Emergence of Azithromycin Resistance Mediated by Phosphotransferase-Encoding mph(A) in Diarrheagenic Vibrio fluvialis

The progressive rise in antibiotic resistance among enteric pathogens in developing countries is becoming a big concern. India is one of the largest consumers of antibiotics, and their use is not well regulated. V. fluvialis is increasingly recognized as an emerging diarrheal pathogen of public health importance. Here we report the emergence of azithromycin resistance in V. fluvialis isolates from diarrheal patients in Kolkata, India. Azithromycin has been widely used in the treatment of various infections, both in children and in adults. Resistance to azithromycin is encoded in the gene mph(A). Emerging azithromycin resistance in V. fluvialis is a major public health challenge, and future studies should be focused on identifying ways to prevent the dissemination of this antibiotic resistance gene.

this pathogen have been reported worldwide (1-4). The molecular characterization of this pathogen has given several insights, including its clonality and multidrug resistance (MDR) features (2,4). The emergence and spread of antibiotic resistance represent one of the most important public health concerns and are correlated with the inappropriate use of antimicrobial agents, which results in increased mortality, morbidity, and health care costs (5). Antibiotic resistance arises through several modes, such as transfer of plasmids, integrons, and transposons, mutations in target genes, and the overexpression of efflux systems (5). The rapid increase and spread of antibiotic resistance in V. fluvialis during the past 20 years are a major cause of concern because of the organism's ability to cause epidemics (6).
We have reported the existence of extended-spectrum ␤-lactamases (ESBLs) and fluoroquinolone-acetylating aminoglycoside-6=-N-acetyltransferase in V. fluvialis, which confers the resistance to fluoroquinolones and ␤-lactam antimicrobials (7), including bla NDM-1 -mediated carbapenem resistance (8). Here we report the identification and characterization of an azithromycin resistance (AR) macrolide 2 P-phosphotransferase I-encoding gene, mph(A), in 28 isolates of V. fluvialis (AR-VF) isolated during 2014 to 2015 from hospitalized patients in Kolkata, India. Azithromycin belongs to the macrolide class of antibiotics, which are primarily used to treat infections caused by Grampositive microorganisms. This antibiotic is active also against several Gram-negative organisms (9). Considering the emergence of MDR enteric bacteria, azithromycin is recommended for the treatment of diarrhea (10,11). Isolates with higher MICs of azithromycin were found to harbor mph(A) (12). From the previous studies, it is known that the mph(A) gene has been disseminated among different pathogens, like Escherichia coli, Salmonella spp., and Shigella spp. (13,14). The present study was undertaken to understand the azithromycin resistance mechanisms in V. fluvialis isolates from diarrheal patients in Kolkata.

RESULTS
During 2014 to 2015, a total of 48 V. fluvialis were isolated from 2,308 (1,135 and 1,173 received in 2014 and 2015, respectively) stool specimens of acute diarrheal patients, of which 28 (58%) were found to be resistant to azithromycin. The isolation rates of AR-VF were 19% in 2014 (9 of 48 isolates) and 39% (19 of 48 isolates) in 2015. Patients from whom V. fluvialis was isolated presented cholera-like diarrhea, i.e., with watery stool (67%), severe dehydration (14%), and abdominal pain (50%) ( Table 1). Out of the 28 isolates of AR-VF (each strain representing a case), 15 (54%) were isolated as the sole pathogen and the remaining 13 (46%) were recovered along with other pathogens, such as Vibrio cholerae, diarrheagenic Escherichia coli (DEC), Shigella spp., Salmonella, Campylobacter spp., parasites, and viruses. Among the polymicrobial etiology cases, V. fluvialis along with V. cholerae O1 were found to be present at a relatively high proportion (14%), followed by DEC and Shigella spp. (11% each). AR-VF infection was more often detected in adults (82%) than in children Ͻ5 years of age (18%). The other enteric pathogens isolated along with AR-VF remained susceptible to azithromycin (data not shown).
Pulsed-field gel electrophoresis (PFGE) was performed to investigate the genetic relationship between AR-VF isolates. Twenty-four different PFGE patterns were detected (data not shown). Distinct lineages were identified between the isolates and MDR patterns, suggesting that the AR-VF isolates were genetically diverse.

DISCUSSION
Globally, multidrug resistance in enteric bacterial pathogens is an emerging threat to public health (5). Infection caused by V. fluvialis has been increasingly reported worldwide (6), with frequent documentation of MDR (15,16). The prevalence of MDR in V. fluvialis organisms varies depending on the country and the source of isolation. A study from South Africa reported the prevalence of V. fluvialis resistant to ampicillin, penicillin G, streptomycin, sulfamethoxazole, trimethoprim, chloramphenicol, erythromycin, ciprofloxacin, and polymyxin B (1). The presence of a relatively high number of   Azithromycin has been recommended for the treatment of acute diarrhea caused by Campylobacter spp., DEC, Shigella spp., nontyphoidal Salmonella, and sometimes V. cholerae (17). Due to the wide use of this antibiotic, resistance or reduced susceptibility to azithromycin has increased in several enteric pathogens, such as Escherichia coli, Salmonella spp., and Shigella spp. (12,18,19).
The AR-VF isolates were characterized using phenotypic and molecular techniques to understand the mechanism of resistance and their clonal relationships. Several studies have already reported that macrolide resistance has been acquired through a variety of mechanisms. In many Gram-negative bacteria, macrolide resistance is primarily involved with target site modification by methylases encoded by erm [erm(A), erm(B), and erm(C)] genes, macrolides inactivated by modifying esterase enzyme [ere(A) or ere(B)] genes, phosphotransferases encoded by mph [mph(A) and mph(B)] genes, and acquisition of efflux pump [mef(A) and msr(A)] genes. The association of these genes with complete cross-resistance of erythromycin and azithromycin has been reported frequently (12,13,20). Considering this, we have targeted these nine genes for macrolide resistance while screening the V. fluvialis isolates that showed resistance to azithromycin. We detected mph(A) in 28 isolates and ere(A) in 1 isolate; no other macrolide resistance-encoding genes were detected. Twenty-eight of 48 isolates examined in this study harbored the mph(A) gene and had azithromycin MICs between 4 and Ͼ256 mg/liter. One isolate that had both the ere(A) and mph(A) genes showed an azithromycin MIC of Ͼ256 mg/liter. These results indicate that carriage of mph(A) alone can confer higher azithromycin resistance in V. fluvialis. This is similar to previous observations made in Shigella spp. and Salmonella spp. (13,14). All the AR-VF isolates possessed class 1 integrons, of which 17 had dfrA1 and orfC. The presence of the dfrA1 gene cassette, coding for trimethoprim resistance in V. fluvialis, has been reported previously (7,15). As reported before (16), the vfspb gene, which encodes periplasmic pectic oligomer binding protein (Sbp), was found in the class 1 integrons of six AR-VF isolates. A 2.5-kb amplicon obtained from 2 AR-VF isolates (IDH 06263 and IDH 06279) showed the presence of arr2 and cm1A5, coding for rifampin and chloramphenicol resistance. Interestingly, the presence of these genes has been reported for the integrons of Acinetobacter baumannii isolates from East Africa (21). The major ESBL genes, like bla OXA-1 (96%), bla OXA-7 (93%), bla TEM-9 (68%), and bla CTX-M-3 (68%), were identified in most of the AR-VF isolates. This observation further suggests that the major ESBL groups are prevalent in V. fluvialis in Kolkata (7). Since these ESBL genes are generally located on antimicrobial resistance plasmids, they can easily be disseminated between different species of bacteria (22). The other enteric pathogens isolated along with AR-VF were susceptible to azithromycin. This indicates the genomic suitability of V. fluvialis in the quick acquisition of the mph(A) gene.
In this study, we have shown that most of the AR-VF organsims isolated in Kolkata harbor various antimicrobial resistance-encoding genes. AR-VF isolates belonged to different lineages, indicating their diverse sources of infection. This is a cause of major concern, as multidrug resistance could pose a challenge both for the treatment and prevention of spread of infections caused by V. fluvialis. It is important to monitor the genes responsible for the resistance to azithromycin and other antimicrobials in V. fluvialis and other enteric bacteria isolated as copathogens.

MATERIALS AND METHODS
Bacterial isolates. Twenty-eight azithromycin-resistant V. fluvialis isolated from patients with cholera-like diarrhea admitted to Infections Diseases, Kolkata, between 2014 and 2015 were included in this study. V. fluvialis was isolated from stool specimens or rectal swabs in Cary-Blair medium using thiosulfate-citrate-bile salts-sucrose agar (TCBS), followed by overnight incubation at 37°C. Yellow colonies on the TCBS plates were examined by oxidase test, string test using 0.5% sodium deoxycholate solution, biochemical testing (ID 32GN; bioMérieux, Marcy l'Etoile, France) and by a species-specific V. fluvialis toxR PCR (4).
Antimicrobial susceptibility testing. Antibiotic susceptibility testing was done using the Kirby-Bauer method according to the Clinical and Laboratory Standards Institute (23). Antibiotic discs (BD, USA) used were meropenem (10 g), ceftazidime (CAZ; 30 g), cefotaxime (CTX; 30 g), cefepime (30 g), ampicillin (10 g), azithromycin (15 g), ceftriaxone (30 g), chloramphenicol (30 g), ciprofloxacin (5 g), co-trimoxazole (25 g), erythromycin (15 g), gentamicin (10 g), kanamycin (30 g), neomycin (30 g), tetracycline (30 g), nalidixic acid (30 g), norfloxacin (10 g), ofloxacin (5 g), and streptomycin (300 g). Each isolate was inoculated in Mueller-Hinton broth (Difco) and incubated at 37°C for 3 h. The turbidity of the suspension was adjusted to 0.5 McFarland standard, and the suspension was then spread on a Mueller-Hinton agar (Difco) plate using a sterile swab. The plates were incubated at 37°C overnight after placement of the antimicrobial susceptibility test discs. The zone of inhibition was measured and interpreted as per the CLSI guidelines (23). Extended-spectrum-␤-lactamase (ESBL) production was confirmed by double-disc synergy testing with CAZ and CTX. An increase of 5 mm in the zone diameter for CAZ or CTX compared to that for clavulanic acid was considered for production of ESBL. The MICs of meropenem, ciprofloxacin, norfloxacin, ceftazidime, cefotaxime, and cefepime were determined using Etest (AB Biodisk, Solana, Sweden), following the manufacturer's instructions. Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as quality control strains.
Sequencing. Amplified PCR products were extracted from the gel after electrophoresis and were purified using a QIAquick gel extraction kit (Qiagen) as per the manufacturer's instructions. Purified DNA was analyzed on a 1.5% agarose gel. Sequencing reactions were performed using a BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). The dye terminators from sequencing reactions were removed using a DyeEx 2.0 spin kit (Qiagen). Purified sequences were analyzed using an