Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 1824
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size

ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 3  |  Page : 339-342
Ventilator-associated pneumonia caused by carbapenem-resistant Enterobacteriaceae carrying multiple metallo-beta-lactamase genes

1 Department of Microbiology, SGPGIMS, Lucknow, India
2 Department of Biochemistry, Lucknow University, India
3 Department of Critical Care Medicine, SGPGIMS, Lucknow, India

Click here for correspondence address and email

Date of Web Publication12-Aug-2009


Context: Ventilator-associated pneumonia (VAP) is a leading nosocomial infection in the intensive care unit (ICU). Members of Enterobacteriaceae are the most common causative agents and carbapenems are the most commonly used antibiotics. Metallo-beta-lactamase (MBL) production leading to treatment failure may go unnoticed by routine disc diffusion susceptibility testing. Moreover, there is not much information on association of MBL-producing Enterobacteriaceae with ICU-acquired VAP. Therefore, a study was undertaken to find out the association of MBL-producing Enterobacteriaceae with VAP. Settings: This study was conducted in a large tertiary care hospital of North India with an eight-bed critical care unit. Materials and Methods: The respiratory samples (bronchoalveolar lavage, protected brush catheter specimens and endotracheal or transtracheal aspirates) obtained from VAP patients (during January 2005-December 2006) were processed, isolated bacteria identified and their antibiotic susceptibilities tested as per standard protocols. The isolates of Enterobacteriaceae resistant to carbapenem were subjected to phenotypic and genotypic tests for the detection of MBLs. Results: Twelve of 64 isolates of Enterobacteriaceae were detected as MBL producers, bla IMP being the most prevalent gene. Additionally, in three strains, simultaneous coexistence of multiple MBL genes was detected. Conclusion: The coexistence of multiple MBL genes in Enterobacteriaceae is an alarming situation. As MBL genes are associated with integrons that can be embedded in transposons, which in turn can be accommodated on plasmids thereby resulting in a highly mobile genetic apparatus, the further spread of these genes in different pathogens is likely to occur.

Keywords: Carbapenem resistance, Enterobacter aerogenes , Enterobacteriaceae, Klebsiella pneumoniae , metallo-beta-lactamase, ventilator-associated pneumonia

How to cite this article:
Dwivedi M, Mishra A, Azim A, Singh R K, Baronia A K, Prasad K N, Dhole T N, Dwivedi U N. Ventilator-associated pneumonia caused by carbapenem-resistant Enterobacteriaceae carrying multiple metallo-beta-lactamase genes. Indian J Pathol Microbiol 2009;52:339-42

How to cite this URL:
Dwivedi M, Mishra A, Azim A, Singh R K, Baronia A K, Prasad K N, Dhole T N, Dwivedi U N. Ventilator-associated pneumonia caused by carbapenem-resistant Enterobacteriaceae carrying multiple metallo-beta-lactamase genes. Indian J Pathol Microbiol [serial online] 2009 [cited 2023 Mar 20];52:339-42. Available from:

   Introduction Top

Ventilator-associated pneumonia (VAP) is one of the most common nosocomial infections among patients admitted to the intensive care unit (ICU), [1] with high morbidity and mortality. [2],[3] Associated mortality and morbidity in VAP is increased in patients with wrong or delayed initial antibiotic treatment, which is frequently associated with the presence of resistant strains. [4],[5],[6],[7] An important group of resistant pathogens are carbapenem-resistant gram-negative bacteria, production of metallo-beta-lactamase (MBL) being one of their major defense mechanisms. MBL-producing Enterobacteriaceae have occasionally been isolated from patients of VAP in this hospital but no attempt was made to differentiate colonization from actual infection. Therefore, this study was undertaken to find out the association of MBL-producing Enterobacteriaceae with VAP.

   Materials and Methods Top

This study was conducted in a large tertiary care hospital of North India with an eight-bed ICU. The study period was January 2005-December 2006. All consecutive patients admitted to this ICU who developed VAP during the ICU stay were included in this study.

VAP was defined as nosocomial pneumonia in patients on mechanical ventilatory support (by endotracheal tube or tracheostomy) for >48 h during their ICU stay, excluding any infection present or in incubation at the time of ICU admission. Diagnostic criteria for pneumonia were radiographic appearance of a new or progressive and persistent pulmonary infiltrate and conjunction with at least two of the following criteria: purulent respiratory secretions, temperature more than 38.5C or less than 35C and leukocyte count >10,000/mm 3 or less than 1500/mm 3 . Only patients exhibiting bacteriologically documented pneumonia were studied; establishment of etiologic diagnosis required isolation of bacteria in significant quantity from samples of lower respiratory tract secretions (endotracheal secretions >10 6 cfu/ml, protected brush catheter >10 3 cfu/ml and bronchoalveolar lavages >10 4 cfu/ml) or isolation of a definitive pathogen from a blood or pleural fluid culture. Patient could not be entered into the study more than once. Consecutively, only the first episode of bacteriologically documented VAP was taken into account.

The microbiological samples were collected and processed according to standard protocols. [8] All the bacteria isolated were identified to the species level by standard biochemical tests and their antibiotic susceptibility testing was performed by the Kirby-Boir disc diffusion method on Muller-Hinton agar (Oxoid, Cambridge, UK) as per the Clinical and Laboratory Standards Institute guidelines. [9]

In order to analyze carbapenemase production phenotypically, carbapenem (imipenem [IPM] or meropenem)-resistant isolates were screened by a modified Hodge test [10] [Figure 1] and IPM-ethylenediaminetetraacetic acid (EDTA) disk synergy test. [11] [Figure 2]. In the modified Hodge test, the surface of a Muller-Hinton agar plate was inoculated with an overnight culture suspension of  Escherichia More Details coli ATCC 25922. In this test, a 10 g IPM disc was placed at the center of an inoculated Muller-Hinton agar plate. Ten microliters of 50 mM zinc sulfate solution (140 g) was added to the disc. The plate was read after overnight incubation at 37C. Presence of a cloverleaf-shaped zone of inhibition was interpreted as positive modified-Hodge test. In the combined disk test, two IPM disks (10 g), one containing 10 l of 0.1 M (292 g) anhydrous EDTA (Sigma Chemicals, St. Louis, MO, USA), were placed 25 mm apart (center to center). An increase in the zone diameter of >4 mm around the IPM-EDTA disk compared with that of the IPM disk alone was considered positive for MBL. [11]

A multiplex polymerase chain reaction (PCR) assay was performed to detect and differentiate five families of acquired MBL genes (VIM, IMP, SPM, GIM and SIM families) in a single reaction. Conserved regions of all available, bla IMP , bla VIM, bla SPM-1 , bla GIM-1 and bla SIM-1 alleles were identified in clustal multiple alignments. [12] Five primer pairs, specific for each family of acquired MBLs, were designed to amplify fragments of 188 bp (IMP), 390 bp (VIM), 271 bp (SPM-1), 477 bp (GIM-1) and 570 bp (SIM-1). [12] Details of the primers used are given in [Table 1].

DNA template was prepared by emulsifying five colonies in 100 l of PCR-grade water and adding 2 l to the PCR reaction mixture before thermal cycling. The cycling conditions were initial DNA release and denaturation at 94C for 5 min, followed by 40 cycles of 94C for 30 s, 52C for 40 s and 72C for 50 s, followed by a single, final elongation step at 72C for 5 min.

   Results Top

During the study period, 103 patients of the 287 patients admitted to the ICU developed VAP. From these patients, 64 isolates of Enterobacteriaceae have been recovered, of which 13 isolates were found to be resistant to either IPM or Meropenem. Modified-Hodge test was positive in 11 isolates while the IPM-EDTA disc synergy test was positive in 12 isolates. Multiplex PCR showed presence of MBL genes in 12 isolates (which were also positive by the IPM-EDTA disc synergy test). Three strains of the Enterobacteriaceae family (two strains of Klebsiella pneumoniae and one strain of Enterobacter aerogenes ) isolated from different patients have shown presence of multiple MBL genes. One each of K. pneumoniae and E. aerogenes isolates were found to contain both the SIM-1 and IMP gene families while one of the K. pneumoniae isolates had shown the presence of VIM and SIM-1 genes. To the best of our knowledge, this is the first report on the identification of any strain of Enterobacteriaceae simultaneously carrying bla IMP and bla SIM-1 or bla VIM and bla SIM-1 . The results of multiplex PCR are shown in [Table 2].

The most common MBL subtype was bla IMP , found in seven of 12 isolates of Enterobacteriaceae. In addition to the presence of multiple MBL genes, another new finding in the study is that three members of Enterobacteriaceae carrying bla SIM have been isolated. Bla SIM is a very rare gene, usually found in Acinetobacter species. Its presence in Enterobacter species itself indicates widespread dissemination of MBL genes.

   Discussion Top

The carbapenem group of antibiotics plays a vital role in the management of VAP because of its broad spectrum of activity and stability to hydrolysis by most of the beta-lactamases, including extended-spectrum-beta-lactamases. Unsurprisingly, VAP caused by carbapenem-resistant gram negative bacteria in critically ill patients is the most difficult infection to treat. The major defense in these bacteria is production of MBLs. [13],[14] Because MBLs can hydrolyze a very broad range of broad-spectrum b-lactams, MBL-producing gram negative bacteria usually display resistance to a variety of broad-spectrum b-lactams, including oxyiminocephalosporins, cephamycins and carbapenems, which are the last remedy to control the infections caused by gram negative bacteria. Thus, MBL-producing gram negative bacteria have been renowned to be among the most important nosocomial pathogens. Clinical infections with MBL-producing organisms pose serious therapeutic challenges, with increasing reports of poor patient outcomes and death. [15]

Five major MBL types have been identified, which include multiple variants of the VIM and IMP families and single members of the SPM, GIM and SIM families. [16] A new MBL family has been recently reported in Pseudomonas aeruginosa from Australia - bla AIM-1 . Among the pathogenic microbes, production of MBLs is commonly seen in P. aeruginosa , Acinetobacter species and, infrequently, among the members of Enterobacteriaceae. [17] These acquired MBL genes are located on integron structures that reside on mobile genetic elements such as plasmids or transposons, [16] thus enabling widespread dissemination.

Members of the family Enterobacteriaceae are among the most important bacterial human pathogens, accounting for the majority of the bacteria isolated from clinical samples.[18] A major concern is that these gram negative bacilli are rapidly acquiring resistance to one or more antimicrobial agents traditionally used for treatment.

To the best of our knowledge, this is the first report of carbapenem-resistant Enterobacteriaceae carrying multiple MBL genes, which is a public health concern in our country and requires efficient detection and intervention to preserve antibiotic options. The presence of the multiple MBL genes in Enterobacteriaceae is of great relevance because their transfer from Enterobacteriaceae to other bacteria will increase the antimicrobial-resistance problem, and this phenomenon is already occurring in some countries with the subsequent rise in health care costs and mortality rates. [19],[20],[21],[22] Tato et al . described an ongoing outbreak of infection and colonization with MBL-producing gram negative bacteria that is resistant to most widely used antibiotics. [19],[20] They report a situation that is worryingly close to a state of endemicity, whereby untreatable organisms are well established in their institution, having a mortality rate of almost 50% among patients who were infected with these organisms [19],[20] Fukigai et al. have reported nosocomial outbreak of genetically related IMP-1 beta-lactamase-producing K. pneumoniae in a general hospital in Japan. [23]

These and several other reports [19],[21],[22] provide an important signal that there is an urgent need for application of stringent prevention strategies, including improving methods for early identification of MBL-producing bacteria, identifying patient populations at high risk for infection with MBL-producers, changes in antibiotic treatment regimens, application of hygiene measures and control of horizontal nosocomial transmission of organisms so as to combat the coming threat of increasing antibiotic resistance in gram negative bacilli. It is very important that MBL screening is routinely made for gram negative bacilli (including Enterobacteriaceae) in clinical laboratories, because failure to control outbreaks involving MBL-producing organisms will eventually lead to a situation where all possible empirical treatment options are ineffective.

   Acknowledgement Top

Mayank Dwivedi acknowledges the financial assistance from the Council of Scientific and Industrial Research, New Delhi, India.

   References Top

1.Chastre J, Fagon JY. Ventilator-associated pneumonia. Am J Respir Crit Care Med 2002;165:867-903.  Back to cited text no. 1    
2.Safdar N, Dezfulian C, Collard HR, Saint S. Clinical and economic consequences of ventilator-associated pneumonia: A systematic review. Crit Care Med 2005;33: 2184-93.  Back to cited text no. 2    
3.Rello J, Ollendorf DA, Oster G, Vera-Llonch M, Bellm L, Redman R, et al . Epidemiology and outcomes of ventilator-associated pneumonia in a large US database. Chest 2002;122:2115-21.  Back to cited text no. 3    
4.Kollef MH, Sherman G, Ward S, Fraser VJ. Inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients. Chest 1999;115:462-74.  Back to cited text no. 4    
5.Kollef MH. Inadequate antimicrobial treatment: An important determinant of outcome for hospitalized patients. Clin Infect Dis 2000;31:S131-8.  Back to cited text no. 5    
6.Rello J, Gallego M, Mariscal D, Sonora R, Valles J. The value of routine microbial investigation in ventilator-associated pneumonia. Am J Respir Crit Care Med 1997;156:196-200.  Back to cited text no. 6    
7.Luna CM, Vujacich P, Niederman MS, Vay C, Gherardi C, Matera J, Jolly EC, et al . Impact of BAL data on the therapy and outcome of ventilator-associated pneumonia. Chest 1997;111:676-85.  Back to cited text no. 7    
8.Wu CL, Yang DIe, Wang NY, Kuo HT, Chen PZ. Quantitative culture of endotracheal aspirates in the diagnosis of ventilator-associated pneumonia in patients with treatment failure. Chest 2002;122:662-8.  Back to cited text no. 8    
9.Clinical Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests. 9 th ed. Vol.26. 2006. p. M2-A9.  Back to cited text no. 9    
10.Lee JH, Choi CH, Kang HY, Lee JY, Kim J, Lee YC, et al . Differences in phenotypic and genotypic traits against antimicrobial agents between Acinetobacter baumannii and Acinetobacter genomic species 13TU. J Antimicrob Chemother 2007;59:633-9.  Back to cited text no. 10    
11.Franklin C, Liolios L, Peleg AY. Phenotypic detection of carbapenem- susceptible metallo-beta-lactamase-producing gram-negative bacilli in the clinical laboratory. J Clin Microbiol 2006;44:3139-44.  Back to cited text no. 11    
12.Ellington MJ, Kistler J, Livermore DM, Woodford N. Multiplex PCR for rapid detection of genes encoding acquired metallo-beta-lactamases. J Antimicrob Chemother 2007;59:321-2.  Back to cited text no. 12    
13.Helfand MS, Bonomo RA. Current challenges in antimicrobial chemotherapy: the impact of extended-spectrum beta-lactamases and metallo-beta-lactamases on the treatment of resistant Gram-negative pathogens. Curr Opin Pharmacol 2005;5:452-8.  Back to cited text no. 13    
14.Nordmann P, Poirel L. Emerging carbapenemases in Gram-negative aerobes. Clin Microbiol Infect 2002;8:321-31.  Back to cited text no. 14    
15.Laupland KB, Parkins MD, Church DL, Gregson DB, Louie TJ, Conly JM, et al . Population-based epidemiological study of infections caused by carbapenem-resistant Pseudomonas aeruginosa in the Calgary Health Region: importance of metallo-beta-lactamase (MBL)-producing strains. J Infect Dis 2005;192:1606-12.  Back to cited text no. 15    
16.Walsh TR, Toleman MA, Poirel L, Nordmann P . Metallo-beta-lactamases: the quiet before the storm? Clin Microbiol Rev 2005;18:306-25.  Back to cited text no. 16    
17.Livermore DM, Woodford N. The beta-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol 2006;14:413-20.  Back to cited text no. 17    
18.Eisenstein BZ, DF, Enterobacteriaceae. 5 th ed. Philadelphia, Pa: Churchill Livingstone; 2000.  Back to cited text no. 18    
19.Paterson DL, Doi Y. A step closer to extreme drug resistance (XDR) in gram-negative bacilli. Clin Infect Dis 2007;45:1179-81.  Back to cited text no. 19    
20.Tato M, Coque TM, Ruνz-Garbajosa P, Pintado V, Cobo J, Sader HS, et al . Complex clonal and plasmid epidemiology in the first outbreak of Enterobacteriaceae infection involving VIM-1 metallo-beta-lactamase in Spain: toward endemicity? Clin Infect Dis 2007;45:1171-8.  Back to cited text no. 20    
21.Bratu S, Landman D, Haag R, Recco R, Eramo A, Alam M, et al . Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch Intern Med 2005;165:1430-5.  Back to cited text no. 21    
22.Peleg AY, Franklin C, Bell JM, Spelman DW. Dissemination of the metallo- beta-lactamase gene blaIMP-4 among gram-negative pathogens in a clinical setting in Australia. Clin Infect Dis 2005;41:1549-56.  Back to cited text no. 22    
23.Fukigai S, Alba J, Kimura S, Iida T, Nishikura N, Ishii Y, et al . Nosocomial outbreak of genetically related IMP-1 beta-lactamase-producing Klebsiella pneumoniae in a general hospital in Japan. Int J Antimicrob Agents 2007;29:306-10.  Back to cited text no. 23    

Correspondence Address:
Afzal Azim
Department of Critical Care Medicine, SGPGIMS, Lucknow - 226014, UP
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.54988

Rights and Permissions


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]

This article has been cited by
1 Molecular prevalence of resistance determinants, virulence factors and capsular serotypes among colistin resistance carbapenemase producing Klebsiella pneumoniae: a multi-centric retrospective study
Aradhana Das, Rajesh Kumar Sahoo, Mahendra Gaur, Suchanda Dey, Saubhagini Sahoo, Anshuman Sahu, Dibyajyoti Uttameswar Behera, Sangita Dixit, Pooja Singhvi Jain, Bhawana Jain, Kundan Kumar Sahu, K. Swapna Kumari, Enketeswara Subudhi
3 Biotech. 2022; 12(1)
[Pubmed] | [DOI]
2 Emergence of blaNDM-1 and blaVIM producing Gram-negative bacilliin ventilator-associated pneumonia at AMR Surveillance Regional Reference Laboratory in India
Mithlesh Kumari, Sheetal Verma, Vimala Venkatesh, Prashant Gupta, Piyush Tripathi, Avinash Agarwal, Suhail Sarwar Siddiqui, Zia Arshad, Ved Prakash, Abdelazeem Mohamed Algammal
PLOS ONE. 2021; 16(9): e0256308
[Pubmed] | [DOI]
3 Multicenter Evaluation of the Xpert Carba-R Assay for Detection and Identification of Carbapenemase Genes in Sputum Specimens
Zhen Cai, Jia Tao, Tianye Jia, Hongyu Fu, Xin Zhang, Mei Zhao, Hong Du, Hua Yu, Bin Shan, Bin Huang, Liang Chen, Yi-Wei Tang, Wei Jia, Fen Qu, Nathan A. Ledeboer
Journal of Clinical Microbiology. 2020; 58(9)
[Pubmed] | [DOI]
4 Double- and multi-carbapenemase-producers: the excessively armored bacilli of the current decade
G. Meletis,D. Chatzidimitriou,N. Malisiovas
European Journal of Clinical Microbiology & Infectious Diseases. 2015; 34(8): 1487
[Pubmed] | [DOI]
5 Predominance of carbapenem-resistant Pseudomonas aeruginosa isolates carrying blaIMP and blaVIM metallo--lactamases in a major hospital in Costa Rica
F. Toval,A. Guzman-Marte,V. Madriz,T. Somogyi,C. Rodriguez,F. Garcia
Journal of Medical Microbiology. 2015; 64(Pt_1): 37
[Pubmed] | [DOI]
6 Molecular epidemiology of carbapenemresistant Enterobacteriaceae from a North Indian Tertiary Hospital
Shahid, M., Umesh, Sobia, F., Singh, A., Khan, H.M., Malik, A., Shukla, I.
New Zealand Journal of Medical Laboratory Science. 2012; 66(1): 5-7
7 An observational study on bloodstream extended-spectrum beta-lactamase infection in critical care unit: Incidence, risk factors and its impact on outcome
Prashant Nasa, Deven Juneja, Omender Singh, Rohit Dang, Akhilesh Singh
European Journal of Internal Medicine. 2011;
[VIEW] | [DOI]
8 Metallo-β-lactamases: a last frontier for β-lactams?
Giuseppe Cornaglia, Helen Giamarellou, Gian Maria Rossolini
The Lancet Infectious Diseases. 2011; 11(5): 381
[VIEW] | [DOI]
9 Editorial: New Delhi metallo beta-lactamase: What is in a name
Nataraj, G.
Journal of Postgraduate Medicine. 2010; 56(4): 251-252


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  

    Materials and Me...
    Article Figures
    Article Tables

 Article Access Statistics
    PDF Downloaded786    
    Comments [Add]    
    Cited by others 9    

Recommend this journal