Comparative prevalence of MDR S. typhi in Northern and Southern parts of Karnataka, South India

Typhoid fever due to infection of MDR Salmonella enterica serovar typhi resistance to Chloramphenicol, ampicillin and cotrimoxazole has posed a serious therapeutic challenge in many parts of the world, including South India. Till recently ciprofloxacin was the drug of choice for the treatment for the MDR S.typhi. However treatment failure to ciprofloxacin has been recorded with decreased susceptibility to nalidixic acid, which is the marker for ciprofloxacin resistance in S.typhi. Alternative drugs for the treatment of MDR S. typhi the cephalosporins; however, emergence of cephalosporins resistance is a matter of concern. This prompted us to study drug resistance patterns among S. typhi isolates from North Karnataka, which is endemic for typhoid fever and compared with South Karnataka which is non endemic region and formulate the appropriate drugs for the management of enteric fever.


Introduction
Typhoid fever caused by Salmonella enterica serovar typhi continues to remain a public health problem in many countries. An estimated 21.7 million illnesses and 2,17,000 deaths are caused worldwide 1 . Incidence of Typhoid in Asia has been reported to be 274 per 1, 00,000 persons 1,2,4 . Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A are the main etiological agents of enteric fever in India and S.typhi is predominant 3 . Many outbreaks have been reported in the developing countries 4 .
The emergence of multidrug resistance in S.typhi has further complicated the treatment and management of enteric fever, mainly resistance to ampicillin, chloramphenicol and cotrimoxazole which are the first line drugs for the treatment of enteric fever. In India multidrug resistant S. typhi (MDRST) was first reported from Calicut 6 . Since then a steady increase in the MDRST over the next two decades has been recorded. There was a decline in MDR S.typhi with the introduction of nalidixic acid. Strains with decreased susceptibility to fluroquinolones and increased incidence of nalidixic acid resistant S.typhi (NAST), especially that exhibit decreased susceptibility to most recent fluroquinolones used for the treatment of Typhoid fevar are reported 7,8 . Interestingly there are reports on the gradual decrease in MDR S.typhi and increased sensitivity to the chloramphenicol 9 . There are also reports on the third generation cephalosporin resistant S.typhi and ESBL S.typhi in India and various parts of the world 10 .
The above background prompted us to determine the antibiotic resistance pattern of outbreak causing as well as sporadic isolates of S.typhi from two geographically different regions namely Northern and Southern parts of Karnataka in India ,and the Northern part of Karnataka is the endemic for S.typhi.

Methodology
The study was carried during April 2010 to October 2012 in Gulbarga and Raichur Districts in North Karnataka and from May 2011 to Sept 2012 in Bangalore, South Karnataka. The study included a total of 105 S.typhi isolates from blood samples of patients suffering from suspected typhoid fever who attended the outpatient clinics or were admitted in the private and government hospitals of these regions. A total of 1500 blood samples, 1200 from Northern part of Karnataka and 300 from Southern part of Karnataka were included in the study.

Isolation and Characterisation
Blood samples from the febrile patients 5 to 10 ml were collected by vein puncture and inoculated directly in to blood culture bottles containing 50 ml of brain heart infusion broth 11 . The enriched samples after visible turbidity were streaked on Mac-Conkey Agar and Wilson Blair Agar plates 12 . The isolates producing characteristic colonies were identified by standard biochemical tests and confirmed by agglutination with salmonella O9 vi specific and Hd antisera (Kings Institute of Preventive Medicine Guindy, Chennai)

ESBL detection
The isolates that were resistant to the third generation cephalosporins by disc diffusion method were selected for ESBL screening by double disc synergy test (DDST) 15 . A disc of amoxyclave (30 µg) was placed on the centre of Mueller Hinton Agar plate. Each disc of cefetazidime, cefoxitine, cefotaxime, and ceftriaxone was placed around the amoxyclave disc, 20 mm apart and incubated for 24 hrs at 37ºC. A perfect formation of edge of the inhibition zone of any of the antibiotics towards the disc containing amoxyclave was interpreted as positive for ESBL production.
Confirmation of ESBL producers was done by phenotypic confirmatory disc diffusion method using cefotaxime with and without clavulanic acid 13 . A clear zone difference of ≥5mm with antibiotic + clavulanic acid disc when compared to antibiotic alone was considered confirmatory. Strains showing resistance to more than three different groups of antimicrobials as quinolones, tetracycline, cephalosporin's etc. were grouped as multi drug resistant strains 14.

Abstract
Typhoid fever due to infection of MDR Salmonella enterica serovar typhi resistance to Chloramphenicol, ampicillin and cotrimoxazole has posed a serious therapeutic challenge in many parts of the world, including South India. Till recently ciprofloxacin was the drug of choice for the treatment for the MDR S.typhi. However treatment failure to ciprofloxacin has been recorded with decreased susceptibility to nalidixic acid, which is the marker for ciprofloxacin resistance in S.typhi. Alternative drugs for the treatment of MDR S. typhi the cephalosporins; however, emergence of cephalosporins resistance is a matter of concern. This prompted us to study drug resistance patterns among S. typhi isolates from North Karnataka, which is endemic for typhoid fever and compared with South Karnataka which is non endemic region and formulate the appropriate drugs for the management of enteric fever. Keywords: Typhoid fever, salmonella typhi, antibiogram, ESBL IJBR (2014) 05 (03) www.ssjournals.com

Results
The study included a total of 1500 blood samples. A total of 105 S.typhi were isolated from the samples showing an isolation rate of 7%, which was slightly higher (7.3%) in South Karnataka as compared to North Karnataka (6.9%). Among the 83 isolates from North Karnataka, 71 were S.typhi and 12 were S.paartyphi A, whereas in South Karnataka among the 22 isolates, 17 were S.typhi, 3 were S.paartyphi A and 2 were S.typhimurium (fig-1).

Figure 1: Isolation of Strain
Antibiotic susceptibility of all the isolates is depicted in fig 2. A total of 67 (80.7%) isolates were MDR in Northern Karnataka and 15 (68.18%) in southern Karnataka. All the isolates of Southern Karnataka were sensitive to chloramphenicol and 71 (86.02%) were resistant to chloramphenicol in Northern region is one of the clear difference in drug resistance pattern between the two different geographical regions of Karnataka.

Figure 2: Antibiotic susceptibility of all the isolates
Out of the 83 isolates from North Karnataka, 4 (4.8%) were ESBL producers while only one isolate (4.5%) from South Karnataka was producing ESBL (Table-2 Table-3, which shows that the frequency of multidrug resistance is higher among ESBL producers than non ESBL producers from both the regions.

Discussion
Enteric fever caused by the S.typhi continues to be the global burden causing considerable morbidity and mortality and 80% of the global burden of S.typhi is mainly seen in the Asian and African countries 4 . It is a serious problem in endemic countries and travelers to these areas. It may be due to a combination of factors including poor sanitation and health care infrastructure 16 . In the present study, the antimicrobial susceptibility of typhoidal Salmonellae isolated from two geographically distinct regions of Karnataka Southern India is reported. Northern part of Karnataka is endemic for typhoid fever and Southern parts mainly Bangalore city which is densely populated and a site of travelers from various regions.
The study highlighted the predominance of multi drug resistance among S. typhi isolates from both geographic areas. Further, increased nalidixic acid resistance is of concern. However, there is a decreased susceptibility to fluroquinolones especially to ciprofloxacin 17 which is the choice of drug for the treatment of MDR typhoid fever. Study also shows the increased drug resistance to first line drugs namely ampicillin, chloramphenicol and cotrimoxazole. A remarkable difference in chloramphenicol resistance has been observed among these two geographical areas. All the 22 isolates from South Karnataka region are susceptible to chloramphenicol where as 71 (86.6%) isolates from Northern Karnataka are resistant to chloramphenicol 17 . This shows a change in the drug resistance pattern between two geographical regions. Increase in the ciprofloxacin resistance has been recorded in both regions from North Karnataka, 27.7% and South Karnataka 86%, when compared with previous reports of 8% 16 , which may be attributed to the increased nalidixic acid resistance. Previous studies have indicated that increased nalidixic acid resistance results in the gradual increase in ciprofloxicillin MIC (≤0.125µg/L>1 µg/L). This indicates that there has been a situation to relook at the break points and the zone diameters for reporting ciprofloxacin resistance for Salmonella 17 . In the present study 4.7% of third generation cephalosporin resistant S.typhi have been isolated, which is a matter of concern 18 . We have screened for the ESBL producers among the third generation cephalosporin resistant strains by DDST phenotypic method. A total of 5 ESBL isolates have been recorded which is alarming in both the regions. And all the organisms from both the regions are susceptible to imipenem which is promising.
To conclude this study demonstrates the increased MDR S.typhi in both the regions and difference in the susceptibility to the chloramphenicol stresses the need for a regular and regional monitoring of the antibiotic susceptibility and check the empirical use of antibiotics which is one of the causes for development of multi drug resistance. Third generation cephalosporin resistant and ESBL S.typhi are alarming which further stresses the necessity for rapid and economical techniques to detect and monitor the ESBL producers.