Prevalence of gram positive and gram negative organisms in various cli

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 99 IJAMSCR |Volume 4 | Issue 1 | Jan – Mar - 2016 www.ijamscr.com Research article Medical research Prevalence of gram positive and gram negative organisms in various clinical samples M.Anitha Sathya Pandurangan DM.Monisha J.Hemapriya Department of Microbiology Shri Sathya Sai Medical College Research Institute Thiruporur Sri Balaji Vidyapeeth University Tamil Nadu India. Corresponding author: M.Anitha ABSTRACT Background The present work was aimed to depict the prevalence of Gram positive and Gram negative organisms isolated from different clinical samples. Materials and Methods Different pathogenic organisms were isolated from Males and females of different clinical samples such as Midstream Urine Miscellaneous Sputum Pus HVS Body fluids endotracheal aspirates Eye swabs and Blood samples during the period of October 2015 to December 2015. Samples were processed and identification of isolates were done by gram staining biochemical test. Antibiotic sensitivity testing of isolated pathogenic organisms was done by a disk diffusion technique using Mueller Hinton Agar plates according to CLSI guidelines. Results Out of total 770 samples analyzed 414 samples showed the presence of bacteria whereas 356 samples showed no growth of organisms on culture medium. Microbial growth revealed the presence of 11 different organisms which are reported here Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Pseudomonas aeruginosa Acinetobacter baumannii Enterococcus faecalis Citrobacter koseri Proteus mirabilis Staphylococcus epidermidis Staphylococcus aureus Streptococcus pyogenes. Conclusion The present study provided the most needed information on the prevalence of both Gram positive and Gram negative bacteria which were also responsible for infections in Urine Miscellaneous and Blood samples. Keywords: Prevalence Disc Diffusion Midstream Urine Miscellaneous Sputum Pus HVS Body fluids Endo Tracheal aspirates Throat swabs Eye swabs and Blood samples. ISSN:2347-6567 International Journal of Allied Medical Sciences and Clinical Research IJAMSCR

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 100 INTRODUCTION Microorganisms are living things ordinarily too small to be seen without magnification in terms of numbers and range of distribution. Microbes are the dominant organisms on earth. A large and diverse group of microscopic organisms that exists as single cells or cell clusters it also includes viruses which are microscopic but not cellular. 1 A nosocomial or hospital acquired infection develops during the patient’s stay in the health care center and is generally not present or incubating at the time of admission. The microbes that grow in health care settings along with patients weakened defense mechanisms help in setting the stage for nosocomial infections which are mostly responsible for lengthening the patient’s stay in the hospital and increasing the risk of death besides making the diagnosis and treatment of such patient’s expensive. 2 Besides this usage of poor aseptic protocols by health care service providers also increases the risk of infections. 3 Nosocomial Infections most commonly invade the body through the urinary tract while other common portals of entry will be through the surgical wounds the respiratory tract and the blood stream. 4 The number of infections caused by microorganisms is increasing significantly over the last few decades. One of the reasons for this increase is a development of microbial resistance to drugs used to treat these infections. About 50-60 of noscomial infections in the United States are now caused by antibiotic resistant strains of bacteria. 5 Nowadays difficulties in treating infectious diseases like Malaria Tuberculosis TB diarrhoea Urinary Tract Infections UTI etc. are because of development of drug resistance in infecting microbes. The threat to health care associated infection has been a matter of concern due to development of multidrug resistant MDR strains among common isolates in hospital. Applying antibiotics caused a breakthrough in treatment stepping out in ills group infections treated in hospital however enlarging resistance the formation of new mechanisms of resistance and/or the spreading of the gene of resistance has become the shortcomings of antibiotic therapy. 6 Urinary Tract Infection UTI remains the most commonly existing bacterial infection in human population especially in nosocomial infected patients. 7 UTIs refer to the presence of microbial pathogens within the urinary tract and it is usually classified by the site of infection such as bladder cystitis kidney pyelonephritis or urine bacteriuria. They are asymptomatic or symptomatic. UTIs that occur in a normal genitourinary tract with no prior instrumentation are considered as “uncomplicated” whereas “complicated” infections are diagnosed in genitourinary tracts that have structural or functional abnormalities including instrumentation such as indwelling urethral catheters and are frequently asymptomatic. 8 Pseudomonas aeruginosa frequently isolates as an opportunistic pathogen in recurrent infections of hospitalized patients and has been isolated from a number of sites in the hospital environment. 9 It is the most relevant resistant and dangerous organism infecting the burn patients. 10 It is the fifth most common pathogen among hospital microorganisms and causes 10 of all hospital acquired infections. 11 Bacteremia and other bloodstream Infections BSIs are a major cause of morbidity and mortality worldwide. Usually the bloodstream is sterile. Individuals with bacteremia may develop Septicemia a life-threatening condition in which multiplying bacteria release toxins into the bloodstream. Microorganisms present in circulating blood whether continuously or intermittently a threat to every organ in the body. Approximately 200000 cases of bacteraemia and fungemia occur annually with mortality rates ranging from 20-50. Blood culture remains a highly specific indicator of bacteriemia. Antimicrobial susceptibility testing assist a great deal in a precise identification of the most appropriate choice of drug to be administered. 12 The present work was intended to depict the prevalence of both Gram positive and Gram negative organisms isolated from various clinical samples. MATERIALS AND METHODS Sample Collection Clinical samples which include Midstream Urine Miscellaneous Sputum Pus HVS Fluids Endo Tracheal aspirates Throat swabs Eye swabs and Blood were collected from both male and female patients. The present study was carried out over a period of 3 months from Oct 2015 to Dec 2015 in Microbiology department SSSMC RI.

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 101 Culturing Characteristics and Identification of bacteria Samples were processed according to their nature type. All the samples were cultured on MacConkey Agar Blood agar chocolate agar plates and incubated according to the sample nature type. The confluent growth of the pathogens isolated was included in this study. Isolates were subcultured and colonies were screened for Gram positive and Gram negative isolates. Identification of isolates were done by gram staining and biochemical test. Result interpretation was done based on the identifying characters as described by Cheesbrough. 1415 Antibiotic Sensitivity Testing Antimicrobial sensitivity was determined by Kirby Bauer disc diffusion method on Mueller Hinton agar MHA. A suspension of each isolate was made so that the turbidity was equal to 0.5 McFarland standards and then plated as a lawn culture onto MHA. Antibiotic discs were placed and plates were incubated at 37°C for 18-24 hrs. Results were interpreted in accordance with central laboratory standards institute CLSI guidelines. 16 RESULTS Table:1 Prevalence of bacterial growth in urine sample: Organism isolated No of isolates No of Male No of female Total percentage Escherichia coli 105 45 60 50 Klebsiella pneumoniae 38 21 17 18 Klebsiella oxytoca 25 11 14 12 Pseudomonas aeruginosa 15 9 6 7 Staphylococcus epidermidis 8 3 5 4 Acinetobacter baumannii 4 0 4 2 Citrobacter koseri 4 0 4 2 Enterococcus faecalis 5 2 3 2 Proteus mirabilis 4 3 1 2 Streptococcus pyogenes 2 0 2 1 Total 210 94 116 100 Percentage 45 55 Table: 2 Prevalence of bacterial growth in miscellaneous samples: Organism isolated No of isolates No of Male No of female Total percentage Escherichia coli 50 29 21 28 Klebsiella pneumoniae 43 27 16 23 Klebsiella oxytoca 27 11 16 15 Pseudomonas aeruginosa 25 16 9 14 Acinetobacter baumannii 16 8 8 9 Enterococcus faecalis 6 3 3 3

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 102 Citrobacter koseri 5 2 3 3 Proteus mirabilis 4 3 1 2 Staphylococcus epidermidis 2 1 1 1 Staphylococcus aureus 1 1 - 1 Streptococcus pyogenes 2 1 1 1 Total 181 102 79 100 Percentage 56 44 Table: 3 Bacterial identification in each sample of Miscellaneous: 0rganism isolated Sputum Pus HVS Fluids Endo Trachael Throat swabs Eye swabs Total Escherichia coli 9 17 20 1 2 1 - 50 Klebsiella pneumonia 23 10 9 1 - - - 43 Pseudomonas aeruginosa 11 10 3 - - - 1 25 Klebsiella oxytoca 9 9 8 - 1 - - 27 Acinetobacter baumannii - 5 5 - - - - 16 Proteus mirabilis - 4 - - - - - 4 Enterococcus faecalis 1 2 2 1 - - - 6 Staphylococcus epidermidis - 1 1 - - - - 2 Citrobacter koseri 2 1 1 - - 1 - 5 Staphylococcus aureus - 1 - - - - - 1 Streptococcus pyogenes 2 - - - - - - 2 Total 63 60 49 3 3 2 1 181 Percentage 34 33 27 2 2 1 1 100 Table: 4 Prevalence of Bacterial Growth in Blood culture Organism isolated No of isolates No of Male No of female Total percentage Staphylococcus epidermidis 8 5 3 37 Klebsiella pneumoniae 4 3 1 17 Acinetobacter baumannii 4 2 2 17 Pseudomonas aeruginosa 2 1 1 9 Staphylococcus aureus 1 1 - 4 Klebsiella oxytoca 1 1 - 4 Salmonella typhi 1 - 1 4 Proteus mirabilis 1 1 - 4 Escherichia coli 1 1 - 4 Total 23 15 8 100 Percentage 65 35

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 103 Fig: 1 Distribution of bacterial growth in Urine sample: Fig: 2 Distribution of Bacterial Growth in Miscellaneous samples: Fig: 3 Distribution of Bacterial Growth in Blood culture: 38 25 105 15 8 4 4 5 4 2 Organisms isolated from urine Klebsilla pneumoniae Klebsiella oxytoca Escherichia coli Pseudomonas aeruginosa Staphylococcus epidermidis 43 27 50 4 25 16 5 2 2 1 6 Organisms isolated from miscellaneous samples Klebsiella pneumoniae Klebsiella oxytoca Escherichia coli Proteus mirabilis Pseudomonas aeruginosa 8 4 4 2 1 1 1 1 1 Organisms isolated in Blood culture Staphylococcus epidermidis Acinetobacter baumannii Klebsiellapneumoniae Pseudomonas aeruginosa Klebsiella oxytoca

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 104 This study was conducted during a period of three months on clinical isolates from indoor patients in order to determine the prevalence of clinically significant Gram-positive cocci and Gram negative bacilli in various clinical samples at SSSMC RI. Between October 2015 and Dec 2015. Midstream Urine Miscellaneous and Blood cultures were examined for the presence of bacteria. Out of total 770 samples analyzed 414 samples showed the presence of infection while in 356 samples no growth of organisms was seen in culture medium. The 3 types of clinical samples such as Midstream Urine Miscellaneous Sputum 34 Pus 33 HVS 27 Fluids 2 Endo Tracheal aspirates 2 Throat swabs 1 Eye swabs 1 and Blood were observed which is shown in Table 3. The commonly isolated organisms from Mid- stream Urine samples include Escherichia coli50 Klebsiella pneumoniae18 Klebsiella oxytoca12 Pseudomonas aeruginosa7 Staphylococcus epidermidis4 Acinetobacter baumannii2 Citrobacter koseri2 Enterococcus faecalis2 Proteus mirabilis2 Streptococcus pyogenes1 whereas in Miscellaneous includes Escherichia coli28 Klebsiella pneumoniae23 Klebsiella oxytoca15 Pseudomonas aeruginosa14 Acinetobacter baumannii9 Enterococcus faecalis3Citrobacter koseri3 Proteus mirabilis2 Staphylococcus epidermidis1 Staphylococcus aureus1Streptococcus pyogenes 1 and in Blood samples include Staphylococcus epidermidis 37 Klebsiella pneumoniae 17 Acinetobacter baumannii17 Pseudomonas aeruginosa9 Staphylococcus aureus4 Klebsiella oxytoca4 Salmonella typhi4Proteus mirabilis4 Escherichia coli4 as shown in the Table 124 Fig 123. Gram negative organisms were dominant compared to Gram positive organisms in the nosocomial infections. E. coli was found to be the most dominant pathogen in the Mid-stream Urine 50 and Miscellaneous samples 28 but in Blood samples Staphylococcus epidermidis was the dominant pathogen. Streptococcus pyogenes 1 were found to be the least responsible in the urine Staphylococcus aureus 1 Staphylococcus epidermidis1 were least in the Miscellaneous samples and E.coli4 was least in the Blood samples. Salmonella typhi 4 was also observed to be present in collected Blood samples. Hence in urine samples female 55 were highly infected than Male 45. Where as in Miscellaneous male was 56 and female was 44 and in Blood samples male was found to be affected more 65 and female 35. DISCUSSION This study was targeted to depict the prevalence of Gram positive and Gram negative organisms isolated from different clinical samples such as Urine Miscellaneous and Blood samples. The most common uropathogens in our study were E. coli 50 and Klebsiella pneumoniae 18. Although the percentage of E. coli is much higher in our study it supports the previous findings indicating that the E. coli is the principal etiological agent of UTI accounting for 46.98 of the screened cases. 171819 In another study it was reported that predominant uropathogens are E. coli followed by Klebsiella species which also support our study. 20 Infections of Pseudomonas aeruginosa 7 Staphylococcus epidermidis 4 Acinetobacter baumannii 2 Citrobacter koseri 2 Enterococcus faecalis 2 Proteus mirabilis 2 Streptococcus pyogenes 1 were found to be very few in this study. The similarities and differences in the type and distribution of uropathogens may result from different environmental conditions and host factors and also from some practices such as health care and education programmers socioeconomic standards and hygiene practices in each country. In Miscellaneous samples S. aureus was found in the most prevalent organism and its prevalence was found to be 36.36 38.78 33.33 and 54.05 during the period 2000- 2003 respectively. It was followed by E. coli and Proteus spp. In Miscellaneous includes Escherichia coli28 Klebsiella pneumoniae23 Klebsiella oxytoca15 Pseudomonas aeruginosa14 Acinetobacter baumannii 9 Enterococcus faecalis3Citrobacter koseri 3 Proteus mirabilis2 Staphylococcus epidermidis 1 Staphylococcus aureus1 Streptococcus pyogenes 1 The most common Gram negative and Gram positive organism isolated from the blood was

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 105 Staphylococcus epidermidis 37 Klebsiella pneumoniae 17 Acinetobacter baumannii 17 Pseudomonas aeruginosa 9 Staphylococcus aureus 4 Klebsiella oxytoca 4 Salmonella typhi 4 Proteus mirabilis 4 Escherichia coli 4. We considered the growth of Staphylococcus epidermidis to be indicative of its presence in the patient as opposed to being a contaminant. Staphylococcus seems to be emerging as the dominant organisms in bloodstream infections. Nosocomial infection due to Staphylococcus aureus constitutes a major part of the total annual nosocomial infection. 21 On the other hand the variation in blood culture positivity depends on several other factors such as the amount of blood taken for screening 22 formulation of the blood culture medium used for bacterial detection or administration of antibiotic therapy prior to taking blood for culture 23. Our study showed that the rate of isolation of Gram negative bacteria is higher than Gram positive bacteria. In contrast to our study Kavitha P et al and Swain B et al 2425 reported Gram positive bacteria were dominant than Gram negative bacteria. Bloodstream infections with Pseudomonas spp. have been associated with increased morbidity in some studies. Another significant finding of the study is the isolation of Salmonella typhi in 4 of the cases. An increasing incidence of Salmonella species has also been reported in some recent studies. 26 27 In most of the studies Gram-negative bacilli have taken over the Gram-positive organisms especially in hospital settings. Mehta et al 28 has reported the incidence of 80.96 for gram-negative and 18 for Gram-positives which was similar to present findings. Our study revealed that Gram- negative bacteria were predominant than Gram positive bacteria. This has been an observation among similar studies done in the patients of developing countries. 29-32 CONCLUSION The present study provided much necessary information based on the prevalence of both Gram positive and Gram negative bacteria were responsible for infections in Urine Miscellaneous and Blood samples. Infections in the patients are notifiable problems. Adherence to infection prevention protocols and the proper monitoring and the judicious use of antibiotics are important in preventing such infections. The solution can be planned by continuous efforts of microbiologist clinician pharmacist and community to promote greater understanding of this problem. Frequent hand washing prevent spread of the organism should be encouraged in a hospital environment. Better medical care should be provided to patients during hospital stays. Acknowledgment The authors wish to acknowledge the support provided by the management to carry out this work. REFERENCES 1. Lynn Maori Vivian Okemena Agbor and Wasa Alibe Ahmed. The prevalence of bacterial organisms on toilet door handles in Secondary Schools Journal of Pharmacy and Biological Sciences Volume 8 Issue 4 Nov. – Dec. 2013 PP 85-91. 2. Pittet D Tarara D and Wenzel R P. Nosocomial blood stream infection in critically ill patients Excess length of stay extra costs and attributable mortality The Journal of the American Medical Association 1994271:1598–1601. 3. Aranaz-Andres J M et al. Terol-Garcia E and the ENEAS workgroup Incidence of adverse events related to health care in Spain results of the Spanish National Study of Adverse Events Journal of Epidemiology and Community Health 200862:1022–1029. 4. Peleg A Y and Hooper D C. Hospital acquired infections due to Gram negative bacteria The New England Journal of Medicine 2010362:1804–1813. 5. Jones RN. Resistance patterns among nosocomial pathogens Trends over the past few years 2001 119 2_suppl: 397S-404S. 6. Kritu Panta Prakash Ghimire et al. Antibiogram typing of Gram negative isolates in different clinical samples of a tertiary hospital Asian journal of pharmaceutical and clinical research Vol 6 Issue 1 2013.

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 106 7. Gastmeir P Kampf G Wischnewski N Hauer T Schulgen G et al. Prevalence of nosocomial infections in representative German hospitals. J. Hosp. Infect1998 38:37-49. 8. Gonzalez CM Schaeffer AJ. Treatment of urinary tract infection: what’s old what’s new and what works. World JUrol. 1999 17:372–82. 9. Vianelli N Giannini MB Quartic C. Resolution of a Pseudomonas aeruginosa outbreak in a haematology unit with the use of disposable sterile water filter Haematol J 2006917:983-5. 10. Estahbanati HK Kashani PP Ghanaatpishels F. Frequency of Pseudomonas aerugunosa serotypes in burn wound infections and their resistance to antibiotics Burns.200228:340-8 11. National Nosocomial Infections Surveillance System. NNIS report data summary from Jan 1990 - May 1999. Am J Infect Control 1999 27:520-32. 12. R. Sharma and S. Gupta. Bacteriological analysis of blood culture isolates with their antibiogram from a tertiary care hospital IJPSR 2015 Vol. 611: 4847-4851. 13. Cheesbrough M. District Laboratory Practice in Tropical countries Cambridge University Press London. 2000 2: 151-4 180-265. 14. Forbes AB Sahm FD Weissfelt SA. Bailey and Scott’s diagnostic Microbiology.12th edition.Mosby publication. 2007. 15. Clinical and Laboratory Standards Institute Performance standards for antimicrobial susceptibility testing 15th informational supplement CLSI document M100-S15. Wayne PA: Clinical and Laboratory Standards Institute 2005. 16. Jha N Bapat SK. A study of sensitivity and resistance of pathogenic micro organisms causing UTI in Kathmandu valley. Kathmandu Univ Med J 2005 3:123-9 17. Ronald A The quinolones and renal infection. Drugs 1999 58:96-8. 18. Ronald A The etiology of urinary tract infection: traditional and emerging pathogens. Am J Med 2002 113:14-9. 19. Olafsson M Kristinsson KG Sigurdsson JA. Urinary tract infections antibiotic resistance and sales of antimicrobial drugs--an observational study of uncomplicated urinary tract infections in Icelandic women Scand J Prim Health Care200018:35-8. 20. Vanitha RN Gopal K Venkata NM Vishwakant D Nagesh VRD Yogitam et al. A retrospective Study on blood stream infections and antibiotic susceptibility patterns in a tertiary care teaching hospital. International Journal of Pharmacy and Pharmaceutical Sciences 2012 41: 543-548. 21. Lee A Mirrett S Reller LB and Weinstein MP. Detection of bloodstream infections in adults: How many blood cultures are needed Journal of Clinical Microbiology 2007 45: 3546-3548. 22. Mehdinejad M. Khosravi AD and Morvaridi A. Study of prevalence and antimicrobial susceptibility pattern of bacteria isolated from blood cultures. Journal of Biological Sciences 2009 93: 249 -253. 23. Kavitha P Sevitha B and Sunil R Bacteriological Profile and antibiogram of blood culture isolates in a pediatric care unit. Journal of Laboratory Physicians 2010 2: 85-88. 24. Swain B and Otta S. Bloodstream infections in a teaching hospital. Annals of Biological Research 2012 34:192 1928. 25. Alam MS Pillai PK Kapur P and Pillai KK. Resistant patterns of bacteria isolated from bloodstream infections at a university hospital in Delhi. J Pharm Bio allied Sci. 2011 34: 525–530. 26. Shrestha S Amatya R Shrestha RK Shrestha R. Frequency of Blood Culture Isolates and their Antibiogram in a Teaching Hospital. J Nepal Med Assoc 2014 52193: 692-6. 27. Sharma PP Halder D Dutta AK. Bacteriological profile of neonatal septicemia. Ind Pediatr 1987 24:1011-1017. 28. 20. Nimra LF Batchoun R. Community-acquired bacteraemia in a rural area predominant bacterial species and antibiotic resistance J Med Microbiology 2004 53:1045-49. 29. Figuera Esparaza M Carballo M Silva M Figuerdo A Avilan J. Microbiological isolates in pati ents with febrile neutropenia and nematological neoplasias Article in Spanish Rev Esp Quimioter 2006 19:247 - 51.

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M.Anitha E et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-41 2016 99-107 107 30. Butt T Afzal RK Ahmad RN Salman M Mahmood A Anwar M. Blood stream infections in febrile neutropenic patients Bacterial spectrum and anti microbial susceptibility pattern. J Ayub Med Coll Abbottabad 2004 16:18-22. 31. Chen Ly Tang JL Hsueh PR You MHuang SY Chen Yc et al. Trends and anti microbial resistance of pathogens causing blood stream infections among febrile neutropenic adults with hematological malignancy J Formos Med Assoc 2004103:526-32. How to cite this article: M.Anitha Sathya Pandurangan DM.Monisha J.Hemapriya Prevalence of gram positive and gram negative organisms in various clinical samples: Int J of Allied Med Sci and Clin Res 2016 41: 99-107. Source of Support: Nil. Conflict of Interest: None declared.

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