Detection of New Delhi Metallo-b-Lactamase Variants NDM-4, NDM-5, and NDM-7 in Enterobacter aerogenes Isolated from a Neonatal Intensive Care Unit of a North India Hospital:A First Report
A total 402 Enterobacteriaceae isolates were recovered from blood and rectal swabs of 1,000 infants admitted to the Neonatal Intensive Care Unit (NICU) of the Jawaharlal Medical College and Hospital Aligarh, India. Car- bapenamase producers were determined by Carba NP phenotype biochemical assay. Out of 402 isolates, it was the first time three of the isolates were identified as Enterobacter aerogenes carrying blaNDM-4, blaNDM-5, and blaNDM-7 genes. These genes were identified by polymerase chain reaction (PCR) and sequence analysis. The isolates were further characterized to know the plasmid type and genetic environment features, including integron and IS elements. All the three E. aerogenes isolates (AK-93, AK-95, and AK-96) were resistant to all b-lactams, including carbapenems. The b-lactamase genes blaOXA-1, blaOXA-9, blaSHV-1, and blaVIM-2 were also found to be coassociated with blaNDM-4 in AK-93, blaOXA-1, blaOXA-9, and blaCMY-149 were found to be coexisted with blaNDM-5 in AK-95, and blaOXA-1; blaOXA-9, and blaCMY-145 were also found to be coassociated with blaNDM-7 in AK-96, identified by PCR analysis. Plasmid-based replicon typing revealed plasmids of different incompatibility in
E. aerogenes in each of the isolates, AK-93 AK-95, and AK-96, respectively. ERIC-PCR was performed for the analysis of genetic relatedness of the strains. We found blaNDM-4, blaNDM-5, and blaNDM-7 producing three E. aerogenes strains, which were not clonally related. Genetic environment analysis revealed the presence of bleomycin resistance gene (bleMBL) to downstream of blaNDM and complete ISAba125 sequence were found upstream of blaNDM in all the three variants of these isolates. This is the first time we have identified blaNDM-4, blaNDM-5, and blaNDM-7 in E. aerogenes species, isolated from the NICU of a tertiary care hospital in India.
Keywords: NDM-4, NDM-5, NDM-7, antibiotic resistance, carbapenemase
Introduction
NfecTIoNs caUsed BY caRBapeNeM-ResIsTaNT entero- bacteriaceae (CRE) are one of the most serious health threats for humans and animals, worldwide.1 Carbapenems are often considered as antibiotics of last resort for treatment of bacterial infections. Emergence of New Delhi metallo-b- lactamase (NDM) variants cause difficult-to-treat infections in clinical settings. NDM-1 was first identified in Klebsiella pneumoniae, isolated from a 59-year-old man who returned to Sweden after hospitalization in India during January 2008.2 The NDM-producing bacteria are resistant to almost all available antibiotics, except polymyxin3,4; however, after the discovery of MCR-1 in animals and humans, identified in China, the hope of polymyxin and colistin as treatment options to control these infections is now limited.5 A total of 17 known variants of NDM have been identified so far (www.lahey.org/Studies/other.asp#table 1). These variants were identified in diversified species of Gram-negative bacteria and were found to have variation either by repla- cing single amino acid or multiple residues at different positions. In India, the most prevalent variants found were, NDM-4, NDM-5, NDM-6, and NDM-7. The NDM-4 was first detected in Escherichia coli isolated from a urinary sample of a patient, previously hospitalized in India. A single amino acid substitution at position 154 (Met/Leu) was found in NDM-4 compared with NDM-1, which may cause increased carbapenemase activity.6 Another variant, NDM-5, was first identified in a multidrug-resistant E. coli isolated from a patient in the United Kingdom, who had a history of hospitalization in India.7 NDM-5 differs from NDM-1 by substitutions at positions 88 (Val/Leu) and 154 (Met/Leu). NDM-6 was identified in E. coli with single amino acid substitution at position 233 (Ala/val).8 NDM-7 was first identified in E. coli with substitutions of two res- idues at position 130 (Asp/Asn) and 154 ( Met/Leu).In view of the current spread of NDM-producing bacterial strains, we have undertaken an initiative to identify and characterize these markers in neonatal intensive care unit (NICU) of one of the tertiary care hospitals of north India.
Materials and Methods
Sample collection
A total of 1,000 Enterobacteriaceae clinical isolates were screened for carbapenem resistance from the NICU at Jawaharlal Nehru Medical College and Hospital ( JNMCH), Aligarh Muslim University, Aligarh, India, during Novem- ber 2015 through October 2016. JNMCH is a tertiary care hospital with a 1,300 bed capacity. NICU is a tertiary care unit for pediatrics and has 20 beds out of a total of 120 beds allocated to pediatric patients. The unit has several current projects on ‘‘safe children’’, in collaboration with UNICEF, USAIDS, and National Health Mission. The treatment given to babies admitted to NICU is protocol based. One of the strains was isolated from the blood of an 18-day-old female child hospitalized in NICU, whereas the other two strains were isolated from rectal swabs collected from two female children 30 days of age.
Ethical statement
All the isolates were collected after the approval of the institution’s ethics committee, the approval number is: 151/ 201517/PDFWM-2015-2017-UTT 31140 (SAII).
Antimicrobial susceptibility tests and metallo-b-lactamase detection
Antimicrobial susceptibility was determined by the stan- dard disc diffusion method using Mueller-Hinton agar as per the Clinical and Laboratory Standards Institute Guidelines.10 More than 50 colonies were picked from MH agar plate for antimicrobial susceptibility testing and MBL detection. De- tection of metallo-b-lactamase ( MBL) activity was per- formed by using two imipenem discs (10 mg), one containing 10 ml of 0.1 M anhydrous ethylenediaminetetraacetic acid. The discs were placed 25 mm apart on Mueller-Hinton plates.11 Minimum inhibitory concentrations (MICs) were determined using broth microdilution method following CLSI guidelines, 2013.10
Carba NP test
Carba NP test is a biochemical method used for the de- tection of carbapenemase activity in Enterobacteriaceae isolates, which was performed, as described earlier.12
Isolate identification
The species-level identification of isolates was performed by using BD Phoenix™-100 automated microbiology system using panel NMIC/ID-55(Gram-negative susceptibility card) and further validated by 16 s rRNA sequencing using primers, as described perversely.13
Polymerase chain reaction amplification and sequencing
Genes were amplified by polymerase chain reaction (PCR; [Applied Biosystems]) using primers as described previous- ly14,15 for blaNDM and other resistant markers (blaVIM, blaOXA-1, blaOXA-9, blaCMY, blaTEM, blaSHV, and blaKPC). Amplicons of NDM were purified from the gel using the Gel Extraction Kit (Thermo Fisher Scientific), following the manufacturer’s protocol. It was then sequenced at DNA sequencing facility at the SciGenom Labs Private Ltd, Cochin, Kerala, India. The nucleotide and deduced protein sequences were analyzed with software available at the National Center for Bio- technology Information website (www.ncbi.nlm.nih.gov).
Conjugation experiment
The plasmidic locations of resistant markers (blaNDM, blaCMY-145, blaCMY-149, blaOXA-1, blaOXA-9, blaVIM-2, and blaSHV-1) were determined by conjugation, using isolates as donors and an azide-resistant E. coli J53 strain as the re- cipient. Transconjugants were screened on Luria Bertani agar supplemented with ceftazidime (10 mg/ml [Sigma- Aldrich]) and sodium azide (100 mg/ml) (HiMedia Labora- tories, India). The transconjugants having resistant markers were confirmed by PCR amplification.
Replicon typing
Plasmid incompatibility group was determined by a PCR- based replicon typing method as described previously.16
Integron analysis
The transconjugants of all the isolates, with blaNDM, were subjected to undergo integron analysis, using PCR amplifi- cation of 3¢/5¢ conserved segment along with Int1 and Sul1, as described previously.17
Molecular typing
Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) assay was performed for the analysis of ge- netic relatedness of the strains as described earlier.18
Genetic environment analysis was performed to detect the genes present at upstream and downstream of blaNDM variants, as reported previously.19
Results
Isolate identification, antimicrobial susceptibility test, and MBL detection Out of 1,000 isolates, 402 isolates were found to be carba- penem resistant. Of 402, 72 were MBL-producing strains and three of them were identified as E. aerogenes, carrying blaNDM-4, blaNDM-5, and blaNDM-7 genes, reported first time by our group. These three isolates (AK-93, AK94, and AK-95) were found highly resistant to frequently used antibiotics in clinical settings vis-a`-vis carbapenems (imipenem and meropenem), extended-spectrum cephalosporins (cefoxitin, ceftazidime, and cefotaxime), aminoglycoside (gentamicin and amikacin), monobactam (aztreonam), tetracycline (minocycline and tige- cycline), fluoroquinolone (ciprofloxacin), polymyxin, and co- listin b-lactam/b-lactamase inhibitor (cefoperazone/sulbactam) (Table 1). MICs data are shown in Table 2.
PCR amplification and sequencing confirmed that AK-93, AK-95, and AK-96 were NDM-4-, NDM-5-, and NDM-7-producing strains of E. aerogenes, respectively. The acces- sion numbers of these sequences submitted to NCBI database are given in Table 2. The blaOXA-1, blaOXA-9, blaSHV-1, and blaVIM-2 were also found to be coassociated with blaNDM-4 in AK-93, blaOXA-1, blaOXA-9, and blaCMY-149 were found to coexist with blaNDM-5 in AK-95 and blaOXA-1, blaOXA-9, and blaCMY-145 were found to be coassociated with blaNDM-7 in AK-96 as shown in Table 1. Conjugation experiment, further confirmed the presence of these resistant markers on the plasmid in each isolates. All three isolates were found to carry class 1 integron on the plasmid.PCR-based genetic environment analysis of blaNDM vari- ants was performed and found bleMBL to downstream of all three blaNDM variants in this study. The complete ISAba125 sequence was present at upstream to blaNDM variants (Table 2).
Molecular typing
NDM variants (blaNDM-4, blaNDM-5, and blaNDM-7) pro- ducing three E. aerogenes strains isolated from NICU, were not clonally related.
Replicon typing
AK-93 were carrying plasmid of incompatibility FIA, FIB, I, F, K, A/C, and FII types, whereas AK-95 were found to have plasmid of incompatibility, FIA, FIB, I, Y, F, K, and FII types. Incompatibility FIA, FIB, I, Y, F, and K types of plasmids were observed in AK-96. All three isolates were carrying different types of incompatibility plasmids (Table 1).
Discussion
Enterobacter is a genus of the family Enterobacteriaceae, consisting of common Gram-negative, facultative anaerobic, rod-shaped, and nonspore-forming bacteria. E. aerogenes is recognized as an important bacterial pathogen in hospital- acquired infections.20 It has already been reported earlier to carry blaNDM-1 along with other resistant markers.21–23 However, our study reveals for the first time the prevalence of these variants in E. aerogenes. It further showed that AK-93 was found to have blaNDM-4, associated with blaOXA-1, blaOXA-9, blaSHV-1, and blaVIM-2, AK-95 was found to carry accordance with earlier reports.11,19 Hence, it depicts that no change was found in the genetic environment of blaNDM in all three clinical strains under investigation.
Conclusion
The study is clinically significant for physicians to un- derstand the spread of resistant markers and infections in hospital settings. We have identified for the first time blaNDM-4-, blaNDM-5-, and blaNDM-7-producing three E. aerogenes strains isolated from a NICU. Hence, it is ex- tremely important to think sensibly about infection control in hospital settings.
Nucleotide Sequence Accession Numbers
The nucleotide sequences of the three NDM variants containing E. aerogenes have been submitted in the GenBank database and Accession No. are KX999120, KX999122, and KX999123 for blaNDM-4,AK 7 blaNDM-5, and blaNDM-7, respectively.