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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 17  |  Issue : 1  |  Page : 14-19

Prevalence and antibiogram pattern of Salmonella enterica serotypes in Garhwal Region: First report from foothills of himalayas


1 Department of Microbiology, Dr. Sampurnanand Medical College, Jodhpur, Rajasthan, India
2 Department of Medicine, AIIMS, Rishikesh, India
3 Department of Microbiology, AIIMS, New Delhi, India
4 Department of Microbiology, Government Doon Medical College, Dehradun, Uttarakhand, India
5 Department of Microbiology, Amaltas Institute of Medical Sciences, Dewas, Madhya Pradesh, India

Date of Web Publication2-Jul-2018

Correspondence Address:
Vikrant Negi
Department of Microbiology, Dr. Sampurnanand Medical College, Shastri Nagar, Jodhpur - 342 001, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ajmhs.ajmhs_55_17

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  Abstract 


Introduction: Enteric fever, caused by Salmonella enterica subsp. enterica serotype Typhi and Paratyphi, is endemic in India with an incidence of 102–2219/100,000 populations. The definitive diagnosis of enteric fever in patients with compatible clinical picture is isolation of Salmonellae from blood, bone marrow, stool or urine, and demonstration of four-fold rise in antibody titer to both O and H antigen of the organism between acute and convalescent-phase sera. Aim: The aim of the study was to study the prevalence of various serotypes of S. enterica and their antibiogram in foot hills of Himalayas. Materials and Methods: During February 2012–January 2013, all clinically suspected patients were screened for enteric fever by Widal tube agglutination test. For the isolation of etiology, venous blood, stool and urine specimen were obtained from patients with antibody titer of ≥80 and 160 for anti-O agglutinin and anti-H agglutinin of Salmonella typhi, respectively, and ≥20 for anti-H agglutinin of S. paratyphi A and S. paratyphi B. Characterization and antibiogram determination of the isolates was done by conventional microbiological methods including Kirby–Bauer's disc diffusion technique. Result: Among 1173 suspected cases, 373 showed a high titer of antibodies against O (≥80), H (≥160), AH (≥20), and BH (≥20) antigens. A total of 81 isolates were obtained from 76 patients (29 from blood and 49 from stool and three from urine), of which 54 were identified as Salmonella typhi, 20 as Paratyphi A and seven as Paratyphi B. Extended-spectrum beta-lactamase production was observed in four isolates of S. typhi. Ciprofloxacin followed by co-trimoxazole was resistant to 46.5 and 36.5% of the isolates, respectively. Conclusion: This report indicates a significant percentage of drug resistance in S. enterica serotypes in Garhwal region. Periodic monitoring of the antibiogram pattern along with the implementation of strict antibiotic policies and patient education is needed.

Keywords: Blood culture, extended-spectrum beta-lactamase, Salmonella typhi, typhoid fever, Widal test


How to cite this article:
Negi V, Pathania M, Prakash R, Juyal D, Sharma MK, Pal S. Prevalence and antibiogram pattern of Salmonella enterica serotypes in Garhwal Region: First report from foothills of himalayas. Afr J Med Health Sci 2018;17:14-9

How to cite this URL:
Negi V, Pathania M, Prakash R, Juyal D, Sharma MK, Pal S. Prevalence and antibiogram pattern of Salmonella enterica serotypes in Garhwal Region: First report from foothills of himalayas. Afr J Med Health Sci [serial online] 2018 [cited 2018 Jul 23];17:14-9. Available from: http://www.ajmhs.org/text.asp?2018/17/1/14/235740




  Introduction Top


Enteric fever includes typhoid and paratyphoid fever, caused by  Salmonella More Details enterica serovars Typhi (S. typhi), Paratyphi A (Salmonella paratyphi A), and Paratyphi B (Salmonella paratyphi B). Typhoid fever is an important cause of morbidity and mortality. S. typhi infects about 12 million people worldwide every year.[1],[2] About 130,000 deaths occur annually due to typhoid fever throughout the globe.[3] Typhoid fever is endemic in developing countries due to overpopulation and poor hygiene. It is a major health problem in India causing more than 200,000 deaths every year.[4] However, the mortality rate remains <1% when antibiotic treatment is given; it can be as high as 30% without antibiotic treatment.[5],[6] In the past two decades, multidrug-resistant (MDR) strains of S. enterica have emerged worldwide.[7] Reduced susceptibility to fluoroquinolones has been reported in India.[8] Changing trends in antibiotic resistance among S. enterica has appeared against ampicillin, co-trimoxazole, and chloramphenicol.[8],[9]

There are no published data for the prevalence of S. enterica and its antibiogram from the Garhwal region of Uttarakhand, India, so the present study was undertaken to assess the prevalence of S. enterica serovars in cases of clinically suspected enteric fever and study their resistogram in a tertiary care hospital of Garhwal region.


  Materials and Methods Top


Study Design

A prevalence-based cross-sectional study was conducted in the Department of Microbiology and Immunology of Veer Chandra Singh Garhwali Government Medical Sciences and Research Institute and attached Hemwati Nandan Bahuguna Base Hospital, Srinagar, Uttarakhand, for a period of 1 year, from February 2012 to January 2013.

Inclusion criteria

All the patients, irrespective of their age and gender that presented to Hemwati Nandan Bahuguna Base hospital with clinical symptoms of enteric fever, were included in this study.

Sample collection

Certificate for ethical clearance was taken from the Institutional Ethical Committee. Written informed consents were obtained from the patients after disclosing to them the protocol and objectives of the study. Under aseptic conditions, 2 ml of blood samples were collected from each patient, in the plain vials without anticoagulants by an expert phlebotomist. Blood was allowed to clot in plain vials and serum separated by centrifugation at 2500 rpm for 10 min. Sera were screened for enteric fever by Widal tube agglutination test.

For the isolation of etiology, venous blood, stool, and clean-catch midstream urine specimen were obtained from patients with antibody titer of ≥80 and 160 for anti-O agglutinin (TO) and anti-H agglutinin (TH), respectively, and ≥20 for AH and BH antigens and tested for culture and antibiotic sensitivity testings as per standard operating procedure (SOP) of the laboratory.

Widal test

Commercially available antigens which contained the S. enterica subsp. enterica serovar Typhi O and H antigens, the S. enterica subsp. enterica serovar Paratyphi AH antigen, and the Paratyphi BH antigen were used (Span Diagnostics Ltd.,). A volume of 0.5 ml of the two-fold serially diluted sera (dilutions from 1:20 to 1:320) in 0.9% normal saline were tested by adding an equal amount of antigen, and the tubes were then incubated overnight at 37°C in a water bath. A negative control was included in every batch of the tests. The results were interpreted and analyzed. The highest dilution that gives a visible agglutination was considered the antibody titer of the test samples and the agglutination titer ≥1:80 for TO, ≥1:160 for TH, and ≥1:20 for AH and BH were considered as significant infection.[10]

Blood culture

Two to three milliliters (from children) and 5–10 ml (from adults) venous blood samples were collected in commercially available brain–heart infusion broth (HiMedia) under aseptic precautions. Bottles were incubated at 37°C. After 24 h of incubation, subcultures were made on 5% sheep blood agar and MacConkey agar plates. If no growth were observed, then subcultures were subsequently performed on day 3, day 5, and day 7 before reporting it as sterile.

Stool culture

Samples were collected in sterile universal containers and subcultured on MacConkey agar plate and Selenite F broth as per laboratory SOP. Subcultures from Selenite F broth, after overnight incubation at 37°C, were made on xylose lysine deoxycholate agar. Growths were looked for after incubation for 24–48 h at 37°C.

Urine culture

Midstream urine was collected in sterile urine containers and plated on 5% sheep blood agar and MacConkey agar by semi-quantitative methods. Streaked plates were incubated at 37°C for 24–48 h.

Identification

Isolates were studied for colony morphology, Gram-staining, hanging drop motility, and routine biochemical testings. Nonlactose-fermenting, Gram-negative bacilli with active motility, catalase producing, oxidase negative, alkaline slant and acidic butt in triple sugar iron agar, indole negative, methyl red positive, Voges–Proskauer negative, urease nonproducing, nitrate reducing, and glucose-fermenting strains were suspected for S. enterica. Antibiotic susceptibility testing was performed by Kirby–Bauer disc diffusion method. Isolates with antibiotic inhibition zone size of <22 mm with ceftazidime (30 μg), <25 mm with ceftriaxone (30 μg), and <27 mm with cefotaxime (30 μg) were suspected for extended-spectrum beta-lactamase (ESBL) producers and were confirmed by combination disc diffusion test using a disc of ceftazidime (30 μg) alone and ceftazidime + clavulanic acid (30/10 μg) placed at a distance of 25 mm center to center. Growths suggestive for S. enterica were confirmed by agglutination test with specific antisera (Remel Europe Ltd., Dartford, UK).


  Results Top


During this study period, a total of 1173 patients were clinically suspected for enteric fever. Significant Tube Widal test with visible agglutination titer above 1:80 for TH/1:160 for TO/1:20 for AH and BH were found positive for 373 patients (males = 206; females = 167) with a higher incidence among children [Table 1]. Out of 373 tube Widal positives, only 76 (male = 49; females = 27) patients had S. enterica subsp. enterica growth in their clinical samples.
Table 1: Age group and gender distribution of patients found positive for Widal tube agglutination

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A total of 81 isolates of S. enterica subsp. enterica yielded from 76 patients, of which 29 growths were from blood samples, 49 from stool, and 3 from urine as represented in [Figure 1]. Blood and stool samples of four patients while stool and urine samples of one patient were found to have growth of S. enterica subsp. enterica, whereas other 71 patients had growth only in a single sample. No common growth was seen in blood and urine samples of the same patients. Fifty-four were identified as S. typhi (blood = 20, stool = 32, and urine = 2), 20 as Paratyphi A (blood = 7, stool = 12, and urine = 1), and seven as Paratyphi B (blood = 2 and stool = 5). Five growths in more than single specimens were of S. typhi only.
Figure 1: Growth pattern of Salmonella enterica from various samples.

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Out of 49 growths in the stool, only 28 grew in direct inoculation on agar plates, whereas all 49 were cultured on agar plates when enrichment for 6–8 h were done in Selenite F broth.

[Figure 2] illustrates the resistance recorded in the isolates. Ciprofloxacin followed by co-trimoxazole was resistant to 44.44% and 40.74% of the isolates, respectively. Four isolates of S. typhi were found to be ESBL producers.
Figure 2: Graphical representation of antibiotic resistance of Salmonella enterica isolates

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  Discussion Top


Low socioeconomic regions with deteriorated public health status always have an impact on people's health status. In such conditions, an increase in certain diseases, such as typhoid fever, is common. Typhoid fever is reported from throughout India, especially in children.[11]

S. enterica causes infections with considerable morbidity and mortality worldwide.[6],[12] R. Ganesh et al. in their study at Chennai reported that 7 out of per 1000 patients presented to hospital were suffering from typhoid fever. Most of those patients with typhoid fever were children with age ranging between 5 and 10 years.[13] Another study from Delhi has reported an incidence rate of typhoid as 27.3/1000 at age under 5 years, 11.7 at 5–19 years, and 1.1 between 19 and 40 years.[14]

In the present study, 31.80% patients among the clinically suspected patients for typhoid and paratyphoid fever were found to have significant raised Widal titer. The male:female ratio in Widal positives was 1.23:1 in which 55.23% were males and 44.77% were females. The Widal test positive was seen highest in pediatric age group, especially 1–14 years old (51.21%).

Among the Widal-positive patients, only 20.38% patients were found culture positive. Nearly 35.80% growth was observed in blood samples; 60.49% in stool and only 3.70% growths seen in urine. This report is similar to other studies where cases screened positive with Widal test were confirmed only in 26.5% blood culture positive.[15] High rate of Widal positive but less confirmed cases with culture can be due to false-positive reaction in Widal test in other diseases such as malaria.[16],[17],[18] Malarial parasites contain some undefined antigenic determinants which can cross-react with those of S. typhi and results in antibodies' production against Salmonella. The high rate of typhoid fever diagnosed in health-care facilities with no culture facilities can be due to this reason.[15] Another reason for low blood culture positives can be initiation of antibiotic therapy before collection for clinical samples as this tertiary care hospital caters a large geographical region and many patients are referred from various primary health care and district hospitals. Such patients are already been prescribed antibiotics at those setups and referred only on treatment failure/under response.

Although stool culture is useful for the diagnosis of typhoid carriers, the isolation of S. typhi from stools, along with clinical presentation, is suggestive of typhoid fever.[19] Stool remains the specimen with highest yield of Salmonella species in this study as most of the cases were referred from primary health care after 2–3 weeks of onset of fever. The Salmonella yield is much higher than the reports from other studies.[18],[20] Most of the growths seen in stool specimen were from the sample enriched for 6–8 h in Selenite F broth compared to direct inoculation on agar plates.

Isolation of S. typhi from urine is very rare even in endemic regions.[21],[22] It can be isolated from urine during the later stage or prodromal stage of typhoid fever, in chronic carrier states involving the urinary system, and occasionally following localized urinary tract infection. Even in this study, isolation of Salmonella from urine was rare. The popular belief is that the shedding of S. typhi in urine occurs following a recent typhoid fever as part of the natural history of typhoid fever or in chronic carrier states.[23] Urine culture positives were seen in young adults. Two cases were of recurrent typhoid fever where the patients experienced low-grade fever in every summer or monsoon for the past few years, with tube Widal test having significant titer but no yield of pathogen from blood and stool samples.

ESBL production leads to cephalosporin resistance which is a serious problem worldwide. ESBL production is reported from S. enterica.[24] Al Naiemi et al. have reported the production of an ESBL, SHV-12, in S. enterica serotype typhi due to the presence of a blaSHV gene.[25] Eight isolates of Salmonella were found to produce ESBL in this study; however, the reason for ESBL detection was not looked for. The emergence of ESBL-producing S. enterica strains i leading to typhoid fever treatment failure cases as in most of the Himalayan and sub-Himalayan regions, the facilities of bacterial culture and antibiotic susceptibility testings are not available. This region of country also has less number of trained medical practitioner with minimum facilities, so most of the time patients are treated by inexpert medical practitioner without any confirmed diagnosis. This leads to inappropriate and indiscriminated antibiotic therapy. Irrational antibiotic therapy and lack of awareness lead to antimicrobial resistance [26] which describes the emerging ESBL S. enterica strains in this part of the country. Higher level of resistance was seen against ciprofloxacin, co-trimoxazole, ampicillin, and third-generation cephalosporins which is a serious concern as this hospital is the only tertiary care facility available in Garhwal region and the presence of antibiotic resistance in this area signals about indiscriminate usage of antibiotics by general practitioners working at rural or semi-urban areas. Usually, the patients with enteric fever visit this tertiary care hospital on treatment failure. They are previously been treated by local general practitioner on the basis of Widal test or typhidot test results as there is no culture and antibiotic susceptibility testing, other than this hospital, available in hills of Garhwal region. The another reason for antibiotic resistance can be the introduction of resistant strains by tourists as Garhwal caters a huge number of tourists from all over India and MDR Salmonella cases are reported from different parts of India.[8],[9],[27] Ampicillin and co-trimoxazole are used among the first line of drugs for the treatment of Salmonella infections and resistance to ampicillin, chloramphenicol, and co-trimoxazole are considered as multidrug resistance strains. Higher number of resistant strains cases against ampicillin and co-trimoxazole suggests increased MDR salmonella prevalence. Ciprofloxacin is considered as drug of choice for MDR salmonella infection. Ceftriaxone (third-generation cephalosporin) is also used for the treatment of MDR Salmonella strains. Ciprofloxacin and ceftriaxone are even being used as the first line of drugs for treating Salmonella infections.[27] Antibiotic resistance against these two drugs raises the concern for treatment failure.

Limitation

Widal test negative but symptomatically positive cases were not tested for culture which could have been important in identifying the negative predictive value of Widal test. Although the bone marrow aspirate was not taken for culture as it is more invasive and painful, the yield of S. enterica is much higher from it in comparison to blood specimen. The institute lacked the facility for genotyping which could have identified the gene responsible for antibiotic resistance.


  Conclusion Top


False-positive titers are common with Widal test, so culture and antimicrobial sensitivity must be performed to confirm the diagnosis. Stool culture along with clinical signs and symptoms can be of a good diagnostic value, so it is recommended that stool culture should be followed in suspected typhoid and paratyphoid fever cases. Stool sample enrichment in Selenite F broth increases the rate of yield of S. enterica. High positivity among children suggests a proper vaccination strategy to be followed.

Isolation of drug-resistant strains specially ESBL producers from this region strongly recommends an initiative for establishing improved hospital antimicrobial policy and antimicrobial prescribing guidelines should be undertaken. Periodic monitoring of the antibiogram pattern along with the implementation of strict antibiotic policies and patient education is needed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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