|Year : 2015 | Volume
| Issue : 3 | Page : 323-327
|Molecular characterization and clinical significance of New Delhi metallo-beta-lactamases-1 producing Escherichia coli recovered from a South Indian tertiary care hospital
Arindam Ckakraborty1, Prabha Adhikari2, Shalini Shenoy3, Shrikala Baliga3, Gopalkrishna Bhat3, Satish Rao2, Dhanashree Biranthabail3, Vishwas Saralaya3
1 Department of Microbiology, Moti Lal Nehru Medical College, Allahabad, Uttar Pradesh, India
2 Department of Medicine, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
3 Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
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|Date of Web Publication||14-Aug-2015|
| Abstract|| |
Context: The increased rate of infection by New Delhi metallo-beta-lactamases-1 (NDM1) producing Escherichia coli is a major concern since they show a high rate of drug resistance and are responsible for mortality and morbidity. Aims: To characterize the NDM1 producing E. coli isolates and their impact on patients' clinical outcome. Settings and Design: This descriptive study was carried out in a multi-specialty tertiary care hospital. Materials and Methods: Three hundred nonrepeat strains of E. coli from inpatients were included in the study. Modified Hodge test and metallo-beta-lactamases (MBL) e-test were performed to detect carbapenemase and MBL activity. Polymerase chain reaction (PCR) technique was performed to detect NDM1. NDM1 positive isolates were further tested for plasmid mediated AmpC, blaCTX , blaSHV , blaTEM genes and also for phylogrouping by PCR methods. Treatment and patients' clinical outcome were also analyzed. Results: Out of 300 isolates, 21 (7%) were MBL producers by phenotypic methods. Of this, 17 (81%) were NDM1 positives, among the NDM1 producers 6 (35%) isolates were belongs to phylogroups D followed by A 5 (29%), B1 4 (24%) and B2 2 (12%), 15 (88%) isolates were blaCTX-M positive suggestive of extended-spectrum beta lactamase producing strain and 7 (47%) were positive with CIT type of AmpC. With the follow-up of the patients, it was found that 12 (71%) recovered and 3 (18%) developed relapses, and mortality was seen in 2 (12%) patients. Conclusions: NDM1 producing isolates showed a high degree of drug resistance but can be treated with suitable antimicrobials, in the majority. Early detection and choice of appropriate antibiotics may help in reducing mortality and morbidity.
Keywords: Drug resistance, Escherichia coli, New Delhi metallo-beta-lactamases-1, phylogroups
|How to cite this article:|
Ckakraborty A, Adhikari P, Shenoy S, Baliga S, Bhat G, Rao S, Biranthabail D, Saralaya V. Molecular characterization and clinical significance of New Delhi metallo-beta-lactamases-1 producing Escherichia coli recovered from a South Indian tertiary care hospital. Indian J Pathol Microbiol 2015;58:323-7
|How to cite this URL:|
Ckakraborty A, Adhikari P, Shenoy S, Baliga S, Bhat G, Rao S, Biranthabail D, Saralaya V. Molecular characterization and clinical significance of New Delhi metallo-beta-lactamases-1 producing Escherichia coli recovered from a South Indian tertiary care hospital. Indian J Pathol Microbiol [serial online] 2015 [cited 2019 Aug 25];58:323-7. Available from: http://www.ijpmonline.org/text.asp?2015/58/3/323/162864
| Introduction|| |
Escherichia More Details coli, one of the most common causative agents of urinary tract infection (UTI) is also responsible for bacteremia, pneumonia, soft-tissue infection and neonatal meningitis.  High degree of antibiotic resistance exhibited by such E. coli strains is a major concern, as they have been developing resistance to even the carbapenems, which are considered to be the last-line of antibiotics for certain E. coli infections. Carbapenem resistance is mainly due to the production of carbapenemase enzymes. Carbapenemase is the most versatile family of beta-lactamases that are increasingly being reported worldwide.  The commonly known carbapenemases in Enterobacteriaceae are IMP, VIM, KPC and OXA-48. In addition, a novel class of New Delhi metallo-beta-lactamases-1 (NDM-1) has been reported worldwide since 2008, originating mostly from the Indian subcontinent.  However, there is still controversy regarding the source and the impact on patient's treatment of infections caused by blaNDM1 producing isolates. The major problem with blaNDM-1 producers is that they show resistance to several classes of antibiotics including beta-lactams, aminoglycosides and fluoroquinolones, which leaves few therapeutic options for the physician. The aim of the present study was to characterize the blaNDM-1 producing E. coli isolates and to know the impact in patient's clinical outcome.
| Materials and Methods|| |
Participants and clinical isolates
The study was conducted during the period August 2010 to January 2012, from hospitalized patients of two tertiary care hospitals from Mangalore, India, after obtaining permission from the Institutional Ethical Committee (IEC/KMC/134/2010). Three hundred nonrepeat isolates of E. coli were included in the study. Details of antibiotics used and clinical outcome of patients were collected. The isolates were identified using automated biochemical system Vitek 2 (bioMerieux, France).
Antimicrobial susceptibility testing
Antibiotic susceptibility testing was done by the modified Kirby-Bauer disk diffusion method in accordance with Clinical Laboratory Standards Institute guidelines.  The antibiotic disks (HiMedia, Pvt. Ltd., Mumbai, Maharashtra, India) used were ampicillin (10 μg), piperacillin (10 μg), piperacillin + tazobactam (100/10 μg), ceftriaxone (30 μg), cefotaxime (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), amikacin (30 μg), gentamicin (10 μg), co-trimoxazole (1.25/23.75 μg), nitrofurantoin (300 μg), tigecycline (15 μg), cefoperazone + sulbactam (75/30 μg), imepenem (IPM;10 μg), meropenem (MRP;10 μg) and ertapenem (ETP;10 μg).
Detection of carbapenemase production
Plates of Mueller-Hinton agar were inoculated with suspensions of test strains adjusted to the turbidity equivalent to 0.5 McFarland standards. A set of discs (HiMedia, Pvt. Ltd., Mumbai, Maharashtra, India) of IPM, MRP and ETP (10 μg each) were applied to the surface of the agar, plates were incubated overnight (18 h) at 35°C aerobically, and diameters of zone of inhibition (≥23 mm indicated sensitivity, 20-22 mm indicated intermediate resistance and ≤19 mm indicated resistance) were recorded. Carbapenemase production was further confirmed by modified Hodge test (MHT). 
Detection of metallo-beta-lactamase producers
Identification of metallo-beta-lactamases (MBL) activity was performed by two methods: A carbapenem - ethylenediaminetetraacetic acid (EDTA) combined disk method and MBL E-test (HiMedia, Pvt. Ltd., Mumbai, Maharashtra, India).  A known MBL producing isolate was used as a positive control for all tests.
Isolates that were MBL producers were further tested for extended-spectrum beta lactamase (ESBL) and AmpC activities by phenotypic methods as described elsewhere. ,
Phylogenetic analysis was performed by triplex polymerase chain reaction (PCR) based methods as described by Clermont et al.  Briefly, a combination of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2), allows the determination of the main phylogenetic groups of E. coli (these being A, B1, B2 and D).
Genotypic detection of extended-spectrum beta lactamase encoding genes
A multiplex PCR assay was performed to detect and differentiate blaTEM , blaSHV and blaCTX-M genes. The primers were chosen from earlier published studies. , Another PCR assay was performed to detect blaCTX-M15 variant of blaCTX-M as described earlier. 
Genotypic detection of plasmid mediated ampc beta-lactamases
All the isolates were tested by a multiplex PCR assay, which identified six family-specific AmpC genes carried on plasmids such as MOX, FOX, EBC, ACC, DHA and CIT, using primers and conditions as described previously. 
Genotypic detection of bla ndm-1 gene
A PCR assay was performed to detect blaNDM-1 gene as per primer and conditions described earlier with minor modification.  Amplification was performed on an Eppendorf thermocycler. Briefly, the program for amplification included a step of initial denaturation at 94°C for 3 min followed by 36 cycles of 94°C for 30 s, 52°C for 40 s and 72°C for 50 s and a final extension step at 72°C for 5 min. The PCR products were loaded in 2% w/v agarose gel prepared in Tris-borate-EDTA buffer at 120V for 1 h and detected by ethidium bromide staining after electrophoresis.
Chi-square test was used to find an association between bla NDM1 , ESBL, AmpC, carbapenemase producers and patient's clinical outcome. Analysis was performed using statistical package SPSS version 17.0 USA.
| Results|| |
Of the total 300 isolates, 29 (10%) isolates were carbapenemase producer by disk diffusion methods as well as positive with MHT. Out of these, 21 (7%) isolates gave a positive result in the E-test MBL assays with IMP-EDTA strips, as well as the carbapenem - EDTA combined disk methods. Of these 21 MBL producers, 17 (81%) isolates were blaNDM1 positive by PCR [Figure 1]. Among the blaNDM-1 positive isolates 13 (76%) were from UTIs and 4 (24%) were from blood-stream infections. Prevalence of blaNDM-1 was seen in all age groups (0-79 years, mean age 49 years, (standard deviation ± 18) of patients with infections occurring both in neonates and older people. Among the 17 blaNDM-1 positive patients, the most common predisposing factor was diabetes (8/17 [47%]) and only two patients had carcinoma. Phylogenetic analysis [Figure 2] of the blaNDM-1 producers showed that 5 (29%) belonged to Group A, 4 (24%) belonged to Group B1, two (12%) were from Group B2 and 6 (35%) were from Group D (commensal strains 9 [53%] belonging to phylogroups A and B1; and 8 [47%] virulent strains, belonging to phylogroups B2 and D respectively). Among the blaNDM-1 producers, all the isolates were phenotypically ESBL producers, on molecular analysis 15 isolates were carrying blaCTX-M gene, one isolate was carrying each of blaTEM and blaSHV. Combination of bla TEM + CTX-M were found in four isolates and bla SHV + CTXM were in two isolates, ten isolates were found to be positive with blaCTX-M15 and among Plasmid encoded AmpC genes belonging to the CIT family were detected in 47% (8/17) isolates all the 8 isolates were also positive with AmpC disk test. All the 17 isolates were resistant to beta-lactam antibiotics although four isolates were sensitive to cefoperazone-sulbactam and one to piperacillin-tazobactam. Out of 17 isolates none of them were sensitive to imapenem and meropenem however one isolates were sensitive to ertapenem. Among the aminoglycosides group, three isolates were sensitive to amikacin. One isolate was sensitive to ciprofloxacin and one strain to norfloxacin. Among the other antibiotics, most strains were sensitive to nitrofurantoin (11/17), tigecycline (10/17) and co-trimoxazole (6/17), respectively. Of the patients infected with blaNDM1 positive organisms, 71% (12/17) recovered with appropriate antibiotic treatment. Relapses were seen in 18% (3/17) patients and 2 (12%) patients only expired [Table 1].
|Figure 1: Gel image of polymerase chain reaction amplification of the metallo-beta-lactamase gene NDM1: A 621 bp fragment specific for NDM1 gene was amplified: Lane 1: 100 bp DNA ladder. Lane 2: Positive control. Lane 3: Negative control. Lane 4 and 5: Test sample|
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|Figure 2: Phylogenetic grouping of UPEC isolates: Phylogenetic Group A ([chuA−, yjaA−, TspE4.C20−] and [yjaA+, chuA−, TspE4.C2−]); Group B1 (chuA−, yjaA−, TspE4.C2+); Group B2 ([chuA+, yjaA+, TspE4.C2−] and [ chuA+, yjaA+, TspE4.C2+]); and Group D ([chuA+, yjaA−, TspE4.C2−] and [chuA+, yjaA−, TspE4.C2+])|
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|Table 1: Different features associated with blaNDM1 positive E. coli isolates|
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| Discussion|| |
The results of our study showed that 6% of our inpatient E. coli infections were due to blaNDM-1 producing strains, blaNDM-1 is a transferable class B MBL. Since its 1 st appearance in 2008, it has been found in different Gram-negative isolates from various parts of the world including UK, Pakistan, Australia and USA, mostly from patients who are epidemiologically linked to the Indian subcontinent. ,, Several reports from India have shown there is 5-8% prevalence of blaNDM-1 , a finding that is similar to our study findings. ,, Among carbapenemase producers, 72% isolates were MBL positive and in that 81% isolates were blaNDM-1 that indicate a significant association between MBL producers with blaNDM-1 positive isolates. In the present study, we isolated blaNDM-1 producers from neonates as well as elderly patients with UTI followed by sepsis and around 90% of our cases were from community-acquired infections, similar results have also been reported by other investigators. , As diabetes was the most common predisposing factor for various infections, there were no exception to infection by blaNDM-1 producing E. coli also, where we found a significant association (P < 0.05) between diabetics and infection with blaNDM-1 producers. On the analysis of phylogenetic group we found, phylogroup D was more common followed by A, B1 and B2 respectively, which indicate that all group of isolates were capable to carrying blaNDM-1 gene, several other studies have found that among the phylogroups B1 is the most common. , Hence, we conclude that commensals as well as virulent strains are capable of carrying the blaNDM-1 plasmid.
In our study, blaNDM-1 producing isolates were observed to exhibit a high degree of multidrug resistant patterns associated with plasmid encoded blaCTX-M , blaTEM , blaSHV and blaCIT genes. Similar results have been obtained by other investigators worldwide where they reported blaNDM-1 encoding plasmids may co-harbor multiple resistance determinants, including blaTEM-1 , blaOXA-10 , blaCMY quinolone resistance genes as well as 16S RNA methylases that alter the ribosomes to block binding of nearly all aminoglycoside antibiotics. ,,, Several studies have reported a significant association of bla NDM-1 with bla CTX-M15 that is similar to our study findings where we got approximately 90% of the blaNDM-1 positive isolates encoding blaCTX-15 gene. ,, Previous studies have reported bla NDM-1 producers exhibiting resistance to many other classes of antibiotics, including fluroquinolones and aminoglycosides, nevertheless most isolates being susceptible to tigecycline and nitrofurantoin. ,, However, a few isolates may retain susceptibility to any one of chloramphenicol, tetracyclines, nitrofurantoin and co-trimoxazole and their clinical efficacy is questionable, as tigecycline is unsuitable for use in urinary tract and in vitro susceptibility of the others is not reliable. In our study, we found similar findings where in most bla NDM-1 producers exhibited sensitivity to tigecycline and nitrofurantoin and a few isolates were sensitive to co-trimoxazole and chloramphenicol. However, regarding treatment of our study population, several groups of antibiotics were used including beta lactam + beta lactamase inhibitor combinations and carbapenems. Follow-up of patients in our study group for a period of 1 year disclosed a better prognosis of infection, that majority of cases (71%) showing recovery, mortality was not different from other E. coli infections, which indicates that most of them may not be virulent. Phylogenetic distribution of our blaNDM1 positive strains indicated that all such strains (both commensal and pathogenic strains) may be present as colonizers and cause infection when patients become compromised. In conclusion, blaNDM-1 producing E. coli Infections are emerging infections. Commensal strains too are capable of producing blaNDM-1 enzyme and also co-expressed plasmid - mediated AmpC as well as ESBL's and can act as vectors to spread antibiotic resistance genes at a high rate, worldwide. This is an alarming public health concern since blaNDM-1 producing isolates are highly resistant to most antibiotic classes and unfortunately new and effective antibiotic as well as an effective vaccine are still not available. Therefore, there is an urgent need to undertake measures which would prevent such infections and development of multidrug resistance among isolates in the absence of effective antimicrobial agents wherein even a common UTI may become a life-threatening infection for those who are immunocompromised.
| Acknowledgement|| |
We would like to thanks Lotte Jakobsen MSc (biology), PhD Statens Serum Institut Microbiology & Infection Control 5 Artillerivej, build 46/202 DK-2300 Copenhagen S for providing us the NDM-1 positive control isolates for the study.
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Dr. Arindam Ckakraborty
Department of Microbiology, Moti Lal Nehru Medical College, Allahabad, Uttar Pradesh
Source of Support: API Karnataka, India, Conflict of Interest: None
[Figure 1], [Figure 2]