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Vol-28, no.-1, june 11 micro.pmd

Bangladesh J Microbiol, Volume 28, Number 1, June 2011, pp 7-11 Prevalence of Ciprofloxacin and Nalidixic Acid Resistant Salmonella enterica
serovar Typhi in Bangladesh
Shirin Afroj1, Mohammad Ilias1, Maksuda Islam2, and Samir K Saha2* 1Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh. 2Department of Microbiology, Dhaka Shishu (Children) Hospital,Dhaka-1207, Bangladesh (Received 3 January 2011; Accepted 9 April 2011)
A total of 1,059 Salmonella enterica serovar Typhi were isolated from blood samples during January 2006 to
October 2007 from urban rural facilities in Dhaka, Bangladesh, of which 980 (92.5%) isolates were nalidixic
acid resistant. The minimum inhibitory concentrations (MIC) of ciprofloxacin (CIP) were determined for 127
nalidixic acid resistant Salmonella enterica serovar Typhi (NARST) strains (every fifth) isolated during 2006.
Nine isolates were found to be resistant against CIP (3%) with high MIC (12 - >32 µg/mL). Only four isolates
were found to be sensitive (MIC <0.125 µg/mL), whereas most of the isolates (N=113) showed reduced
susceptibility (MIC 0.125 – 2 µg/mL) to CIP. All these isolates were subjected to molecular typing by multiplex
PCR on VNTR (variable number tandem repeats) loci, which revealed eight different VNTR patterns. Almost
all CIP resistant strains had similar genetic organization, identical to the most common VNTR type. Restriction

fragment length polymorphism (RFLP) analysis of the gyrA gene revealed point mutations at Ser-83 and Asp-
87 in all CIP resistant strains.
Key words: Salmonella enterica serovar Typhi, fluoroquinolone resistant, VNTR
determining region (QRDR) of the gyrA gene in Salmonella Diseases caused by Salmonella enterica serovars are especially usually leads to resistance against nalidixic acid and to decreased prevalent in developing countries. Typhoid fever is sometimes a susceptibility to ciprofloxacin7, 15-16. Turner et al.17 reported that fatal infection to adults and children that causes bacteremia and two substitutions in gyrA (Ser-83 → Phe or Tyr and Asp-87 → imflammatory destruction of the intestine and other organs. This Asn) and one in parC (Glu-84 → Lys) confer complete requires an urgent treatment by the administration of appropriate fluoroquinolone resistance in S. typhi. Emergence of these antibiotics. Emergence of multidrug-resistant (MDR) S. enterica resistant strains are associated with treatment failure or delayed serovar Typhi strains in Asia in the beginning of 1990s, led to the response to cipropfloxacin9,18.
widespread use of fluoroquinolones for treating enteric fever1-2.
There are few reports on the occurrence of typhoid by absolute MDR typhoid fever endemic started in Bangladesh in 1990s, which fluoroquinolone resistant strain in Bangladesh. Here, we report reached to peak in 1994, and then declined and re-emerged in nine cases of typhoid fever in Dhaka, Bangladesh caused by S. 2001 and 20023. During the last decade, several treatment failures enterica serovar Typhi with complete resistance to ciprofloxacin of S. typhi strains with decreased susceptibility to ciprofloxacin3- during 2006.
5 have been reported and some studies also confirmed the Materials and Methods
presence of fluoroquinolone resistant S. enterica serovar Typhi Specimen collection and identification of Salmonella enterica strains6-8. Antibiotic-resistant Salmonella strains pose a serovar Typhi significant threat to the development of reliable therapies. S.
serovar Typhi with resistance to nalidixic acid and The patients were selected for specimen collection based on the decreased susceptibilities or resistance to fluoroquinolones have clinical manifestation of typhoid fever diagnosed by physician been increasingly reported in several countries including from different sites at Dhaka City, Bangladesh. Blood samples Bangladesh7, 9-13. Such strains have also been reported from were collected along with patient's demographic data from all other parts of the world, most of them have travel history to age group of typhoid patients during 2006 to October 2007, as a Asian countries particularly South East Asia6,14.
part of routine diagnosis.
The mechanisms of fluoroquinolone resistance have been well Blood cultures were performed by the lysis centrifugation method studied. Single point mutation in the quinolone resistance- as described in our previous report19. Positive cultures were *Corresponding author:
Samir K. Saha, Department of Microbiology, Dhaka Shishu (Children) Hospital, Dhaka-1207, Bangladesh. Tel: 9138594; E-mail: [email protected]

Afroj et al. subsequently plated on blood, choocolate and MacConkey agar amplified product contains two HinfI restriction sites at the codon plates. Colonies of Salmonella enterica serovar Typhi were corresponding to Ser-83 and Asp-87 of gyrA. Restriction identified by standard biochemical and serological procedures20.
digestions were performed in a total of 15 µL reaction mixture Determination of antibiotic sensitivity and MIC of ciprofloxacin containing 8 µL of the above-mentioned PCR product, 1.5 µL of10x digestion buffer and 10 U of HinfI for overnight at 37ºC. The Antibiotic susceptibility testing was performed by disk diffusion restriction digests were electrophoresed onto a 3% agarose gel.
method using discs (Oxoid, UK) containing ampicillin (10 µg), HinfI-digested PCR products of gyrA gene from a known nalidixic ceftriaxone (30 µg), cotrimoxazole (25 µg), chloramphenicol (30 acid sensitive S. enterica serover Typhi strain, and the undigested µg), ceftazidime (30 µg), ciprofloxacin (5 µg) and nalidixic acid (30 product from known ciprofloxacin resistant strain, were run as µg). Based on the date of isolation, every fifth nalidixic acid resistant S. enterica serovar Typhi (NARST) isolated during theyear 2006 were selected to determine the MIC of ciprofloxacin (CIP) by E-strip. All results were interpreted according to Clinical During the period of the study (2006 to October 2007), a total of and Laboratory Standards Institute (CLSI) guidelines21 and were 1,059 Salmonella enterica serovar Typhi were isolated. The analysed by SPSS version 11.5. The trend of multidrug resistance, majority (57%; 605 out of 1059) of the S. enterica isolates were which is defined as resistance to ampicillin, chloramphenicol and isolated in the year 2006. A significant higher proportion of cases co-trimoxazole was also analysed during 2006 to October 2007.
(43%) occurred in patients younger than 5 years of age than in Multiplex PCR on VNTR loci those 5 years of age (9%) or older. The highest rate (13%) of S.
serovar Typhi isolation was in the second year of life All the isolATES whose MIC was determined by E-strip test were subjected to molecular typing by multiplex PCR on variablenumber tandem repeats (VNTR) using primers flanking threeVNTR loci (TR1, TR2 and TR3) (Table 1), as described by Liu etal.22. In brief, each 25 µL of reaction mixture contained 1.5 µL ofthe bacterial lysate suspension and 10 pmol each of the forwardand reverse primers for TR1 and TR3, as well as 12.5 pmol each ofthe corresponding primers for TR2, in addition to 22 µL of theTaq PCR mastermix (QIAGEN GmbH, Hilden, Germany). Afterinitial denaturation at 94°C for 5 min, the PCR reaction was run for35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min,followed by a final extension at 72°C for 7 min. The PCR products,along with a 100-bp DNA marker (Invitrogen, USA), weresubjected to electrophoresis on a 2 % agarose gel. In every case,positive and negative controls were run simultaneously.
Restriction fragment length polymorphism (RFLP) analysis PCR-RFLP was performed to detect common mutations related tofluoroquinolone resistance at the codon Ser-83 and Asp-87 of Figure 1. Age distribution of typhoid fever cases caused by S.
gyrA following the procedure described by Hirose et al.23. PCR enterica serovar Typhi identified at a diagnostic referral center was performed with the primers, gyrA-F, 5´ TGT CCG AGA TGG and Dhaka Shishu Hospital, Bangladesh during 2006-2007 (n CCT GAA GC 3´ and gyrA-HinfI-as, 5´ATG TAA CGC AGC GAG = 1059). Numbers above the bars show the total numbers of AAT GGC TGC GCC ATA CGA ACG CTG GAG 3´. The 195-bp Table 1. PCR primers flanking the three VNTR loci of S. enterica serovar Typhi22.
Nucleotide positions Sequence (5´-3´) AGA ACC AGC AAT GCG CCA ACG A CAA GAA GTG CGC ATA CTA CAC C CCC TGT TTT TCG TGC TGA TAC G CAG AGG ATA TCG CAA CAA TCG G CGA AGG CGG AAA AAA CGT CCT G TGC GAT TGG TGT CGT TTC TAC C Prevalence of Ciprofloxacin and Nalidixic Acid Resistant Salmonella enterica Antibiotic susceptibility patterns of 1,059 S. enterica serovar All the NARST isolates were subjected to molecular typing on Typhi were analysed for seven antibiotics. Among them only 52 VNTR loci through multiplex PCR. A total of 8 VNTR types (type (4.9%) isolates were sensitive to all antimicrobial agents tested.
A to type H) based on the number and size of bands were identified Although no resistance to cefriaxone (CRO) or ceftazidime (CAZ) among 127 isolates (Table 2). Most of the isolates 73% (93/127) was observed, 92.5% isolates were nalidixic acid (NA) resistant.
belonged to the VNTR type A and eight of the nine ciprofloxacin Multidrug resistance (MDR) was noted in around 56% (593 out resistant isolates were also included in this group.
of 1059) of the isolates. Moreover, MDR strains were more likely The fragments containing the gyrA QRDR of nine ciprofloxacin to be resistant to NA, in comparison to sensitive strains (96.7% resistant isolates were amplified by PCR. The 195 bp PCR products of CIP resistant strains were not digested with HinfI The results of molecular typing and MIC for ciprofloxacin are (Fig. 3), suggesting point mutations at both Ser-83 and Asp-87 of presented in Table 2 and Figure 2 respectively. Although not gyrA. In contrast, the PCR product from nalidixic acid susceptible considered resistance by current CLSI standard breakpoints, 89% control strain cleaved at both HinfI sites at the codon (113 out of 127) isolates demonstrated a "reduced susceptibility" corresponding to Ser-83 and Asp-87 of gyrA (Fig. 3).
to ciprofloxacin (MIC value 0.125 to 2 µg/mL) and 9 isolates werecompletely resistant to CIP (MIC value 12 - >32 µg/mL) (Fig. 2).
Table 2. Different VNTR types and their corresponding amplicon

Number of isolates Amplicon size (bp) > 200, > 600 150, >200, 600 >250, >300, 600 200, < 400, > 600 Figure 3. PCR-RFLP Pattern of S. enterica serovar Typhi based
on digestion of gyrA gene (195 bp) with HinfI restriction enzyme.
Lanes 1-9: Treated gyrase PCR product of CipR strains; Lanes
10: Treated gyrase product of NAS strain; Lanes 11: Non-treated
gyrase PCR product of CipR strain; Lanes 12: 25 bp DNA Ladder

Typhoid fever is still endemic in developing countries with fatalinfection in children and occasionally in adults. In the presentstudy the disease was found highest in young children rangedwithin 2-3 years. This result corroborates with our previous studyin which we reported the highest incidence of the disease inchildren younger than 5 years of age and more than one fourth ofcases occurred in the first two years of life24. The prevalence ofS. typhi infection in younger children (<5 years) has also beenreported by several other groups25-27.
Using the breakpoints recommended by the CLSI21, nine typicalS. enterica serovar Typhi isolates resistant to ciprofloxacin wereisolated in this study. However, most of the isolates (89%) were Figure 2. MIC of ciprofloxacin of some nalidixic acid resistant
found with decreased susceptibility to ciprofloxacin (MICs ranged Salmonella enterica serovar Typhi (n = 127) isolated in 2006. from 0.125 - 2.0 µg/mL). The isolates with decreased ciprofloxacin MIC value <0.125 µg/mL, Sensitive; MIC value 0.125 to 2 µg/ susceptibility were also uniformly resistant to nalidixic acid.
ml, decreased susceptibility; MIC value >2 to <4 µg/mL, Recently, S. enterica serovar Typhi isolates with decreased intermediate; MIC value 12 - >32 µg/mL, resistant ciprofloxacin susceptibility (MIC, >0.125 µg/mL) have become Afroj et al. the subject of worldwide attention28. Although none of these isolates were phenotypically "resistant" according to the current The study was partially supported by The Child Health Research CLSI guidelines, treatment failure in a substantial proportion of Foundation and Popular Diagnostic Centre Ltd, Bangladesh. We our patients treated with ciprofloxacin has been observed. The gratefully acknowledge these donors for their support and documentation of clinician as well as microbiological failure commitment to this investigation.
suggests a need to re-evaluate the interpretive MIC breakpointsof fluoroquinolones, since the current reference standards are unable to distinguish between fluoroquinolone susceptible Rahman MM, Haq JA, Morshed MA, Rahman MA. 2005. Salmonellaenterica serovar Typhi with decreased susceptibility to ciprofloxacin- isolates and isolate with reduced susceptibility7,29.
an emerging problem in Bangladesh. Int J Antimicrob Agents. 25:
The multiplex PCR based VNTR profiling revealed eight different types of VNTR patterns, among them 93 (73%) isolates showed Renuka K, Sood S, Das BK, Kapil A. 2005. High-level ciprofloxacinresistance in Salmonella enterica serotype Typhi in India. J Med two bands corresponding to 200, >600 bp (type A). Eight out of Microbiol. 54: 999-1000.
nine ciprofloxacin resistant isolates also gave the same banding Rahman M, Siddique AK, Shoma S, Rashid H, Salam MA, Ahmed QS, pattern as type A. As most of the isolates gave the same banding Nair GB, Breiman RF. 2006. Emergence of multidrug-resistant pattern like resistant strains and MIC of these strains ranged Salmonella enterica serotype Typhi with decreased ciprofloxacinsusceptibility in Bangladesh. Epidemiol Infect. from 0.125 to 2 µg/mL (reduced susceptible group), therefore indicating all of these isolates are prone to resistance to Launay O, Nguyen Van JC, Buu-Hoi A, Acar JF. 1997. Typhoid feverdue to a Salmonella typhi strain of reduced susceptibility to fluoroquinolones. Clin Microbiol Infect. 3: 541-544.
Our previous report identified three ciprofloxacin resistant isolates Slinger R, Desjardins M, McCarthy AE, Ramotar K, Jessamine P, in 2005, afterward we identified nine resistant strains with high Guibord C, Toye B. 2004. Suboptimal clinical response to ciprofloxacinin patients with enteric fever due to Salmonella spp. with reduced MIC value (12 - >32 µg/mL) in 2006. These results suggest that fluoroquinolone susceptibility: a case series. BMC Infect Dis. 4: 36.
NARST with reduced susceptibility to ciprofloxacin is now Morita M, Hirose K, Takai N, Terajima J, Watanabe H, Sagara H, endemic in Bangladesh. Only a few cases of fluoroquinolone Kurazono T, Yamaguchi M, Kanazawa Y, Oyaizu T, Izumiya H. 2010.
resistant isolates have been reported up to now, but the Salmonella enterica serovar Typhi in Japan, 2001-2006: emergence
of high-level fluoroquinolone-resistant strains. Epidemiol Infect 138:
increasing number of isolates resistant to nalidixic acid with decreased susceptibility to ciprofloxacin is a matter of concern.
Saha SK, Darmstadt GL, Baqui AH, Crook DW, Islam MN, Islam M, There is also an urgent need to reevaluate fluoroquinolones Hossain M, El Arifeen S, Santosham M, Black RE. 2006. Molecular breakpoints for S. enterica serovar Typhi.
basis of resistance displayed by highly ciprofloxacin-resistantSalmonella enterica serovar Typhi in Bangladesh. J Clin Microbiol.
More recently, Salmonella spp. isolates, exhibiting reduced susceptibility to ciprofloxacin but sensitive to nalidixic acid has Yanagi D, de Vries GC, Rahardjo D, Alimsardjono L, Wasito EB, De been observed30. Therefore, screening method for detection of I., Kinoshita S, Hayashi Y, Hotta H, Osawa R, Kawabata M, Shirakawa reduced susceptible group to ciprofloxacin by nalidixic acid disc T. 2009. Emergence of fluoroquinolone-resistant strains ofSalmonella enterica in Surabaya, Indonesia. Diagn Microbiol Infect diffusion need reconsideration. Until then, laboratory should Dis. 64: 422-426.
include MIC method along with standard disc diffusion, Asna SM, Haq JA, Rahman MM. 2003. Nalidixic acid-resistant particularly in endemic region.
Salmonella enterica serovar Typhi with decreased susceptibility to This study has the limitation that patient information was collected ciprofloxacin caused treatment failure: a report from Bangladesh.
Jpn J Infect. Dis. 56: 32-33.
retrospectively, only after confirmation that the isolates were S. Ahmed D, D'Costa LT, Alam K, Nair GB, Hossain MA. 2006.
enterica serovar Typhi. Furthermore, we could not confirm the Multidrug-resistant Salmonella enterica serovar typhi isolates with mutation in gyrA, which contain the quinolone resistance high-level resistance to ciprofloxacin in Dhaka, Bangladesh.
determining region, by DNA sequencing. The emergence of highly Antimicrob Agents Chemother. 50: 3516-3517.
ciprofloxacin-resistant isolates may be due to the overuse of this Shirakawa T, Acharya B, Kinoshita S, Kumagai S, Gotoh A, Kawabata drug in a population with high prevalence of NARST, as predicted M. 2006. Decreased susceptibility to fluoroquinolones and gyrA genemutation in the Salmonella enterica serovar Typhi and Paratyphi A by Hirose et al.23. Therefore, now it is a prime concern to consider isolated in Katmandu, Nepal, in 2003. Diagn Microbiol Infect Dis. new, effective and alternative choice of drug in forthcoming days 54: 299-303.
to combat against typhoid caused by ciprofloxacin resistant S. Chau TT, Campbell JI, Galindo CM, Van Minh Hoang N, Diep TS, enterica serovar Typhi. All isolates investigated here including Nga TT, Van Vinh Chau N, Tuan PQ, Page AL, Ochiai RL, Schultsz C, ciprofloxacin-resistant isolates were susceptible to ceftriaxone Wain J, Bhutta ZA, Parry CM, Bhattacharya SK, Dutta S, Agtini M,Dong B, Honghui Y, Anh DD, Canh do G, Naheed A, Albert MJ, and azithromycin (data not shown), which might indicate that Phetsouvanh R, Newton PN, Basnyat B, Arjyal A, La TT, Rang NN, these antibiotics could still provide an appropriate therapy for Phuong le T, Van Be Bay P, von Seidlein L, Dougan G, Clemens JD, typhoid fever. But the increased therapeutic cost, together with Vinh H, Hien TT, Chinh NT, Acosta CJ, Farrar J, Dolecek C. 2007.
difficulties in intravenous administration can be a serious Antimicrobial drug resistance of Salmonella enterica serovar typhiin asia and molecular mechanism of reduced susceptibility to the handicap in developing countries like Bangladesh.
fluoroquinolones. Antimicrob Agents Chemother. 51: 4315-4323.
Prevalence of Ciprofloxacin and Nalidixic Acid Resistant Salmonella enterica Chuang CH, Su LH, Perera J, Carlos C, Tan BH, Kumarasinghe G, So Liu Y, Lee MA, Ooi EE, Mavis Y, Tan AL, Quek HH. 2003. Molecular T, Van PH, Chongthaleong A, Hsueh PR, Liu JW, Song JH, Chiu CH.
typing of Salmonella enterica serovar typhi isolates from various 2009. Surveillance of antimicrobial resistance of Salmonella enterica countries in Asia by a multiplex PCR assay on variable-number tandem serotype Typhi in seven Asian countries. Epidemiol Infect. 137:
repeats. J ClinMicrobiol. 41: 4388-4394.
Hirose K, Tamura K, Watanabe H. 2003. Screening method for Dimitrov T, Dashti AA, Albaksami O, Udo EE, Jadaon MM, Albert Salmonella enterica serovar Typhi and serovar Paratyphi A with MJ. 2009. Ciprofloxacin-resistant Salmonella enterica serovar typhi reduced susceptibility to fluoroquinolones by PCR-restriction fragment from Kuwait with novel mutations in gyrA and parC genes. J Clin length polymorphism. Microbiol Immunol. 47: 161-165.
Microbiol. 47: 208-211.
Saha SK, Baqui AH, Hanif M, Darmstadt GL, Ruhulamin M, Nagatake Ouabdesselam S, Tankovic J, Soussy CJ. 1996. Quinolone resistance T, Santosham M, Black RE. 2001. Typhoid fever in Bangladesh: mutations in the gyrA gene of clinical isolates of Salmonella. Microb. implications for vaccination policy. Pediatr Infect Dis J. 20: 521-
Drug Resist. 2: 299-302.
Piddock LJ, Ricci V, McLaren I, Griggs DJ. 1998. Role of mutation in Sinha A, Sazawal S, Kumar R, Sood S, Reddaiah VP, Singh B, Rao M, the gyrA and parC genes of nalidixic-acid-resistant Salmonella Naficy A, Clemens JD, Bhan MK. 1999. Typhoid fever in children serotypes isolated from animals in the United Kingdom. J. aged less than 5 years. Lancet 354: 734-737.
Antimicrob. Chemother. 41: 635-641.
Turner AK, Nair S, Wain J. 2006. The acquisition of full Mahle WT, Levine MM. 1993. Salmonella typhi infection in children fluoroquinolone resistance in Salmonella typhi by accumulation of younger than five years of age. Pediatr Infect Dis J. 12: 627-631.
point mutations in the topoisomerase targets. J Antimicrob Brooks WA, Hossain A, Goswami D, Nahar K, Alam K, Ahmed N, Chemother. 58: 733-740.
Naheed A, Nair GB, Luby S, Breiman RF. 2005. Bacteremic typhoid Threlfall EJ, Ward LR, Skinner JA, Smith HR, Lacey S. 1999.
fever in children in an urban slum, Bangladesh. Emerg Infect Dis. 11:
Ciprofloxacin-resistant Salmonella typhi and treatment failure. Lancet Brown NM, Millar MR, Frost JA, Rowe B. 1994. Ciprofloxacin Saha SK, Khan WA, Saha S.1992. Blood cultures from Bangladeshi resistance in Salmonella paratyphi A. J Antimicrob Chemother. 33:
children with septicaemia: an evaluation of conventional, lysis-direct plating and lysis-centrifugation methods. Trans R Soc Trop Med Hyg. Chitnis S, Chitnis V, Hemvani N, Chitnis DS. 2006. Ciprofloxacin 86: 554-556.
therapy for typhoid fever needs reconsideration. J Infect Chemother.
Cheesebrough M. 1998. Medical laboratory manual for tropical 12: 402-404.
countries. Tropical health technology and butterworth-Heinemann, Hakanen AJ, Lindgren M, Huovinen P, Jalava J, Siitonen A, Kotilainen London, United Kingdom.
P. 2005. New quinolone resistance phenomenon in Salmonella CLSI. 2006. Performance standards for antimicrobial susceptibility enterica: nalidixic acid-susceptible isolates with reduced testing. CLSI Document M100-S16. CLSI, Wayne, PA.
fluoroquinolone susceptibility. J Clin Microbiol. 43: 5775-5778.


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Safety Data Sheet according to Regulation (EC) No1907/2006 SDS No. : 76601 TEROSON SB 2444 known as TEROKAL 2444 TB 175GR Revision: 17.09.2014 printing date: 21.11.2014 SECTION 1: Identification of the substance/mixture and of the company/undertaking 1.1. Product identifier TEROSON SB 2444 known as TEROKAL 2444 TB 175GR