Pattern of antibiotic sensitivity among klebsiella isolates from sputum of hospital acquired respiratory tract infection

Klebsiella Pneumoniae is one of the most common causes of nosocomial respiratory tract infections all over the world. Unfortunately, it is developing resistance to many antibiotics. This study was carried out in a teaching hospital for determining the prevalence and pattern of antibiotic sensitivity among Klebsiella pneumoniae isolated from sputum samples of hospital acquired respiratory tract infection. Materials and Methods: A total of 154 samples were included in the present study. Klebsiella Pneumoniae were isolated in (21.52%) samples and identiﬁed by standard microbiological techniques. Antibiotic susceptibility testing was done by the Kirby-Bauer disc diffusion method and interpreted as per CLSI guidelines. Results: Isolated strains were sensitive to Amikacin (70%) Levoﬂoxacin (70%) Gentamicin (65%) Chloramphenicol (65%) and resistant to Ampicillin (98.72%) Co-trimoxazole (87.5%) Ceftriaxone (85%) Tigecycline (85%) Tobramycin (85%). Conclusion: This study may help to formulate local antibiotic policy as a part of rational antibiotic therapy for hospitals; this may reduce the


Introduction
Klebsiella Pneumoniae, a gram-negative opportunistic bacterium, ubiquitously present and isolated worldwide causing nosocomial infections. 1 Unfortunately it develops resistance to a various class of antibiotics including betalactam group. Indiscriminate prescription and overuse of beta-lactam group of antibiotics are responsible for drug resistance. 2 Multi-drug resistant (MDR) Klebsiella Pneumoniae isolates were defined by non-susceptibility to at least one agent in three or more antibiotic categories. 3 Klebsiella Pneumoniae is a gram-negative, encapsulated, non-motile, lactose fermenting, facultative anaerobe belonging to the Enterobacteriaceae family. This was isolated by Friedlander from the lungs of patients who died of Pneumonia. The organism was named after him as Friedlander's bacillus.
Most of the Klebsiella Pneumoniae infections are associated with hospitalization mainly in immune-compromised and those with severe underlying diseases. Klebsiella Pneumoniae had been identified as a causative agent in nosocomial pneumonia (7 to 14%), septicemia (4 to 15%), wound infections (2 to 4%) and neonatal pneumonia (3 to 30%). 4 Antibiotic resistance of Klebsiella Pneumoniae is due to spread of transmissible plasmids and acquiring resistant genes. This occurs by horizontal gene transfer that may carry virulence determinants. 5 Capsule which is known as K antigen that protect bacteria from phagocytosis. Lipo-polysaccharide, siderophore (iron scavenging system), fimbriae (type1 &3) are virulence factors that contribute to the pathogenicity of Klebsiella Pneumoniae. Production of ESBL by Klebsiella Pneumoniae renders resistant to many classes of antibiotics such as beta-lactam/ beta-lactamase inhibitors combinations, aminoglycosides and quinolones.
Evaluating anti-biotic susceptibility pattern of Klebsiella Pneumoniae which varies geographically is a must for effective control and prevention of the rapid spread of drug resistance. 7 The prevalence of nosocomial infection due to Klebsiella Pneumoniae at our hospital is 21.52% and routinely beta-lactam group of antibiotics are used as a treatment. Random use of this drug may lead to MDR so to prevent and have a specific antibiotic policy of the hospital the present study of evaluating the susceptibility pattern of K. pneumoniae was undertaken.

Materials and Methods
The present study was approved by the Institutional Ethics Committee. The samples were processed in the Department of Microbiology of D.Y. Patil Medical College, Kolhapur. Sputum samples [n=158] from hospitalized patients developing respiratory tract infection were collected over a period of six months (Jan 2018 to June 2018) Written consent from volunteering participants was collected. For isolation of bacteria, the sputum samples were aseptically inoculated on blood, Mac-Conkey, and chocolate agar plates. The plates were incubated at 37 0 C for 24 hours. Klebsiella Pneumoniae were identified by colony morphology which showed large dome-shaped colonies on blood and chocolate agar while colonies on Mac-Conkey agar were mucoid and lactose fermenting. Gram staining of sample smear revealed gram-negative, short, stout and blunt rods. Further confirmation was done by bio-chemical reactions which show indole negative, H 2 S negative, V-P positive, Citrate utilization positive, Urease positive. Sugar fermentation tests exhibited production of abundant gas and acid from lactose, sucrose, glucose, mannitol and maltose. 8 Among 158 sputum samples, 34 positive isolates were subjected to antibiotic sensitivity by Kirbi Bauer disc diffusion method as per CLSI guidelines. 9 A log-phase broth culture inoculums of isolates with turbidity equivalent to Mc-Farlands 0.5 standard (1.5 x10 8 CFU/ml) was prepared. It was lawn cultured on Mueller-Hinton agar and allowed to dry. The antibiotic discs were applied on the surface with sterile forceps. Antibiotics selected were Gentamicin (10mcg), Amikacin (30mcg), Co-trimoxazole (30mcg), Tobramycin (10mcg), Aztreonam (30mcg), Ceftriaxone (30mcg), Chloramphenicol (30mcg), Nitrofurantoin (300mcg), Tetracycline (30mcg), Tigecycline (15mcg), Polymyxin B (30mcg) Levofloxacin (5mcg), Ciprofloxacin (30mcg) Piperacillin-tazobactam (100/10mcg) Amoxiclav(20/10mcg).

Discussion
This study was carried out to find susceptibility for 16 different commonly prescribed antibiotics against Klebsiella Pneumoniae from Sputum samples [n=158] of hospitalized patients developing respiratory tract infections.
Present study indicates nosocomial infection due to Klebsiella Pneumoniae was common among male (25.59%) as compared to female(16.67%) our finding is consistent with other studies conducted by Anu Sharma 10 and Gauri Kulkarni (2017) 11 Male preponderance may be due to the habit of smoking making them susceptible to the development of COPD. 12 Without any gender bias individuals having more than 60 of age were found susceptible to Klebsiella Pneumoniae infections, this may be due to the opportunistic nature of the organism. 13 Culture positivity of 21.52% for Klebsiella Pneumoniae found in our study is slightly less than Ravichitra et al 8 and Manikandan et al 14 which may be due to rational use of antibiotics in the early stage of the disease. However, our result matches with Lal et al. 15 Out of 16antibiotics tested, the present study shows a sensitivity of Klebsiella Pneumoniae to only a few antibiotics. Isolates were sensitive to mainly aminoglycoside and quinolones.
Due to irrational use of beta-lactam group of antibiotics for respiratory tract infections the isolates of Klebsiella Pneumonia exhibited maximum resistance to them. High resistance observed for Klebsiella Pneumoniae to Tobramycin (85%), Piperacillin-tazobactam (71%), and Amoxiclav (70%) may be due to the production of betalactamase enzyme by bacteria which causes hydrolysis of beta-lactam ring, rendering them ineffective. 16 Isolated Klebsiella Pneumoniae were sensitive to Amikacin (70%) 15,17 Chloramphenicol (65%), Gentamicin (65%) 15 these being older, less commonly prescribed antibiotics have shown good sensitivity. But routine and improper  exposure of isolates to newer antibiotics have shown higher resistance to them. Isolates exhibiting a mixed reaction to Fluro-quinolones may be due to ESBL production by them which were sensitive to Levofloxacin (70%) 15 while resistant to Ciprofloxacin (80%). 10,18

Conclusion
This study concludes that Amikacin, Chloramphenicol, Gentamicin and Levofloxacin antibiotics having average 65% sensitivity should be recommended under the local antibiotic policy as a part of rational antibiotic therapy for hospitals which may help to reduce the development of multi-drug resistance.
The hospital should follow uniform policy regarding the use of antibiotics which include education of patients and physicians about an appropriate drug, dose and duration of treatment. Constant surveillance of anti-microbial resistance and anti -microbial use at the hospital will help to reduce antibiotic resistance.

Acknowledgement
Authors would like to thank Chancellor Sanjay D. Patil, D.Y, Patil University, Kolhapur for kind support.

Source of Funding
None.

Conflict of Interest
None of the author have claimed any conflict of interest.