ANTIBIOTIC RESISTANCE PATTERN OF BACTERIAL ISOLATES FROM POST-TRACHEOSTOMISED PATIENTS ATTENDING TERTIARY CARE HOSPITAL IN NEPAL

The bacterial profile and their drug susceptibility pattern was studied in post-tracheostomised patients admitted in National Institute of Neurological and Allied Sciences. Identification of organisms was done by standard microbiological techniques and antibiogram was performed by Kirby Bauer disc diffusion method according to Clinical and Laboratory standard (CLSI) guidelines. Significant growth was observed in 58.6% samples with polymicrobial growth in 19.5% samples among the total positive cultures. Pseudomonas aeruginosa was the predominant organism (34.2%) followed by Acinetobacter spp. (31.8%), Escherichia coli (8.9%), Klebsiella pneumoniae (6.0%), Citrobacter fruendii (5.6%), Klebsiella oxytoca (4.0%), Providencia spp. and Staphylococcus aureus (2.8% each), Proteus mirabilis (2.4%) and Proteus vulgaris (1.2%) . More than 90% of Gram negative bacterial isolates were found resistant to Ampicillin, Cefixime, Cephotaxime. The most effective drugs against S. aureus were Vancomycin, Rifampicin, Amoxycillin and Clavulanic acid showing 100% sensitivity. Highest resistance rate was observed for Ampicillin (85.71%) and low for Gentamicin (14.28%). Findings of this study show emerging threat of multidrug resistant bacteria. Thus, periodic monitoring and assessing drug susceptibility pattern of bacteria and rational use of antibiotics in post tracheostomised patients were recommended.


INTRODUCTION
Tracheostomy is an invasive procedure aimed to provide a secure airway to ventilate and aspirate the patient in the critical care setting (Pignatti et al. 2009).It is performed in many clinical cases such as decreased level of consciousness, poor airway protective reflexes, severe alterations in physiology associated with trauma and medical illness and the most common being prolonged respiratory failure (Durbin 2010).Tracheostomy is associated with a number of clinical complications out of which airway colonisation and infection is the most frequent complication in patients with chronic tracheaostomy (Apostolopoulou et al. 2003).The colonisation route is either endogenous or exogenous which generally causes lower airway infection such as tracheobronchitis and bronchopneumonia.Nosocomial bacteria such as Pseudomonas aeruginosa, Acinetobacter spp., methicillin resistant Staphylococcus aureus, Serratia spp.and members of Enterobacteriaceae are the frequently encountered infection causing bacteria.However, others like Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, and methicillin sensitive S. aureus also colonise and infect the lower respiratory tract (Ionas et al. 2001, Morar et al. 2002, Erdem et al. 2008).This work was undertaken to assess the antibiotic susceptibility of bacteria isolated from samples associated with tracheostomised patients.

MATERIALS AND METHODS
This study was conducted at National Institute of Neurological and Allied Sciences, Bansbari, Kathmandu, Nepal from March 2014 to August 2014.A total of 350 clinical specimens including tracheal aspirates (180), urine (73), blood (22), pus (13), wound swabs (4), Cerebrospinal fluid (23), Catheter tip (13), CVP tip (19), Foley's tip (1) and ICP tips (2) from tracheostomised patients that were sent to microbiology laboratory for routine analysis were processed via standard microbiological procedure.Identification of organisms was done on the basis of colony characteristics, staining, biochemical tests such as catalase, oxidase, sulfide indole motility, citrate, urea hydrolysis, triple sugar iron agar test and coagulase tests.Antibiotic susceptibility test was performed using the Kirby Bauer disk diffusion method and results were interpreted according to CLSI guidelines (CLSI 2007).The approval to conduct this work was obtained from the hospital.

RESULTS
Among the samples processed, significant bacterial growth was observed in 205 (58.6%) samples with polymicrobial growth in 19.5% of the total positive cultures.From these 205 positive samples 248 bacterial isolates were obtained out of which 2.8% were Gram positive cocci and remaining 97.2% were Gram negative rods and coccobacilli.Gram positive cocci were identified as S. aureus whereas majority (34.2%) of the Gram negative isolates were identified as P. aeruginosa (Table 1).Most of the S. aureus isolates were found to be resistant to Ampicillin while moderate numbers were resistant to Cotrimoxazole, Cephoxitin and Erythromycin.Few isolates were found resistant against Gentamicin whereas none were resistant against Vancomycin, Rifampicin and Amoxycillin-Clavulanic acid (Table 3).

DISCUSSION
Results of this study showed that the bacterial growth is high in number, which is in the harmony with another study conducted in the same hospital that reported 60.88% positive growth (Bhandari et al. 2015).Polymicrobial growth was reported in 19.5% samples.Most of the polymicrobial growths in this study were associated with Acinetobacter spp.and P. aeruginosa, the two frequently acquired nosocomial pathogens.This high growth rate with polymicrobial growth may be either due to the fact that the patients were critically ill and thus had poor immune system to combat against pathogenic microbes or due to presence of these bacteria in the hospital environment.
The dominant isolates accounted were Gram negative bacteria.High prevalence of Gram negative rods in tracheostomised patients was also reported in other study (Siddiqui et al. 2011).P. aeruginosa was the predominant organism which is also analogous to other studies (Morar et al. 2000, Nseir et al. 2002).Factors such as their ubiquity in hospital environment, ability to form incredibly hard biofilms on many seemingly uninhabitable surfaces and calcium alginate capsules may have contributed to the highest prevalence of P. aeruginosa (Inglis et al. 1989).
Besides, some of the isolated bacteria were resistant to more than one antibiotic.It was found that more than 90% of Gram negative bacterial isolates were resistant to Ampicillin, Cefixime, Cephotaxime.Low resistance was observed for Imipenem with resistance rate ranging between 3.52-9.09%.For aminoglycosides such as Amikacin and Gentamicin, the resistance rate was between 8.23-100%.Resistance for Ciprofloxacin was observed between 42.85-100%, for Cotrimoxazole between 57.14-100% and for Piperacillin-Tazobactam between 28.57-85.71%.Similar pattern of degree of resistance was witnessed in different studies (Kumari et al. 2007, Mohammadimehr & Feizabadi 2011).Contrary, a study by Hossein et al. (2012) reported resistance of 45-75% to Cefixime.Gram negative organisms adopt different resistance mechanism to neutralize the affects of many commonly used antibiotics.This may be contributed by the production of -lactamases primarily ESBL, AmpC enzymes, metallo-carbapenemases, aminoglycoside modifying enzymes, loss of porin proteins and the presence of efflux pumps like MexABOpr M.An increase in antibiotic resistance among Gram negative bacterial isolates has been a great concern in hospitals.Overuse of antimicrobial agents and problems with infection control practices have led to the development of multidrug resistant Gram negative bacterial infections (Falagas et al. 2006).
A notorious nosocomial bug, Acinetobacter spp.
showed high resistance to a number of antibiotics.This resistance mechanism is furnished via altered penicillin-binding proteins, low/decrease permeability of the outer membrane to antibiotics or increase in the active efflux of the antibiotics, target site mutations and inactivation (Jain & Danziger 2004).The type of species, antibiotic and geographical location is also conducive to resistance mechanism (Vila et al. 2002, Jain & Danziger 2004).

CONCLUSION
We conclude that there is a significant prevalence of Gram negative microorganisms in post tracheostomised patients with P. aeruginosa being the most common.Most of the bacterial isolates were resistant to multiple antibiotics, which is a threat to the emergence of Multi Drug Resistant bacteria making treatment of infections extremely difficult.Hence, early detection of infection, empirical use of antibiotics and restriction in use of broad spectrum antibiotics are obligatory.Since majority of isolates are nosocomial pathogens, aggressive cleaning and monitoring of hospital environment is necessary to minimize the spread of theses bugs to other immune-compromised patients.