ventilator - associated pneumonia ( vap ) is defined as pneumonia occurring more than 48 hours after patients have been intubated and received mechanical ventilation .
vap is the most common nosocomial infection in the intensive care unit ( icu ) with an incidence ranging from 8% to 28% in intubated mechanically ventilated patients .
ventilator - associated pneumonia complicates the course of patients receiving mechanical ventilation inspite of major advances in techniques for its diagnosis and treatment . in the absence of a gold standard , vap
is assumed to be diagnosed more accurately by bronchoscopic sampling and microbiological cultures of the lower respiratory tract .
bronchoscopy , being invasive , is not uncommonly associated with complications , especially in patients on high respiratory supports .
this has paved the way for less invasive tests such as endotracheal aspirates ( eta ) and quantitative eta cultures with a threshold of 10 to 10 bacteria per milliliter of exudates that is considered as optimal for the microbiological confirmation of vap.[46 ] the american thoracic society ( ats ) guidelines recommend that quantitative cultures can be performed on eta or samples collected either bronchoscopically or nonbronchosopically .
more importantly , recent small trials have repeatedly shown that there is no advantage of bronchoscopic cultures over quantitative eta cultures when mortality was considered as the end - point further strengthening the case for quantitative eta as a diagnostic tool.[811 ] detection of causative organisms and their antibiotic susceptibility is crucial for diagnosis of vap in order to initiate the appropriate antibiotic treatment thereby reducing the adverse effects of inadequate antibiotic treatment on the patient prognosis .
hence , the present study is undertaken to isolate , identify and quantitate bacteria and to perform the antibiotic susceptibility testing from the endotracheal aspirates of the clinically suspected patients of vap .
the prospective study was carried out during the period from january 2010 to december 2010 in department of microbiology and medical intensive care unit ( micu ) on 54 patients with clinical suspicion of ventilator - associated pneumonia .
the approval of the institutional review board was obtained during the planning phase of the study and each patient ( or his / her caregivers ) gave informed consent prior to participation in the study .
all the patients 18 years and above who were under mechanical ventilation for more than 48 hours and clinically suspected of having contracted vap were included in this study .
the diagnosis of vap was established using clinical pulmonary infection score ( cpis ) , which was evaluated on a daily basis until the patient was on ventilator support .
clinically diagnosed ventilator - associated pneumonia were observed and clinical parameters were recorded from their medical records and bedside charts .
endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .
the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .
chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions .
only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing .
the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .
samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min
. 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .
the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .
all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .
growth of any organism below the threshold was assumed to be due to colonization or contamination .
organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .
escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .
isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test .
as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .
isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method .
all the patients 18 years and above who were under mechanical ventilation for more than 48 hours and clinically suspected of having contracted vap were included in this study .
the diagnosis of vap was established using clinical pulmonary infection score ( cpis ) , which was evaluated on a daily basis until the patient was on ventilator support .
clinically diagnosed ventilator - associated pneumonia were observed and clinical parameters were recorded from their medical records and bedside charts .
endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .
the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .
chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions . only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing .
the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .
samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min
. 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .
the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .
all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .
growth of any organism below the threshold was assumed to be due to colonization or contamination .
organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .
escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .
isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .
isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method .
endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .
the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .
chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions .
only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing .
the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .
samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min
. 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .
the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .
all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .
growth of any organism below the threshold was assumed to be due to colonization or contamination .
organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .
escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .
isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .
isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method .
this prospective study was done in the period from january 2010 to december 2010 in our hospital icu .
the meansd age of patients was 46.418.45 years ( range 18 to 86 years ) , having a predominance of male population .
bimodal distribution of age was observed with the first peak between 18 - 28 years of age group and second peak at around 59 years of age(28.89% ) .
distribution of total number of cases was done depending on the duration of mechanical ventilation into  early - onset vap  developing within 4 days of intubation and  late - onset vap  developing after 4 days of intubation .
the percentage of patients with early onset vap was 39.62% and late onset vap was 60.38% .
in our study , acinetobacter baumannii caused predominantly bilateral and right sided pneumonia whereas pseudomonas aeruginosa caused more of bilateral bronchopneumonia [ table 1 ] .
chest x - ray finding in relation to the organism isolated of 53 patients diagnosed as vap based on a cpis score of more than six , 51 ( 96.23 % ) patients had monomicrobial infection and 2 ( 3.77% ) had polymicrobial infection . among the 27 isolates of acinetobacter baumannii , 1 ( 3.70% ) was resistant to all group of antibiotics tested in the study , including carbapenems .
other 25 ( 92.59% ) isolates of acinetobacter baumannii were resistant to cotrimoxazole , ciprofloxacin and amikacin , 24 ( 88.89% ) to imipenem , 23 ( 85.18% ) to ceftazidime , 11 ( 40.74% ) to doxycycline , 10 ( 37.04% ) to piperacillin - tazobactam .
all the 27 ( 100% ) isolates of acinetobacter baumannii were multidrug resistant ( mdr ) i.e. resistant to three or more class of antibiotics .
pseudomonas aeruginosa was resistant to gentamicin ( 100% ) , aztreonam ( 88.23% ) , ciprofloxacin and amikacin ( 82.35% ) , imipenem ( 47.06% ) , ceftazidime ( 35.29% ) and piperacillin - tazobactam ( 23.53% ) .
figure 1 shows that late - onset vap was more common than early - onset vap . among the early onset
vap common organisms isolated were acinetobacter baumannii , pseudomonas aeruginosa , klebsiella pneumoniae , citrobacter freundii and staphylococcus aureus while in case of late onset vap common organisms were acinetobacter baumannii , pseudomonas aeruginosa and methicillin - resistant staphylococcus aureus .
organisms causing vap ( early and late onset vap ) eight ( 47.06% ) of the 17 pseudomonas aeruginosa isolates were mbl producers . among 27 isolates of acinetobacter baumannii ,
12 ( 44.44% ) were ampc producers while 14 ( 51.85% ) were mbl producing strains .
the co - existence of ampc and mbl was reported in 9 ( 33.33% ) isolates of acinetobacter baumannii and 2 ( 11.76% ) isolates of pseudomonas aeruginosa [ figure 2 ] .
different enzymes produced by the isolated strains in the present study it was found that the mortality rate was 45.28%.rate of mortality was less in early - onset vap ( 23.80% ) as compared to late onset vap group ( 59.37% ) and the difference was found to be statistically significant ( x1=6.473 ; df=1 ; p=0.011 ) .
out of 53 cases of ventilator - associated pneumonia , 16 ( 30.19% ) were female and 37 ( 69.81% ) were male .
aetiological agents widely differ according to the population of the patients in the intensive care unit , duration of hospital stay and prior antimicrobial therapy .
the increase of acinetobacter baumannii ( 49.09% ) infections is due to its great resistance to the environment which enables it to spread , its limited virulence and its extraordinary ability to develop resistance to all the antimicrobials and spread by aerosols .
one of them was methicillin - resistant staphylococcus aureus ( mrsa ) , but mrsa is generally associated with late onset vap .
this may be due the fact that this case showed mixed infection with predominant organism being citrobacter freundii ( 1.8  10 cfu / ml ) while mrsa was showing 2.1  10 cfu / ml in quantitative cultures .
secondly , the patient had history of prior antibiotic therapy which increases risk of mrsa .
found x - ray pattern in acinetobacter baumannii vap is non - specific . in half of the cases it causes lung infiltration and a diffuse bilateral pattern
some enzymes of pseudomonas aeruginosa have invasive properties , causing thrombosis of pulmonary vessels and pulmonary infarction ; this produces nodular bilateral lesions , predominantly in inferior lobes , and it is such a characteristic image that it should make the presence of pseudomonas aeruginosa suspected .
the antibiotic resistance pattern of nonfermenters was almost the same in both early- and late - onset vap .
many of the early - onset vap cases had the risk factors such as prior antibiotic therapy and current hospitalization for five days or more for infection with mdr pathogens .
that could be the reason for almost similar susceptibility pattern of the isolates from early- and late - onset vap .
even the american thoracic society guidelines support the same reasoning by suggesting that patients with early - onset vap who have received prior antibiotics or who had the prior hospitalization within the past 90 days are at greater risk for colonization and infection with mdr pathogens and should be treated similarly to patients with late - onset vap .
ampc beta - lactamases hydrolyze cephamycins and are not inhibited by commercially available beta - lactamase inhibitors .
bacteria , mostly klebsiella pneumoniae and e. coli , producing plasmid - mediated ampc beta - lactamases have been responsible for nosocomial outbreaks of infection and colonization .
twelve ( 44.44% ) out of 27 isolates of acinetobacter baumannii have shown production of ampc beta lactamase enzyme , and no other enterobacteriaceae showed production of ampc beta lactamase .
presently , there is concern about the acquisition of plasmid - mediated metallo - beta lactamases active against carbapenems , penicillins and cephalosporins . in this study
, 14 ( 51.85% ) isolates of acinetobacter baumannii and 8 ( 47.06% ) isolates of pseudomonas aeruginosa were plasmid - mediated metallo - beta lactamases enzyme producing strains detected by imipenem - edta combined disc method which corresponds to a study were metallo - beta lactamases were produced by 21.74% of acinetobacter spp . and 50% of pseudomonas aeruginosa .
early - onset vap in our study was found to be 39.62% while in various study it was found to be around 40% .
the low incidence of early onset vap in our study may be due to antibiotic use before admission to the icu .
studies have shown that previous antibiotic use decreases early - onset vap but markedly increases multidrug - resistant ( mdr ) pathogens , which is also reflected in our study . in the present study , it was found that the mortality rate was 45.28% which was in accordance with previous studies such as those by gupta a et al .
the bacteriological approach for the management of vap avoids the problem of overtreatment by separating colonizers from infecting pathogens .
this study showed that quantitative culture of eta is a useful test for early diagnosis of vap .
the antibiotic susceptibility pattern of these isolates will also help the clinicians to choose the appropriate antimicrobial agents for prophylactic as well as treatment purposes .
 background : ventilator - associated pneumonia ( vap ) is a common complication of ventilatory support for patients with acute respiratory failure and is associated with increased morbidity and mortality.aim of the study : the present study was undertaken to do quantitative cultures of aerobic bacteria , perform the antibiotic susceptibility testing from the endotracheal aspirates and clinical outcome of the clinically suspected patients of vap.methods:a prospective study was performed over a period of one year in a tertiary care hospital , enrolling patients on mechanical ventilation ( mv ) for 48 hr . endotracheal aspirates ( eta ) were collected from patients with suspected vap , and direct gram 's stain criteria was used to accept the sample . 
 quantitative cultures of eta were performed with the threshold for microbiological diagnosis of vap was taken as 105 colony forming units ( cfu)/ml.results : out of 53 cases , 2 ( 3.77% ) were polymicrobial . 
 multidrug resistant bacteria , mainly acinetobacter baumannii 49.09% ( 27/55 ) and pseudomonas aeruginosa 30.91% ( 17/55 ) were the most common pathogens isolated . 
 metallo - beta lactamases ( mbls ) was produced by 47.06% ( 8/17 ) of pseudomonas aeruginosa and 62.96% ( 17/27 ) of acinetobacter baumannii.conclusion:the bacteriological approach for the management of vap helps the clinicians in choosing the appropriate antibiotics . 
 this study showed that quantitative cultures of endotracheal aspirate at a cutoff point of 105 cfu / ml is one of the alternative to bronchoscopy in the diagnosis of clinically suspected ventilator associated pneumonia . 
INTRODUCTION
METHODS
Inclusion criteria
Exclusion criteria
Collection of endotracheal aspirates
Microbiological processing
RESULTS
DISCUSSION
CONCLUSION