Carbapenem Resistance in Clonally Distinct Clinical Strains of Vibrio fluvialis Isolated from Diarrheal Samples

These strains might acquire the blaNDM-1 gene without exposure to antimicrobial drugs.

hours, accompanied by dehydration, nausea, vomiting, abdominal cramping, fever, chills, muscle aches, and fecal urgency. A questionnaire that collected demographic information, illness onset and symptoms, medical care sought, and food/drink consumed was completed by the patient or a family member. Patients with other associated illness and who used antibiotic drugs before hospitalization were not included in this study.
Fecal specimens were collected in McCartney bottles (KM Enterprises, Kolkata, India) by using sterile catheters or rectal swabs in Cary Blair medium (Difco, Sparks, MD, USA) and were examined within 2 hours for enteric pathogens comprising bacterial, viral, and parasitic pathogens by using a combination of conventional, immunological, and molecular methods (16). Patients were observed until their discharge from the hospital. The patients lived in different areas of the Kolkata Municipal region.
We performed the modified Hodge test on Mueller-Hinton agar (Difco) plates, using E. coli ATCC 25922 as the indicator organism and a 10-μg imipenem disk (21). The modified Hodge test is a phenotypic assay for the detection of carbapenemase enzyme-producing bacteria. This assay is based on the inactivation of a carbapenem by carbapenemase-producing test isolates that facilitate a carbapenemsusceptible indicator strain (E. coli ATCC 25922) to spread its growth toward a carbapenem-containing disc along the streak of inoculum of the test isolate. A positive test result produces a cloverleaf-like hollow.
We used the Kado and Liu method (22) to extract plasmid DNA from donors, recipients, and transconjugants and analyzed it by gel electrophoresis using 0.8% agarose. We used a PCR-generated DNA probe by the chemoluminescent method (ECL nucleic acid detection system; GE Healthcare Life Sciences, Buckinghamshire, UK) to make Southern hybridization to confirm the presence of bla NDM-1 in the plasmids. Plasmid-mediated transfer of antibiotic resistance from a NDM-1-positive V. fluvialis isolate (IDH 04744) to E. coli J53 (having dual resistant markers for nalidixic acid and sodium azide [Na-Az R ]) was tested on MacConkey agar plates (Difco) containing sodium azide (100 mg/L) and meropenem (5 mg/L). Another plasmidmediated transfer of antibiotic resistance from a NDM-1-positive V. fluvialis isolate (IDH 04744) has also been tested with diarrheagenic E. coli, Salmonella spp., and Shigella spp. and V. parahaemolyticus on meropenem (5 mg/L) supplemented MacConkey, xylose lysine deoxycholate, and thiosulfate citrate bile sucrose agar (Difco) plates.
We determined the replicon types of bla NDM-1 harboring plasmids from the wild isolates and transconjugants by PCR using published methods (25). Sequencing of the bla NDM-1 and its flanking regions were made from a wild isolate of V. fluvialis (IDH 05720) by primer walking. The DNA sequence reported in this study has been deposited in GenBank (accession no. KR733543).
Pulsed-field gel electrophoresis (PFGE) analysis of No-tI-digested genomic DNA of bla NDM-1 -harboring V. fluvialis isolates (NDM-VF) was performed by using a CHEF-Mapper (Bio-Rad Laboratories, Hercules, CA, USA) according to the PulseNet standardized protocol for subtyping of V.
cholerae (26). The PFGE image was captured by using a Gel Doc XR system (Bio-Rad). The PFGE image was normalized by aligning the peaks of the XbaI size standards of Salmonella enterica serovar Braenderup (H9182) in each gel and was analyzed by using BioNumerics software version 4.0 (Applied Maths, Sint-Martens-Latem, Belgium). The similarities between isolates were evaluated by using the cluster analysis with the UPGMA method and the Dice correlation coefficient with a position tolerance of 1.5%.

Results
A total of 115 V. fluvialis were isolated from the acute diarrheal patients (each isolate represent a case), of which 27 (23.5%) were resistant for carbapenem and harbored The rest of the pathogens tested in this study were susceptible to carbapenem.
The bla NDM-1 -harboring V. fluvialis isolates carried multiple plasmids ranging from 5 kb to 150 kb. In Southern hybridization, the large plasmids extracted from the transconjugants were positive for bla NDM-1 . In the transconjugants, only a single plasmid of ≈80-90 kb was detected. The transconjugant (TC-J53) also showed resistance to ampicillin, erythromycin, streptomycin, ceftriaxone, cefotaxime, cefuroxime, and imipenem (MIC 2 mg/L), indicating the possibility that the NDM-1 plasmid also harbored genes encoding resistance to these antibiotics. The transconjugant was susceptible to ciprofloxacin, tetracycline, trimethoprim, chloramphenicol, and azithromycin, suggesting that the genes encoding resistance to these drugs are not carried by the bla NDM-1 -harboring plasmid. Most of the other enteric pathogens used as transconjugants showed resistance to ampicillin, ceftriaxone, cefotaxime, and sulfamethoxazole. The transfer frequencies ranged from 1.4 × 10 3 to 8.7 × 10 5 (Table 2).
In replicon typing, plasmids of NDM-VF isolates were untypeable. To gain insight into the genetic background of bla NDM-1 , the flanking regions of this gene were examined in a representative V. fluvialis isolate (IDH 04744). bla NDM-1 flanking sequences of IDH 04744 V. fluvialis were identical to the ones reported in the E. coli isolates from Hong Kong, China (pNDM-HK; GenBank accession no. HQ451074), and from a Spanish traveler returning from India (DVR22; GenBank accession no. JF922606.1) (Figure 1). The left junction of the sequences starts upstream of the bla NDM-1 with a truncated ISAba125 region, whereas the right junction possessed different genes such as ble MBL (bleomycinresistance encoding gene), trpF, bla DHA-1 , and ampR.
Eighteen different patterns that could be grouped into 2 distinct clusters (A-C; Figure 2) were obtained in the PFGE analysis. Most of the isolates in cluster B had ≈90%-100% similarity. Nearly identical PFGE profiles were obtained for 11 isolates (cluster B). These isolates were isolated over a span of 1 year (May 2011-May 2012), without any epidemiologic link. We also found no correlation between the PFGE and antimicrobial drug resistance patterns.

Discussion
Since its discovery, global distribution of bla NDM-1 in different bacterial species has been extensively documented (27). NDM-1 producers are reported not only from patients epidemiologically linked to the Indian subcontinent but also from several indigenous cases all over the world with no such link. Previously, we reported on the emerging trend of V. fluvialis among the diarrheal cases in the Kolkata region (14). However, NDM-VF emerged in Kolkata during 2011, and 25%-50% of the V. fluvialis isolates harbored bla NDM-1 each year until 2013. It is difficult to epidemiologically link the isolates because of the wide difference in the dates of isolation of NDM-VF, lack of common food sources, and variation in the proximity of the residential area of the patients; antibiogram and PFGE patterns are also widely divergent.
NDM-1 producers have been found to be highly resistant to several classes of antibiotics (28)(29)(30), related to their unusual genetic assemblage, which helps in the acquisition and transfer of many resistance genes. Environmental strains of Aeromonas caviae and V. cholerae were found to carry bla NDM-1 on the chromosomes (12). In contrast, we found that, in NDM-VF, bla NDM-1 is present on the large plasmids. Generally, the emergence of NDM-1 producers is associated with excessive use of carbapenems in patients with nonintestinal infections that necessitate a prolonged stay in a hospital. However, none of the patients in this study had a history of using carbapenem drugs. Most NDM-VF isolates remained susceptible to azithromycin, which is currently used in the treatment of diarrheal patients in Kolkata.
We found that a large plasmid from NDM-VF was effectively transferred to E. coli J53 and other enteric pathogens.    The resistance profiles of ampicillin, ceftriaxone, trimethoprim/sulfamethoxazole, cefuroxime, and cefotaxime have been transferred to all the transconjugants. This indicates that the bla NDM-1 -positive isolates may carry similar plasmids with the uniform resistance genes and, hence, confer the same resistance phenotype. Generally, the conjugative plasmids carrying bla NDM-1 have been classified into several replicon types, including IncA/C, IncFII Y , IncHI1b, IncX3, and IncT (32). However, the NDR-VF isolates were negative for all the NDM-1 plasmids in the PCR-based replicon typing. These results suggest that the NDM-1-encoding genes move with several plasmid scaffolds or as the same Inc type, which might not be covered by the currently used replicon typing scheme of Enterobacteriaceae. In many bacterial species from India, the bla NDM-1 -harboring plasmids were found to belong to A/C-type, an uncommon group for conferring multidrug-resistant phenotypes (3).
Analysis of the genes adjoining the bla NDM-1 in V. fluvialis isolate IDH 04744 revealed a high homology with E. coli NDM-HK and DUR-22 (GenBank accession nos. HQ451074 and JF922606) (33,34). Insertion sequences (IS) IS26 and ISAba125 have been identified upstream of the bla NDM-1 gene, and these sequences have been reported in other organisms. In most of the NDM-1-positive bacteria, the IS elements are detected in the flanking regions of bla NDM-1 . We detected the IS26 and ISAba125 in the upstream of the bla NDM-1 gene. The presence of IS26, ISCR1, and transposases have been increasingly implicated in interspecies and intraspecies dissemination of antimicrobial drug resistance genes (35,36). These IS elements probably help in the mobility of bla NDM-1 .
We also identified the ble MBL gene downstream of bla NDM-1 . In most of the Enterobacteriaceae, bla NDM-1 has been detected between a truncated ISAba125 located upstream and ble MBL at the downstream. This genetic arrangement suggests an en bloc acquisition of bla NDM-1 and ble MBL through the ISAba125-related mobilization system. The presence of ble MBL appears to be an added advantage to the bla NDM-1 -positive bacteria, because both genes are expressed under the control of single promoter; therefore, the presence of ble MBL may help the bla NDM-1 -bearing plasmids to spread in other bacterial species (37).
V. fluvialis is increasingly being detected in our setting and among diarrheal patients (14). These V. fluvialis isolates are capable of readily acquiring antibiotic resistance genes through mobile genetic elements (38). Our findings indicate that V. fluvialis might acquire the bla NDM-1 gene without any antibiotic selective pressure. This pathogen also has the potential to transfer this gene to other enteric pathogens. PCR-based identification of the NDM-1 regions in suspected pathogens will be very useful. The V. fluvialis isolates harboring bla NDM-1 are mostly susceptible to doxycycline and azithromycin. Considering the pathogenicity of V. fluvialis to humans and its ubiquitous presence in the environment, the need for constant monitoring of this Vibrio species is ongoing.