phenotypic confirmatory combined-disc test. Results: Out of the 100 samples processed, culture positivity was observed in 72 specimens. Among them, Staphylococcus aureus 19(26%) was the predominant isolate and 44 (61%) are biofilm producers. 17 (89%) were MRSA (Methicillin resistance Staphylococcus aureus) strains and 15(37%) were ESBL producing strains. Conclusion: The appropriate pre- and post- operative care should be taken to prevent orthopaedic implant associated infections. Staphylococcus spp was the commonest isolate and its ability to produce biofilm indicates the need for an appropriate antibiotic policy and screening for MRSA carriers to reduce the infection. Keywords: Orthopaedic implant, Infection, Biofilm, MRSA, ESBL

Background: Plants have been the basis for many medicinal compounds that are used in modern-day medicine, screening for medicinal plants for bioactive compounds is a way of identifying and developing new antimicrobials that are less expensive and with improved safety and efficacy. Objective: The objective of this study was to investigate the in vitro antimicrobial activity of Azadirachta indica (neem) using leaf extracts of Ethanol, Petroleum ether, and Ethyl acetate and testing them on E.coli, Klebsiella, S.aureus, MRSA and C.albicans. Materials & Methods: Minimum inhibitory concentration (MIC) was found using the serial dilution method and antimicrobial susceptibility testing (AST) was done using the disc diffusion method. Results: Ethanolic extract showed better results against Klebsiella and S.aureus, Ethyl acetate extract showed better results against E.coli and MRSA, whereas both ethanolic and petroleum ether extract showed activity against C.albicans. 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay showed that all the extracts have antioxidant property and variations were noted in the antimicrobial activity on comparing samples taken from different locations. Conclusion: Further studies are needed into the bioactive compounds of neem in order to explore their antimicrobial activity. Keywords: Antimicrobial susceptibility, Azadirachta indica, Ethanol, Ethyl acetate, Disc diffusion, MIC, Petroleum ether

The sequential pathology of Kyasanur forest disease (KFD) in mouse brain was assessed in this study. Kyasanur forest disease virus (KFDV) strain P9605 used in this study was confirmed by real-time reverse transcriptase-polymerase chain reaction targeting the NS5 gene. Mouse Lethal Dose 50 (MLD50) of the virus was determined by in-vivo mice inoculation test. One MLD50 of the KFDV was inoculated intra-cerebrally into 36 mice aged 2-3 weeks. Another group of 36 age-matched mice that served as control group were inoculated with plain media. Six mice each from infected and control groups were euthanized every 24 hrs intervals for six days. Brain tissues were collected in 10% NBF. The collected brain tissues were processed and subjected to histopathological studies by Hematoxylin and Eosin staining. Grossly, the infected mice showed symptoms of dullness, hunched back appearance, weakness, sluggish movements with indication of hind quarter paralysis on day four post-infection. These symptoms got aggravated with complete paralysis of the hind quarters, inability to move and death on 5th and 6th day post-infection. Microscopically, the brain showed apoptosis of neurons, perivascular cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic neurons. The real-time RT-PCR on hippocampus of the KFDV-infected mouse brain showed three-fold higher expression levels of Caspase 3, a crucial mediator of apoptosis. The cerebral cortex, cerebellum and hippocampus that control the motor neuron activities and muscle tone were primarily affected, possibly correlating with the gross symptoms of hind quarter paralysis, ataxia, and other motor neuron dysfunctions noticed. Taken together, these findings reveal that KFDV induces apoptosis of neurons in the cerebrum and hippocampus of KFDV infected mice. Further studies are needed to confirm if the lesions noticed in mice brain simulate the brain lesions in humans since gross motor-neuron symptoms are similar in mice as well as humans.

We evaluated the safety and potency of the Kyasanur Forest disease (KFD) vaccine inactivated with different formalin concentrations in mice, since the side effects due to higher formalin concentrations have been a major reason for vaccine refusal. Furthermore, with an objective to reduce the use of mice in vaccine testing, we performed quantification of the KFD virus by real-time PCR and compared it with in vivo titration in mice. The KFD vaccine prepared in chicken embryo fibroblast cells was inactivated with 0.04%, 0.06%, and 0.08% concentrations of formalin. The vaccine inactivated with 0.04% and 0.06% formalin failed the safety test, whereas the KFD vaccine inactivated with 0.08% formalin was safe and potent with a log protective index of 5678 in mice. This reduced formalin content may induce no/lesser side-effects of pain/swelling which may increase the vaccine acceptance. The real-time PCR on individual KFD vaccine harvests interpreted that when the CT value of each harvest is 50 of the vaccine lies in the tenfold dilution that yields CT values between 31 and 34.

Neonatal septicaemia is one among the leading causes of neonatal mortality in India. For the last 20 years CoNS has been identified as a major cause of neonatal septicaemia in NICU. Different studies show 70-75% of CoNS as resistant to methicillin. Material and methods: Blood samples from neonatal septicaemia cases during Dec 2007 to Dec 2010 were processed & isolates were identified. CoNS were biotyped & methicillin resistance was detected using cefoxitin 30 µg disk. Results: Of 2256 blood samples from neonatal septicaemia cases, 587(36.98%) were CoNS. Staphylococcus epidermidis 234(39.86%) was the commonest isolate. Methicillin resistance was noted in 286 (48.72%). MRCoNS prevalence during 2008, 2009 & 2010 were 41.57%, 47% and 57.36% respectively. Conclusion: Our study showed increased trends of MR-CoNS in neonatal septicaemia leading to increased usage of vancomycin & other glycopeptides, resulting in emergence of multidrug resistant strains, thus narrowing the treatment options in neonates.

Introduction: The emergence of Methicillin Resistant (MR) staphylococcal infections had led to fewer therapeutic options. Ceftaroline fosamil is the only cephalosporin approved by United States of America (USA) Food and Drug Administration (FDA) till now which has activity against MR staphylococcal isolates. Until now, studies on in-vitro activity of ceftaroline are limited. Aim: To know the susceptibility pattern of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Resistant Coagulase Negative Staphylococci (MRCoNS) against ceftaroline in a rural tertiary health care centre. Materials and Methods: The present cross-sectional study was conducted in a tertiary care hospital in rural part of Karnataka between two months (July and August 2019). A total of 50 consecutive, non-repetitive clinical isolates of MRSA and MRCoNS were obtained. Antibiotic susceptibility testing was done according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The Minimum Inhibitory Concentration (MIC) of ceftaroline was detected using E-strips (Biomerieux). The CLSI breakpoints applied for the interpretation of ceftaroline MIC- Sensitive: ≤1 μg/mL; Susceptible Dose Dependent (SDD): 2-4 μg/mL and resistant ≥8 μg/mL). Staphylococcus aureus American Type Culture Collection (ATCC) 29213 was used as a quality control. Statistical analysis was done using microsoft excel. Percentages were used in this study to analyse variables. Results: Out of 50 MR staphylococcal isolates, 10 (20%) were MRSA and 40 (80%) were MRCoNS. Of the MR staphylococcal isolates tested, 49 (98%) were sensitive to ceftaroline. The MIC 50 and MIC 90 for the 50 MR staphylococci was 0.25 μg/mL and 1 μg/mL, respectively. Conclusion: Ceftaroline demonstrated a potent in-vitro activity against MR staphylococci. So it can be used as an effective drug in the treatment of such infections.

We describe the molecular epidemiology of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) involved in outbreaks and massive spread of Porcine Reproductive and Respiratory Syndrome (PRRS) in Karnataka state of India during the year 2017. The study involved investigation of outbreaks in three districts.viz, Udupi, Dakshina kannada and Bengaluru in Karnataka. The disease was characterised by large scale piglet mortality with severe respiratory distress and abortions in pregnant sows. The study recorded death of 394 piglets, 131 adults and abortions in 82 pregnant sows. The organ samples collected from dead pigs were found negative for Classical swine fever virus by 5’UTR gene based Reverse transcription polymerase chain reaction (RT-PCR). RT-PCR targeting full length ORF5 gene of PRRSV on spleen and lung samples of dead pigs yielded specific amplicon of 803 bp indicating the presence of PRRSV. The phylogenetic analysis of the nucleotide sequences derived from ORF5 gene of PRRSV involved in the current outbreaks revealed 99.99% sequence homology with the highly pathogenic PRRSV of genotype 2 (North American type) from China and India (Mizoram state). Since pig husbandry plays a significant role in socio-economic upliftment of the poor and marginalised farmers in the country, it’s time to put in place effective prevention and control measures for PRRS, before it cripples pig industry in India and its surrounding world. Present study is the first epidemiological report of PRRS outbreaks in South India.

This study evidences that, stress due to vaccination can cause re-activation and clinical outbreak of bovine herpesvirus-1 in latently infected cattle. Three to four days following mass-vaccination of cattle against Foot and Mouth Disease in Karnataka of India, symptoms of acute respiratory distress, conjunctivitis and vulvo-vaginitis were observed in vaccinated cattle in many villages of the state. Nasal and ocular swabs were collected from 25 ailing cattle in six villages of Hassan district of Karnataka. Upon bacteriological analysis, the samples were found negative for Pasteurella multocida. The serum samples from ailing animals were positive for bovine herpesvirus- 1 (BoHV-1) antibodies by Indirect-ELISA. The swab samples were found for BoHV-1 by PCR targeting glycoprotein- C gene of BoHV-1. The swab samples when subjected to virus isolation in MDBK cells yielded characteristic CPE of bunch of grape like clustering of cells by fifth passage. PCR targeting conserved region on glycoprotein-C gene on DNA extracted from cell-culture supernatants showing CPE confirmed the presence of BoHV-1. Nucleotide sequencing of the PCR amplicon showed that the BoHV-1 isolated during this study shared 100% sequence identity with BoHV-1 isolates from India, Switzerland and Brazil. This study emphasizes that stress due to vaccination can cause re-activation and clinical outbreak of bovine herpesvirus-1 in latently infected cattle.

Introduction: Urinary tract infection (UTI) is one of the leading causes of morbidity encountered in clinical practice. Biofilm produced by the urinary pathogens leads to recurrent and recalcitrant UTI there by contributing to longer stay in hospital and increased cost of treatment. Aim: The present study was conducted to evaluate Congo Red Agar method (CRA) and Tube Method (TM) in detection of biofilm formation in uropathogens with respect to Tissue Culture Plate method (TCP). Materials and Methods: Study was conducted in the Department of Microbiology from May 2016 to May 2017. Midstream clean catch urine collected from patients with symptoms of UTI was processed by standard methods. A total of 264 randomly selected urinary isolates were subjected to biofilm detection by CRA method, tube method and TCP method. TCP was considered as gold standard. Results were expressed in terms of percentages, sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV). Results: Biofilm was detected in 105 (39.77%) isolates by TCP method, in 124(46.97%) by CRA method and 101(38.26%) by tube method. CRA method and TM method showed a sensitivity of 80% & 63.81%, specificity of 75.47% & 78.62%, PPV of 68.29% & 66.34%, and NPV of 85.11% and 76.69% respectively. Conclusion: Congo red agar and tube methods can be considered for detection of biofilms in resource constraint conditions.

Introduction: Enterococci are normal commensals in the gastrointestinal tract, oral cavity, vagina etc. They are organisms of low virulence, but are known to cause various clinical infections. Enterococcus is considered as an important nososcomial pathogen because of its intrinsic as well as acquired antibiotic resistance. The increasing importance of Enterococcus is largely due to their resistance to many antimicrobials particularly intrinsically resistant Enterococci, which is the cause of changing pattern of Enterococcal infection resulting in treatment failures. Objectives: 1. Isolation, Identification and speciation of Enterococci from clinical specimens by conventional method. 2. Determination of Antibiogram of such isolates of Enterococci. Materials and Methods: Over a period of one year, 3,197 various clinical specimens were processed and a total of 80 strains of Enterococci isolated. Such isolates were identified and speciated by recommended conventional tests and biochemical reactions. Their antibiotic susceptibility pattern was also determined to the common antibiotics by disc diffusion method as per the recommendations. Results: Among the total 80 strains of Enterococci, 69 were E.faecalis, 10 were faecium and one was durans and all the isolated strains were identifiable to the species level by conventional method. Maximum numbers of isolates were from urine specimen (38, 1.97%), followed by exudates (32, 5.54%) and blood (10, 1.40%), and with an overall incidence of isolation of Enterococci from the above said specimens is 2.5%. Among 80 strains of Enterococci 35% were β hemolytic & 63.75% isolates were non hemolytic. More than 50% of the total isolates were resistant to Ampicillin (E.faecalis – 49.27%, E.faecium -90%),Penicillin(E.faecalis–49.27%, E.faecium -80%),Tetracycline(E.faecalis–57.97%, E.faecium -70%), Erythromycin(E.faecalis –53.62% , E.faecium -60%) and Gentamicin (E.faecalis –59.42% E.faecium -80%) and the most useful antibiotic was ciprofloxacin to which 61.25% (E.faecalis – 62.31%, E.faecium -50% ) of the total isolates were sensitive. Conclusion: From this study it was possible to identify all the isolates of Enterococci to the species level by following conventional methods. It was observed in our study that there is more drug resistance to the tested antimicrobials among E.faecalis isolates.

Neonatal septicemia is one of the leading causes of neonatal mortality and morbidity worldwide. Hence, the present study was undertaken to isolate the bacteria causing neonatal sepsis and determine their antibiotic susceptibility pattern. Fifty neonates suspected to have septicemia were screened for 2 months (July and August 2014). Out of 50 specimen, 15 (30%) were blood culture positive. Coagulase-negative staphylococci was the most common isolate (10, 66.6%), with 60% (6 isolates) methicillin resistance. In view of the increasing antibiotic resistance, periodic surveillance should be conducted to control the emergence and spread of antimicrobial resistance.