International Journal of Research in Medical Sciences

Ranjan N et al. Int J Res Med Sci. 2014 Feb;2(1):228-233 pISSN 2320-6071 | eISSN 2320-6012

DOI: 10.5455/2320-6012.ijrms20140244
Research Article

Antimicrobial resistance in bacteria causing ventilator-associated
pneumonia in a tertiary care hospital: one year prospective study
Neelima Ranjan1, K. P. Ranjan1, Uma Chaudhary2, Dhruva Chaudhry3
Department of Microbiology, Gajra Raja Medical College, Gwalior, Madhya Pradesh, India
Department of Microbiology, Postgraduate Institute of Medical Sciences, Rohtak, Haryana, India
Department of Critical Care and Pulmonary Medicine, Postgraduate Institute of Medical Sciences, Rohtak, Haryana,

Received: 4 November 2013
Accepted: 13 November 2013

Dr. K. P. Ranjan,

© 2014 Ranjan N et al. This is an open-access article distributed under the terms of the Creative Commons Attribution
Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.


Background: Ventilator-associated pneumonia (VAP) is the most common infection diagnosed in intensive care
units (ICUs). The causative organisms of VAP vary among different populations and are increasingly associated with
resistance against various antimicrobial agents. Objective of current study was to determine the bacteriological
etiology of VAP, antimicrobial susceptibility pattern of the isolates and detect the presence of extended-spectrum -
lactamases (ESBL), metallo β-lactamases (MBL) and AmpC -lactamases in multidrug resistant isolates causing VAP
in the medical ICU.
Methods: A prospective study was carried out over a year to know the various etiological agents of VAP and their
drug susceptibility patterns. ESBL, MBL and AmpC -lactamases were detected in various isolates by combination
disk method, imipenem-EDTA combined disk method and AmpC disk method respectively.
Results: The majority of bacterial isolates causing VAP were found to be gram negative bacilli. Acinetobacter spp
accounted for 34.28% of VAP cases followed by Pseudomonas aeruginosa which was responsible for 25.71% cases.
Other gram negative bacilli isolated were Klebsiella pneumoniae, Citrobacter freundii, Enterobacter spp, and
Escherichia coli. Out of the total 70 isolates, 67 (95.7%) were multidrug resistant and not even a single isolate was
sensitive to all the drugs tested.
Conclusions: Most of the pathogens causing VAP in our institute were multidrug resistant and in many isolates this
resistance was due to production of ESBL, MBL, and AmpC β-latamases. Polymixin-B and colistin were found to be
highly effective against multidrug resistant Acinetobacter spp and P. aeruginosa.

Keywords: Ventilator-associated pneumonia, Intensive care unit, ESBL, MBL, AmpC -lactamases

INTRODUCTION ventilation, usually carries a better prognosis, and is more
likely to be caused by antibiotic sensitive bacteria. Late
Ventilator associated pneumonia (VAP) is the most onset VAP occuring 5 days or more after mechanical
common infection diagnosed in intensive care units ventilation is more likely to be caused by multidrug
(ICUs). VAP is defined as pneumonia that occurs 48 resistant (MDR) pathogens, and is associated with
hours or more after endotracheal intubation or increased patient mortality and morbidity.2 The specific
tracheostomy, caused by infectious agents not present or microbial causes of VAP are many and varied. Gram
incubating at the time mechanical ventilation was negative bacteria, including Pseudomonas aeruginosa,
started.1 It can be of two types. Early-onset VAP, defined Acinetobacter spp and enteric gram negative rods are
as occurring within the first 4 days of mechanical implicated in 55-85% of VAP cases. High rates of

International Journal of Research in Medical Sciences | January-March 2014 | Vol 2 | Issue 1 Page 228

In threshold for endotracheal aspirates while growth >104 the monomicrobial episodes gram negative isolates CFU/ml was taken as cutoff for BAL.12 etiology of VAP and the antimicrobial susceptibility pattern of the isolates. Gram staining was done after making smears Klebsiella pneumoniae. The diagnosis was confirmed when significant growth The majority i. 13 MBL detection was done and Acinetobacter spp. Other gram negative bacilli isolated were processing. In relation to gender the incidence of VAP was VAP was based on clinical and microbiological criteria. In critically ill patients requiring flattening or indentation of the cefoxitin inhibition zone prolong mechanical ventilation in ICUs.2(1):228-233 Haemophilus influenzae. account for 30-40% of VAP. 3 considered to be ESBL producer if there was ≥5 mm Prompt usage of appropriate antibiotics is essential to increase in zone diameter of ceftazidime-clavulanate disk optimize the outcome of VAP. The pneumonias per 1.4 Appropriate increase in inhibition zone with the imipenem and EDTA choice of antibiotics requires awareness of relevant disk was ≥7 mm than the imipenem disk alone.5 Patients who incidence of VAP in our study was 57. The diagnosis of group. isolate was identified using standard Methicillin sensitive Staphylococcus aureus (MSSA). P value less than 0. aeruginosa which was responsible for immediately to the laboratory for microbiological 25. Out of the 60 cases 21 (35%) were categorized infection score (CPIS) was followed as a screening under early onset group and 39 (65%) under the late onset method to clinically diagnose VAP. quantitative cultures were done. Streptococcus pneumoniae.7% of bacterial isolates causing VAP was obtained in the culture of the samples. We also aimed to detect the Statistical analysis presence of extended-spectrum -lactamases (ESBL). If the antibiotics.28% of VAP cases samples of the patients were collected and sent followed by P. more among males (65%) than females (35%) and in A clinical suspicion of VAP was made in patients with different age groups the incidence of VAP was highest in modified CPIS score >6. tests.7 per 1000 ventilator were included in the study. Fisher’s exact test was applied when two or more set of variables were compared. If the inhibition zone evolution of antibiotic resistance.8. and multi drug resistant gram negative bacteria detected by combination disk method. Int J Res Med Sci.12 Amp C β-lactamases detection was the past decade and has created obstacles to effective done by Amp C disk method. only 3 isolates were gram positive bacteria. 95. aeruginosa.71% cases.10 Antibiotic testing was done susceptible Enterobactericeae were constantly found in by Kirby Bauer disk diffusion method for each isolate. VAP rate was study. Enterobacter of the samples and the samples were then inoculated on spp. P.7 The MacConkey plates Among them 2 isolates were of Staphylococcus aureus were incubated at 370C while blood agar and chocolate and 1 was Enterococcus spp. ESBL was (MRSA). whereas P.9 Samples showing accounted for 96% (48/50) and even in polymicrobial growth less than these thresholds were assumed to be due episodes of VAP gram negative bacilli were predominant to colonization or contamination. growth.000 ventilator days.14% and the received mechanical ventilation for more than 48 hours incidence density of VAP was 31. Acinetobacter An isolate was considered as MDR. Ranjan N et al.05 METHODS was considered statistically significant. as compared to zone diameter of disk containing antimicrobial resistance has escalated dramatically within ceftazidime alone. and the host considered as MBL positive. or microbiological techniques. Modified clinical pulmonary days. Endotracheal were found to be gram negative bacilli. A positive test appeared as antibiotic choices. if it was resistant to spp. Citrobacter freundii. Organism was were significantly more frequent in late onset VAP. 11 early onset VAP. which are resistant to many by imipenem-EDTA combined disk method. and Escherichia coli (Table 1).14 MRSA detection was done and demographic factors that may lead to infection and/or by cefoxitin disk diffusion method. metallo β-lactamases (MBL) and AmpC -lactamases in The statistical analysis was performed using standard multidrug resistant isolates causing VAP in the ICU. 10 episodes of VAP were dioxide. MacConkey agar and chocolate agar. 2014 Feb. aeruginosa in the vicinity of the test disk. isolates. Among the total 60 episodes agar were incubated at 370C in presence of 5-10% carbon of VAP reported. In case of significant accounting for 90% of etiological agents. A prospective study was conducted in Department of RESULTS Microbiology in association with the multidisciplinary ICU of the Pulmonary and Critical Care Medicine A total of 105 patients who were on mechanical Department of our institute for a period of one year ventilation for more than 48 hours were included in the extending from June 2009 to May 2010. Among gram International Journal of Research in Medical Sciences | January-March 2014 | Vol 2 | Issue 1 Page 229 . Out of the total 70 blood agar. Unfortunately. it was pathogens. Semi.6 patients more than 55 years of age (73. Acinetobacter spp aspirates (ETA) and bronchoalveolar lavage (BAL) accounted for a maximum 34. antimicrobial resistance patterns.68%). Methicillin resistant Staphylococcus aureus at least three classes of antimicrobial agents. Sixty patients fulfilled the clinical and defined as the number of ventilator-associated microbiological criteria for the diagnosis of VAP. around the cefoxitin disk was >22 mm then the isolate was considered MSSA and if the zone was <21 mm then The aim of our study was to determine the bacteriological it was considered as MRSA.e. Growth >105 CFU/ml was taken as the cutoff polymicrobial and 50 episodes were monomicrobial.

maximum by A. Vancomycin (Va).2% isolates. The single isolate of Enterococcus spp was in K. AmpC β-lactamase and MBLs within these pathogens. of Ce E Cd G Cf Va Do Lz Cp Gf Ac Pm isolates isolates Staphylococcus 1 0 0 0 1 0 1 0 2 0 0 1 2 aureus (50) (0) (0) (0) (50) (0) (50) (0) (100) (0) (0) (50) Enterococcus 1 1 1 0 1 0 0 1 1 1 1 1 - spp (100) (100) (100) (0) (100) (0) (0) (100) (100) (100) (100) Cefoxitin (Ce). The ESBL pristinamycin (Table 2). ciprofloxacin.It was also found to be highly resistant to various drugs such as also produced by 66. Gram negative bacteria were production was highest in case of E.42 bacteria Pseudomonas aeruginosa 18 25.28 Escherichia coli 1 1. produced MBL (Table 4). and in C. 12 (50%) isolates produced AmpC β-lactamase. to the presence of various degradative enzymes like freundii. Erythromicin (E). No.71 Klebsiella pneumoniae 15 21. doxycycline. one was MRSA spp.43 Total 70 100 Table 2: Antibiotic susceptibility pattern of gram positive bacteria isolated from VAP patients. freundii.86 Gram negative Acinetobacter lwoffii 1 1.67% isolates. amikacin. In case of Acinetobacter spp.67% isolates. meropenem. 67 (95. Cephalexin (Cp). Clindamycin (Cd). In P.52% of early Out of the total 70 isolates. piperacillin/ isolates. the drugs tested. doxycycline and aeruginosa. Ciprofloxacin (Cf). it sulbactam combination were found to be quite effective was 20. Gatifloxacin (Gf).85 Enterococcus spp 1 1. Gentamicin (G). Linezolide (Lz). C. Int J Res Med Sci. International Journal of Research in Medical Sciences | January-March 2014 | Vol 2 | Issue 1 Page 230 . pneumoniae ceftazidime. pneumoniae. which was resistant to cephalexin. aureus accounted for 9.34% of K. 3 4. The MBL production was maximum in case of tazobactam. Some of this rsistance can be attributed baumannii followed by P. and Pristinamycin (Pm). aeruginosa (27. it was seen to be produced by 66. also found to be resistant to vancomycin. Colistin. co-trimoxazole. aeruginosa. (%) of resistant strains Bacterial No.83% and in K. polymixin-B and cefoperazone / P. 16.18%). 2014 Feb.58 Enterobacter spp. Doxycycline (Do).67% of Enterobacter spp isolates. ESBLs. aztreonam. gatifloxacin. Table 1: Distribution of organisms isolated from samples in VAP patients. it was seen to be produced by 22.67% of C.7%) were multidrug onset VAP. coli (100%). and Enterobacter spp.2(1):228-233 positive bacteria S. Majority of late onset VAP episodes were resistant and not even a single isolate was sensitive to all also caused by gram negative bacteria. freundii and 13. K. ciprofloxacin. Out of the total 24 isolates of Acinetobacter Out of the two isolates of S. aureus.43 Acinetobacter baumannii 23 32. Bacterial isolates Number Percentage Gram positive bacteria Staphylococcus aureus 2 2. pneumoniae by 26. Ranjan N et al. pneumoniae only one isolate (Table 3). Amoxyclav (Ac).43 Citrobacter freundii 6 8.

The incidence density of resistant. Aztreonam (Ao).7 0 increased among a number of gram negative pathogens.3) (100) (0) (33. Ranjan N et al.7 13.3) (100) (26) (78) (0) (0) (8. including the penicillins. Ceftazidime (Ca). 2014 Feb. Amikacin (Ak).7 16. it was observed that vancomycin Diagnosing VAP requires a high clinical suspicion and linezolide were the most effective antibiotics for S. radiographic aureus.7) (83.2(1):228-233 Table 3: Antibiotic susceptibility pattern of gram negative bacteria other than pseudomonas aeruginosa isolated from VAP patients. and Ticarcillin/clavuanalate (Tc) Table 4: Distribution of AmpC.14%. and MBL in the study and the study duration was less as compared to bacterial isolates from VAP patients. The higher incidence of VAP in our study can 27.3) (20) (13.7) (26) Klebsiella 10 8 8 10 14 14 3 2 0 0 0 0 15 pneumoniae (66. Escherichia coli 0 100 0 especially P. doxycyline and ciprofloxacin were found to understanding local factors leading to VAP and the be effective against Enteroccus spp. aureus was MRSA.7) (100) (66.2 aeruginosa bacteria is common in ICUs mainly because of heavy use Klebsiella of antibiotics.7 per 1000 ventilator days highly effective. Int J Res Med Sci. Colistin (Cl). Ciprofloxacin (Cf).7 0 aminoglycosides.3) Escherichia 1 1 1 0 0 1 1 0 0 0 0 0 0 coli (100) (100) (0) (0) (100) (100) (0) (0) (0) (0) (0) (0) Acinetobacter 0 0 0 0 0 1 0 0 0 0 0 0 1 lwoffii (0) (0) (0) (0) (0) (100) (0) (0) (0) (0) (0) (0) Co-trimoxazole (Co). Cefoperazone/ Sulbactam (Cfs). Acinetobacter spp.3) (50) (50) (66.6) (93. Citrobacter freundii 66.6) (87) (91. examination.e.3) (0) (0) (0) (33.7) (100) (0) (33. All strains of P. When considering gram negative bacteria Acinetobacter In our study the incidence of VAP was 57. other studies showing fewer incidences. ESBL. cephalosporins.7 pneumoniae antibiotics. Acinetobacter spp 50 0 20.8% strains showed resistance to meropenem. (%) of strains resistant Bacterial No of Co Do Ak Cf Ca Ao Mr Pt Pb Cl Cfs Tc isolates isolates Acinetobacter 22 19 19 20 21 23 6 18 0 0 2 6 23 baumannii (95.3) (53.3) (93. Polymixin-B and colistin were found to be VAP in our study was 31. K. International Journal of Research in Medical Sciences | January-March 2014 | Vol 2 | Issue 1 Page 231 . combined with bed side examination.3) Enterobacter 3 3 2 3 2 3 0 1 0 0 0 1 3 spp (100) (100) (66.3) (66. secretions.6) (53. No.3 6. This resistance microbiologic milieu of a given unit. aeruginosa. In our study the incidence of MRSA was 50% i.16 DISCUSSION On performing the antimicrobial susceptibility testing of VAP is the most frequent ICU acquired infection gram positive bacteria and studying their antibiotic occurring in 10% to 65% of the ventilated patients.8 Pseudomonas The development and spread of antibiotic resistant 22.17 plague ICUs and critically ill patients.3) (0) (0) (0) (33.3) (0) (0) (0) (0) Citrobacter 5 3 3 4 5 6 0 2 0 0 0 2 6 freundii (83. pneumoniae and Enterobacter spp. Meropenem (Mr). One more reason for this high incidence can be the lack of adequate AmpC ESBL MBL nursing staff (which should ideally be 1:1 as compared to Bacterial isolates producer producer producer 4:1 in our institute) which may have adversely affected (%) (%) (%) the quality of care given to the patients. has gradually Enterobacter species 33. Multiple antibiotic resistance to useful 26. 15 susceptibility pattern. Doxycycline (Do).6) (82. This spp was the most common isolate and its resistance figure is at the higher end of the range of 15-58% as pattern showed that majority of isolates were multi-drug reported by other investigators. and microbiological analysis of respiratory one of the two isolates of S. In other be attributed to the fact that the total number of cases in gram negative bacilli also resistance to multiple drugs was seen.2 0 27. Aggressive surveillance is vital in Linezolide.6) (82. Polymixin B (Pb). Judicious antibiotic pattern of gram positive bacteria isolated from patients of usage is essential as resistant organisms continue to VAP was in accordance with other studies.3 66. Piperacillin/Tazobactam (Pt). aeruginosa were also which were high but compatible with ICUs in developing uniformly sensitive to polymixin-B and colistin but countries. and fluoroquinolones.

their drug susceptibility patterns.9(4):211-6. Arch Intern Med. which can be of great 11. Ventilator associated and health care 15. Prevalence of extended spectrum beta Conflict of interest: None declared lactamase and AmpC beta lactamase producers Ethical approval: The study was approved by the among Escherichia coli isolates in a tertiary care institutional ethics committee hospital in Jaipur.45:493-6. 2010.171:388-416. Sherris J et al. Yum JH. Cefotetan) as well as Medical Microbiology: 14th ed. and five isolates of Acinetobacter spp (20. Am Rev Respir Dis. ESBL production was seen in members of family 8. eds. Sherman G. Simmons A.160:1926-36. Fagon JY.a prospective cohort study. M2-A7.148:138-44. Bauer AN.2(1):228-233 An important group of resistant VAP pathogens are 3.83%) associated pneumonia (VAP) Rate per 1. US: Wayne PA. and Treatment. of MBL being one of their major defence mechanisms. coli but may also occur in other gram-negative 6. Jean Yves Fagon. ventilator days. Incidence. Ranjan N et al. New York: other extended-spectrum cephalosporins. MBL producing P. Rossolini GM. to be highly effective against multidrug resistant Simmons A. MBL. Experience with a clinical guideline susceptible enzymes that hydrolyze penicillins. aeruginosa. CLSI microbiology and antibiotic susceptibility data is essential Document 20th ed. Chest patients of VAP. 2011. In: Collee JG. different geographic regions. Miles RB. Lynch III. Shin HB.51:367-9. They are typically plasmid-mediated clavulanate VJ. Fraser AG. Jean Chastre. Marimion BP. Marquette CH. aeruginosa also. New York: information regarding the pathogens causing VAP and Churchill Livingstone. Alaka K Deshpande. Pinto S. 2002. Lee K. J Clin Microbiol 2002. 1996: 131-49. Accessed 2 Feb 2011. Marimion BP. Polymixin B and colistin were found of bacteria. Funding: No funding sources Pathak D. Ward S. Watt B.ihi. In: Collee JG. Imipenem-EDTA disk method for 1. 1966. Fraser bacteria. Acinetobacter spp and 4. most of the pathogens causing VAP in our cultures in intubated patients with suspected institute were multidrug resistant and in many isolates pneumonia. Crit Care Med 2001. Approved standards. Mackie and McCartney Practical hydrolyze cephamycins (Cefoxitin. Fraser AG.67%).18%). http://www. Marimion BP. Int J Res Med Sci. associated pneumonia. five isolates of P. 165: 867-903. Ibrahim EH. These findings are similar to the studies done by previous 2000. individual pathogens varies substantially between 12. clinical outcome. REFERENCES 14. Microbiology. Sharma R.000 were metallo β. Schaiff R. ventilator associated pneumonia. They can be syndromes. Joseph P. Available at: detected by imipenem-EDTA disk method.132:621-30. Vidya S Nagar. ceftriaxone.lactamases enzyme producing strains. Collee JG. authors. Chastre J et al. differentiated from other ESBLs by their ability to Simmons A. one isolate of Klebsiella spp 5.20 9. Antibiotic assistance to the physicians for the prophylaxis and susceptibility testing by a standardized single disk treatment of VAP patients. Hospital-Acquired Pneumonia: other gram negative bacilli have been isolated from Risk Factors. Panwar R. Clinical and Laboratory Standards Institute. and 10. Kirby WMM. aztreonam. clinical isolates of Pseudomonas spp and 2000. Sinha P. Collee JG. 2001. aeruginosa. and risk stratification of 2005. International Journal of Research in Medical Sciences | January-March 2014 | Vol 2 | Issue 1 Page 232 . 13. cefepime and others) and 7.19 In our study Churchill Livingstone. Georges H et al. Fraser AG. 2014 Feb. eds.40:3798- 2. AmpC β-lactamases are cephalosporinases Laboratory strategy in the diagnosis of infective that are poorly inhibited by clavulanic acid. production associated Topics/CriticalCare/IntensiveCare/Measures/Ventila ESBLs are most commonly produced by Klebsiella spp tor Associated Pneumonia.119:373S-84S. Guidelines for management of adults with hospital 801. for the treatment of ventilator associated expanded-spectrum cephalosporins (cefotaxime. This study provided Medical Microbiology: 14th ed. Morehead R. pneumonia. Chong Y. Am J Respir Crit Care Med.29:1109-15.18 In our study. Am J Clin Pathol. aeruginosa (27. Sood S. Ann Intern Med. different institutions and Performance standards for antimicrobial disk even different units in the same hospital. this resistance was due to production of ESBL. Local susceptibility testing. 30(1): for making informed antibiotic treatment choices. The relative prevalence of method. Indian J Pathol Microbiol 2008. and E. acquired. Invasive and non-invasive Enterobacteriaceae and AmpC β-lactamases were strategies for management of suspected ventilator detected in Acinetobacter spp and P. Mackie and McCartney Practical Acinetobacter spp and P. Yong D. ceftazidime. Test for identification AmpC β-latamases. I J Crit Care Med 2005. Sharma R. Ventilator-associated differentiation of metallo-β-lactamases producing pneumonia: review article. Institute for Healthcare Improvement: Ventilator- (6. 1993. associated pneumonia. Ventilator carbapenem-resistant gram negative bacteria. Kollef M. Diagnostic efficacy of endotracheal aspirates with quantitative bacterial In conclusion. Acinetobacter spp. Rishi S. Am J Respir Crit Care Med. 1996: 53-95.

ijrms20140244 2010 April. Bairy I. Int J Res care areas in a tertiary care hospital. Ann Thorac Med 2007.2(1):228-233 16. Tarun Kumar Antimicrobial susceptibility of Acinetobacter Dutta. Baveja SM. Noyal Mariya Joseph. De AS. 2014 Feb. Ranjan KP.5455/2320-6012. species. Dey A. Incidence of multidrug-resistant of mechanical ventilation & development of organisms causing ventilator-associated pneumonia multidrug resistant organisms in hospital acquired in a tertiary care hospital: A nine months' pneumonia. Chaudhry D. Antimicrob Agents Chemother Subhash Chandra Parija. Chaudhary 18. Ashok Shankar Badhe. Desdemona Rasitha. Cite this article as: Ranjan N. A Ayyagari. December. A Bhargava.14:217-9.2:228-33.2:52-7.37(4):750-3. pneumonia in a tertiary care hospital in India: role of multi-drug resistant pathogens. Pulverer G. Indian J Crit Med Sci 2014. 20. C Mukhopadhyay. Schulze A. Role 19. Antimicrobial resistance in bacteria metallo-β-lactamase producing Pseudomonas causing ventilator-associated pneumonia in a tertiary aeruginosa and Acinetobacter species in intensive care hospital: one year prospective study. Sujatha Sistla.4(04):218-25. Indian J Med Res 2003 prospective study. Care Med 2010.118:229-35. Baginski R. 17. Ventilator-associated 1993. Kumar SH. International Journal of Research in Medical Sciences | January-March 2014 | Vol 2 | Issue 1 Page 233 . JIDC DOI: 10. Prevalence of U. Ranjan N et al. Int J Res Med Sci. Seifert H.