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Vol-28, no.-1, june 11 micro.pmdBangladesh 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.
enterica 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@example.com
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.
enterica 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:
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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