BACTERIOLOGICAL STUDY OF VENTILATOR-ASSOCIATED PNEUMONIA IN A TERTIARY CARE HOSPITAL.

to detect commonly associated and determine their of a for more than 48 hours, (21.0%) clinically as cases. The VAP was found to be cases per 1000 ventilator days. (38.11%), (26.74%), K. (14.85%), S.aureus (11.38%), E. coli (3.96%), C. A. and P. mirabilis were Amongst the gram negative isolates, 15.64% were ESBL, 11.73% were AmpC and 13.96% were MBL producers. Early diagnosis of VAP along with their sensitivity pattern will help as an epidemiological marker for initial prophylaxis and treatment planning for mechanically ventilated patients.

Ventilator-associated pneumonia (VAP) is the most common nosocomial infection diagnosed in the intensive care units (ICUs). Microorganisms responsible for ventilator associated pneumonia vary from place to place. VAP requires a rapid diagnosis and initiation of the antibiotic treatment.The present study was carried out to detect bacteria commonly associated with VAP and determine their susceptibility patterns including beta-lactamases production. Out of a total 1438 patients intubated for more than 48 hours, 302 patients (21.0%) were clinically diagnosed as VAP cases. The VAP rate was found to be 2.06 cases per 1000 ventilator days. A. baumannii (38.11%), P. aeruginosa (26.74%), K. pneumoniae (14.85%), S.aureus (11.38%), E. coli (3.96%), C. freundii (1.98%), A. lwoffi (1.98%) and P. mirabilis (0.99%) were isolated. Amongst the gram negative isolates, 15.64% were ESBL, 11.73% were AmpC and 13.96% were MBL producers. Early diagnosis of VAP along with their sensitivity pattern will help as an epidemiological marker for initial prophylaxis and treatment planning for mechanically ventilated patients.

Introduction:-
Ventilator-associated pneumonia (VAP) is the most common nosocomial infection diagnosed in the intensive care units (ICUs). 1 It is defined as pneumonia occurring more than 48 hours after endotracheal intubation and initiation of mechanical ventilation (MV). 2 VAP is the most frequent infection occurring in 9 to 24% of the intubated patients admitted in ICU. 3,4 It has been associated with an attributable mortality of approximately 30% depending on the pathogen isolated, 5,6 which may exceed 50%. 7,8 Microorganisms responsible for ventilator associated pneumonia vary from place to place. Common pathogens include aerobic gram-negative bacilli (GNB), such as Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Acinetobacter species; 9 gram positive cocci such as Staphylococcus aureus. 5 There have been reports of increased occurrence of multi-drug resistant pathogens including carbapenem resistance bacteria in health care settings in recent times.
To date, however, there are very few studies in India evaluating VAP. Thus, this study was carried out to detect the bacteria commonly associated with VAP and determine their susceptibility patterns.

Material and methods:-
The present study was carried out at a tertiary care hospital and approved by the Ethical committee of Indira Gandhi Government Medical College, Nagpur.
During the two and a half year study period, a total of 1438 patients admitted to the ICUs were intubated for more than 48 hours. Out of them, 302 patients were clinically diagnosed to have VAP. Thus, patients on MV for more than 48 hours having temperature of >38 o C or <36 o C, leukopenia or leukocytosis or having purulent tracheal secretions were included in the study.
Endotracheal tube aspirates were obtained with sterile precaution using a 22 inch No.14F suction catheter and collected in a mucous collector. 10 The samples were processed and the isolates were identified by standard microbiological procedures. 11 Antimicrobial susceptibility testing was done by Kirby-Bauer disk diffusion method 10 as per CLSI guidelines. 12 All the gram negative isolates (Enterobacteriaceae group, Acinetobacter spp. and P. aeruginosa) were tested for extended spectrum β-lactamases (ESBL) and AmpC β-lactamases production 10,13 by initial screening and phenotypic confirmatory test (PCT) as per CLSI guidelines. 10 ESBL production was detected by combined disk test using both cefotaxime and ceftazidime, alone and in combination with clavulanic acid, was performed. 10 Amp C production was detected by AmpC disk test. 14 Carbapenemases production in Enterobacteriaceae 10 and non-fermentative gram negative 15 isolates were tested by performing both initial screening test and phenotypic confirmatory test (Modified Hodge test). MBL production was detected by minimum inhibitory concentration (MIC) testing by agar dilution method using meropenem; and MIC reduction testing using meropenem and meropenem-EDTA as per CLSI guidelines. 10,16,17 MBL testing was also done using double disk synergy test (Imipenem, Meropenem and Ceftazidime). 18,19 Methicillin resistant S. aureus (MRSA) production was detected by Cefoxitin disk diffusion testing 10,20,21 and Minimum inhibitory concentration testing of oxacillin 10,13,22 by agar dilution method using oxacillin-salt screen agar containing 6μg/ ml oxacillin and 4% NaCl. 25

Statistical analysis
Data analysis was done by Chi square test with appropriate (yate's) correction to see the significance of difference using SPSS software; p ≤0.05 was considered significant.

*Colistin resistance was detected by MIC using agar dilution method
Non-fermenters (66.83%) were the most predominant pathogens causing VAP, members of Enterobacteriaceae and gram-positive bacteria (S. aureus) causing VAP were 42.99% and 11.38% respectively. VAP episodes due to grampositive bacteria (11.38%) were relatively less common as compared to gram-negative bacilli (88.61%).

Detection of ESBL, AmpC β-lactamase and Metallobetalactamase
ESBL production was detected in 25% of Enterobacteriaceae isolates and 12.59% of the Non-fermenters. AmpC βlactamases were produced by 34.09% and 4.44% of the members of Enterobacteriaceae and non-fermenters respectively. MBL production was seen in 11.36% of the Enterobacteriaceae isolates while 14.81% of the nonfermenters were MBL producers (Table II).

Discussion:-
VAP is an important nosocomial infection among ICU patients receiving MV along with multi-drug resistant pathogens causing VAP are a major concern in any kind of ICU set up. The rate of VAP obtained in our study is 2.06 cases per 1000 ventilator days similar to a study conducted by Bowton DL et al. 23  Most of the Enterobacteriaceae isolates were sensitive to meropenem, piperacillin-tazobactum and amikacin; most non-fermentors being sensitive to piperacillin-tazobactum and colistin; and S. aureus being sensitive to vancomycin. Thus, the use of these antibiotics can be advocated in our area. This finding also emphasises the need for stringent preventive measures for VAP, as the treatment of an established VAP becomes very expensive. 25 We observed that the most effective antibiotic for gram negative isolates was meropenem, while for S.aureus isolates was vancomycin. Piperacillin-tazobactam was highly active against Acinetobacter spp., while colistin has good activity against Pseudomonas spp. Our findings were similar to the other studies. 26,27,28 MBL was produced by most of the non-fermenters, especially A. baumannii consistent with other studies. 29 Some K. pneumoniae isolates also showed MBL production similar to a study by Galani et al. 30 Similarly ESBL and AmpC β-lactamases were produced by a large proportion of the Enterobacteriaceae. 25  The presence of these MDR pathogens highlights the need for treatment of the VAP cases with second-line antibiotics effective against them.
When the results of our study were compared with the findings of other studies, it was clear that the incidence of VAP and its resistance pattern have been changing with due course of time. Geographical variation and difference in patient population studied could be the possible factor for variability. The emergence of multi-drug-resistant pathogens causing VAP has made its treatment very difficult and, in some cases, impossible. The observed high number of GNB showing ESBL, AmpC, MBL production; S. aureus with methicillin resistance in VAP cases reflects its emerging resistance pattern. Thus, the antimicrobial policy needs to be updated from time to time.
Early diagnosis of VAP along with their sensitivity pattern will help as an epidemiological marker for initial prophylaxis and treatment planning for mechanically ventilated patients. In recent years, there has been a rapid increase in the incidence of VAP among the patients admitted to the ICUs for various clinical conditions and so is the increase in the multi-drug resistant pathogens due to their prolonged stay in the ICU leading to increased mortality and morbidity.
Hence, it is the hour of need to prevent ventilator associated pneumonia caused by hospital-acquired multi-drug resistant strains by formulating appropriate hospital antibiotic policy and proper infection control practices.