Aerobic Bacteriological Profile and its Antimicrobial Sensitivity Pattern From Blood Culture Specimens in A Tertiary Care Hospital

Background :Blood stream infections (BSIs) are important cause of mortality and morbidity and are among most common health-associated infections.Blood stream infection constitutes one the most serious situations with increased cost of care, morbidity, mortality and thus, timely detection and identification of blood stream pathogen is important.Sothe present study was undertaken to describe aerobic bacteriological profile and its antibiotic sensitivity pattern from blood culture specimen in a tertiary care setting. Method: The study was carried out in the Department of Microbiology, Indian Institute of Medical Sciences, Jalna, Maharashtra for a period of 8 months (July 2015 to February 2016). A total 1920 samples were evaluated from clinically suspected cases of bacteremia. Blood was collected depending upon age group with aseptic precaution and inoculated on brain heart infusion broth (BHIB).Subculture were made on blood agar and Mac-conkey agar plates. Organisms were identified and antibiotic sensitivity test of isolates were performed. Result : During 8 month study period out of 1920 blood culture, 369(19.21%) yielded growth of different organisms. Out of this 369 organisms gram positive bacteria 199 (53.9%) were isolated more often than gram negative bacteria 170 (46.1%). Staphylococcus aureus 49.05 % was leading pathogens isolated followed by enterobactericae group 21.00% (Escherichia coli and Klebsiella spp) and Salmonella typhi & S. Paratyphi A 13.82%. Conclusion: This study provides information on antibiotic sensitivity pattern of blood isolates which may be useful to guide clinicians to initiate empiric therapy and will help in formulation of antibiotic therapy strategy in this part of country.


Introduction
Blood stream infections (BSIs) are important cause of mortality and morbidity and are among most common health-associated infections [1] .Bacteremia signifies the presence of bacteria in the blood stream [1] .Bacteremia may be transient, continuous or intermittent. Micro-organisms present in the circulating blood are a threat to every organ in the body [1] .
It can have serious consequences like shock, multiple organ failure, disseminated intravascular coagulation, etc. thus, the blood stream infection constitutes one the most serious situations with increased cost of care, morbidity, mortality and , as a result,timely detection and identification of blood stream pathogen is important. Blood culture plays an integral role in the evaluation of sepsis [1,2] .
Increasing antimicrobial resistance is worldwide concern. The prevalence of resistance in both out-patients and hospitalised patients with septicaemia is increasing, and its varies in accordance with geographical and regional location. In almost all cases, antimicrobial therapy is initiated empirically before the results of blood culture are available [3] .
Selecting appropriate antimicrobial for treating BSI is multifaceted, including possible cause and source of infection, in vitro activity of drug according to microbiological susceptibility testing results, pharmacokinetics and adverse effects of the drug [2] .
However, before considering these aspects, the choice of antibiotic mainly relies on knowledge of the pathogen likely involved.Monitoring and analysing the antimicrobial suscepitibility pattern of most frequently isolated microorganisms according to local epidemiology which helps clinicians to choose empirical therapies and develop rational prescription policy for antibiotics [4,5] .
Annals of Pathology and Laboratory Medicine, Vol. 04, No. 01, January -February, 2017 Indian Institute of Medical Sciences Badnapur , Jalna, Maharshtra after approval from institutional ethics committee.

Sample Size:
The study was carried out during the period of July 2015-February 2016; a total 1920 samples were evaluated from clinically suspected cases of bacteremia.
Inclusion Criteria: All patients with unexplained fever/ undiagnosed fever, whose blood culture specimens were sent to department of microbiology were included in the study.
Exclusion Criteria: Contaminated, duplicate and repeat specimens from patients were excluded from study.
Collection of Sample and Processing : In adults 5 ml of blood for culture was drawn in sterile syringe after skin preparation by a two step process with 70% alcohol andpovidone iodine application and then dried for 1 min. Blood collected was aseptically incubated into blood culture bottle containing 50 ml of Brain Heart Infusion Broth (BHIB). In paediatric cases 1-2ml of blood was inoculated in 5-10 ml of BHIB.These bottles will be incubated at 37° C temperature under aerobic conditions in the incubator maximum for 7 days. Subculture will be made on Blood agar and MacConkey's agar daily from 1st to 7th day. However if the growth was observed further sub cultures were not done. Growth was processed according to standard microbiological techniques which includes Gram staining, colony characteristics and biochemical properties described in WC. Koneman's Colour Atlas and text book of Diagnostic Microbiologyand Bailey and Scott's Diagnostic Microbiology [6,7] . Blood culture broth which showed no microbial growth after 7 days were reported as culture negative.
Methicillin resistance in Staphylococcus aureus (MRSA) was tested using Muller Hinton Agar with Cefoxitin disc (30mcg) by Kirby-bauerdisc diffusion methods as per CLSI guidelines [8] . Suspected extended-spectrum beta lactamases (ESBLs) producing Enterobactericae were confirmed by double disk synergy test as as per CLSI guidelines [8] .
Staphylococcus aureus (ATCC 25923), E. coli (ATCC 25922) and P. aeruoginosa (ATCC 27853) were used as quality control throughout the study for culture and antimicrobial susceptibility testing.

Statistical analysis
The results were expressed as percentages for analysis of various epidemiological details and for analysing the distribution of different bacterial isolates and their sensitivity pattern. Microsoft excel was used for the interpretation of these results.

Results
During 8 month study period 1920 blood culture were analysed. 369(19.21%) yielded growth of different organisms. Of all isolates 291(76.8%) were isolated from hospitalised patients while 78 (23.2%) were from those who attended out-patients department. Majority of the patients were males 285(77.23%); male to female ratio was 3.4:1.
The detailed microbiological data of pathogens and their antimicrobial susceptibility causing blood stream infection is shown in table 1, 2, 3 &4

Discussion
The varying microbiological pattern of bacteremia/ septicaemia warrants the need for an ongoing review of causative organisms and their antimicrobial susceptibility pattern.
Out of 1920 suspected cases of bacteremia, in our study, 369 were culture positive with blood culture positivity rate of 19.21%. Similar positivity rates were reported by other studies [9,10] .
Our study highlights that gram positive septicaemia was encountered in 53.9% culture positive cases which was in concordance with studies done by China et al; and Gupta et.al; which shows increase incidence of gram positive bacteria especially Staphylococcus aureus in producing BSIs [11,12] .
www.pacificejournals.com/apalm eISSN: 2349-6983; pISSN: 2394-6466 OPD-out patient department, Med-medicine ward, Paeds-paediatric ward, ICU-intensive care unit, Surg-surgery ward, Othersobstetrics &gynaecology ward, orthopaedic ward, etc   Gram negative septicaemia was encountered in 46.1% of culture positive cases which is lower as compared to other studies which had reported increased incidence of gram negative bacteria ranging from 50% to 78% [12,13] . Commonest gram negative bacteria isolated in our study was E.coli , Klebsiella spp and followed by Salmonella spp and non-fermenters (Pseudomonas and Acinetobacter spp) which was in concordance with other studies carried out in different parts of India [14,15] .
The gram positive organism especially Staphylococcus aureus showed 41.4% and 55.24% sensitivity to penicillin and erythromycin respectively but were 100 sensitive to Vancomycin and Linezolid similar sensitivity pattern were seen in other studies [11,14,15] . 41(22.65%) were detected as methicillin resistant Staphylococcus aureus (MRSA). Studies by Indian researchers [16] , reported a similar prevalence of 41% MRSA. Our study also point out increase isolation of Enterococci spp as many studies had not isolated any single case of Enterococci spp except for few [17,18] . All Enterococci Spp were 100% sensitive to Vancomycin and showed 88.88% and 83.33% sensitivity to Ampicillin and Gentamicin (high level) respectively.
Most of gram negative bacteria especially Enterobactericae (except Salmonella spp )showed 100 sensitivity to imipenem and high resistance to ciprofloxacin and ceftazidime , similar trends has been reported by an Indian study [19] and many other researchers [11,12] highlighting higher resistance to third generation cephalosporins. Our study found out around 30% Enterobactericae isolates were ESBL producers which is alarmingly on higher side as compared with other studies [13] .
While member of nonfermenter (Pseudomonas and Acinetobacter spp) shown 100% sensitivity to Imipenem and Pipercillin-tazobactam this results are comparable to work done at other centers [19,20] and also revealed better sensitivity to third generation Cephalosporin and Gentamicin as compared with other studies [18,20] .

Conclusion
The detection, identification and susceptibility testing of causative species of bacteria are essential for proper treatment and better prognosis of patient in case of BSIs. Blood culture still remains as one of the most important microbiological investigation available to clinicians for diagnsosis of BSIs. Gram positive organisms were predominant in our setup for producing BSIs with higher incidence of MRSA. Our study also showed alarmingly higher incidence of ESBL producing Enterobacterciae group which points towards judicious uses of third generation Cephalosporins.