Concentration Dependent Effect of Azadirachta indica (Neem) Seed Oil and Neem Bark extract on Planktonic and Established Biofilm Growth of Pseudomonas aeruginosa and Staphylococcus aureus

Azadirachta indica Juss (Neem) is well documented for its antimicrobial activity. The effect of varying concentrations (0.1 to 50% v/v) of Azadirachta indica derived neem seed oil (NSO), neem seed oil with tween 20 and neem bark extract was evaluated on planktonic, biofilm formation and mature biofilms of multiple drug resistant Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 25923 using the crystal violet assay and scanning electron microscopy. NSO showed antimicrobial activity at 25% v/v for P. aeruginosa but not S. aureus in zone of inhibition assay. Neem bark extract on the contrary showed antimicrobial activity against both the isolates at 50% v/v concentrations. Interestingly, in biofilm formation assay, low concentrations of NSO (3.5 to 0.2% v/v) induced biofilm formation while inhibition of both planktonic and biofilm was seen in concentration dependent manner from 12.5% v/v onwards. Complex of NSO and tween in comparison of NSO alone caused low induction in S.aureus biofilm formation, while inhibiting biofilm formation of P. aeruginosa at all the concentrations. In biofilm eradication assay, NSO induced biofilm of both P. aeruginosa (50 to 0.1%v/v) and S. aureus (50 to 3.13%v/v). Eradication effect of neem bark extract was found on P. aeruginosa biofilm in a dose dependent fashion from 50 to 20% v/v followed by 0.2 to 0.1%v/v concentration respectively. S. aureus biofilm were eradicated at 50 to 25%v/v concentrations. At low concentrations, both the neem derivatives induced biofilm mediated growth of the pathogenic organisms. The data also indicate that neem seed oil was more effective against Gram negative P. aeruginosa while neem bark extract was effective against Gram positive S. aureus. This study highlights the crucial but variable effects of concentration dependent effect of phytochemicals and their composition on biofilm induction as well as eradication, the primary growth form in clinical settings. This challenges the notion that all herbal products are safe as antimicrobial activities differ as per microbial growth modes. Hence, concentration dependent effect of medicinal plant derived products requires thorough investigation prior to their use as antimicrobial agents

MOLECULAR DETECTION OF HUMAN RHINOVIRUS IN RESPIRATORY SAMPLES OF SWINE FLU NEGATIVE NORTH INDIAN CHILDREN WITH FLU-LIKE ILLNESS

Objectives: Flu-like illness may also be caused by different respiratory viruses other than influenza. Human rhinovirus (HRV) shows almost flu-likesymptoms. The purpose of this study is the molecular detection of HRV in throat swab of swine flu negative North Indian children during the years2012 and 2013. Reverse transcriptase (RT) - polymerase chain reaction (PCR) amplification of 5'non-coding region (NCR) was used for HRV detectionfollowed by cell culture isolation of HRV.Methods: PCR confirmed swine flu negative throat swab samples were collected from the Department of Microbiology, Sanjay Gandhi Post GraduateInstitute of Medical Sciences, Lucknow, Uttar Pradesh, India. The RNA isolation of samples was done using the QIAampViral RNA Mini Kit (Qiagen),followed by single step RT-PCR amplification (AgPath-ID, Life Technologies). All PCR positive HRV samples were cell cultured in HeLa and HEp-2 celllines for viral isolation.®Results: 135 swine flu negative throat swab samples were examined. Out of which 34 samples (25.2%) were found HRV positive by RT-PCR, while onlyfour samples (11.8%) were culture positive on HeLa cell line. Younger children (0-4 year) were found more susceptible to HRV infection. This studyindicated the highest prevalence of HRV (37.0%) during the months (September-October) of the Autumn season in 2012 and 57% in Winter-springseason (February-March) during 2013.Conclusion: HRV may be a cause of flu-like symptoms in swine flu suspected North Indian children with a higher rate during Autumn and Springseason. Molecular detection of HRV using RT-PCR is more sensitive than cell culture assay.Keywords: Human rhinovirus, Swine flu, Influenza-like illness, Lower respiratory tract infections

High Prevalence of Multiple Drug Resistant and Biofilm Forming Staphylococcus aureus among HIVInfected Patients with Suspected Pneumonia

Staphylococcus aureus is one of the major causes of life threatening pneumonia, especially in immunocompromised population. In HIV positive patients, S. aureus associated pneumonia can be either health care associated or community acquired and responsible for high rate of mortality. In this study total 102 throat swab samples of HIVInfected Patients with suspected pneumonia were collected during 2014-2016, out of them 46 samples (45.1%) were found positive for S. aureus by biochemical tests. 38 (82.6%) isolates were found multiple drug resistant while 9 (19.6%) strains showed resistance to cefoxitin antibiotic, were considered as methicillin resistant Staphylococcus aureus (MRSA). Only one strain (2%) was found vancomycin intermediate (VISA), remaining 98% isolates were sensitive to vancomycin antibiotic. In PCR test, all cefoxitin resistant strains were found positive for the presence of MecA gene. Biofilm former S. aureus were screened by tissue culture plat (TCP) methods. In TCP assay, 21 (45.6 %) isolates were confirmed as high biofilm formers (OD value > 0.250), 16 (34.8 %) were moderate biofilm formers (OD values- between 0.150 to 0.250), while 9 (19.6 %) were low biofilm formers (OD value < 0.150). A significant association was found among multiple drug resistance and high biofilm formation (p value < 0.05). High prevalence of biofilm forming MDR isolates in airways of pneumonia suspected HIV patientsis matter of great concern as poor antibiotic response may cause more severe diseases with increasing cost and duration of treatment. The MecA gene might be a cause of methicillin resistance among MRSA isolates