Identification of Bacterial Isolates from Endotracheal Aspirate of Patients in Intensive Care Unit and Their Antimicrobial Susceptibility Pattern

Background: Nosocomial infections have been discussed as a critical issue among intubated patients leading to significant morbidity and mortality. Tracheal colonization of different bacteria may be responsible for added or superinfections and may increase the risk of mortality. Irrational use of antibiotics also increases the emergence of drug-resistant bacteria. Objectives: We aimed to investigate the bacterial isolates in the endotracheal aspirates of ICU patients and to see the pattern of antibiotic susceptibility. Materials and Methods: This cross-sectional study was based on 40 specimens of endotracheal aspirates which were collected from ICU patients of Dhaka Medical College Hospital. All the specimens were processed and cultured on MacConkey and blood agar media. The isolated organisms were identified by different biochemical tests. Results: Among the 40 specimens, 38 (95%) yielded growths of different bacteria. Of them, Acinetobacter baumanni were 13 (34%), Pseudomonas aeruginosa were 6 (16%), Klebsiella pneumoniae were 3 (8%), Klebsiella oxytoca were 3 (8%) and Staphylococcus aureus were 3 (8%). All the isolated bacteria were sensitive to colistin and most of the Acinetobacter baumanni were resistant to different antibiotics. Among the 32 isolated gram-negative bacteria, 10 (31.25%) were ESBL producers. Conclusion: Most of the bacteria showed antibiotic resistance to different common antibiotics, which is very alarming for the ICU patients.


Introduction
Hospital-acquired infection (HAI) is a serious and burning problem worldwide and is responsible for high rate of morbidities and mortalities. 1It has been shown that in developed countries 5−15% patients in general wards and 50% patients in intensive care units (ICU) suffered from HAI. 2 In developing countries this burden is somewhat underestimated, which may be due to lack of knowledge of proper surveillance, proper resources and proper guidance. 2In ICU most of the patients suffer from urosepsis, post-surgical infection and lower respiratory infection. 3The modern apparatus responsible for HAI are endotracheal tube, catheter and different surgical appliances. 4Tracheal colonization of different bacteria may be responsible for added or superinfections and at the same time, increases the risk of mortality. 5Again due to irrational use of antibiotics, there is increasing emergence of drug-resistant bacteria. 6These gram-negative drug resistant bacteria are prevalent all over the world. 7he aim of our study was to detect different pathogens from endotracheal aspirate in ICU patients and to see their antimicrobial susceptibility pattern.

Materials and Methods
This cross-sectional study was carried out on 40 specimens of endotracheal aspirates collected from ICU patients of Dhaka Medical College Hospital.This study was done in the department of Microbiology, Dhaka Medical College (DMC), Dhaka from July 2013 to June 2014.Patients with clinically suspected respiratory tract infection hospitalized in ICU of Dhaka Medical College Hospital (DMCH) irrespective of age, sex and antibiotic used were included in this study.Informed written consent was taken from all patients or their legal guardians before specimen collection.

Specimen collection
Endotracheal tube aspirates were collected from clinically suspected respiratory tract infected patients in ICU of DMCH.With all aseptic precautions, endotracheal aspirates were collected by using a 50 cm and 14 Fr suction catheter.The suction catheter was gently introduced through the endotracheal tube for a distance of approximately 25−26 cm.The endotracheal aspirate was obtained by suction, without instilling saline and the catheter was withdrawn from the endotracheal tube.After the catheter was withdrawn, 2 mL of 0.9% sterile normal saline was injected into the suction catheter with a sterile syringe to flash the exudates.The aspirates were collected into a sterile falcon tube and were transported immediately to the laboratory for further processing. 8

Specimen processing
Endotracheal aspirates were mechanically liquefied and homogenized by vortexing for one minute. 9After vortexing specimens were centrifuged at 2000 g for 10 minutes.Two mL supernatant was discarded using a sterile pipet and the deposit was further mixed by vortexing.The processed specimen was used for culture.

Gram staining of aspirate and microscopic examination
Smears were prepared from the aspirate, gram staining was done and then examined under microscope for the presence of gram-positive or gram-negative organisms.

Isolation of organisms
Specimens were inoculated by using a sterile wire loop on the blood agar medium and MacConkey agar medium (three sectors consecutively).After inoculation, all the media were incubated overnight at 37°C aerobically.The microbiological growths of endotracheal aspirates were classified as rare, light, moderate or heavy, based on the number of colonies in each of two sectors shown in the table (Table I). 10 Moderate to heavy growth was considered as significant. 11For confirmation of Staphylocoocus spp.subculture was done on mannitol salt agar medium from blood agar medium and incubated at 37ºC for 24 hours and examined after 24 hours.

Identification of organisms
All the isolated organisms were identified by their colony morphology, staining characters and further confirmed by relevant biochemical tests.

Screening of ESBL producers by double disc synergy assay
The test was performed on Muller-Hinton agar medium.Disc containing 30 µg of ceftazidime and a disc containing amoxicillin plus clavulanic acid (20 µg + 10 µg) were placed 20−25 mm apart (center to center).The Muller-Hinton agar plate was incubated at 37ºC for 24 hours.A clear extension of the edge of inhibition zone of cephalosporin disc towards amoxiclav disc was interpreted as ESBL production. 13

Data analysis
After compiling data were analyzed using 'Microsoft Office Excel 2007' program and c 2 test was used to compare the results.
According to antimicrobial resistance pattern to different antibiotics, none of the isolates were resistant to colistin and 12 (92%) Acinetobacter baumanni were resistant to amoxiclav, ceftazidime, ciprofloxacin and cefoxitin.Among the 3 isolated Staphylococcus aureus, 2 (66.67%) were resistant to ciprofloxacin and all were sensitive to linezolid, oxacillin, cefoxitin and vancomycin (Table III).No MRSA and VRSA were isolated.

Discussion
Infections are the most important and the leading cause of mortality and morbidity in ICU. 14 Endotracheal tubes are susceptible to infection and therefore it is important to be aware of the relevant factors and responsible organisms to take prompt action. 5The findings of this study would be helpful in selection of appropriate antibiotics.In this study, all the positive colonies of significant growth obtained from endotracheal aspirates were considered.
In this study, 40 endotracheal aspirates were studied and organisms were isolated from 38 specimens.Among the isolated organisms, 13 (34%) were Acinetobacter baumannii, 6 (16%) were    15 reported that gram-negative bacilli accounted for 83% among all isolates.The most commonly identified organism was Acinetobacter spp.followed by Pseudomonas aeruginosa in that study.
In another study, gram-negative bacilli accounted for 86% among all isolates and the most commonly identified organism was Acinetobacter spp.followed by Klebsiella pneumoniae. 16Klebsiella pneumoniae (34%) was the most common isolate, followed by Pseudomonas aeruginosa (20%) and Acinetobacter species (18%) in another study. 9In a study done by Dominic et al 17 in Kasturba Medical College Hospital, Mangalore, it was reported that the majority of bacteria in their study were gram-negative bacilli (81.14%), among them Pseudomonas spp.accounted for 41.14%, Klebsiella spp.15.43%, Acinetobacter spp.10.28%.In this study, 7.5% Staphylococcus aureus was found but incidence of Staphylococcus aureus was 15.2% in another study which is significantly higher than findings in our study. 18This variation in the pattern of bacterial isolates may be due to the fact that the studies were done in different geographical areas.
Other factors are differences in patient population, exposure to antibiotic, type of ICU patient, length of ICU stay and the method used for diagnosis of ventilator-associated pneumonia (VAP). 15 this study, most (85%) of the Acinetobacter baumannii were resistant to imipenem.High percentages (29%) of Acinetobacter baumannii strains resistant to imipenem were reported in another study. 19A study done by Shaheda 20 in BSMMU found that 62% of the Acinetobacter were imipenem sensitive.Since last 10 years, resistance to imipenem has been increasingly reported worldwide in non-fermenting gram-negative bacilli (NFGNB) including Acinetobacter spp.. 21 In this study, most (92%) of the Acinetobacter baumannii were resistant to ceftazidime and amoxiclav and amikacin.
Acinetobacter baumannii was found highly resistant (89%) to ceftazidime in another study that was almost in agreement with the present study. 22Infection by metallo-β-lactamase (MBL) producing organism including bla NDM-1 producers are increasing in the last few years in Bangladesh, which are resistant to most of the commonly used third generation cephalosporin including imipenem. 23,24 the present study, among the 13 Acinetobacter baumannii, 12 (92%) were multidrug-resistant.Two previous studies reported 85.4% and 68.1% multidrugresistant Acinetobacter spp.were isolated from ICU patients. 25,26These results are very much similar to the present study.
In In the present study, among the 32 isolated gramnegative bacteria 10 (31.25%) were ESBL producers.Among them Klebsiella pneumoniae were 50%.
Another study carried out in Bangladesh in 2012 reported that among 169 gram-negative bacteria isolated from wound swab and urine, 42 (25%) were ESBL producers, which is slightly lower than the present study. 23In that study, most of the isolates of Klebsiella pneumoniae (83.33%),Klebsiella oxytoca (33.33%),Enterobacter aerogenes and Proteus spp.produced ESBL, which is in accordance with the present study.In the present study, out of 13 Acinetobacter baumannii isolates, none were found positive for ESBL production.Higgins et al 28 reported that 9.09% ESBL producer was Acinetobacter baumannii in their study.
In conclusion, this study presents the most common microorganisms colonized from endotracheal tube of hospitalized patients and their pattern of antibiotic resistance.This study shows that Acinetobacter are the most common bacteria and most of them are resistant to common antibiotics, which is alarming for developing countries like Bangladesh.The antibiotic colistin was found sensitive for all the organisms in the present study.

Table I :
Semi-quantitative reporting of microbial growth in culture performed in petri-dishes

Table II :
Distribution of bacteria isolated from endotracheal aspirate samples (n=38)

Table III :
Antibiotic resistance pattern of major isolated bacteria to different antibiotics

Table IV :
Distribution of ESBL producing gram-negative bacteria identified by DDS test Pseudomonas aeruginosa, 6 (16%) were Klebsiella pneumoniae, 3 (8%) were Klebsiella oxytoca, 3 (8%) were Staphylococcus aureus.Out of 38 significant growths in semiquantitative culture positive cases, gram-negative bacteria were isolated in 32 (84.21%) cases.Amini et al 27e present study, all the six isolated Pseudomonas aeruginosa (100%) were resistant to amoxiclav and gentamicin.Imipenem was found most sensitive against Pseudomonas aeruginosa.Isolated Pseudomonas aeruginosa were imipenem sensitive in 83.44% and piperacillin sensitive in 67.67% cases.In contrast, Pseudomonas was most sensitive to amikacin (52.78%) and ceftazidime (47.22%) in a study done by Taj et al27in Pakistan.Klebsiella spp.were sensitive to colistin and 77.78% to imipenem.Dominic et al 17 reported that 66.67% Klebsiella spp.were sensitive to amikacin, which is similar to the present study.Among the three isolated Staphylococcus aureus, 2 (66.67%) were resistant to ciprofloxacin.Taj et al27reported 63.7% Staphylococcus aureus were resistant to ciprofloxacin, which is close to the present study.In this study, all (100%) isolated Staphylococcus aureus were sensitive to linezolid.Another study also found that all the Staphylococcus was sensitive to linezolid.27