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Background: The bla (New Delhi Metallo-β-lactamase-1) gene has disseminated around the globe. NDM-1 NDM-1 producers are found to co-harbour resistance genes against many antimicrobials, including fluoroquinolones. The spread of large plasmids, carrying both bla and plasmid-mediated fluoroquinolone resistance (PMQR) markers, is NDM one of the main reasons for the failure of these essential antimicrobials. Methods: Enterobacteriaceae (n = 73) isolated from the blood of septicaemic neonates, admitted at a neonatal intensive care unit (NICU) in Kolkata, India, were identified followed by PFGE, antibiotic susceptibility testing and determination of MIC values for meropenem and ciprofloxacin. Metallo-β-lactamases and PMQRs were identified by PCR. NDM-positive isolates were studied for mutations in GyrA & ParC and for co-transmission of bla and PMQR NDM genes (aac(6′)-Ib-cr, qnrB, qnrS) through conjugation or transformation. Plasmid types, integrons, plasmid addiction systems, and genetic environment of the bla gene in NDM-positive isolates and their transconjugants/ NDM transformants were studied. Results: Isolated Enterobacteriaceae comprised of Klebsiella pneumoniae (n = 55), Escherichia coli (n = 16), Enterobacter cloacae (n = 1) and Enterobacter aerogenes (n = 1). The rates of ciprofloxacin (90%) and meropenem (49%) non-susceptibility were high. NDM was the only metallo-β-lactamase found in this study. NDM-1 was the predominant metallo-β-lactamase but NDM-5, NDM-7, and NDM-15 were also found. There was no significant difference in ciprofloxacin non-susceptibility (97% vs 85%) and the prevalence of PMQRs (85% vs 77%) between NDM-positive and NDM-negative isolates. Among the PMQRs, aac(6′)-Ib-cr was predominant followed by qnrB1 and qnrS1. Twenty-nine isolates (40%) co-harboured PMQRs and bla , of which 12 co-transferred PMQRs along with NDM bla in large plasmids of IncFIIK, IncA/C, and IncN types. Eighty-two percent of NDM-positive isolates possessed NDM GyrA and/or ParC mutations. Plasmids carrying only bla were of IncHIB-M type predominantly. Most of the NDM isolates had ISAba125 in the upstream region of the bla gene. NDM Conclusion: We hypothesize that the spread of PMQRs was independent of the spread of NDM-1 as their co- transfer was confirmed only in a few isolates. However, the co-occurrence of these genes poses a great threat to the treatment of neonates. Keywords: Ciprofloxacin, NDM, Enterobacteriaceae, Neonates, PMQRs, India * Correspondence: firstname.lastname@example.org; email@example.com Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases, P33, CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 2 of 15 Background carbapenems . A thorough evaluation of their resist- Fluoroquinolones are considered as critically important ance level also makes this study clinically relevant. antimicrobials by the World Health Organization . They are used extensively to treat gram-negative and Materials and methods some selective gram-positive bacteria. Quinolones (Nali- Identification of strains dixic acid) and fluoroquinolones (Ciprofloxacin, gatiflox- Enterobacteriaceae (n = 73) obtained from blood cultures acin etc.) are bactericidal antimicrobials that selectively of 66 septicaemic neonates (new-borns less than 28 days target the action of gyrase and topoisomerase IV disab- of life), admitted to the neonatal intensive care unit of ling the DNA replication . The classical mechanisms IPGMER and SSKM Hospital, Kolkata, India, during of fluoroquinolone resistance are the accumulation of January 2012 to June 2014, were included in this study. mutations in the target enzymes and upregulation of the The isolates were identified by 5 biochemical tests which efflux pumps. Both these mechanisms are mutational include Triple Sugar Iron test, Mannitol motility test, and are passed vertically to the surviving progeny. Add- Simmons citrate agar test, Urease test, Indole test, and ing fuel to this fire are the plasmid-mediated quinolone discrepancies were resolved by Vitek2 system (bioMe’r- resistance (PMQR) genes which raise greater concern ieux, Marcy l’E’toile, France). Due to unavoidable cir- because of their transmissibility. PMQRs include penta- cumstances, isolates were not collected between 2012 peptide Qnr protein genes (qnrA, qnrB, qnrS, qnrC, June to 2012 December qnrD) which give protection to gyrase and topoisomer- ase IV, fluoroquinolone modifying enzyme aac(6′)-Ib-cr Antimicrobial susceptibility testing and determination of which is a variant of the acetyltransferase of aminoglyco- MIC values sides, and plasmid DNA encoded efflux pumps qepA The antimicrobial susceptibility testing for different anti- and OqxAB. Although PMQRs confer low-level resist- biotic agents (piperacillin (100 μg), cefotaxime (30 μg), ance, they facilitate the selection of mutations in gyrase cefoxitin (30 μg), aztreonam (30 μg), meropenem (10 μg), and topoisomerase genes which results in high-level ciprofloxacin (5 μg), ofloxacin (5 μg), amikacin (30 μg), resistance . gentamicin (10 μg), tigecycline (15 μg), and trimetho- With the emergence of carbapenem resistance in prim/sulfamethoxazole (1.25 μg /23.75 μg) (BD Diagnos- Enterobacteriaceae, treatment options have been se- tics, Franklin Lakes, NJ, USA) was done by the Kirby- verely jeopardized. Though a number of carbapenemases Bauer standard disk diffusion method. The MIC values (IMP, VIM, SIM, SPM, GIM, KPC, SME) have been (mg/L) of meropenem and ciprofloxacin were deter- identified in Enterobacteriaceae, the advent of NDM-1 mined using Etest (AB Biodisk, Solna, Sweden). All the has been the ‘last straw’ in this growing problem. This values were interpreted according to CLSI guidelines  study focuses on bla instead of other carbapene- except for tigecycline, which was interpreted according NDM-1 mases because it is widely prevalent in India, to EUCAST guidelines 2013 . MIC and MIC 50 90 Bangladesh, and Pakistan . It is a metallo-β-lactamase (MIC at which 50 and 90% of the isolates were inhibited that contains zinc at its active site and can hydrolyze not respectively) were calculated for meropenem and only carbapenems but almost all hydrolyzable β-lactams ciprofloxacin. except aztreonam . Apart from resistance to β-lactam antibiotics, most bla carrying Enterobacteriaceae Genotypic detection of resistance markers NDM-1 are also resistant to a wide range of non-β-lactam antibi- PCR assays were performed on all isolates for the detec- otics such as aminoglycosides, fluoroquinolones, sulpho- tion of carbapenemase genes (bla , bla , bla , NDM VIM IMP namides, trimethoprim, chloramphenicol . bla , bla , bla bla bla )[8, 11–13], SPM GIM SIM, KPC, OXA-48 Both PMQRs and NDM-1 are present on transmissible other β-lactamase genes (bla , bla , bla , CTX-M TEM SHV elements and several studies have shown the presence of bla )[14, 15] and PMQR genes (qnrA, qnrB, qnrS, OXA-1 PMQRs with bla [5, 6]. With increasing resistance to qnrC, qnrD, aac(6′)-Ib-cr, qepA, oqxA, oqxB)[3, 16]. The NDM carbapenems, and concurrent resistance to fluoroquino- qepA and aac(6′)-Ib-cr genes were analyzed by a multi- lones in NDM-possessing isolates, a better understanding plex PCR with a buffer suitable for GC rich sequences as of this association is necessary. This study focuses on the GC content of qepA gene is high (70%). The fluoroquinolone non-susceptibility and prevalence of aac(6′)-Ib-cr was differentiated from its wild-type allele by PMQRs in NDM-positive and NDM-negative Enterobac- digestion with BtsCI enzyme (New England Biolabs, teriaceae isolated from cases of neonatal septicaemia. It Massachusetts) . Primers used do not discard the also highlights the possibility of co-transmission of these presence of the non-ESBL variants of bla and bla . TEM SHV resistance genes in single large conjugative plasmids. As the study focuses on bla and PMQRs, the PCR NDM-1 In developing countries neonates are prescribed fluor- products of bla and bla genes were not further TEM SHV oquinolones for life-threatening infections  and so are sequenced and this remains a shortcoming of the study. Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 3 of 15 Sequencing Plasmid addiction systems (pemKI, ccdAB, relBE, parDE, All bla and qnrB amplified products were sequenced vagCD, hok–sok, pndCA, srnBC) were investigated by PCR NDM using primers described previously [17, 18]. qnrS was assays . Plasmid types were also determined by PBRT amplified with a pair of primers designed in this kit (Diatheva srl, Cartoceto, Italy). Presence of class 1, study:- qnrSF5’- TCTAGCCCTCCTTTCAACAAG-3′ class 2, and class 3 integrons was investigated . and qnrSR:5′- TGAGCGTTTAAAATCACACATCA-3′. The upstream and downstream regions of bla NDM Additionally, in NDM-positive isolates, quinolone resist- were amplified and sequenced with a series of primers ance determining region (QRDR) of gyrA and parC genes which were designed previously . were sequenced . Sequencing was carried out using Big-Dye terminator v3.1 cycle sequencing kit (Applied Statistics Biosystems, Foster City, USA) in an automated DNA se- Determination of significant differences between NDM- quencer (Applied Biosystems 3730DNA Analyzer, Perkin positive isolates and NDM-negative isolates and between Elmer, USA). organisms Escherichia coli and Klebsiella pneumoniae was calculated using the chi-square test of independence Pulsed-field gel electrophoresis (PFGE) by comparing the variables. All statistical testing was Genetic relatedness of the isolates was examined by two-tailed and all comparisons were unpaired. Statistical PFGE in a CHEF-DRIII apparatus (Bio-Rad Laboratories, significance was defined as P ≤ 0.05. Hercules, and CA) following digestion of genomic DNA with XbaI enzyme (New England Biolabs, Massachu- Results setts) according to Tenover et al. . The PFGE images Isolates were processed and the dendrogram was calculated by Seventy-three isolates were identified as Enterobacteria- FPQuest software v4.5 (Biorad laboratories inc, ceae which included Klebsiella pneumoniae (75%, 55/ Hercules, California, USA.) using Dice coefficient and 73), Escherichia coli (22%, 16/73), Enterobacter cloacae UPGMA (unweighted pair group method using arith- (1%, 1/73) and Enterobacter aerogenes (1%, 1/73). metic averages). Isolates having more than 95% similarity were considered identical. Antibiotic susceptibility pattern Ninety-seven percent (71/ 73) of the isolates were multi- Molecular characterization of NDM-positive isolates with drug resistant (MDR) i.e. non-susceptible to three or a focus on fluoroquinolone resistance more groups of antibiotics. Thirty isolates were resistant Transmissibility of bla was studied by conjugation to 7 groups of antibiotics. Isolates were highly resistant NDM experiment. In the solid mating assay, donor strain and to most of the antibiotics except meropenem and tigecyc- recipient strain (Escherichia coli J53 azide resistant) were line, resistance was generally higher in K. pneumoniae plated in a ratio of 1:5 on Luria Agar plates and incu- than E. coli. Non-susceptibility to different antibiotics for bated at 37 °C. Transconjugants were selected on two all isolates is depicted in Table 1 and Additional file 1. En- types of agar plates containing: (A) cefoxitin (10 mg/L) terobacter aerogenes and Enterobacter cloacae were and sodium azide (100 mg/L) and (B) ciprofloxacin non-susceptible to piperacillin, cefotaxime, cefoxitin, cip- (0.06 mg/L) and sodium azide (100 mg/L), as recom- rofloxacin, and aztreonam. Additionally, Enterobacter mended by earlier studies [21, 22]. Isolates which could aerogenes was non-susceptible to meropenem and Entero- not transfer their plasmid through conjugation were bacter cloacae isolate was non-susceptible to gentamicin. subjected to electro-transformation using E. coli DH10B Since this study focuses on carbapenem and fluoro- as host cells. Transformants were selected in LA plates quinolone resistance, analysis of the isolates was carried containing cefoxitin (5 mg/L). In one case where there out in terms of these two antibiotics separately. Forty-nine was no colony on cefoxitin plate, transformants were se- percent (36/73) of the total isolates were non-susceptible lected on ampicillin (50 mg/L) agar plate. The transcon- to meropenem and nearly all (97%, 35/36) meropenem jugants /transformants were screened for the presence non-susceptible isolates, were non-susceptible to cipro- of the bla gene, PMQRs (aac(6′)-Ib-cr, qnrB and floxacin, which included E. coli (n =6),K. pneumoniae (n NDM qnrS), β-lactamases (bla , bla , bla , bla ), = 29) and Enterobacter aerogenes (n = 1). Eighty-four CTX-M TEM SHV OXA-1 and 16S rRNA methylases (armA, rmtB, rmtC, rmtA, percent (31/37) of the meropenem susceptible isolates npmA, rmtD). were also non-susceptible to ciprofloxacin. Overall, cipro- Plasmid DNA was isolated from wild-type and trans- floxacin non-susceptibility (90%) was higher than merope- conjugants/transformants by modified Kado and Liu nem non-susceptibility (49%). plasmid isolation technique  and was sized by Quan- The range of MIC against meropenem in E. coli was tity One® 1-D analysis software (Biorad) comparing with 0.032 mg/L to > 32 mg/L and in K. pneumoniae was 0.023 plasmids of E. coli V517 and Shigella flexineri YSH6000. mg/L to 32 mg/L. Whereas, MIC against ciprofloxacin in Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 4 of 15 Table 1 Antibiotic susceptibility pattern of the studied isolates Antibiotics Non-susceptible isolates Non-susceptible E. coli isolates Non-susceptible K. pneumoniae P value (E. coli / no. (%) no.(%) isolates. no. (%) K.pn.) Piperacillin 72 (99%) 15 (94%) 55 (100%) 0.5080 Cefotaxime 68 (93%) 13 (81%) 53 (96%) 0.1274 Cefoxitin 55 (75%) 9 (56%) 44 (80%) 0.1160 Aztreonam 65 (89%) 13 (81%) 50 (91%) 0.5311 Meropenem 36 (49%) 6 (38%) 29 (53%) 0.4306 Ciprofloxacin 66 (90%) 12 (75%) 52 (95%) 0.0670 Ofloxacin 58 (79%) 12 (75%) 46 (84%) 0.6753 Amikacin 55 (75%) 9 (56%) 45 (82%) 0.0756 Gentamicin 65 (89%) 11 (69%) 52 (95%) 0.0154 Sulfomethoxazole / 62 (85%) 11 (69%) 49 (89%) 0.1126 trimethoprim Tigecycline 16 (22%) 1 (6%) 15 (27%) 0.1523 Statistically significant P values are underlined E. coli was 0.25 mg/L to > 32 mg/L and in K. pneumoniae product revealed that most of them were bla , NDM-1 was 0.064 mg/L to > 32 mg/L. The MIC of meropenem in except 3 which were bla bla and bla NDM-5, NDM-7 NDM-15. Enterobacter aerogenes and Enterobacter cloacae were 4 The bla was a novel variant and the sequence NDM-15 mg/L and 0.047 mg/L respectively whereas MIC of cipro- was submitted to GenBank (accession no. KP735848). floxacin were 4 mg/L and 15 mg/L. In E. coli isolates Two isolates non-susceptible to meropenem, yet lacking MIC50 and MIC 90 of meropenem were 0.125 mg/L and bla , possessed bla . bla was not present in NDM OXA-48 KPC 24 mg/L respectively and in K. pneumoniae isolates MIC any of the isolates. β-lactamase genes bla , 50 CTX-M and MIC of meropenem were 1.5 mg/L and 10 mg/L bla , bla , bla were present in 66% (48/73), 90 SHV TEM OXA-1 respectively. In both organisms, MIC and MIC of 49% (36/73), 42% (31/73) and 66% (48/73) isolates 50 90 ciprofloxacin were > 32 mg/L. respectively (Table 2, Additional file 1). Overall 81% (59/73) isolates were confirmed to carry Prevalence of various β-lactamases and PMQRs at least one of the PMQRs which included 7 E. coli Since bla genes can persist even in cells exhibiting isolates, 50 K. pneumoniae isolates and both the Entero- NDM-1 very low-level resistance to meropenem , all isolates bacter sp. Overall, 40% (29/73) isolates co-harboured were screened for bla and other carbapenemases. NDM and PMQRs. Among qnr genes, qnrB and qnrS NDM Forty-seven percent (34/73) isolates were bla -posi- were present in 51% (37/73) and 3% (2/73) of isolates NDM tive which included 6 E. coli,27 K. pneumoniae, and 1 respectively. Other qnr genes qnrA, qnrC, and qnrD Enterobacter aerogenes isolate. No other metallo-β-lacta- were absent. All qnrB and qnrS genes found were qnrB1 mase was found. Sequencing of bla amplified and qnrS1 respectively. Seventy-one percent (52/73) NDM Table 2 Distribution of resistance genes in studied organisms Resistance Markers Total (n = 73) E. coli (n = 16) K. pneumoniae (n = 55) Enterobacter sp. (n = 2) P value (E. coli/ K.pn.) bla 34 (47%) 6 (38%) 27 (49%) 1 0.5938 NDM PMQR (number) 59 (81%) 7 (44%) 50 (91%) 2 0.0001 aac(6′)-Ib-cr 52 (71%) 7 (44%) 44 (80%) 1 0.0117 qnrB 37 (51%) 0 (0%) 35 (64%) 2 0.0001 qnrS 2 (3%) 0 (0%) 2 (4%) 0 0.4391 oqxAB 55 (75%) 2 (13%) 53 (96%) 0 0.0001 bla 48 (66%) 8 (50%) 38 (69%) 2 0.2671 CTX-M bla 31 (42%) 11 (69%) 20 (36%) 0 0.0442 TEM bla 36 (49%) 2 (13%) 34 (62%) 0 0.0014 SHV bla 48 (66%) 7 (44%) 39 (71%) 2 0.0883 OXA aac(6′)-Ib 18 (25%) 0 (0%) 18 (33%) 0 0.0202 Statistically significant P values are underlined Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 5 of 15 isolates were positive for modifying enzyme coding largest cluster which included 9 isolates. The presence of aac(6′)-Ib-cr gene. Fourteen isolates carried both the a higher number of clonal isolates in K. pneumoniae aac(6′)-Ib and aac(6′)-Ib-cr alleles. None of the isolates may have contributed to the higher rate of fluoroquino- carried plasmid-mediated efflux pump gene qepA. MDR lone non-susceptibility and prevalence of PMQRs in K. family efflux pump genes oqxA and oqxB were found in pneumoniae compared to E. coli. 53 K. pneumoniae and 2 E. coli isolates. Prevalence of aac(6′)-Ib-cr in K. pneumoniae 80% (44/55) was signifi- Detailed molecular characterization of NDM-possessing cantly higher than E. coli 44% (7/16) (P-value 0.0117). In isolates with a focus on co-transfer of bla and PMQRs NDM K. pneumoniae prevalence of qnrB and qnrS were 64% Study of the mutations in GyrA and ParC in NDM- (35/55) and 4% (2/55) respectively but these genes were possessing isolates absent in E. coli (Table 2). Since fluoroquinolone resistance in Enterobacteriaceae results also from the accumulation of mutations primar- Distribution of PMQRs in NDM-positive and NDM- ily in DNA gyrase (GyrA) and then in topoisomerase IV negative isolates (ParC), sequences of the QRDR of NDM-positive isolates An analysis of the distribution of PMQRs was carried were studied for mutations in gyrA and parC genes. All out in NDM-positive and NDM-negative isolates. PMQR 6 E. coli isolates carrying NDM had mutations in GyrA genes were highly abundant in both NDM-positive iso- at codons 83 (Ser > Leu) and 87 (Asp>Asn) as well as in lates (85%, 29/34) and NDM-negative isolates (77%, 30/ ParC at codon 80 (Ser > Ile). An additional mutation in 39) (Table 3). Ninety-seven percent (33/34) ParC was present at codon 88 (Leu > Gln) in one iso- NDM-positive isolates were non-susceptible to cipro- late and at codon 84(Glu > Val) in two isolates which floxacin against 85% (33/39) of the NDM-negative iso- were clonally indistinguishable (EN5132, EN5141) lates. Prevalence of aac(6′)-Ib-cr (82%,) and qnrB (56%) (Fig. 2a). was higher in NDM-positive isolates than K. pneumoniae isolates possessed varied mutations: NDM-negative isolates (62 and 46% respectively). Of all Ser83Phe and Asp87Ala in GyrA along with Ser80Ile in isolates, qnrS was found only in 2 isolates which also ParC (n = 10); Ser83Ile mutation in GyrA and Ser80Ile possessed NDM (Table 3). Since oqxA and oqxB genes ParC (n = 4) and Ser83Tyr mutation only in GyrA (n =8). are mostly chromosomally located in K. pneumoniae Five isolates had no mutation in the QRDR region , we have excluded this from the calculation of the (Fig. 2b). The Enterobacter aerogenes isolate possessed total percentages of PMQRs. no mutation in GyrA or ParC. In general, isolates accumulating mutations in both Relatedness of the studied isolates based on PFGE GyrA and ParC had higher MIC values (> 32 mg/L) patterns than isolates possessing mutations in only GyrA According to the cladogram, majority (12/16) of the E. (1.5–32 mg/L) (Fig. 2aand b). coli isolates were diverse (Fig. 1a), except 4 isolates Analysis of the interplay between chromosomal muta- which were indistinguishable and grouped as cluster A. tions and PMQRs and its effect on the MIC of ciproflox- However, the cladogram of K. pneumoniae showed (Fig. acin is represented in Fig. 2b. Four K. pneumoniae 1b) that many isolates were indistinguishable. They were isolates (EN5129, EN5135, EN5142, and EN5181) which grouped into 6 clusters (cluster B – G). Cluster B, C, D, had acquired PMQRs but lacked QRDR mutations were F, and G include 2–4 identical isolates. Cluster E was the non-susceptible to ciprofloxacin according to CLSI cri- teria. Their MIC values were 12, 6, 2, and 14. Whereas one isolate (EN5123) which lacked both PMQRs and Table 3 The difference between NDM-positive and NDM- mutation in QRDR had a very low MIC (0.094 mg/L). negative isolates with respect to ciprofloxacin non-susceptibility Enterobacter aerogens [EN5131] also lacked the chromo- and prevalence of PMQRs somal mutations but carried PMQRs (aac(6′)-Ib-cr and Characteristics NDM-positive NDM-negative P value qnrB) and MIC against ciprofloxacin was 4 mg/L. Over- isolates [n = 34] isolates [n = 39] all, 82% NDM-positive isolates possessed GyrA and/or Ciprofloxacin 33 (97%) 33 (85%) 0.1607 nonsusceptibility ParC mutation. Isolates carrying 29 (85%) 30 (77%) 0.5430 PMQR Transfer of resistance genes by conjugation / transformation assays and characterization of plasmids aac(6′)-Ib-cr 28 (82%) 24 (62%) 0.0890 Genetic transference of bla and PMQRs was studied NDM qnrB 19 (56%) 18 (46%) 0.5521 by conjugation (n = 28) or transformation (n = 5) assays. qnrS 2 (6%) 0 (0%) 0.4139 Out of the 29 isolates co-harbouring PMQRs and oqxAB 27 (79%) 28 (72%) 0.6305 blaNDM, 12 (41%) isolates including E. coli and K. Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 6 of 15 Fig. 1 Genetic relatedness of (a) E. coli and (b) K. pneumoniae isolates. Analysis of PFGE of XbaI digestion pattern based on Dice’s similarity co- efficient and UPGMA (the position tolerance and optimization were set at 1.5 and 1.5% respectively). More than 95% similarity in PFGE band pattern was interpreted as indistinguishable pneumoniae co-transferred the resistance markers in [EN5129] possessed both bla and PMQRs but did NDM large plasmids. Aac(6′)-Ib-cr co-transferred with bla not transfer the bla gene. Detailed molecular char- NDM NDM in 18% (5/28) isolates which co-harboured the genes. acteristics of these isolates and their transconjugants/ Similarly, qnrB co-transferred with bla in 47% (9/ transformants are presented in Tables 4 and 5. An ana- NDM 19) cases. One of the two isolates co-harbouring qnrS lysis of the transconjugants of E. coli and K. pneumoniae transferred the gene along with bla One isolate is presented below separately. NDM. Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 7 of 15 Fig. 2 Genetic relatedness, the presence of PMQRs and chromosomal mutations and MIC values of meropenem and ciprofloxacin in NDM- positive (a) E. coli and (b) K. pneumoniae isolates. Analysis of PFGE of XbaI digestion pattern based on Dice’s similarity co-efficient and UPGMA (the position tolerance and optimization were set at 1.5 and 1.5% respectively). More than 95% similarity in PFGE band pattern interpreted as indistinguishable. MEM: meropenem, CIP: ciprofloxacin All bla -positive E. coli isolates (n = 6) carried bla and PMQR genes in single large plasmids of NDM NDM-1 multiple plasmids and were able to conjugally trans- IncFIIK, IncA/C and IncN type. It was noted that 8 of the fer their plasmid(s) carrying bla in selective 10 isolates co-transferring the genes on an IncFIIK NDM plates containing cefoxitin (10 mg/L) and sodium plasmid were clonal (indistinguishable PFGE pattern) and azide (100 mg/L) but no transconjugants in ciproflox- this particular clone was isolated from the neonates acin (0.06 mg/L) and sodium azide (100 mg/L) plates. between 2013 September to 2014 June (Table 4,Fig. 2b). Among these, 3 possessed aac(6′)-Ib-cr but only in Isolate EN5174 transferred both bla and PMQRs NDM-1 one case this gene co-transferred with bla in a but multiple plasmids were isolated from the transconju- NDM single large 212 kb IncA/C type plasmid which also gant. Hence, it was hard to determine whether PMQRs carried IntI1 and various other resistance genes co-transferred with bla in a single plasmid or not. NDM-1 (bla bla bla armA). In other bla However, one isolate (EN5175) yielded different transcon- CTX-M, TEM, OXA, NDM- positive E. coli isolates that did not possess PMQRs, jugants on cefoxitin-sodium azide and ciprofloxacin-so- bla -harbouring plasmids were of varied replicon dium azide plates. EN5175.T1 (selected on cefoxitin- NDM types such as IncFII, IncFIIS, IncHIB-M, IncI1A, sodium azide plate) harboured bla in an IncHIB-M NDM-1 IncF1A, and IncFIB. Study of the upstream region of plasmid whereas EN5175.T2 (selected on ciprofloxacin- bla revealed that 4 carried the complete ISAba125 sodium azide plate) harboured qnrB in IncFIIK or IncN NDM and 2 carried a truncated version of it. One isolate plasmid. This showed that bla and qnrB were carried NDM [EN5169] possessed IS5 element followed by a truncated on different plasmids. Rest of the K. pneumoniae isolates ISAba125 in the upstream region. ble was present (n = 14) only transferred bla in plasmids of type MBL NDM in the downstream region of bla in all E. coli IncHIB-M (n = 8), IncA/C (n = 2), IncFIIK (n =1) and NDM isolates (Fig. 3). untypable (n = 3). One K. pneumoniae did not transfer Twenty-six of 27 bla -positive K. pneumoniae bla via conjugation or transformation. NDM-1 NDM successfully transferred this gene either through conju- Out of 27 K. pneumoniae isolates, 20 isolates had gation (n = 22) or transformation (n = 4). Ninety-three ISAba125 upstream bla either complete (1/16) or NDM-1, percent (25/27) K. pneumoniae isolates co-harboured truncated (15/16). Isolate EN5181 possessed IS630 trans- bla and at least one of the PMQRs. Among these, 11 posase, isolate EN5127 possessed ISKpn26 transposase, NDM K. pneumoniae yielded transconjugants co-harbouring and isolates EN5135 and EN5174 possessed IS5 element bla and PMQRs. On analysis of the transconjugants, followed by a truncated ISAba125 upstream bla . NDM NDM-1 it was revealed that 10 of these isolates co-transferred the The upstream region of 7 isolates could not be determined. Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 8 of 15 Table 4 Genotypic characterization of NDM-positive isolates co-transferring bla and PMQRs NDM-1 Strain no. Organism NDM PMQR Other Resistance genes MEM MIC CIP MIC Plasmid size Plasmid type Plasmid addiction system Integron EN5132 Escherichia coli bla aac(6′)-Ib-cr bla 2 > 32 212, 6, 3 FIB,A/C,FIIK,FII,N PemK, CcdAB, Hok-Sok intI1 NDM-1 CTX-M, TEM, OXA armA EN5132.T1 E. coli J53 Fox-azide bla aac(6′)-Ib-cr bla 2 0.047 212 A/C – intI1 NDM-1 CTX-M, TEM, OXA armA EN5127 Klebsiella pneumoniae bla aac(6′)-Ib-cr, qnrS aac(6′)-Ib, bla rmtC 24 > 32 210, 4,3 A/C,FIIK,N – intI1 NDM-1 SHV, EN5127.T1 E. coli J53 Fox-azide bla aac(6′)-Ib-cr aac(6′)-Ib, rmtC 2 0.016 210 N, A/C – IntI1 NDM-1 EN5127.T2 E. coli J53 Cip-Azide bla aac(6′)-Ib-cr aac(6′)-Ib, rmtC 6 0.5 210 N, A/C – IntI1 NDM-1 EN5150 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB aac(6′)-Ib, bla 4 > 32 248 FIIK, PemK IntI1 NDM-1 CTX-M, SHV, OXA EN5150.T1 E. coli J53 Fox-azide bla aac(6′)-Ib-cr,qnrB aac(6′)-Ib bla 4 0.25 248 FIIK, – IntI1 NDM-1 CTX-M, OXA EN5150.T2 E. coli J53 Cip-Azide bla aac(6′)-Ib-cr,qnrB aac(6′)-Ib bla 4 0.25 248 FIIK, IntI1 NDM-1 CTX-M, OXA EN5151 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB aac(6′)-Ib bla 2 > 32 248, 128 FIIK, – IntI1 NDM-1 CTX-M, SHV,OXA EN5151.T1 E. coli J53 Fox-azide bla qnrB aac(6′)-Ib, 1 0.064 248 FIIK, – IntI1 NDM-1 EN5151.T2 E. coli J53 Cip-Azide bla qnrB aac(6′)-Ib 0.5 0.047 248 FIIK, IntI1 NDM-1 EN5163 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla aac(6′)-Ib 6 > 32 266, 154 FIIK – IntI1 NDM-1 CTX-M, SHV, OXA, EN5163.T1 E. coli J53 Fox-azide bla qnrB aac(6′)-Ib 3 0.125 266 FIIK – IntI1 NDM-1 EN5163.T2 E. coli J53 Cip-Azide bla qnrB aac(6′)-Ib 2 0.125 266 FIIK IntI1 NDM-1 EN5165 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB aac(6′)-Ib bla 12 > 32 266, 154 FIIK – IntI1 NDM-1 CTX-M, SHV, OXA EN5165.T1 E. coli J53 Fox-azide bla qnrB aac(6′)-Ib 0.38 0.75 266 FIIK IntI1 NDM-1 EN5165.T2 E. coli J53 Cip-Azide bla qnrB aac(6′)-Ib 2 0.064 266 FIIK IntI1 NDM-1 EN5166 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB aac(6′)-Ib bla 4 > 32 266, 154 FIIK – IntI1 NDM-1 CTX-M, SHV, OXA EN5166.T1 E. coli J53 Fox-azide bla qnrB aac(6′)-Ib 0.5 0.047 266 FIIK – IntI1 NDM-1 EN5166.T2 E. coli J53 Cip-Azide bla qnrB bla 3 0.064 266 FIIK IntI1 NDM-1 CTX-M, OXA aac(6′)-Ib EN5170 Klebsiella pneumoniae bla aac(6′)-Ib-cr, qnrB aac(6′)-Ib, bla 4 > 32 260, 200 FIIK – IntI1 NDM-1 CTX-M, SHV, OXA EN5170.T1 E. coli J53 Fox-azide bla qnrB aac(6′)-Ib, 0.5 0.064 260 FIIK – IntI1 NDM-1 EN5170.T2 E. coli J53 Cip-Azide bla qnrB aac(6′)-Ib 0.5 0.25 260 FIIK IntI1 NDM-1 EN5173 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla 1.5 > 32 116, 90 FIIK, – IntI1 NDM-1 CTX-M, SHV, OXA aac(6′)-Ib EN5173.T1 E. coli J53 Fox-azide bla qnrB aac(6′)-Ib 0.5 0.094 116 FIIK – IntI1 NDM-1 EN5173.T2 E. coli J53 Cip-Azide bla qnrB aac(6′)-Ib 1 0.047 116 FIIK IntI1 NDM-1 EN5174 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla , armA 3 16 260, 7, 5, 4 FIIK, N, HIB-M VagC/D IntI1 NDM-1 CTX-M, SHV,OXA EN5174.T1 E. coli J53 Fox-azide bla aac(6′)-Ib-cr,qnrB bla , armA 3 6 260, 7, 5, 4 FIIK, N, HIB-M Vagc/D IntI1 NDM-1 CTX-M, SHV,OXA EN5181 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrS bla 2 14 220 FIIK, FII PemK – NDM-1 CTX-M, SHV, TEM, EN5181.T1 E. coli J53 Fox-azide bla qnrS bla 0.5 0.19 220 FIIK –– NDM-1 CTX-M, TEM Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 9 of 15 Table 4 Genotypic characterization of NDM-positive isolates co-transferring bla and PMQRs (Continued) NDM-1 Strain no. Organism NDM PMQR Other Resistance genes MEM MIC CIP MIC Plasmid size Plasmid type Plasmid addiction system Integron EN5181.T2 E. coli J53 Cip-Azide bla qnrS bla 0.75 0.19 220 FIIK –– NDM-1 CTX-M, TEM EN5185 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla 3 > 32 266, 154 FIIK – IntI1 NDM-1 CTX-M, SHV, OXA aac(6′)-Ib EN5185.T1 E. coli J53 Fox-azide bla aac(6′)-Ib-cr,qnrB aac(6′)-Ib 0.5 0.032 266 FIIK – IntI1 NDM-1 EN5185.T2 E. coli J53 Cip-Azide bla aac(6′)-Ib-cr,qnrB aac(6′)-Ib 1.5 0.19 266 FIIK - IntI1 NDM-1 MEM: meropenem, CIP: ciprofloxacin, (-): absent or untypable (in case of plasmid types), E. coli J53 Fox-azide or ‘.T1’ : transconjugants selected in cefoxitin (10μg/ml)-sodium azide (100μg/ml), ), E. coli J53 Cip-Azide or ‘.T2’ : transconjugants selected in ciprofloxacin (0.06 μg/ml)-sodium azide (100 μg/ml) Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 10 of 15 Table 5 Genotypic characterization of NDM-positive isolates transferring only bla gene NDM Strain no. Organism NDM PMQR Other Resistance MEM MIC CIP MIC Plasmid size in kb Plasmid type (Inc) Plasmid addiction Integron genes (approximately) system EN5134 Escherichia coli bla aac(6′)-Ib-cr bla 24 > 32 225, 158,4 FIA, FII, I1A PemK, CcdAB, Hok-Sok, intI1 NDM-15 CTX-M, TEM, OXA rmtB PndCA EN5134.T1 E. coli J53 Fox-azide bla – bla rmtB 2 0.012 121 FII – intI1 NDM-15 TEM, EN5141 Escherichia coli bla aac(6′)-Ib-cr bla 4 > 32 246, 134, 6 FIB, HIB-M, FII PemK, Hok-Sok – NDM-1 CTX-M, TEM, OXA, armA EN5141.T1 E. coli J53 Fox-azide bla – bla 1.5 0.008 246, 6 HIB-M –– NDM-1 TEM, EN5143 Escherichia coli bla – bla 4 > 32 105,56, 9 FIA, I1 , FII, I1a PndC/A intI1 NDM-1 TEM γ rmtB EN5143.T1 E. coli J53 Fox-azide bla – bla 1.5 0.008 105 FIA, FII – intI1 NDM-1 TEM rmtB EN5169 Escherichia coli bla – bla > 32 > 32 200, 5, 2 I1A, FIA, FIB, FIIS, PndCA, Hok-Sok, - NDM-7 TEM rmtB R, FII ccdAB, SrnBC, PemK EN5169.T1 E. coli J53 Fox-azide bla – bla 1 0.012 200,5 I1A, Pnd C/A - NDM-7 TEM, EN5177 Escherichia coli bla – bla 4 > 32 163,126,55,5, 2 FIB, FIIS PemK IntI1 NDM-5 TEM rmtB EN5177.T1 E. coli J53 Fox-azide bla – bla 1 0.012 163,126 FIB, FIIS – IntI1 NDM-5 TEM rmtB EN5114 Klebsiella pneumoniae bla aac(6′)-Ib-cr bla 1.5 1.5 248 HIB-M PemK intI1 NDM-1 CTX-M, SHV, OXA armA EN5114.T1 E. coli J53 Fox-azide bla – bla 1.5 0.012 248,180 HIB-M – - NDM-1 CTX-M armA EN5117 Klebsiella pneumoniae bla aac(6′)-Ib-cr bla 1.5 1.5 248 HIB-M PemK intI1 NDM-1 CTX-M armA EN5117.T1 E. coli J53 Fox-azide bla – bla 1 0.016 248 HIB-M – - NDM-1 CTX-M, SHV, OXA armA EN5123 Klebsiella pneumoniae bla – bla aac(6′)-Ib 3 0.094 210, 20,7,5 FIA, R, FIIK – intI1 NDM-1 CTX-M, EN5123.T1 E. coli J53 Fox-azide bla – bla aac(6′)-Ib 1 0.008 210,5 FIIK – - NDM-1 CTX-M, EN5129 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla aac(6′)-Ib 2 12 263,230,15.6,6.7, FIIK, R – intI1 NDM-1 CTX-M, EN5129.T1F-A E. coli DH10B - aac(6′)-Ib-cr,qnrB bla 0.023 0.19 230 FIIK – - CTX-M EN5130 Klebsiella pneumoniae bla aac(6′)-Ib-cr bla 8 > 32 270,205,29,13, 8, 7, FIIK, FIA, X2 – intI1 NDM-1 CTX-M, OXA 6,5, 3, 2 EN5130.T1F E. coli DH10B bla – bla 2 < 0.002 270, 205, 29, 13, 8,–– - NDM-1 CTX-M, OXA - 7,5, 3, 2 EN5135 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla 1.5 16 230, 5, 4, 2, 0.1 FIIK, HIB-M VagCD, intI1 NDM-1 CTX-M, TEM, SHV, OXA EN5135.T1 E. coli J53 Fox-azide bla – bla 1.5 0.004 230 HIB-M –– NDM-1 CTX-M EN5136 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla 3 4 340, 212, 9, 6, 4 FIIK, HIB-M PemK, CcdAB, VagCD, intI1 NDM-1 CTX-M, TEM, OXA Hok-Sok Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 11 of 15 Table 5 Genotypic characterization of NDM-positive isolates transferring only bla gene (Continued) NDM Strain no. Organism NDM PMQR Other Resistance MEM MIC CIP MIC Plasmid size in kb Plasmid type (Inc) Plasmid addiction Integron genes (approximately) system EN5136.T1 E. coli J53 Fox-azide bla – bla 0.75 0.012 340 HIB-M – intI1 NDM-1 CTX-M, TEM EN5137 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla 1.5 32 310,162, 8, 6, 4 FIIK, HIB-M PemK, CcdAB, VagCD, intI1 NDM-1 CTX-M, TEM, OXA Hok-Sok EN5137.T1 E. coli J53 Fox-azide bla – bla 0.75 0.012 310 HIB-M – - NDM-1 CTX-M, TEM EN5139 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla 1 16 250,112,5, 4 FIIK, HIB-M PemK, VagCD intI1 NDM-1 CTX-M, TEM, OXA EN5139.T1 E. coli J53 Fox-azide bla – bla 1.5 0.23 250 HIB-M – - NDM-1 CTX-M, TEM, EN5142 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla 8 2 248,104 FIIK, FIB-M – intI1 NDM-1 CTX-M, TEM, OXA rmtC EN5142.T1 E. coli J53 Fox-azide bla – - 1.5 0.008 248, 104 –– intI1 NDM-1 rmtC EN5144 Klebsiella pneumoniae bla aac(6′)-Ib-cr,qnrB bla 2 6 227, 52, 5, 4 HIB-M – - NDM-1 CTX-M, SHV, OXA armA EN5144.T1 E. coli J53 Fox-azide bla – bla 0.5 0.016 227 HIB-M – - NDM-1 CTX-M, SHV, OXA armA EN5146 Klebsiella pneumoniae bla aac(6′)-Ib-cr bla rmtC, aac(6′)-Ib 10 > 32 192,164, 7 FIA, A/C, FIIK – - NDM-1 CTX-M, EN5146.T1F E. coli DH10B bla – bla aac(6′)-Ib 6 < 0.002 164 A/C – - NDM-1 CTX-M, EN5154 Klebsiella pneumoniae bla aac(6′)-Ib-cr bla 8 > 32 241, 201, 34, 29,8, FIIS, R, FIIK PemK, VagCD IntI1 NDM-1 CTX-M armA 6, 4,3 EN5154.T1F E. coli DH10B bla – bla 6 0.012 240, 4 FIIS – IntI1 NDM-1 CTX-M, EN5175 Klebsiella pneumoniae bla aac(6′)-Ib-cr, qnrB bla 3 4 123, 88,7,5,3 N, FIIK, HIB-M PemK, VagCD IntI1 NDM-1 CTX-M, TEM, OXA armA EN5175.T1 E. coli J53 Fox-azide bla – bla 1.5 0.012 123 HIB-M - - NDM-1 CTX-M, SHV, OXA armA EN5175.T2 E. coli J53 Cip-Azide – qnrB armA 0.023 0.125 96, 50 N, FIIK VagCD - EN5180 Klebsiella pneumoniae bla – armA,rmtC, bla 32 > 32 293,208,5,4 A/C, FIIK, FIB-M, VagCD IntI1 NDM-1 TEM aac(6′)-Ib HIB-M EN5180.T1 E. coli J53 Fox-azide bla – bla 1.5 0.016 208 A/C IntI1 NDM-1 TEM rmtC EN5186 Klebsiella pneumoniae bla qnrB armA, rmtC 6 > 32 235,188,47,5, 3, FII – IntI1,IntI2 NDM-1 EN5186.T1 E. coli J53 Fox-azide bla – bla 1.5 0.008 188 - - IntI1 NDM-1 CTX-M, TEM rmtC EN5131 Enterobacter aerogenosa bla aac(6′)-Ib-cr,qnrB bla rmtC 4 4 205,27,17,7,5 – ccdA/B, hok -sok intI1 NDM-1 CTX-M, OXA, EN5131.T1 E. coli DH10B bla – bla rmtC 2 < 0.002 205,27,7 –– NDM-1 CTX-M, MEM: meropenem, CIP: ciprofloxacin, (-) : absent or untypable (in case of plasmid types), E. coli J53 Fox-azide or ‘.T1’ : transconjugants selected in cefoxitin (10μg/ml)-sodium azide (100μg/ml), E. coli DH10B or ‘.TF’: Transformants selected in cefoxitin (5μg/ml) , ‘.TF-A : Transformants selected in ampicillin (50μg/ml), E. coli J53 Cip-Azide or ‘.T2’ : transconjugants selected in ciprofloxacin (0.06 μg/ml)-sodium azide (100 μg/ml) Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 12 of 15 Fig. 3 Upstream and downstream regions of the bla gene. Structure a was present in 5 isolates, structure b was present in 17 isolates, NDM structure c and d were present in one isolate each and structure e was present in 3 isolates All of the K. pneumoniae isolates had a ble gene which markers and PMQRs . PMQRs do not confer MBL confers resistance to bleomycin in the downstream region high-level resistance to fluoroquinolones, however, their of the bla gene (Fig. 3). presence in clinical isolates is of concern as it in- NDM The Enterobacter aerogenes isolate acquired PMQRs creases the risk of selecting mutations in gyrase and but they were not transferred along with bla topoisomerase genes which results in high-level resist- NDM-1 through transformation. This isolate carried a truncated ance . With the increasing use of fluoroquinolones ISAba125 in the upstream region and ble gene in the both in hospital settings and the community, PMQRs MBL downstream region. Various other resistance determi- can be a palpable threat. In addition to this is the nants (β-lactamases and 16 rRNA methylases) were escalating presence of genes such as bla which NDM-1 transferred along with bla in all the organisms stud- can facilitate the spread of other plasmid-mediated NDM ied (Table 4). genesastheymay be present in the same plasmidor Amongthe plasmidaddictionsystems found(pndC/A, integrons. To the best of our knowledge, this is the first pemKI, ccdA/B, hok-sok, srnB/C, vagC/D,)only pndC/A was study which compares NDM-positive and NDM-negative present in a plasmid which carried bla in one case. Enterobacteriaceae isolates with respect to fluoroquino- NDM-1 lone non-susceptibility and prevalence of PMQRs. Clonality of NDM-possessing E. coli and K. pneumoniae In the studied isolates, fluoroquinolone non-susceptibility isolates was very high (90%). Other studies from India also show a NDM-possessing E. coli isolates (n = 6) were predomin- very high rate of non-susceptibility to ciprofloxacin [30, 31]. antly diverse except for 2 isolates which were indistin- A recent report from India shows that ciprofloxacin guishable [cluster H (EN5132, EN5141] (Fig. 2a). resistance was 15% at Day 1 and 38% in Day 60 in the However, in case of K. pneumoniae, 3 clonal clusters gut flora of antibiotic naïve and exclusively breastfed [cluster I (EN5136, EN5137, EN5139), cluster J (EN5150, neonates . For treatment of neonatal infections, EN5151, EN5163, EN5165, EN5166, EN5170, EN5173, fluoroquinolones are used only as salvage therapy . EN5185) and cluster K (EN5114, EN5117)] were identi- The high prevalence of fluoroquinolone resistance fied and the rest were diverse. (Fig. 2b). Many identical observed in the study is probably a reflection of the isolates expressed different genotypic characteristics high usage of fluoroquinolones to treat other infections (Fig. 2, Tables 4 and 5). such as urinary tract infections (UTI) , as this drug used to be sold in India over the counter without Discussion prescription before 2014 . It is also known that the The spread of antimicrobial resistance is primarily mother’s vaginal flora may be a cause of sepsis (particu- caused by the dissemination of large plasmids carrying larly early onset, the onset of sepsis within 72 h of multiple antibiotic resistance genes . Antibiotic-resist- birth) and mothers may be already harbouring such ant genes, such as bla , are plasmid mediated and resistant organisms . NDM-1 often co-harboured with different antibiotic resistance Forty-seven percent (34/73) of the isolates were markers such as ESBL genes, aminoglycoside resistance NDM-positive. Majority of these possessed bla but NDM-1 Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 13 of 15 isolates harbouring bla bla and a novel in K. pneumoniae, plasmids carrying both bla and NDM-5, NDM-7, NDM variant bla were also detected. The prevalence of PMQRs were of replicon type IncFIIK followed by IncA/C NDM-15 bla is high in India [35, 36] and bla variants and IncN. IncF group plasmids are highly conjugative and NDM-1 NDM have also been reported . are widely distributed in Enterobacteriaceae  and pres- NDM-positive isolates exhibited a higher percentage ence of any gene in this group of plasmids will only escal- (97%) of non-susceptibility towards ciprofloxacin than ate its spread to other organisms. However, plasmid type NDM-negative (85%) but the difference was not statisti- IncHIB-M or an untypable plasmid was mostly associated cally significant. In this study, a significant number of with plasmids carrying bla but not any of the PMQRs. NDM isolates (81%) carried at least one of the PMQRs. However, in E. coli, there were varied plasmid types, no Analysis of the data also revealed that the prevalence of particular type of plasmid predominated. aac(6′)-Ib-cr was highest (71%) followed by qnrB (51%) Fluoroquinolone resistance in Enterobacteriaceae is and qnrS (3%). Earlier studies also support that also caused by the accumulation of mutations, primarily aac(6′)-Ib-cr is the most prevalent PMQR in India [30, 38]. in DNA gyrase (GyrA), and then in topoisomerase IV Although there are currently 81 variants of qnrB and 14 . In our study, most NDM-positive isolates exhibited variants of qnrS according to https://www.ncbi.nlm.nih. mutations in the QRDR region of GyrA and ParC. All of gov/bioproject/PRJNA313047 , we have exclusively these mutations were reported earlier in various studies found only qnrB1 and qnrS1. Theprevalenceof . Four K. pneumoniae isolate and one Enterobacter aac(6′)-Ib-cr was significantly higher in K. pneumoniae cloacae carried PMQRs but lacked mutations in the than E. coli. qnrB and qnrS were absent in E. coli. The QRDR region of GyrA and ParC, yet the isolates exhib- higher prevalence of PMQRs in K. pneumoniae compared ited non-susceptible MIC values against ciprofloxacin. to E. coli can betheresult ofthepresenceofmore clonal This indirectly points to the well-studied phenomenon isolates of K. pneumoniae than E. coli. Theprevalenceof that in the absence of chromosomal mutations PMQRs OqxAB was quite high as they are mostly chromosomally plays an important role in increasing the MIC against located in K. pneumoniae . ciprofloxacin, thus providing an opportunity to the Co-occurrence of PMQRs and bla were reported bacteria to generate chromosomal mutation . NDM in many earlier studies [5, 6, 21]. In this study, 40% (29/ 73) isolates co-harboured NDM and PMQRs. Although Conclusion the prevalence of aac(6′)-Ib-cr, qnrB, and qnrS were This study indicates that fluoroquinolone resistance is generally higher in NDM-positive isolates than NDM- high in neonatal septicaemic isolates. PMQRs are highly negative isolates the difference was not statistically prevalent, aac(6′)-Ib-cr and qnrB are predominant. Car- significant. Hence, probably the spread of PMQRs is not bapenem resistance in the same set of isolates is primar- dependent on the bla spread. The higher prevalence NDM ily due to bla However, we infer that the spread of NDM-1. of PMQRs (81%) per se in comparison to NDM (47%) is PMQRs is independent of the spread of bla as the NDM-1 also indicative of this. The occurrence of PMQRs along prevalence of PMQRs in non-NDM isolates were nearly with β-lactamases has also been reported in several similar to the NDM isolates. The possibility of indis- studies [6, 40]. It is to be noted that β-lactamases are criminate fluoroquinolone use in escalating the spread highly prevalent in the study isolates and could have of bla cannot be ruled out. Co-occurrence of NDM-1 contributed to the spread of PMQRs. PMQRs with bla in an isolate does not necessarily NDM Co-transfer of PMQRs along with bla in single NDM result in co-transfer of the resistance genes due to their large plasmids co-harbouring many other resistance presence mostly in different plasmids. However, the genes have been shown in other studies [6, 21, 27]. The presence of genes such as bla and PMQRs shows NDM-1 transfer of bla along with qnrB, qnrS, aac(6′)-Ib-cr NDM that the window for treatment options are gradually and various other resistance markers (16S rRNA methyl- decreasing and transmissible genes are a threat. ases and other β-lactamases genes) were studied. This study showed that of the 29 isolates which co-harboured Additional file NDM and PMQRs, only 12 isolates showed co-transmis- sion of these genes which indicates that not all isolates Additional file 1: Detailed information of all studied possessing PMQRs co-transferred the gene with bla Enterobacteriaceae : MIC values of meropenem and cirpfloxacin, antibiotic NDM susceptibility pattern and distribution of different resistance genes. (XLSX because of their probable location on different plasmids. 28 kb) Worldwide studies on the plasmid types show that IncFII, IncN, IncL/M, IncHIB-M/IncFIB-M, IncA/C, and Abbreviations untypable plasmids carry blaNDM . PMQRs are NDM: New Delhi metallo-β-lactamase; PBRT: PCR-based replicon typing; associated with IncN, IncL/M, IncFII, IncHI1, IncI1, IncR, PFGE: Pulsed-field gel electrophoresis; PMQR: Plasmid-mediated quinolone colE type plasmids . In this study, we have found that resistance; QRDR: Quinolone resistance determining region Mitra et al. Antimicrobial Resistance and Infection Control (2019) 8:46 Page 14 of 15 Acknowledgments 8. Roy S, Viswanathan R, Singh AK, Das P, Basu S. Sepsis in neonates due to We extend our thanks to George A. Jacoby for providing PCR controls. We imipenemresistant Klebsiella pneumoniae producing NDM-1 in India. J also thank the staff of the Department of Neonatology, SSKM Hospital, who Antimicrob Chemother. 2011;66:1411–3. cared for the neonates included in the study and Mr. Subhadeep De for his 9. Clinical and Laboratory Standards Institute. Performance standards for laboratory assistance. antimicrobial susceptibility testing; twenty-fourth informational supplement. 2014. Transparency declarations 10. European Committee on Antimicrobial Susceptibility Testing. Breakpoint None to declare. tables for interpretation of MICs and zone diameters v 3.1, 2013. 11. Ellington MJ, Kistler J, Livermore DM, Woodford N. Multiplex PCR for rapid Funding detection of genes encoding acquired metallo-β-lactamases. J Antimicrob The study was supported by Department of Science and Technology, West Chemother. 2007;59:321–2. Bengal extramural funding and Indian Council of Medical Research (ICMR) 12. Woodford N, Tierno PM Jr, Young K, Tysall L, Palepou MF, Ward E, Painter intramural funding. S. M and S. N were recipients of Senior Research RE, Sube DF, Shungu D, Silver LL, Inglima K, Kornblum J, Livermore DM. Fellowship from Council of Scientific and Industrial Research, India and Outbreak of Klebsiella pneumoniae producing a new carbapenem- Junior Research Fellowship from the Department of Science and Technology, hydrolyzing class a β-lactamase, KPC-3, in a New York medical center. West Bengal respectively. Antimicrob Agents Chemother. 2004;48:4793–9. 13. Poirel L, Héritier C, Tolün V, Nordmann P. Emergence of oxacillinase- Availability of data and materials mediated resistance to imipenem in Klebsiella pneumoniae. Antimicrob The dataset supporting the conclusions of this article is included within the Agents Chemother. 2004;48:15–22. article and its Additional file 1. 14. Saladin M, Cao VTB, Lambert T, Donay JL, Herrmann JL, Ould-Hocine Z, et al. Diversity of CTX-M β-lactamases and their promoter regions from Authors’ contributions Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett. Conceived and designed the experiments: SB, Performed the experiments: 2002;209:161–8. 1 1 2 SM , SN. Analysed the data: SM ,SM , SD, SB. Contributed reagents/ 15. Colom K, Pérez J, Alonso R, Fernández-Aranguiz A, Lariño E, Cisterna R. materials/analysis tools: SD, SB, SM , PC. Contributed to the writing of the Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHVand manuscript: SM , SB and SD. 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