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Year : 2014  |  Volume : 57  |  Issue : 1  |  Page : 65-68
Multidrug resistant NDM-1 metallo-beta-lactamase producing Klebsiella pneumoniae sepsis outbreak in a neonatal intensive care unit in a tertiary care center at central India

1 Department of Microbiology, Armed Forces Medical College, Pune, Maharashtra, India
2 Department of Microbiology, AIIMS, Bhubaneswar, Odisha, India

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Date of Web Publication17-Apr-2014


Objective: The objective of the following study is to detect genes encoding carbapenem resistance in Klebsiella pneumoniae sepsis outbreak in a neonatal intensive care unit (NICU). Materials and Methods: Antibiotic sensitivity test was performed by standard Kirby Bauer disc diffusion technique and minimum inhibitory concentrations of antibiotics was determined by VITEK-2. Polymerase chain reaction (PCR) assays and sequencing was used to determine the presence of beta-lactamase encoding genes. Conjugation experiments were performed to determine the transferability of beta-lactamase. Isolate relatedness were determined by repetitive-element PCR (REP), enterobacterial repetitive intergenic consensus (ERIC) PCR and random amplified polymorphic deoxyribonucleic acid (RAPD). Results: All the isolates were completely resistant to the second and third generation cephalosporins tested as well as carbapenems. Susceptibility profiling of the isolates indicated that 100% retained susceptibility to tigecycline and colistin. Conjugation experiments indicated that blaNDM-1 was transferable and likely through a plasmid-mediated event. All the isolates showed the presence of blaNDM-1 with co association of bla CTX-M-15 . REP-PCR, ERIC-PCR and RAPD revealed a single clonal type circulating in NICU environment. Conclusion: Co-production of NDM-1 with CTX-M-15 in K. pneumoniae isolates was detected for the first time in our NICU. Transmission of plasmid carrying these resistant genes to other members of Enterobacteriaceae will increase the incidence of multidrug resistance. Early detection of these genes will help in prevention and adequate infection control by limiting the spread of these organisms.

Keywords: bla NDM-1, Klebsiella pneumonia outbreak, multidrug resistance, neonatal intensive care unit

How to cite this article:
Khajuria A, Praharaj AK, Kumar M, Grover N, Aggarwal A. Multidrug resistant NDM-1 metallo-beta-lactamase producing Klebsiella pneumoniae sepsis outbreak in a neonatal intensive care unit in a tertiary care center at central India. Indian J Pathol Microbiol 2014;57:65-8

How to cite this URL:
Khajuria A, Praharaj AK, Kumar M, Grover N, Aggarwal A. Multidrug resistant NDM-1 metallo-beta-lactamase producing Klebsiella pneumoniae sepsis outbreak in a neonatal intensive care unit in a tertiary care center at central India. Indian J Pathol Microbiol [serial online] 2014 [cited 2022 Aug 16];57:65-8. Available from: https://www.ijpmonline.org/text.asp?2014/57/1/65/130900

   Introduction Top

Klebsiella pneumoniae is one of the most common pathogen of Enterobacteriaceae family responsible for nosocomial infections, especially in intensive care units (ICU) and among neonates. [1] Carbapenems are the beta-lactam antibiotics which bind to the bacterial penicillin-binding proteins responsible for elongation and cross link the peptidoglycan of the bacterial cell wall resulting in impaired cell wall synthesis and cell death. Incidence of multi drug resistance in organisms is increasing due to dissemination of resistance determinant genes mediated by transposons, plasmids and gene cassettes in integrons. However, of late, due to the presence of extended-spectrum beta-lactamase and AmpC enzymes in these Gram-negative bacilli, carbapenems have become the drug of choice to treat such infections. Carbapenems, most commonly meropenem and imipenem (IP), have been considered as most potent beta-lactams against multi drug resistant Gram-negative bacteria. However, the increased use of carbapenems has led to the emergence of resistant strains and outbreaks due to these organisms which have been associated with higher morbidity and mortality. Due to upsurge of carbapenemases in clinical isolates of Enterobacteriaceae, the treatment of ICU patients is becoming difficult. Resistance to carbapenems due to carbapenemase production poses serious challenges in the treatment of such infections with pan-resistant phenotypes. [2] Mobile genetic elements are being associated with carbapenemases production. The genetic environment of blaNDM-1 likely facilitates the rapid dissemination of this gene within K. pneumoniae isolates. Spreading of NDM-1 producing K. pneumoniae in a hospital may be a complex event involving several modes of spread, such as dissemination of several unrelated strains or the propagation of a single clone from patient to patient and from the environment to patients. Therefore, practical and accurate phenotypic and genotypic approaches are urgently needed to differentiate the horizontally acquired mechanisms of reduced susceptibilities to carbapenems among Enterobacteriaceae in clinical laboratory. The present study was conducted to investigate the epidemiology and molecular characterization of multi drug resistant NDM-1 producing K. pneumoniae strains associated with an outbreak of bloodstream infection in a busy neonatal intensive care unit (NICU) in a tertiary care hospital in Pune, India.

   Materials and Methods Top

The bacterial isolates

A total of six non-duplicated clinical isolates of K. pneumoniae were recovered from six different neonates admitted to the NICU of tertiary care hospital in Pune, India from February 2012 to March 2012. A volume of 1 ml of blood was drawn from the neonates suspected to have sepsis and inoculated in BACTEC Peds Plus /F (BD, Ireland) and processed by using the BACTEC 9120 culture system. To find the source of the outbreak during the investigation, four body sites of the neonates were sampled: Umbilicus; groin; mouth; and rectum. In addition, swabs from hands and anterior nares were collected from doctors and nursing staff in the NICU and environmental cultures were performed using a swab pre-moistened with sterile saline to identify other potential reservoirs for NDM-1 producing K. pneumoniae. Cultured sites included bed railing, stethoscope, weighing machine, milk powder, suction apparatus and wash basin, radiant warmer, washing area, electric switch, medicine tray, door handles, knobs, ventilator machine and air conditioner. Bacterial identification was performed by routine conventional microbial culture and biochemical tests using standard recommended techniques. [3] The organisms were identified up to the species level using VITEK-GNI cards (bioMérieux, Marcy l'Etoile, France).

Antimicrobial susceptibility testing

Antibiotic sensitivity test was performed by standard Kirby Bauer disc diffusion technique as per the guidelines of the Clinical Laboratory Standards Institute (CLSI) with commercially available discs (Hi Media, Mumbai, India) on Mueller Hinton agar plates. [4] The antibiotics tested shown in [Table 1].  Escherichia More Details coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 strains were used for quality control. Minimum inhibitory concentrations (MIC) of antibiotics was determined by VITEK-2 as per CLSI break points in isolates showing resistance to carbapenems. [4] Furthermore, MICs were further determined by the E-test method against IP, meropenem, tigecycline and colistin (bioMérieux, Marcy l'Etoile, France) [Table 2].
Table 1: Antibiotics tested against Klebsiella pneumoniae Scientific Name Search  isolates (potency in μg/disc

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Screening for the carbapenemase production

K. pneumoniae isolates with a reduced susceptibility to meropenem and IP (diameter of zones of inhibition ≤13 mm) by disc diffusion method were screened for the production of carbapenemase. The phenotypic detection of the carbapenemase production was performed by the modified Hodge test by using a meropenem disc (10 µg) as per CLSI guidelines. [4] The screening of metallo-beta-lactamase (MBL) production was performed by the double-disc synergy tests (DDST) and combined-disc synergy test (CDST) as described previously. [5] Furthermore MBL (IP/imipenem-inhibitor [IPI]) E-test was carried out to detect MBL as per manufacturer's instructions.

Molecular detection of the MBL genes

Deoxyribonucleic acid (DNA) was extracted using the spin column method (QIAGEN; GmbH, Hilden, Germany) as per manufacturer's instructions. Polymerase chain reaction (PCR)-based detection of beta lactamase genes (bla CTXM, bla SHV and bla TEM ), Ambler class (B and D) MBLs bla IMP , bla VIM , bla SPM , bla GIM , bla SIM, bla NDM-1, bla OXA-23 , bla OXA-24 and bla OXA48 and for serine carbapenemases (bla KPC, bla GES and bla NMC ) were carried out on the isolates by using Gene Amp 9700 PCR System (Applied Biosystems, Singapore). [6] PCR products were run on 1.5% agarose gel, stained with ethidium bromide visualized under ultraviolet (UV) light and photographed. The amplicons were purified using QIAquick PCR purification kit (QIAGEN; GmbH, Hilden, Germany).

DNA sequencing and sequence analysis

Automated sequencing was performed on an ABI 3730XL DNA analyzer using the Big Dye system (Applied Biosystems Foster City, CA, USA). Sequences were compared with known sequences using the BLAST facility (http://blast.ncbi.nlm.nih.gov).

Conjugation experiments

Transfer of resistance genes by conjugation was assayed by mating experiments in Luria-Bertani broth using K. pneumoniae isolates (Parental strains) as donors and an azide-resistant E. coli J53 as the recipient strain using 1:10 ratio. The transconjugants were selected on Luria-Bertani agar with selection based on growth on agar in the presence of ceftazidime (30 mg/L) and sodium azide (100 mg/L). Plasmids were separated by co-electrophoresis on horizontal 0.4% agarose gel at 50 V for 3 h. The size of the plasmids were compared by co-electrophoresis with plasmid of known sizes from E. coli (V517and 39R861). Bands were visualized with UV transilluminator after staining with 0.05% ethidium bromide.

Strain molecular typing

Repetitive-element based PCR (REP-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) assays were performed as described to characterize bla NDM-1 positive K. pneumoniae strains recovered from patients. [7] Randomly amplified polymorphic deoxyribonucleic acid (RAPD) analysis was also carried out to detect relatedness of the strains. [8]

Plasmid analysis

Plasmid from the parental strains and their transconjugants was extracted by using Qiagen plasmid mini kit (GmbH, Hilden, Germany) as per manufacturer's Instructions. Extracted plasmid DNA were subjected to plasmid based replicon incompatibility (Inc) typing by using eighteen pairs of primers to perform five multiplex and three single PCRs which recognized F, FIA, FIB, FIC, HI1, HI2, I1-Ic, L/M, N, P, W, T, A/C, K, B/O, X, Y and FII replicons as described previously. [9] Plasmid replicons were determined for carbapenemase producing clinical isolates.

   Results Top

As determined by disc-diffusion antibiotic susceptibility testing, all six K. pneumoniae clinical isolates from neonatal blood culture and one from the weighing machine exhibited the same pattern of resistance to β-lactam agents demonstrating resistant to third, fourth generation cephalosporins and were found to be carbapenem (IP, meropenem and ertapenem) resistant. They were resistant to penicillin/inhibitor combinations such as amoxicillin/clavulanate, ampicillin/sulbactam and piperacillin/tazobactam. The carbapenem resistant isolates exhibited MIC against meropenem: 64 µg/ml, ertapenem: 8 µg/ml and IP: 64 µg/ml. All the isolates were found to be susceptible to trimethoprim/sulfamethoxazole, tigecycline and colistin as per MIC breakpoints (for trimethoprim/sulfamethoxazole was <20 µg/ml, for tigecycline was 1 µg/ml and for colistin <0.5 µg/ml). Antibiogram of these isolates shown is in [Table 2].
Table 2: MICs of blaNDM-1-positive multidrug resistant Klebsiella pneumoniae isolates including those from weighing machine showed identical MIC in μg/ml as depicted

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Carbapenemase production was confirmed by Modified Hodge test. Production of MBL was confirmed by positive DDST, CDST and MBL (IP/IPI) E-test method.

Molecular detection of the beta-lactamase genes and DNA sequencing

All the six carbapenem resistant isolates of K. pneumoniae from blood culture and isolate from weighing machine were found to be positive for bla NDM-1 and bla CTX-M-15 by PCR.

Conjugation experiments

Conjugation experiments indicated that blaNDM-1 was transferable through a plasmid. Passaging over 10 days in media with and without meropenem 0.5 mg/L showed that all the isolates, carried stable blaNDM-1 plasmids. Plasmid profiling of the isolates showed that blaNDM-1 was carried on 50 kb size plasmid and bla CTXM-15 was carried on 140 kb size plasmids.

Strain molecular typing

REP-PCR, ERIC-PCR and RAPD assays showed closed relationship and confirmed the presence of a single clone as per banding pattern [Figure 1] and [Figure 2].
Figure 1: Enterobacterial repetitive intergenic consensus electrophoretic banding pattern on 1.5% agarose gel

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Figure 2: Repetitive-element based polymerase chain reaction electrophoretic banding pattern on 1.5% agarose gel

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Plasmid replicon typing

Plasmids purified from the clinical isolates were typed by PCR based replicon typing. The blaNDM-1 gene in K. pneumoniae was located on IncA/C plasmid. Bla CTX-M-15 was associated with multiple replicons of plasmid (IncFIA, IncFIB).

   Discussion Top

This study describes an outbreak due to multi drug resistant metallo-beta-lactamase producing K. pneumoniae in an NICU in the month of February and March 2012 and subsided at the end of March. Antibiotic-resistant Gram-negative organisms are a significant risk to severely ill neonates in NICUs and in many instances; these are imported into the unit or rapidly acquired from environmental reservoirs. Nosocomial infections in NICUs are the most difficult and tedious to manage and control. The outcome of neonatal infections can be improved if the illness is recognized early and appropriate agents are promptly administered. It is a common practice to use beta-lactams and aminoglycosides in neonatal septicemia. Usually prescribed antimicrobials fail to inhibit these pathogens and the most effective antimicrobial against carbapenemase producing K. pneumoniae is colistin. Early intimation of culture and sensitivity results to the treating neonatologist resulted in saving four neonates while two neonates lost their life. With the world-wide increase in incidence of MBL, producing organisms early detection is important as the co presence of genes in multidrug resistant organisms makes them refractory to the common antibiotics used in clinical practice. Hospital colonization by metallo-beta-lactamase producing bacteria is usually a complex phenomenon involving different mechanisms including dissemination of resistance determinant genes from one species to another. Investigation of the outbreak revealed that the index case was a neonate who was transferred in from a peripheral hospital to our center. From this child, the organism contaminated the weighing machine resulting in the spread of infection. The outbreak was successfully controlled by isolating the neonates having bla NDM-1 , fumigation of the existing NICU; shifting of neonates to new prefumigated NICU, taking strict asepsis measures of disinfecting weighing machines after every use.

   Conclusion Top

Neonatal septicemia is a serious condition and most of them are premature. Multidrug resistant bacteria in NICU set up have become a major cause of neonatal septicemia. These organisms can be transferred to the neonates from inanimate objects through health care workers and through medical equipment's such as ventilators. Early detection of these organisms and resistant genes by molecular methods and with strict preventive measures this type of catastrophe can be prevented.

   References Top

1.Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998;11:589-603.  Back to cited text no. 1
2.Livermore DM, Woodford N. The beta-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol 2006;14:413-20.  Back to cited text no. 2
3.Collee JG, Miles RS, Wan B. Tests for the identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14 th ed. Edinburgh: Churchill Livingstone; 1996. p. 131-50.  Back to cited text no. 3
4.Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty Second Informational Supplement M100-S22. Wayne, PA, USA: CLSI; 2012.  Back to cited text no. 4
5.Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, et al. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009;53:5046-54.  Back to cited text no. 5
6.Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: A molecular, biological, and epidemiological study. Lancet Infect Dis 2010;10:597-602.  Back to cited text no. 6
7.Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 1991;19:6823-31.  Back to cited text no. 7
8.Vogel L, Jones G, Triep S, Koek A, Dijkshoorn L. RAPD typing of Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens and Pseudomonas aeruginosa isolates using standardized reagents. Clin Microbiol Infect 1999;5:270-6.  Back to cited text no. 8
9.Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005;63:219-28.  Back to cited text no. 9

Correspondence Address:
Atul Khajuria
Departments of Microbiology, AFMC, Pune - 40
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.130900

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