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 ¤  Material and Methods
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ORIGINAL CONTRIBUTION
Year : 1999  |  Volume : 53  |  Issue : 6  |  Page : 259-266
 

Multidrug resistant Salmonella*


Department of Pathology, Sir Hurkisondas Nurratumdas Hospital, Raja Rammohan Roy Road, Mumbai 400004., India

Correspondence Address:
R Gandhi
C/o. Prof. Santok Singh, 401-B, New Sathi, Sarronagar, Phase 2 Housing Coop. Society, Louis Wadi, Thane (West) 400 804
India
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PMID: 10776507

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How to cite this article:
Gandhi R, Banker D D. Multidrug resistant Salmonella*. Indian J Med Sci 1999;53:259-66

How to cite this URL:
Gandhi R, Banker D D. Multidrug resistant Salmonella*. Indian J Med Sci [serial online] 1999 [cited 2014 Sep 2];53:259-66. Available from: http://www.indianjmedsci.org/text.asp?1999/53/6/259/12224


 Salmonellosis More Details is one of the most widespread infections in India causing typhoid and paratyphoid fevers, gastroenteritis and diar­rhoea. [1]


 ¤ Material and Methods Top


During 1992-1994 a total of 326  Salmonella More Details strains was obtained from suspected enteric fever and other patients, admitted to Sir Hurkisondas Nurrotumdas Hospi­tal, Nanavati Hospital, Nair Ho­spital, Hinduja Hospital and Jag­jivan Ram Hospital in Mumbai. Of these 326 strains, 318 were isolat­ed from 9121 cases of blood cul­ture, 3 were from urine, 2 each from CSF and bile and 1 was from stool. For blood cultures, 3-5 ml of blood was collected with ase­ptic precautions and inoculated into trypticase soya broth (TSB) and biphasic MacConkey media. TSB was incubated at 37°C for 48 hours and subcultured on to Salmonella Shigella (SS) agar and MacConkey agar. If there was no growth in 48 hours subculturing was repeated after another 24 hours. Culture on biphasic Mac­Conkey medium was incubated at 37 C for one week before discard­ing it if there was no growth. Stool and bile samples were inoculated into selenite broth and incubated at 37°C for 48 hours. MacConkey agar and SS agar were used for subculturing. CLED (cystine lac­tose electrolyte deficient) medium and MacConkey agar were used for urine samples. Incubation was carried out for 24-48 hours at 37°C.

All the non-lactose fermenting colonies on MacConkey and SS agar were identified by gram stain, motility and a set of established biochemical tests. The biochemi­cals used were glucose, xylose, maltose, mannitol (all 1% sugars), irate, indole, methyl red and triple sugar iron (TSI). Biotyping of S.typhi was done by xylose fer­mentation. [2] Xylose positive were considered to be biotype l and xyloss negative were biotype ICI. The serological identification was done by slide agglutination test using polyvalent and monovalent antisera obtained from Wellcome Diagnostics, U.K. and from Natio­nal Salmonella Serotyping Centre, Kasauli (H.P.) The salmonella isolates were subjected to antibio­tic susceptibility testing on Muel­ler Hinton agar using Kirby Bauer disc diffusion tests. [3] The anti­biotics used were ampicillin, aug­mentin, cephalexin, cephazoline, cefuroxime, cefotaxime, ceftazi­dime, gentamicin, amikacin, netil­micin, ciprofloxacin, pefloxacin, norfloxacin, cotrimaxazole, oxy­tetracycline, chloramphenicol and furazolidone. The sensitivity re­sults were reported in reference to the Oxford Staphylococcus NCTC 6571. [4]

The S.typhi, S.paratypni A and S. typhlmurlum cultures were taken in lab-lemco agar butts to National Bacteriophage Typing Centre, Lady Hardinge Medical College, New Delhi for bacteriophage typing. The serotyping of S.typhimurium, S.havana, S.worthington and S.en­teritidis was done at National Sal­monella and  Escherichia More Details Coli Centre Kasauli and at Lady Har­dinge Medical College New Delhi.


 ¤ Results Top


Various serotypes of 326 Sal­monella isolates are shown in [Table 1] A large number of cultures were multidrug resistant (MDR) accord­ing to the antibiotic sensitivity re­sults [Table 2]. see on page No. 263.

Bacteriophage typing of 295 strains was carried out. The re­sults are shown in [Table 3]. The commonest bacteriophage type of S.typhi was type El (68.29%), of S. paratyphi A was type - 1 (64.51%).

In the 40 multidrug resistant (MDR) Salmonella strains which we screened for R-plasmid by con­jugation, antibiotic resistance pat­terns were transferred by direct conjugation, and enbloc transfer of resistance pattern was seen ex­cept in a few S.typhi and S.wor­thington strains where there was partial transfer. No transcon­jugants were obtained when conjugation was carried out bet­ween MDR Sl typhi and drug sen­sitive S.aureus and also between transconjugant E.coil K12 and drug sensitive S. typhi.


 ¤ Discussion Top


S.typhi is the major serotype responsible for enteric fever in our country. [5] A close examination of the data of the National Salmo­nella and E. coli Centre, Kasauli from 1958 to 1991 reveals that S. typhi was the predominant sero­type and continues to be so. [6] In this study too S. typhi was the most predominant serotype followed by S. paratyphi A. S. typhimurium, S.worthington. S.havana , and S.enteritidis. Chopra [7] and Agarwal [8] have also reported S.typhi to be the commonest serotype in their study with biotype I prevalent over biotype II. Bacteriophage typin has been particularly important in support of epidemiological in­vestigations of S. typhi [9] S.para­typhi A, [10] S.paratyphl B and ­S. typhimurium [11] In the present study phage type El of S.typhi was the most predominant followed by A and O. Pillai and Prakash [3] have also reported phage type El to be the commonest. The commonest phage type of S.paratyphl A in this study was type 1 which is in concurrence with the study of Agarwal et al [12] . Roche and Murthi have also reported similar findings. 88.88% strains of S.typhimurium in cur study were untypable. Roche et al have reported 90% of their S.typhimurium strains to be untypable. [13]

The other serotypes isolated and studied were S.worthington, S.havana and S.enteritidis. S.wor­thington has been reported by Saxena et al from human sources in 1986-89 from the National Sal­monella and Escherichia Cenre, asauli. [14] One of the strains of S.worthington was isolated by us from a 5 day old infant from its specimens of blood, stool as well as CSF. An outbreak of meningi­tis caused by S.worthington was reported from Chandigarh in 1988 by Aayagiri et al. [15] S.havana has been reported by Fule and Saoji from purulent material from human source. [16] It has also been report­ed from animals like sheep, goat, igs end from meat. [17],[18] Khan et al in 1976 have reported S.enteriditis along with other serotypes from sporadic case of gastroanteritis, septicaemia and meningitis. [19] Anti­biotic sensitivity test showed that these Salmonella strains have be­come resistant to a number of anti­biotics. The problem of therapy has worsened due to increasing number of drug resistant strains re­ported throughout India. [20],[21],[22],[23] The antibiotic sensitivity of S.typhi showed a 67.47% resistance to chloramphenicol, 65.44% to ampi­cillin, 37.80% to augmentin. Resis­tance to oxytetracycline and cotri­maxazole was 66.26%, and 74.79% respectively. Studies in Calcutta in 1990 showed that S.typhi iso­lates gave a 49.1 % resistance to ampicillin, 78.3% and 89.1%, to chloramphenicol and cotrimoxa­zole respectively. [22] Resistance to chloramphenicol, ampicillin and cotrimaxazole has been observed in salmonella species in India, Africa, Korea, Mexico and Eng­land. [23],[24] The S. typhi strains we studied also showed a 2.43%,, 2.03% and 2.43% resistance to Ciprofloxacin, pefloxacin and nor­floxacin respectively. Renu et al have reported, S. typhi strains to be resistant to ciprofloxacin in vary­ing degrees. S. typhimuriunt, S.havana, and S.worthington showed high resistance to ampicil­lin, chloramphenicol, cotrimaxazole and oxytetracycline [Table 3]. A 100%, sensitivity to quinolones, was observed with strains of S.para­typhi A, S. havana and S. worthington and S. enteritidis except a 27.77% resistance of S. typhimu­rium to norfloxacin. Sensitivity to fluoroqunolones has been noticed to be 100% by most authors. [24] Anti­biotic resistance in salmonella strains is generally encoded by plasmids . The existence of R. fac­tor was first reported in Japan in 1950-1960. [25] The gram negative bacilli like salmonella have become MDR probably due to the transfer of plasmids onto them by the mechanism of conjugation. In this study the resistance patterns were transferred by direct conjugation, and enbloc transfer was observed in most cases. Sharma et al in 1979-82 reported 21.89% of their MDR S.typhimurlum strains to carry conjugative R plasmids. [26] Resistance to ampicillin, oxytetra­cycline and chloramphenicol is in­creasing over the years due to the indiscriminate use of antibiotics, even in trivial infections. Until 1960 all salmonellae were sensitive to a wide range of antimicrobial agents. [14] In the present study it was observed that the salmonella strains had acquired MDR espe­cially to ampicillin, cotrimoxazole, oxytetracycline and chlormapheni­col. The quinolone derivative es­pecially ciprofloxacin is consider­ed a useful alternative in the treatment of drug resistant typhoid fever. In this study 58.82% of the patients were successfully treated with ciprofloxacin for an average of 7 days. In spite of it not been officially recommended it has been widely used in the paediatric age group. [23] Since the salmonella develop resistance to the antibiotics used indiscriminately, the quino­lenes should be used only in MDR cases and not for mild infections.


 ¤ Summary Top


A total of 326 salmonella strains was isolated and studied from sus­pected enteric fever cases in Mumbai (Bombay) during a 2 year period from May 1992 to July 1994. These were identified using stan­dard biochemical and serological tests. Bacteriophage typing, anti­biotic sensitivity and conjugation experiments were also carried out. S. typhi was the most common serotype accounting for 75.46% of the strains. Among S.typhi strains 87% were biotype I and 13% were biotype II 9.5% strains were of S. paratyphi A, 5.52%, of S. typhimurium, 4.60% of S. wor­thington, 4.30% of S. havana and 0.62% of S.enteritidis. The com­monest bacteriophage type of S.typhi was E1, and of S. para­typhi A type 1, whereas 88.88% strains of S.typhimurium were un­typable. Most of the strains were multidrug resistant including com­monly used antibiotics such as chloramphemicol, ampicillin, and cotrimaxazole. Quinolone deriva­tives such as Ciprofloxacin were found to be the most effective drugs. In the conjugation experi­ments there was direct transfer of resistance pattern and enbloc transfer of resistance was observ­ed in most strains. Salmonella typhi is still the most commonly encountered species. There is an alarming increase in multidrug resistance.


 ¤ Acknowlegdgements Top


We are grateful to the Medical Director - Dr. P. M. Bhujang, Sir H.N. Hospital, for the financial help to carry out this research work. We are also grateful to the authorities of Nanavati Hospital, Hinduja National Hospital and Jagjivanram Hospital for allowing us to collect Salmonella cultures from their Pathology departments. We also thank Dr. P. K. Pillai from Dept. of Microbiology, Lady Har­dinge Medical College, New Delhi for carrying out the bacteriophage typing of our Salmonella strains, and Director. Central Research institute, Kasauli for serotyping.

 
 ¤ References Top

1.Mehta PJ, Hakim A, Karnat S. The changing races of Salmonellosis. JAPI 1992;40:713-714.  Back to cited text no. 1    
2.Pillai PK, Prakash K. Current sta­tus of drug resistance and phage types of Salmonella typhi in India. Indian J Med Res 1993;117:154-158.  Back to cited text no. 2    
3.Bauer AW, Kirby WMM, Sherris MD, Turck M. Antibiotic suscepti­bility testing by a standardised single disc method. American J Clin Pathol 1966:45:493-496.  Back to cited text no. 3    
4.Holder HK, Saha DB, Ghose E, Sanayal S. Chloramphenicol resis­tant Salmonella typhi ; The cause of recent outbreak of enteric fever in Calcutta. Ind J Pathol Micro­biol 1992;35:11-17.  Back to cited text no. 4    
5.Paul K, De PK. Transferable chlormaphenicol resistance in Sal­monella typhi isolated during an outbreak of enteric fever in Cal­cutat, West Bengal. Ind J Med Microbiol 1991;9:10-12.  Back to cited text no. 5    
6.Graue K. Alarming increase in multidrug resistant Salmonella typhimurium in Southern India. Ind J Med Res 1981;74:635-641.  Back to cited text no. 6    
7.Chopra GS, Basu SK, Bhattacharya SR. Present phage types and anti­biotic susceptibility of Salmonellae. Ind J Pathol Microbiol 1992:35:345­350.  Back to cited text no. 7    
8.Agarwal SC. Salmonella serotypes identified at National Salmonella and Escherichia Centre, Kasauli, 1950-1960. Ind J Med Res 1962:50: 567-572.  Back to cited text no. 8    
9.Felix A. Laboratory control of the enteri , i fevers Brit Med Bulletin 1951:7:158-162.  Back to cited text no. 9    
10.Agarwal DS, Gupta NP, Gupta SP. Salmonella paratyphi A biochemi­cal reactions and phage types. Ind J Pathol Bactiol 1959;2:198-202.  Back to cited text no. 10    
11.Mitchell E, O'Mahoney M, Lynch D, et al. Large outbreak of fool poisoning caused by Salmonella typhimurium definitive type 49 in mayonnaise. BMJ 1989;298:99-101.  Back to cited text no. 11    
12.Agarwal SK. Goel MM, Misra AB. Prevalence of drug resistance among S.typhi and S.paratyphi A strains isolated from human sour­ces at Lucknow. Ind J Pathol Microbiol 1990:33:41-44.  Back to cited text no. 12    
13.Roche RA, Murthi PK. Salmonella phage types in Bombay from 1983­1987. Ind J Pathol Microbiol 1991; 34:17-21.  Back to cited text no. 13    
14.Saxena SN, Jayashela M, John PC, Soni NK. Salmonella serotypes in India 1986-1989. Ind J Med Micro­biol 1991:9:118-131.  Back to cited text no. 14    
15.Ayyagari A, Jagdish C, Narang A, et al. Outbreak of Salmonella worthington meningitis and septi­caemia in a hospital at Chandigarh. Ind J Med Res 1990;91:15-17.  Back to cited text no. 15    
16.Fule RP, Saoji AM. Isolation of Salmonella havana (13 . 23 : f, g : -) A rare serotype from human source. Ind J Pathol Microbiol 1988;31: 67-68.  Back to cited text no. 16    
17.Saxena SN, Mago ML, Rao LN, Ahuja S, Singh H Salmonella sero­types prevalent in India during 1978-1981. Ind J Med Res 1983;77: 10-18.  Back to cited text no. 17    
18.Khera SS. Animal Salmonellosis in India. Ind J Med Res 1962:50: 569-579.  Back to cited text no. 18    
19.Khan AM, Mehrotra RML, Agarwal SK. Occurrence of uncommon sal­monella serotypes in human infec­tions. Ind J Med Res 1976:64:1571­-1572.  Back to cited text no. 19    
20.Ayyagari A. Multidrug resistant Salmonella typhimurium in Chandi­garh. Ind J Med Res 1960;72:169­-173.  Back to cited text no. 20    
21.Sharma KB, Prakash K, Pillai PK. Epidemiology of MDR Salmonella in India. APUA News letter 1984; 24.  Back to cited text no. 21    
22.Anand AC, Kataria VK, Singh W, Chatterjee SK. Epidemic multi-resistant enteric fever in eastern; India. Lancet 1990;336:252.  Back to cited text no. 22    
23.Renu P, Kasbekar GP, Kinikar AA, Sanjay L, Nila PV. Changing pat­terns of typhoid fever in paediatric uractice. Ind J Internal Med 1992; 3:3-7.  Back to cited text no. 23    
24.Jesudason MV, John TJ. Multi­resistant Salmonella typhi in India. Lancet 1990;336:252.  Back to cited text no. 24    
25.Akiba T, Koyana K, Ishiki Y, Kinura SF. On the mechanism of multiple drug resistant clones of Shigella. Japan J Microbiol 1960: 4:219-227.  Back to cited text no. 25    
26.Sharma PL, Sharma KB, Prakash K. Incidence of conjugative plasmid amongst multidrug resistant Sal­monella typhimurium strains isolated from different parts of India. 1979-1982. Ind J Med Res 1984;79:473-478.  Back to cited text no. 26    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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