Original article: Study of antibiotic sensitivity pattern of salmonella typhi in tertiary care centre 1 Dr Rajashri Patil, 2 Dr Amar Patil 1Assistant Professor, Department of Microbiology, Dr D Y Patil Medical College and Vidyapeeth, Pune 2 Assistant Professor, Medicine Dept, Dr D Y Patil Medical college and Vidyapeeth, Pune Corresponding author: Dr Amar Patil, Assistant Professor, Dr D Y Patil Medical college and Vidyapeeth, Pune Abstract: Introduction: Enteric fever is transmitted via faeco-oral route usually through consumption of contaminated food & water. The incubation period is 7-14 days but may range from 3-56 days & appear to be related to the dose of infection. The clinical course may vary from a mild undifferentiated pyrexia (ambulant typhoid) to a rapidly fatal disease. Materials and methods : It was a descriptive study with sample size 300 clinical isolates. The sample size was estimated with the help of expert statistician. Results: Out of 300 samples 187 patients were with history of taking antibiotics during collection. Out of 203 Widal positive samples 152 were with history of taking antibiotics during collection. Conclusion: Major factors identified by WHO in initiating and promoting antimicrobial resistance include, a) the unnecessary use of antibiotics by humans; b) the misuse of antibiotics by health professionals; c) over-the-counter availability of antibiotics in many countries; d) patient failure to follow the prescribed course of treatment; and e) the use of antibiotics in animal feeds as growth hormones. Individual education about these aspects would be foremost important in control of salmonellosis. Introduction: Enteric fever is transmitted via faeco-oral route usually through consumption of contaminated food & water. The incubation period is 7-14 days but may range from 3-56 days & appear to be related to the dose of infection. The clinical course may vary from a mild undifferentiated pyrexia (ambulant typhoid) to a rapidly fatal disease. Onset is usually gradual, with headache, malaise, anorexia, coated tongue,& abdominal discomfort with either constipation or diarrhea. The typical features are a step ladder pyrexia with relative bradycardia, toxemia, rose spots. Soft palpable spleen is a constant feature. 1 The most important complications are intestinal perforation, hemorrhage,& circulatory collapse. 2 In 2000, it was estimated that over 2, 16 million of typhoid occurrences worldwide, resulting in 216,000 deaths, and that more than 90% of this morbidity and mortality occurred in Asia 3. A report from World Health Organization in 2008 on typhoid fever in five Asian countries showed the annual typhoid incidence (per 100,000 persons years) among 5-15 years age group varied from 24.2 and 29.3 in Vietnam and China, to 180.3 in Indonesia; and to 412.9 and 493.5 in Pakistan and India, respectively. 4 The emergence of multi drug resistance among the enteric fever group of Salmonella to the first line antibiotics such as Ampicillin, Chloramphenicol and Cotrimoxazole has been a concern. The problem only worsened with the 75
advent of NARST (Nalidixic acid resistant Salmonella Typhi) making ciprofloxacin a doubtful drug of choice for the treatment of enteric fever. With the changing patterns in antibiogram it is necessary to continually monitor the drug resistance pattern and understand the mechanisms involved. Hence this study was under taken to characterize the prevalent serotypes and their resistance patterns and analyze the molecular mechanisms involved, so that appropriate strategies can be adopted in the management of enteric fever. 5 Materials and methods: It was a descriptive study with sample size 300 clinical isolates. The sample size was estimated with the help of expert statistician. Inclusion criteria 1. All patients with sustained or continuous fever (38 0 c &above )that has lasted for>3days. 2. Blood samples of all clinically suspected cases of enteric fever sent by clinician. Exclusion criteria Febrile patients not meeting case definition criteria of enteric fever. Venous blood was collected under aseptic precautions from patients clinically evaluated for enteric fever. Clean the puncture site with povidone iodine or 70% ethanol solution using aseptic technique in a circle(starting from center to periphery) approximately 5 cm in diameter. Allow 1-2 minutes for the disinfectant to dry. 5ml from children and 10 to 12 ml from adults respectively were collected in BacT/ALERT bottle and sent to the laboratory. Antibiotic susceptibility testing of the isolates was done by Kirby-Bauer disc diffusion method according to CLSI guidelines. 3-4 isolated, morphologically similar colonies were taken with a sterile loop and inoculated into peptone water and incubated at 37 0 C for 2 hours. Turbidity was adjusted to 0.5 McFarland standard and a lawn culture was made on Mueller-Hinton agar and antibiotic discs like ampicillin(10 µg), ciprofloxacin(5µg), cotrimoxazole (25µg), chloramphenicol(30µg), ceftriaxone(30µg), Nalidixic acid(30µg ) were used. Plates were incubated at 37 0 C for 16-18 hours. The zones of inhibition were measured and interpreted according to CLSI guidelines. Quality control was done using ATCC E.coli 25922. 76
Results: A. Antibiotic susceptibility pattern on disc diffusion method Drugs Sensitive n(%) Intermediate Resistant Ampicillin Chloramphenicol Co-trimoxazole Ciprofloxacin Ceftriaxone Nalidixic Acid 18(81.81%) 3(13.63%) 1(4.5%) 0 0 22(100%) In list of sensitive strain one was of Salmonella paratyphi A which was also Nalidixic acid resistant B. MIC of Ciprofloxacin : Serial no. No. of strains MIC µg/ml 1 2 3 5 4 12 0.25 5 0.5 3 0.12 1 8 0.06 1 77
C. MIC of Nalidixic acid: Serial no. No. of strains MIC µg/ml 1 21 >64 2 1 16 D. Pattern of duration of fever among those 203 were as follows Serial no. Duration of fever No. of patients 1 1 to 2 wks 126 Serial no. Duration of fever No. of patients 12 <1wk 2 to 3 wks 5164 23 1 to <1 2 wks 148 9 34 2 to >4 3 wks 774 4 >4 Total wks 24 203 Total 300 Out of 300 samples 187 patients were with history of taking antibiotics during collection. Out of 203 Widal positive samples 152 were with history of taking antibiotics during collection. Discussion: Antibiotic resistance is one of the world s most pressing public health problems. The antibiotic-resistant organisms can quickly spread and so threaten communities with new strains of infectious disease that are more difficult to cure and more expensive to treat 6 Since 1948, chloramphenicol was the drug of choice for the treatment of typhoid fever, and ampicillin and trimethoprim sulphamethoxazole were useful adjuncts. In May 1972 an outbreak of chloramphenicol-resistant S. Typhi occurred in Kerala, India, where seven of 13 isolates. 78
were resistant to chloramphenicol in vitro 7. In the late 1980s and early 1990s multidrug resistance became established in the Indian subcontinent. In 1990-91, Kolkata, which had curlier witnessed an outbreak due to MDR Salmonella Typhi, reported that nearly all the isolates were MDR. In South India MDR isolates were predominant with rates of 78% being reported 8. In most cases resistance to chloramphenicol, ampicillin and cotrimoxazole was transferable on plasmids, either individually or enbloc. The plasmids found in S. Typhi are of two major types. The first is phcm2, a so-called cryptic plasmid. phcm2 has been found in S. Typhi from Asia but not Africa. Second, antimicrobial resistant strains carry large (approximately 140-180 kbp) self-transferable plasmids 7 The increasing reports of multidrug resistance led to the phasing out of chloramphenicol, trimethoprim sulphamethoxazole, and ampicillin for the treatment of typhoid fever. Multidrug resistance in Delhi fell from 70% in 1991 to 53% in 1993 and the decline was reported to continue in 1999-2004. In central India multidrug resistance decreased from 91% in 1991 to 22% in 2OO2 " In a study from Kolkata, India, the situation underwent a reversal from 100% chloramphenicol resistance in 1991 to 100% sensitivity in 2002 " Increased chloramphenicol sensitivity has been reported from South India as well. Till date, this trend has become established over much of India. The reason for resurgence of strains sensitive to first line drugs may be that,antibiotics like ampicillin and chloramphenicol are rarely used for the treatment of typhoid fever these days thus relieving the selection pressure. Loss of plasmids such as the R-plasmid that confer multi drug resistance, or even may be due to emergence of de novo susceptible strains 8, In this study, none of the isolates were resistant to ceftriaxone, ampicillin, cotrimoxazole, & chloramphenicol that means all are non MDR strains. The MICs for all the isolates were found to be within the breakpoint of sensitivity by CLSI criteria i.e. 1µg/ml for ceftriaxone, 8 µg/ml for ampicillin, 8 µg/ml for chloramphenicol. As no resistance to Ceftriaxone was noted for either S. Typhi or S. paratyphi A in this study & in the recent past, Cephalosporins have gained importance for the treatment of enteric infection. Third generation Cephalosporins in particular are effective in the treatment of typhoid fever. Ceftriaxone, administered either orally or intramuscularly and Cefixime, which is administered orally, are both effective and are the preferred choice in NAR infection. MDR paved the way for the discovery of quinolones such as Nalidixic acid and it s improved derivatives, flouroquinolones ( Ciprofloxacin, Ofloxacin ) etc. In India, since 1990, chloramphenicol was generally replaced by ciprofloxacin as the drug of choice for Typhoid Fever 9. However indiscriminate use of flouroquinolones not only for enteric fever but also for other systemic infections soon led to the development of resistance in Salmonella Typhi and paratyphi A. There is a region called QRDR (Quinolone resistancedetermining region) in both DNA gyrase (gyra,gyrb )and topoisomerase IV (parc,pare ) where mutations usually occur. It has been suggested that a low level resistance to ciprofloxacin, probably arising from one point mutation in the gyra gene, In vitro resistance to nalidixic acid can be used to detect this low level resistance. In this study,on disc diffusion method about 82% (18 isolates out of 22 including one isolate of S.Paratyphi A ) were sensitive to ciprofloxacin i. e. zone diameter >31 mm (as per CLSI guideline), 3 were intermediate sensitive(diameter between 21 30mm), & one was resistant to ciprofloxacin (diameter between 20mm). Escherichia coli ATCC 25922 was included on each test occasion, and all results were within the 79
recommended limits, indicating the validity of our test procedures. MIC was determined by broth microdilution method, which indicate 12 isolates had MIC 0.25 µg/ml, 5 (out of 5 one was S.Paratyphi A) had MIC 0.5 µg/ml (3 isolates out of these 5 are intermediate sensitive on disc diffusion test), 3 isolates had MIC 0.125 µg/ml, 1 isolate had high level ciprofloxacin resistance with MIC 8 µg/ml which is resistant to ciprofloxacin on disc diffusion also & only 1 strain has MIC 0.06 µg/ml. Conclusion: Major factors identified by WHO in initiating and promoting antimicrobial resistance include, a) the unnecessary use of antibiotics by humans; b) the misuse of antibiotics by health professionals; c) over-the-counter availability of antibiotics in many countries; d) patient failure to follow the prescribed course of treatment; and e) the use of antibiotics in animal feeds as growth hormones. Individual education about these aspects would be foremost important in control of salmonellosis. References: 1. Collier L, Balows A, Susman M. Salmonella. In :Topley and Wilson s Microbiology and Microbial infections. Chapter 17, 9th edition, Georgina bentliff, Great Britain.1998; (2): 969-997. 2. Paniker CK,.Anantnarayan & Paniker s Textbook of Microbiology. Chapter 32 Salmonella 8th edition. Universities press India Private Limited. Chennai. 2009; 288-30. 3. Fauci A,. Kasper D L. Harrison s Principles of Internal Medicine. 17th Edition. USA: The McGraw Hil, Companies; 2010. 521-530 4. Ralph A.Gianella by Salmonella.ed. Samuel Baron. Medical Microbiology.4th edition. Galvestone (TX):university of Texas Medical branch at Galvestone;1996. 5. B D Jones. Host responses to pathogenic Salmonella infection. Genes Dev. Cold Spring Harbor Laboratory Press; 1997. 11: 679-687. http://genesdev.cshlp.org/content/11/6/679.full.html#ref-list-1 6. Patrick AD, Francois Xavier weil, WHO Collaborating Centre for Reference and Research on Salmonella. Antigenic formulae of the. Salmonella serovars. 2007. 9th edition. 7. Ntusi N, Aubin L, Oliver S, Whitelaw A, Mendelson M. Guideline for the optimal use of blood cultures. S Afr Med J 2010; 100: 839-843. 8. Lee A, Mirrett, S, Reller LB,. Weinstein MP. Detection of Bloodstream Infections in Adults: How Many Blood Cultures Are Needed? J Clin Microbiol. 2007 November; 45(11): 3546 3548. 9. Harish BN, Menezes GA. Antimicrobial resistance in typhoidal salmonellae. Indian J medical microbiology, 2011 Jul- Sep; 29(3): 223-9. 80