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| Year : 2009 | Volume
: 52
| Issue : 3 | Page : 339-342 |
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| Ventilator-associated pneumonia caused by carbapenem-resistant Enterobacteriaceae carrying multiple metallo-beta-lactamase genes |
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Mayank Dwivedi1, Anshuman Mishra2, Afzal Azim3, RK Singh3, AK Baronia3, KN Prasad1, TN Dhole1, UN Dwivedi2
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 Publication | 12-Aug-2009 |
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 Abstract | | |
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: https://www.ijpmonline.org/text.asp?2009/52/3/339/54988 |
| Introduction | |  |
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 | | |
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 38.5°C or less than 35°C 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 µg IPM disc was placed at the center of an inoculated Muller-Hinton agar plate. Ten microliters of 50 mM zinc sulfate solution (140 µg) was added to the disc. The plate was read after overnight incubation at 37°C. Presence of a cloverleaf-shaped zone of inhibition was interpreted as positive modified-Hodge test. In the combined disk test, two IPM disks (10 µg), one containing 10 µl of 0.1 M (292 µg) 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 µl of PCR-grade water and adding 2 µl to the PCR reaction mixture before thermal cycling. The cycling conditions were initial DNA release and denaturation at 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 52°C for 40 s and 72°C for 50 s, followed by a single, final elongation step at 72°C for 5 min.
| Results | | |
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 | | |
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 | | |
Mayank Dwivedi acknowledges the financial assistance from the Council of Scientific and Industrial Research, New Delhi, India.
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Correspondence Address:
Afzal Azim
Department of Critical Care Medicine, SGPGIMS, Lucknow - 226014, UP
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0377-4929.54988
[Figure 1], [Figure 2]
[Table 1], [Table 2] |
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