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 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 3  |  Page : 361-368
 

Discriminatory power of three typing techniques in determining relatedness of nosocomial Klebsiella pneumoniae isolates from a tertiary hospital in India


1 Department of Hospital Infection Control, Narayana Health City, Bengaluru, Karnataka, India
2 Department of Microbiology, Narayana Health City, Bengaluru, Karnataka, India
3 Mazumdar Shaw Centre for Translational Research, Narayana Health City, Bengaluru, Karnataka, India

Date of Web Publication12-Oct-2017

Correspondence Address:
Vasan K Sambandamurthy
Mazumdar Shaw Centre for Translational Research, Narayana Health City, 258/A, Anekal Taluk, Hosur Road, Bommasandra, Bengaluru - 560 099, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_16_308

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 ~ Abstract 

Purpose: The purpose of this study was to evaluate the discriminatory power of two DNA-based technique and a protein-based technique for the typing of nosocomial isolates of Klebsiella pneumoniae. A second objective was to determine the antimicrobial susceptibility pattern and characterise the presence of genes encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. Materials and Methods: Forty-six K. pneumoniae isolates from patients with bloodstream infections at a tertiary care hospital in India between December 2014 and December 2015 were studied. All isolates were typed using enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) analysis and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility profiles and ESBLs were detected using the BD Phoenix system. The types of ESBL and carbapenemase genes present were detected using PCR. Results: Isolates were subtyped into 31, 30 and 33 distinct genotypes by ERIC-PCR, RAPD and MALDI-TOF, respectively. Several isolates displaying identical DNA fingerprints were binned into different branches based on their proteomic fingerprint. Antimicrobial susceptibility tests revealed that 33/46 strains were multidrug resistant (MDR); a majority of the strains (83%) were sensitive to colistin. PCR-based analysis demonstrated 19 strains to harbour two or more ESBL and carbapenemase genes. Conclusion: ERIC-PCR was the most reproducible method for typing K. pneumoniae isolates and could not be substituted by MALDI-TOF for clonality analysis. A high degree of genetic diversity and the presence of MDR genes highlight the challenges in treating K. pneumoniae-associated infections.


Keywords: Carbapenemases, enterobacterial repetitive intergenic consensus-polymerase chain reaction, extended-spectrum beta-lactamases, matrix-assisted laser desorption ionisation - time-of-flight, randomly amplified polymorphic DNA analysis


How to cite this article:
Purighalla S, Esakimuthu S, Reddy M, Varghese GK, Richard VS, Sambandamurthy VK. Discriminatory power of three typing techniques in determining relatedness of nosocomial Klebsiella pneumoniae isolates from a tertiary hospital in India. Indian J Med Microbiol 2017;35:361-8

How to cite this URL:
Purighalla S, Esakimuthu S, Reddy M, Varghese GK, Richard VS, Sambandamurthy VK. Discriminatory power of three typing techniques in determining relatedness of nosocomial Klebsiella pneumoniae isolates from a tertiary hospital in India. Indian J Med Microbiol [serial online] 2017 [cited 2018 Jul 18];35:361-8. Available from: http://www.ijmm.org/text.asp?2017/35/3/361/216618



 ~ Introduction Top


Klebsiella pneumoniae is an important nosocomial pathogen, frequently implicated in pneumonia, urinary tract, wound and bloodstream infections. Nosocomial isolates of K. pneumoniae are often associated with the ability to produce extended-spectrum beta-lactamases (ESBLs), carbapenemases such as K. pneumonia e carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM), thereby making treatment extremely challenging.[1] Moreover, these isolates are often multidrug resistant (MDR), making it difficult to distinguish between strains based on their antibiotic susceptibility patterns alone.

Genetic methods to establish relatedness of isolates at a molecular level have transformed the ability to investigate nosocomial infection outbreaks. Typing methods that provide differentiation within a genetic lineage and epidemiologically unrelated isolates are critical tools to document a transmission event.[2] The DNA-based typing method pulsed-field gel electrophoresis (PFGE) is considered as a gold standard for molecular typing of isolates. However, this method is very laborious, technically demanding and is prone to subjectivity for reliable comparison of data across laboratories.[2] To overcome these limitations, a number of molecular techniques that are rapid and cheap have been developed for bacterial strain typing.[3] Two polymerase chain reaction (PCR)-based typing techniques, randomly amplified polymorphic DNA (RAPD) analysis and enterobacterial repetitive intergenic consensus sequence -PCR (ERIC-PCR) have shown great promise in typing diverse microorganisms including K. pneumoniae that are implicated in nosocomial outbreaks.[4],[5]

Recently, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry has been proposed as a powerful tool for the rapid identification and clonality analysis of a broad range of bacterial pathogens.[6] A major disadvantage of MALDI-TOF is the lack of standard protocols for sample preparation and analysis which is likely to impact the dendrogram grouping.[7] Despite the availability of many techniques, the decision on the choice of method depends on cost, speed, reliability and assay throughput.

Nosocomial outbreaks caused by MDR strains of K. pneumoniae are responsible for a high number of therapeutic failures leading to numerous deaths.[8],[9] During an outbreak, it is important to establish the molecular fingerprint of the causative agents in real-time to identify and document a transmission event. Given that a majority of DNA fingerprinting methods are PCR-based, results can be obtained rapidly and compared between laboratories. In this study, we have also evaluated the potential of MALDI-TOF to establish strain relatedness by comparing the proteomic fingerprints with the genetic fingerprints derived from the DNA-based methods.

The objectives of this study were to determine the antimicrobial resistance profiles of K. pneumoniae isolates from a tertiary care hospital in India and to study their genetic diversity using three fingerprinting methods. The presence of several ESBL-encoding genes and carbapenemases was determined by PCR. Finally, we present the discriminating power of ERIC-PCR, RAPD and MALDI-TOF analysis for studying the genetic relatedness of K. pneumoniae isolates.


 ~ Materials and Methods Top


Bacterial isolates

Forty-six nonreplicate K. pneumoniae isolates from blood were collected from patients between December 2014 and December 2015. Isolates were identified and characterised using the MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) and liquid cultures of each isolate were stored at −70°C.

Antimicrobial susceptibility testing and screening for extended-spectrum beta-lactamases

In vitro susceptibility testing and ESBL screening were performed on all isolates and interpreted using the Phoenix™ (BD Diagnostic Systems) for the following antibiotics: ceftazidime, amikacin, tobramycin, ciprofloxacin, gentamicin, ticarcillin-clavulanic acid, tazobactam-piperacillin, colistin, cefepime, levofloxacin, meropenem, ceftriaxone, ampicillin, aztreonam, ertapenem, imipenem, cefoxitin, trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid and tigecycline.[10] Minimum inhibitory concentration values obtained by the above methods were categorised according to the National Committee for Clinical Laboratory Standards breakpoints as susceptible (S), intermediate (I) or resistant (R).  Escherichia More Details coli ATCC 39922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains for the antimicrobial susceptibility test.[11]

Molecular typing

Isolates were identified as K. pneumoniae based on their MALDI-TOF profile. Molecular methods were used for typing and establishing genetic relatedness among the isolates.

DNA extraction

A single bacterial colony from a pure culture was resuspended in 100 μl of sterile distilled water in a 0.5 ml microcentrifuge tube. The cultures were heated for 15 min at 95°C, cooled on ice, centrifuged at 10,000 g for 20 s to remove the cell debris. The crude cell lysates were frozen at −20°C until further evaluation. 1 μl of the boiled extract was used in a 25 μl PCR mixture to amplify the target genes using specific primers as described below.

Enterobacterial repetitive intergenic consensus-polymerase chain reaction

The PCR amplification was performed by adding a mixture (25 μl per reaction) of 18 μl of sterile distilled water, 2.5 μl of 10X PCR buffer, 1 μl of 10 mM dNTPs, 1 μl of each of primers, ERIC 1R: 5'-ATG TAA GCT CCT GGG GAT TCA-3' and ERIC 2: 5'-AAG TAA GTG ACT GGG GTG AGC G-3', 0.5 μl of Taq polymerase and 1 μl of the template DNA to the reaction.[12] The amplifications were done as follows – initial denaturation for 3 min at 94°C and 30 cycles of 1 min at 94°C, 3 min at 55°C and 2 min at 72°C followed by a final step of amplification of 10 min at 72°C.

Randomly amplified polymorphic DNA analysis

RAPD analysis was performed using the primer 793.[13] The amplification reaction was carried out in a 25 μl volume containing 18.5 μl of sterile distilled water, 2.5 μl of 10X PCR buffer, 1 μl of 10 mM dNTPs, 1 μl of primer 793 (5'-GACCGACCCA-3'). The cycling conditions were 40 cycles of 1 min at 94°C, 1 min at 36°C and 2 min at 72°C followed by a final amplification of 7 min at 72°C.

Amplified products were separated by electrophoresis on 1.5% agarose gel and photographed under an ultraviolet transilluminator. A 100 kb DNA ladder was used as the molecular size marker.

Molecular pattern analysis

Isolates were categorised according to their PCR banding pattern as identical, similar or unrelated. A similarity index was calculated using the Dice coefficient and cluster analysis of the matrices was generated using the unweighted pair group method using arithmetic averages. Dendrograms of isolates were generated with the tree option using the PAST program.[14] The discriminatory (D) index was calculated according to the method of Hunter and Gaston (1988).[15] The index of discrimination represents the probability that two randomly chosen isolates, sampled consecutively, would be distinguished by the test and range from D = 0 to D = 1.

Detection of genes conferring resistance to carbapenems and β-lactams

Crude DNA extracts were used to detect the presence of the following genes – blaNDM, blaKPC, blaOXA, blaCTX-M, blaSHV and blaTEM. PCR assays were carried out using specific primers as described earlier.[16],[17],[18]K. pneumoniae ATCC 700603 and K. pneumoniae ATCC 1705 were used as a standard positive strains for blaSHV and blaKPC, respectively. For the other genes, in-house strains known to harbour blaTEM, blaCTX and blaNDM(detected using whole genome sequencing) were used as positive controls for PCR analysis. A non-ESBL-producing organism (E. coli ATCC 39922) was used as negative control.

Mass spectrometry

MALDI-TOF analysis was performed on a Bruker microflex LT benchtop instrument controlled by the FlexControl software (version 2.0; Bruker Daltonics GmbH, Leipzig, Germany). Spectra were acquired as per the manufacturer's recommendation by utilising the BioTyper preprocessing and the BioTyper MSP identification standard method.


 ~ Results Top


Antimicrobial susceptibility

Antimicrobial susceptibility profiles of all 46 K. pneumoniae revealed a low degree of diversity in terms of activity to various classes of antimicrobials tested [Figure 1]. Among 83% of the K. pneumoniae isolates (38/46), colistin was the most active antimicrobial agent. All isolates except one (45/46) were resistant to ceftazidime, ceftriaxone and ampicillin. A remarkably large group of isolates were resistant to amikacin (80%), tobramycin, ciprofloxacin and amoxicillin–clavulanic acid (93%), gentamicin (87%), ticarcillin-clavulanic acid (96%), piperacillin + tazobactam (85%), cefepime (93%), levofloxacin (76%), meropenem (83%), aztreonam (96%), ertapenem, imipenem, cefoxitin and trimethoprim + sulfamethoxazole (85%) [Figure 1].
Figure 1: Antimicrobial susceptibility profiles of all 46 Klebsiella pneumoniae isolates interpreted using the Phoenix™ (BD Diagnostic Systems). The minimum inhibitory concentration values obtained were categorised according to the National Committee for Clinical Laboratory Standards breakpoints as susceptible (S), intermediate (I) or resistant (R).

Click here to view


Of the total 46 K. pneumoniae isolates, 39 (85%) were found to be ESBL producers and 7 (15%) were non-ESBL producers as determined by the BD Phoenix system.[10] All of the 39 ESBL producers were resistant (100%) to ceftazidime, tobramycin, ciprofloxacin, cefepime, ceftriaxone, aztreonam, ampicillin, amoxicillin–clavulanic acid. As expected, resistance to third-generation cephalosporin and non-β-lactam antibiotics was higher in EBSL-producing strains as compared to the non-ESBL-producing strains. Based on the resistance profile to three or more antibiotic classes, 33 strains were identified as MDR in this study.

Polymerase chain reaction-based detection of extended-spectrum beta-lactamases and carbapenemase-encoding genes

Using specific primers, all 39 K. pneumoniae isolates scored as ESBL producers were evaluated using a PCR-based method to determine the type of ESBL gene present. The following genes known to encode ESBLs were evaluated- blaTEM, blaSHV, blaCTX-M and blaOXA. Agarose gel electrophoresis revealed a distinct electrophoretic pattern for blaCTX-M, blaTEM, blaOXA and blaSHV PCR products. Among the 39 isolates evaluated, blaSHV gene was present in 4 isolates, the blaTEM gene in 3 isolates and the blaCTX-M gene in 13 isolates [Table 1]. No isolates harboured the blaOXA gene alone; however, four strains harboured this gene along with CTX-M and SHV (B990, B4605, B11425 and B14412) gene. Out of the isolates positive for CTX-M, six tested positive for SHV and TEM. Overall, six isolates (B290, B309, B1169, B1840, B1841 and B14335) were found to harbour all 3 ESBL-encoding genes.
Table 1: Molecular genotyping and determination of antimicrobial resistance determinants in Klebsiella pneumoniae isolates

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For the 33 MDR isolates, the presence of carbapenemase genes - blaKPC and blaNDM in addition to ESBLs were examined using a PCR-based method. Majority of strains showed the presence of blaKPC alone (15/33). This was followed by blaNDM(11/33) and four strains carrying both the genes. In three of the MDR isolates (B872, B3590 and B11425), no PCR product encoding either carbapenemase (blaKPC or blaNDM) was obtained. This outcome is likely due to the absence of these two genes [Table 1] or possibly to the presence of a variant carbapenemase gene that was not tested in this study.[9]

Molecular fingerprinting by enterobacterial repetitive intergenic consensus-polymerase chain reaction and randomly amplified polymorphic DNA

All the 46 K. pneumoniae were characterised by RAPD and ERIC-PCR to determine their molecular fingerprint and phylogenetic relationship. Using ERIC-PCR, we obtained a banding pattern ranging from 0.2 to 3 kb. The ERIC-PCR categorised the 46 strains into 31 unique subtypes (level of similarity ≥75%) [Figure 2]a. Using primer 793 for RAPD analysis as a second molecular method, we detected a banding pattern ranging from 0.2 to 3 kb resulting in thirty unique subtypes (level of similarity ≥75%) [Figure 2]b. Unlike the RAPD method, which displayed a low degree of variability between repeat experiments, the ERIC-PCR yielded robust and reproducible results in multiple repeat experiments. These data establish the overall reproducibility and discriminatory power of ERIC-PCR (D = 0.976) compared to the RAPD (D = 0.959) [Figure 2]b.
Figure 2: Cluster analysis based on molecular typing of 46 Klebsiella pneumoniae isolates using (a) enterobacterial repetitive intergenic consensus-polymerase chain reaction and (b) randomly amplified polymorphic DNA analysis typing using primer 793(B). Isolate numbers are shown on the right. Dendrograms were obtained by unweighted pair group method using arithmetic averages group analysis using similarity coefficient of dice.

Click here to view


Interestingly, three isolates of K. pneumoniae (B290, B990, B160) were categorised as a unique subtype (based on DNA banding pattern) using both techniques. To further investigate the observed genetic relatedness, the epidemiological data related to these patients were gathered. These three patients were in the same postoperative Intensive Care Unit (ICU). The potential source of this transmission could not be ascertained as we were unable to culture the causative agent from the ICU and operating room environment.

Matrix-assisted laser desorption ionisation time-of-flight analysis

All 46 K. pneumoniae isolates included in this study were accurately identified to the species level using the MALDI-TOF hierarchical cluster analysis. The resulting dendrograms revealed the presence of 33 unique fingerprints at a level of similarity ≥75% (D = 0.985) [Figure 3]. Interestingly, one of the three suspected outbreak isolates (B990) shown to be identical by both PCR-based typing methods was demonstrated to be unrelated to the other two isolates (B160, B990) by the MALDI-TOF analysis. The discriminatory power of MALDI-TOF was higher (D = 0.985) compared to the DNA fingerprinting techniques. However, among the isolates that were classified as very closely related based on their MALDI-TOF dendrogram, the ERIC-PCR banding patterns showed lack of genetic relatedness, thereby highlighting the difference in clustering the isolates based on genomic versus proteomic signatures. Overall, we found a great degree of heterogeneity among the 46 isolates based on their antibiogram profile and ESBL production. Our study highlights a lack of clear association between the antibiogram profiles, ESBL phenotypes and fingerprints determined by three independent typing methods.
Figure 3: Dendrogram of all 46 Klebsiella pneumoniae isolates generated by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry using the direct colony method. The dendrogram was generated using a hierarchical clustering algorithm with a Euclidean distance analysis.

Click here to view



 ~ Discussion Top


The widespread use of broad-spectrum antibiotics coupled with the ease of transmissibility of resistance determinants among bacteria mediated by plasmids, transposons and integrons have worsened the problem of antimicrobial resistance.[1] In addition, the emergence and spread of MDR strains of K. pneumoniae have resulted in numerous treatment failures. The prevalence of ESBL-producing K. pneumoniae varies worldwide, with a reported prevalence of 12.9%–26.8% in European countries, 7.5% in North America, 22.4% in Asia-Pacific Rim, 44% in Latin America, 48.5% in Turkey, 51% in China, 71.4% in Mexico and 72% in India.[19],[20],[21] Given the high prevalence of MDR K. pneumoniae, it is essential to understand and establish the clonal relatedness among isolates to prevent and control K. pneumoniae outbreaks in a health-care setting.

Bacterial typing using molecular methods has greatly contributed to an increase in effectiveness and impact of surveillance systems during a nosocomial outbreak. DNA-based typing methods such as plasmid analysis, PFGE and PCR fingerprinting have been employed to study the molecular epidemiology of nosocomial pathogens.[3] In recent times, the MALDI-TOF has found widespread use for the rapid identification of bacteria in clinical laboratories.[6],[22],[23] In this study, we chose to apply ERIC-PCR and RAPD to establish genetic relatedness among K. pneumoniae isolates and to compare the molecular profiles to a MALDI-TOF-based analysis. To the best of our knowledge, there are no published reports comparing the discriminatory power of MALDI-TOF, ERIC-PCR and RAPD methods for typing K. pneumoniae isolates in India. In addition, we sought to utilise these methods to gain insights into the differences between ESBL positive and ESBL negative K. pneumoniae isolates.

Molecular typing methods such as RAPD and ERIC-PCR have been mostly used as epidemiological tools to discriminate between K. pneumoniae isolates and have shown a great degree of heterogeneity among these organisms.[24],[25],[26] As reported by MacCannell,[3] we found low-molecular weight bands in RAPD to be irreproducible and faint, thereby, introducing a high degree of subjectivity. On the contrary, the ERIC-PCR produced banding patterns that were reproducible in multiple repeat studies (data not shown). Consistent with earlier published studies, we found a high degree of heterogeneity among the 46 isolates regardless of their potential to produce ESBLs. We also demonstrated a lack of correlation between the antibiogram profiles and the molecular fingerprints.

The MALDI-TOF spectra from bacteria are mainly composed of ribosomal and surface-expressed proteins, thereby allowing a strain-specific fingerprint to be generated. A recent study demonstrated a lack of direct correlation between the MALDI-TOF, PFGE or RAPD method.[27],[28] In our study, the MALDI-TOF method yielded 33 distinct genotypes. However, the clustering pattern was different compared to that seen with the two PCR-based techniques. Isolates with different DNA fingerprints were grouped into the same cluster based on their MALDI-TOF spectra in our study. Only two of the three isolates (B160, B290) from the suspected outbreak were categorised to the same cluster based on their MALDI-TOF spectra.

The challenge with comparing proteomic and genomic-based methods is that the presence of a genotype does not necessarily correlate with the expressed phenotype in a bacterial strain. However, a good correlation between MALDI-TOF and genetic typing techniques has been observed for few bacterial species but not for others.[29] Since current epidemiological tools for strain classification are mainly based on DNA fingerprints, further investigation is warranted to establish the widespread applicability of MALDI-TOF method for clonality studies across a wide variety of bacteria implicated in nosocomial infections.


 ~ Conclusion Top


Overall, we were unable to see ESBL production-based clustering using any of the three techniques. As in the present study, Aladag et al.[30] found no association between the antibiogram profiles with specific RAPD fingerprints in ESBL-producing K. pneumoniae isolates. Lim et al.[31] showed a high degree of heterogeneity among 51 Malaysian K. pneumoniae isolates and found no correlation between DNA fingerprints and their antibiogram profiles. Similar to our findings in this study, blaCTX-M in K. pneumoniae isolates in India has been reported to be the most prevalent ESBL gene worldwide, replacing SHV and TEM β-lactamases in India.[32],[33],[34] The rate of infection caused by MDR K. pneumoniae strains is relatively high in the Indian subcontinent.[35],[36] Similarly, the emergence of NDM-1 and KPC-producing K. pneumoniae has been reported in many countries, such as India, Israel, Egypt, Germany, Spain, Switzerland, the United Arab Emirates and China.[37],[38]

Given that ESBL-encoding genes are mostly located on transmissible plasmids, the influence of integrons on DNA fingerprints, and therefore, heterogeneity needs to be established. The association between mobile element carriage and antibiotic resistance has already been established.[39],[40]

Our results highlight the need to build a robust data set to reliably apply and establish MALDI-TOF to investigate nosocomial outbreaks. The present study demonstrates that the PCR-based fingerprinting techniques (ERIC-PCR and RAPD) are rapid, reproducible and have the discriminatory power for effective epidemiological surveillance of K. pneumoniae involved in nosocomial infections. The ERIC-PCR was shown to be the most reliable of the methods studied for typing K. pneumoniae isolates.

Acknowledgements

We would like to thank Dr. Devi Shetty for his valuable guidance and support throughout the course of this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04