Antimicrobial resistance, virulence profiles and molecular subtypes of salmonella enterica serovars typhi and paratyphi a blood isolates from kolkata, india during 2009

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Antimicrobial Resistance, Virulence Profiles và Molecular Subtypes of Salmonella enterica Serovars Typhi và Paratyphi A Blood Isolates from Kolkata, India during 2009-2013
Antimicrobial Resistance, Virulence Profiles & Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013 Shanta Dutta, Surojit Das, Utpala Mitra, Priyanka Jain, Indranil Roy, Shelley S. Ganguly, Ujjwayini Ray, Phalguni Dutta, Dilip Kumar Paul

Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was lớn characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 lớn June 2013 và were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance và virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) & 25 Salmonella enterica serovar Paratyphi A (S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to lớn chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) và absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non-IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (blaTEM-1, catA, sul1, sul2, dfrA15, strA-strB) và class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR & sequencing. Most of the study isolates were likely to be virulent due khổng lồ the presence of virulence markers. Major diversity was not noticed among S. Typhi và S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles và S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR & molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology.

Citation: Dutta S, Das S, Mitra U, Jain P, Roy I, Ganguly SS, et al. (2014) Antimicrobial Resistance, Virulence Profiles và Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013. binhphap.vn ONE 9(8): e101347. Https://doi.org/10.1371/journal.pone.0101347

Editor: Nicholas J. Mantis, thủ đô new york State Dept. Health, United States of America

Received: November 19, 2013; Accepted: June 5, 2014; Published: August 6, 2014

Funding: The study was funded by the Indian Council of Medical Research (ICMR) intramural fund. Senior Research Fellowships from Indian Council of Medical Research to lớn Mr. S. Das & Ms. P Jain are gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision lớn publish or preparation of manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Enteric fever caused by Salmonella enterica serovar Typhi (S. Typhi) & Salmonella enterica serovar Paratyphi A (S. Paratyphi A) still continues lớn be a major public health problem in developing countries lượt thích India. Typhoid fever was estimated lớn have caused 21.6 million illnesses and 216,500 deaths globally, và the less severe paratyphoid fever caused an estimated 5.4 million illnesses in 2000 <1>. The incidence of typhoid fever was estimated as 214.2 per 100,000 people/year during 2003–2004 in a population-based study conducted at different urban slums of Kolkata <2>. Although S. Typhi was observed khổng lồ be the predominant serovar worldwide, a shift in the most prevalent Salmonella serotype from S. Typhi lớn S. Paratyphi A has been reported recently by many researchers <3>–<6>.

Antimicrobial therapy is the mainstay for treatment of enteric fever and typhoid fever may become fatal in 30% cases due lớn complications in absence of appropriate antibiotics. Since the late eighties, increased isolation of multidrug resistant (MDR, resistant to lớn ampicillin, chloramphenicol and co-trimoxazole with or without tetracycline) S. Typhi was reported from different parts of India limiting the use of these drugs in the treatment of typhoid <7>, <8>. Subsequently ciprofloxacin was used as the drug of choice in adults. But frequent use of ciprofloxacin led to lớn the global emergence of nalidixic acid resistant S. Typhi & S. Paratyphi A associated with decreased susceptibility lớn ciprofloxacin, causing increased treatment failure cases <6>, <9>–<12>. Thereafter emergence of ciprofloxacin resistant S. Typhi and S. Paratyphi A further decreased the number of treatment options <10>, <13>, <14>. Sporadic reports of Salmonella enterica isolates resistant khổng lồ higher generation cephalosporins lượt thích ceftazidime, cefotaxime, cefuroxime erased even the last option for treatment of enteric fever <15>–<17>. Azithromycin has been used as an effective alternative in uncomplicated cases, but resistance lớn this antibiotic has been reported from India & other countries <18 19, 20>. Knowledge on antimicrobial resistance (AMR) profiles of Salmonella enterica blood isolates is important for predicting the best possible treatment option for enteric fever patients of any region. Many genes involved in AMR are found on mobile elements <21>, <22>, & are therefore likely khổng lồ be transferred between bacterial pathogens, which has a significant public health impact.

S. Typhi & S. Paratyphi A are intracellular pathogens possessing a myriad of virulence factors which contribute in disease progression & severity. The virulence genes are generally distributed on Salmonella pathogenicity islands (SPIs), which are large genomic regions of 3–150 kb acquired horizontally <23>. Virulence ren profiles of the local isolates could shed light on the virulence types of the organisms & predict clinical sequels of the disease.

Molecular typing of any organism is necessary to determine the relatedness among the isolates và to study the molecular epidemiology of the organism. Pulsed Field Gel Electrophoresis (PFGE) is used as a standard and most popular molecular tool for outbreak investigations và tracking the route of transmission <24>. Only a few studies have shown association of AMR profiles and PFGE subtypes of Salmonella enterica isolates <5>, <25>–<27>, but reports are lacking from the Indian subcontinent.

In view of this background information, the present study was undertaken khổng lồ characterize the Salmonella enterica blood isolates from Kolkata during 2009 to lớn 2013 with respect to their AMR profiles, plasmid profiles, virulence genes profiles and PFGE subtypes.

Ethics statement

The present study was reviewed và approved by the Institutional Ethical Committee of National Institute of Cholera và Enteric Diseases (NICED), Kolkata. Blood samples were collected from febrile children after receiving informed written consent from their parents or guardians on behalf of the children enrolled.

Collection of Salmonella enterica blood isolates from a hospital based prospective study.

Children (<28>. Serovar confirmation was done by serological agglutination using Salmonella poly- & monovalent antisera (Denka Seiken co Ltd., Tokyo, Japan). On 7th day of incubation, blood culture was read as negative for Salmonella, if there was no visible growth.

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Collection of Salmonella enterica blood isolates from other hospitals in Kolkata.

Salmonella enterica blood isolates were also collected for the study from the following three other hospitals in Kolkata during the study period: Calcutta Medical Research Institute (CMRI), Apollo Gleneagles Hospital (AGH) và Advance Medical Research Institute (AMRI) located in different parts of Kolkata. All the collected isolates were confirmed as Salmonella enterica by microbiological methods as described earlier.

Antimicrobial susceptibility testing và determination of Minimum Inhibitory Concentration

Antimicrobial susceptibility testing was performed by Kirby Bauer disc diffusion method using the following 17 antimicrobial discs: ampicillin (10 µg), chloramphenicol (30 µg), co-trimoxazole (25 µg), tertracycline (30 µg), streptomycin (10 µg), amoxicillin/clavulanic acid (20/10 µg), amikacin (30 µg), gentamicin (10 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), levofloxacin (5 µg), ceftriaxone (30 µg), ceftazidime (30 µg), cefotaxime (30 µg), aztreonam (30 µg) and azithromycin (15 µg). Interpretation was done according lớn the Clinical & Laboratory Standards Institute (CLSI) 2013 guidelines <29>. Escherichia coli ATCC 25922 was used as control. The minimum inhibitory concentrations (MICs) of the antibiotics were determined by E-test (AB Biodisk, Solna, Sweden). Decreased susceptibility khổng lồ ciprofloxacin was defined as isolates having MIC of ciprofloxacin 0.125–0.5 µg/ml. Strains with MIC ≥1 µg/ml were defined as ciprofloxacin resistant. MIC breakpoints of streptomycin và azithromycin were not published for Enterobacteriaceae in CLSI 2013. Streptomycin resistance was defined at MIC ≥64 µg/ml according to lớn the US National Antimicrobial Resistance Monitoring System-USDA (http://www.ars.usda.gov/Main/docs.htm?docid=6750). The “epidemiological cutoff” for resistance khổng lồ azithromycin in Salmonella was documented at >16 µg/ml <30>.

Plasmid extraction & incompatibility typing

Extraction of heavy plasmids of all the study isolates was done by Kado và Liu method <31>, & E. Coli V517 và Shigella flexneri YSH6000 reference strains were used as molecular weight markers. GenElute Plasmid Miniprep kit (Sigma, St. Louis, MO) was used for extraction of smaller plasmids and supercoiled plasmid DNA (2 to 10 kb) was used as the standard marker. Plasmids were separated by electrophoresis on 0.8% agarose gels và were visualized with a UV-transilluminator after staining the gel with ethidium bromide (0.5 µg/ml). Plasmid incompatibility typing of S. Typhi isolates was performed by PCR using published primers of the 18 major incompatibility groups (FIA, FIB, FIC, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, FIIA including IncHI1 type) among Enterobacteriaceae <32>.

Determination of antimicrobial resistance gen markers by PCR

MDR Salmonella study isolates were screened for the presence of following resistance genes by PCR using published primers (Table 1): blaTEM, blaSHV and blaOXA genes for β-lactamase; cat ren for chloramphenicol resistance; sul1, sul2, sul3 & dfr genes for sulphonamide và trimethoprim resistance; tetA và tetB for tetracycline resistance; aadA, strA and strB genes for streptomycin resistance and integrase int1 ren for the presence of class 1 integron. The ren cassette within the variable region of class 1 integron & the presence of qacEΔ1 gen at 3′ over of the conserved segment were also determined <22>, <33>, <34>. Alleles of blaTEM, cat, dfr, aadA genes and the resistance gene cassettes of class 1 integron were determined by direct sequencing of the PCR products using a 3730 DNA analyzer (Applied Biosystem, Foster City, CA, USA). Sequences obtained were analyzed by comparison with the National Centre for Biotechnology Information (NCBI) database sequences using BLAST program. Suitable positive & negative controls were included in each run.

Conjugation experiment for antimicrobial resistance transfer

We performed conjugation experiments khổng lồ determine whether antimicrobial resistance markers were located on conjugative plasmids by using wild MDR Salmonella isolates as donor & commonly used E. Coli K-12 MG1655 (susceptible khổng lồ all antibiotics) as recipient strain <34>, <35>. Donor và recipient strains at exponential phase were grown in Trypticase soy broth (Difco, Sparks, MD, USA) & were mixed & incubated at 37°C for đôi mươi h. Transconjugants were selected as lactose fermenting colonies on MacConkey agar plates containing following antibiotics: ampicillin (50 µg/ml), chloramphenicol (50 µg/ml) or tetracycline (16 µg/ml). Antimicrobial susceptibilities of the transconjugants were tested by disc diffusion, MICs by E-test and resistance markers were determined by PCR as stated earlier.

Detection of virulence ren markers by PCR

A total of 12 virulence markers, located on và outside SPIs were screened by PCR using suitable published primers or primers designed in this study. The virulence genes selected for screening the isolates were invA, ssaQ, spi4D, viaB, stn, hilA, ssrB, mgtC, sopB, sciN, safB and pltA <23>, <36>.

Pulsed Field Gel Electrophoresis

PFGE was performed following the PulseNet one-day standard protocol <37>. Briefly, bacterial cells were suspended in 1 ml cell suspension buffer (100 mm Tris, 100 milimet EDTA, pH 8.0). The cell suspension (200 µl) was mixed with 10 µl proteinase K (20 mg/ml; Sigma) & molten 1% Seakem agarose (200 µl) (Lonza, USA) for preparing solid agarose plugs. The plugs were treated with 5 ml cell lysis buffer (50 milimet Tris, 50 milimet EDTA, pH 8.0, 1% Sarcosyl) và 25 µl proteinase K (20 mg/ml) và incubated at 54°C for 2 h in a shaking water bath. The plugs were washed thoroughly with pre-warmed water for two times & pre-warmed Tris-EDTA buffer (pH 8.0) for four times at 10 mins interval at 50°C. The DNA plugs were digested with 40 U of XbaI (New England Biolabs, MA) at 37°C for 18 h. The digested DNA was run on 1% pulsed field certified agarose gel (Bio-Rad, Hercules, Calif.) prepared in 0.5x TBE buffer (Sigma) using CHEF DRIII (Bio-Rad) apparatus with an initial switch time of 2.2 sec and a final switch time of 63.8 sec at 6 V/cm for 24 h. The gel was stained with ethidium bromide (1 µg/ml, Sigma), destained with deionized water & PFGE profiles were observed with the UV trans-illuminator using GelDoc (Bio-Rad). Salmonella enterica serovar Braenderup H9812 was used as reference strain.

Analysis of PFGE patterns

The PFGE patterns were analyzed using FPQuest software version 4.5 (Bio-Rad) by methods described earlier <38>. Similarity analysis was done by using Dice coefficient with 1.5% optimization & tolerance levels & clustering of matched bands was done using Unweighted Pair Group Method with Arithmetic mean (UPGMA). The isolates with identical PFGE patterns were described as genetically indistinguishable (Dice coefficient of similarity of 100%); isolates with PFGE patterns differing by three or less bands were designated as related (Dice coefficient of similarity of >80%).

Isolation of Salmonella enterica from prospective samples and collection of strains from other hospitals

A total of 422 hospital (Dr. B. C. Roy Post Graduate Institute of Pediatric Sciences) attending febrile children of which 257 (60.9%) were male và 165 (39.1%) were female with clinical diagnosis of enteric fever were enrolled in the study. Median age of the patients was 5 years (range, 1 month to 15 years) and median duration of fever was 7 days (range, 2 to lớn 90 days). Out of 422 blood samples processed, 34 (8%) were positive for Salmonella enterica of which 27 (6.4%) were S. Typhi & 7 (1.6%) were S. Paratyphi A. Additionally a total of 68 (50 S. Typhi và 18 S. Paratyphi A) Salmonella enterica blood isolates were included in the study, which were collected from the other three hospitals in Kolkata: 31 isolates from CMRI, 22 isolates from AGH và 15 isolates from AMRI. In other hospitals, median age of the enteric fever patients was 24.5 years (range, 1 to lớn 63 years) of which 44 (64.7%) were male & 24 (35.3%) were female. Therefore a total of 77 S. Typhi và 25 S. Paratyphi A were characterized in this study.

Distribution of antimicrobial resistance in the study isolates

All Salmonella enterica isolates were categorized into four groups based on the major AMR patterns: multidrug resistant (MDR), Nalidixic acid resistant (NaR), Decreased susceptibility to ciprofloxacin (DCS) và ciprofloxacin resistant (CiR) Salmonella. Year wise distribution of each AMR group in both the serovars are shown in Table 2. An increase in isolation of MDR S. Typhi was noticed from 2009 (13.6%) khổng lồ 2013 (25%), but MDR isolates were not observed in S. Paratyphi A. Isolation of NaR isolates remained constant (>85%) in both the serovars throughout the study period & all NaR isolates were either CiR or showed DCS. Overall around 20% isolates of both the serovars were resistant to lớn ciprofloxacin. Figure 1 shows the percentage distribution of antimicrobial resistance in S. Typhi & S. Paratyphi A Kolkata isolates during 2009–2013. It was observed that all S. Typhi isolates were susceptible to lớn azithromycin và 7 (28%) S. Paratyphi A showed resistance lớn the drug. All study isolates were susceptible lớn third-generation cephalosporins (ceftriaxone, ceftazidime, cefotaxime) and aztreonam.

MIC values & distribution

MIC Ranges, MIC50 và MIC90 of various antimicrobials are shown in Table 3. It is worth noting that the MIC50 of ciprofloxacin was 0.38 µg/ml for S. Typhi and 0.5 µg/ml for S. Paratyphi A, whereas MIC90 of ciprofloxacin was 32 µg/ml for S. Typhi and 0.75 µg/ml for S. Paratyphi A. MIC distributions of fluoroquinolones (FQs) like ciprofloxacin, ofloxacin & levofloxacin and azithromycin among the study isolates are shown in Figure 2. Interestingly, the majority of the study isolates of both the serovars showed MICs within reduced susceptibility zones of ciprofloxacin, ofloxacin & levofloxacin. Levofloxacin resistance was not observed in S. Paratyphi A. In S. Typhi isolates, MIC values of azithromycin were found below the resistance cutoff (>16 µg/ml) (Figure 2).

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