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Plasmid Profile Analysis and Antimicrobial Resistance Pattern of Shigella flexneri Strains Isolated From Azad Kashmir Pakistan.

Byline: Basharat Ahmed K. Al- Ghanim Shahid Mahboob Farah R. Shakoori and A. R. Shakoori

Abstract This study was carried out to determine the antimicrobial susceptibility patterns of 584 Shigella flexneri isolated from diarrheal patients admitted in hospitals in Azad Kashmir Pakistan and to evaluate their changing trends against twenty antibiotics. The isolates showed highest resistance against penicillin followed by carbenicillin ampicillin tetracycline erythromycin ceftizoxime kanamycin co-trimoxazole piperacillin amoxicillin amikacin streptomycin nalidixic acid gentamicin chloramphenicol cephalothin and ceftriaxone. All S. flexneri isolates were sensitive to cefixime ciprofloxacin and enoxacin. Multiple drug resistance (MDR) was observed against 3-10 drugs and was resistant against three or more antibiotics was at the level as high as 300g/ml. The resistant isolates showed different patterns of antibiotics resistance. The most common pattern was PCaA.

The plasmids were observed in 32.8% MDR strains of S. flexneri which were found resistant against three or more antibiotics. The number of plasmids varied from one to seven. Analysis of plasmid DNA of S. flexneri revealed that all the strains contained a heterogeneous population of plasmids ranging between greater than 23.1 kb to less than 2.0 kb. Based on molecular weight the pattern of different plasmids was also very diverse. Depending on the number of plasmids individual strains were grouped into nine different plasmid patterns. Some of the antibiotic resistance determinants were cured by acridine orange indicating that widespread antibiotic resistance is mediated through plasmid. Transformation experiments showed that the factors for resistance against ampicillin chloramphenicol and sulfamethoxazole-trimethoprim resided in greater than 23.1 Kb and 23.1 Kb plasmids.

Key words: S. flexneri antibiotic resistance R-plasmid plasmid curing multiple drug resistance diarrhoea Shigellosis

INTRODUCTION

Shigella is a rod shaped non-motile nonspore-forming facultative anaerobic Gram- negative bacterium. It is lactose-fermenting bacterium causing dysentery (Yang et al. 2005). In developing countries shigellosis is widespread and causes extensive outbreaks. In industrialized countries this disease has become rare and it currently occurs as sporadic cases in migrant workers or those who travel to developing countries and is limited to epidemic episodes among children in daycare centers individuals in custodial institutions and homosexual men (Niyogi 2005). Consequently although shigellosis is a major public health concern there is a great disparity between developing countries (over 163.2 million cases each year) and developed ones (1.5 million cases). Epidemics usually occur in areas with crowding and poor sanitary conditions where transmission from person to person is common or when the organisms contaminate the food or water (Kotloff et al. 1999).

Dramatic outbreaks may also occur particularly in the context of humanitarian disasters (wars refugee camps). Shigellosis is not the most frequent cause of diarrhoeal disease but its dysenteric form is the most severe; each year it kills between 600000 and 1 million people mostly children in the developing countries. Most that are infected with Shigella develop diarrhoea fever and stomach cramps starting a day or two after they are exposed to the bacterium. Shigellosis usually resolves in 5 to 7 days. In some persons especially young children and the elderly the diarrhoea can be so severe that the patient needs to be hospitalized. A severe infection with high fever may also be associated with seizures in children less than 2 years old.

Shigellosis can usually be treated with antibiotics. The antibiotics commonly used for treatment are ampicillin trimethoprim/ sulfamethoxazole nalidixic acid or ciprofloxacin. Appropriate treatment kills the Shigella bacteria that might be present in the patient's stools and shortens the illness. Unfortunately some Shigella bacteria have become resistant to antibiotics and using antibiotics to treat shigellosis can actually make the germs more resistant in the future. Persons with mild infections will usually recover quickly without antibiotic treatment. Therefore when many persons in a community are affected by shigellosis antibiotics are sometimes used selectively to treat only the more severe cases. Indiscriminate use of the drugs and horizontal gene transfer has led to Shigella species becoming resistant to commonly used antibiotics (Noriega et al. 1999).

Shigellosis is one of the major diarrhoeal diseases in Bangladesh and several other countries and is responsible for a significant number of deaths especially among children (Kotloff et al. 1999). In areas where diarrhoeal disease is endemic S. flexneri is usually the most prevalent species. Until recently at least 47 serotypes of Shigella have been recognized; of which 15 belong to S. flexneri. More elaborately S. flexneri has eight serotypes of which serotypes 1 to 5 are further classified into 12 subserotypes (Talukder et al. 2001). This classification scheme for S. flexneri is not comprehensive because atypical strains or newer subserotypes are being isolated from different parts of the world including Bangladesh (Talukder et al. 2002). Among serotype 1 isolates the prevalence of provisional serotype S.flexneri 1c increased from 0 to 56% from 1978 to 2001 in Bangladesh (Talukder et al. 2003).

The indiscriminate use of antibiotics is considered the main factor in emergence and dissemination of antibiotic resistance. In order to ensure appropriate treatment continual surveillance is required to determine which antibiotics are still active. The people in Azad Kashmir Pakistan face health hazards because of poor sanitation practices. The present study was aimed at determining the status of antibiotic resistance in S. flexneri in Azad Kashmir and to investigate the virulence factors in the local isolates and their possible role in infection.

MATERIALS AND METHODS

Bacterial strains

Shigella flexneri strains were isolated from stools of patients suffering from diarrhoea admitted in different hospitals of Azad Kashmir (Pakistan) over a 5-year period. The samples were obtained from children (aged 0-5 years) and adults. The study subjects were both male and female. For the isolation of S. flexneri a loop full of stool was mixed with 10 ml of sterile buffered peptone water and incubated at 37C for 24 h. After incubation a loop full of culture was streaked on the Salmonella- Shigella agar (SSA) and MacConkey agar plates and incubated at 37C for 24 h. Non-lactose fermenting colonies (i.e. colorless) on MacConkey agar plates were inoculated on xylose lysine deoxycxholate agar and incubated at 37C for 24 h. After incubation red colonies with 2-4 mm diameter were marked and suspected colonies were subjected to subsequent Gram staining to locate Gram negative short rods. All plates were incubated aerobically at 37C for 24 h.

From amongst the suspected S. flexneri from both SSA and MacConkey agar the non-lactose-fermenting colonies were biochemically identified on urea triple sugar iron sulphide- sulphide-indol and motility medium and siminous citrate tests. Serotyping was determined by Kligler iron agar (DIFCO).

Chemicals and media

Chemicals and antibiotics used in this study were obtained from Sigma Chemicals Co. and were of molecular biology grade. The culture media were purchased from DIFCO (USA). LB medium was used for the cultivation of bacteria and Muller Hinton agar (DIFCO) was used for susceptibility testing. Antibiotic susceptibility discs used were from OXOID England and also prepared in the cell and molecular biology laboratory. Antibiotics used in these studies were amikacin (Ak) amoxicillin (Am) ampicillin (A) carbenicillin (Ca) cefixime (Cfm) ceftizoxime (Cxm) ceftriaxone (Cz) cephalothin (Cl) chloramphenicol (C) ciprofloxacin (Cip) co-trimoxazole (Co) enoxacin (E) erythromycin (Er) gentamicin (G) kanamycin (K) nalidixic acid (Na) penicillin (P) streptomycin (S) sulfamethoxazole-trimethoprim (SxT) and tetracycline (T). All solutions were sterilized by Millipore (0.45m) filters and refrigerated.

Antimicrobial sensitivity testing

Antibiotic susceptibility tests of the collected isolates of S. flexneri were performed by antibiotic disc diffusion method (Bauer et al. 1966) using filter paper discs. The minimum inhibitory concentrations (MICs) using a specific range of concentrations of fifteen commonly used antibiotics (25 50 100 and 300g/ml) were determined by agar dilution method. In this study the MIC was defined as the lowest concentration of antibiotic at which no growth was visible. Reference strains Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were tested regularly as controls according to the National Committee for Clinical Laboratory Standards (1993).

Plasmid DNA isolation

Plasmid DNA was isolated from the multiple antibiotics resistant strains according to Birnboim and Doly (1979) and was done to separate identify and purify the plasmid DNA through agarose gel electrophoresis (Meyers et al. 1976). The plasmid DNA was purified by removal of RNA with the help of RNase. To estimate the size of plasmid DNA DNA Marker ( DNA cut with Hind-III) was used. After gel electrophoresis various plasmid DNA bands were cut out of the gel individually DNA extracted and purified by the usual molecular biological techniques (Weislander 1979).

Plasmid curing

The method of Hirota (1960) was followed for this purpose. Inocula (2x10 - 5x10 bacteria) was added to varying concentrations of acridine orange broth and incubated at 37C overnight. Culture containing the highest concentration of acridine orange in which growth was clearly visible was diluted and spread on nutrient agar plates containing appropriate antibiotic.

Transformation

E. coli HB101 (plasmid less and sensitive to antibiotics) were transformed with different individually isolated plasmids. For this 5 l of plasmid DNA of MDR S. flexneri was added to competent cells of E. coli HB101 incubated on ice for 30 min and then at 42C for two min. One ml of pre-warmed LB broth was then added to this mixture and re-incubated at 37C at 60 rpm for 80 min. The whole mixture was then spread on two different Luria-Bertani agar plates containing ampicillin (100 g/ml) chloramphenicol (100 g/ml) sulfamethoxazole-trimethoprim (100 g/ml) and incubated at 37C overnight (Sambrook et al. 1989).

RESULTS

Prevalence vs age groups In this study 584 strains of S. flexneri were isolated during the study period 149 (66.2%) were isolated in 2004 94 (55.9%) in 2005 125 (65.8%) in 2006 81 (66.4%) in 2007 and 135 (65.5%) in 2008. S. flexneri decreased from 66.2% in 2004 to 65.5% in 2008.

Over all the highest proportion of stool specimens infected with S. flexneri was observed in the age group of 6-10 years and 21-30 years (66.7%) followed by 0-5 years (66.5%) 11-20 years (63.2%) greater than 60 years (46.1%) 51-60 years (45.4%) and 31-40 years (44.1%). The lowest infestation was observed in the age group 41-50 years (42.8%) as against 67% in children of 0-5 years of age.

Antimicrobial sensitivity testing

Five hundred and eighty four clinical isolates of S. flexneri were screened for commonly used antibiotics resistance. The resistance pattern of the isolates is shown in Table I. Overall 65.1% S. flexneri isolates were resistant to penicillin (P) followed by 56.3% to carbenicillin (Ca) 55.6% to ampicillin (A) 51.9% to tetracycline (T) 50.5% to erythromycin (Er) 47.9% to ceftizoxime (CXM) 43.3% to kanamycin (K) 42.9% to co-trimoxazole (Co) 41.8% to piperacillin (Pa) 39.0% to amoxicillin (Am) 37.8% to amikacin (Ak) 31.2% to streptomycin (S) 29.3% to nalidixic acid (Na) 27.4% to gentamicin (G) 26.1% to chloramphenicol

Table I Occurrence of antibiotics resistance of 584 S. flexneri isolates at four different concentrations.

Antibiotics###No. of resistant isolates at

###25 g/ml###50 g/ml###100 g/ml###300 g/ml

Amikacin (Ak)###221(37.8%)###186 (31.8%)###82 (14.0%)###16 (2.7%)

Ampicillin (A)###325(55.6%)###270 (46.2%)###164 (28.1%)###71 (12.1%)

Amoxicillin (Am)###228(39.0%)###197 (33.7%)###88 (15.0%)###13 (2.2%)

Carbenicillin (Ca)###329(56.3%)###304 (52.0%)###187 (32.0%)###75 (12.8%)

Cefixime (Cef)###00(0.0%)###00 (0.0%)###00 (0.0%)###00 (0.0%)

Ceftizoxime (CXM)###280(47.9%)###252 (43.1%)###141 (24.1%)###52 (8.9%)

Ceftriaxone (Cz)###114(19.5%)###87 (14.9%)###36 (6.2%)###3 (0.5%)

Cephalothin (Cl)###130(22.3%)###118 (20.2%)###40 (6.8%)###7 (1.2%)

Chloramphenicol (C)###153(26.1%)###132 (22.6%)###51 (8.7%)###12 (2.0%)

Ciprofloxacin (Cip)###00(0.0%)###00(0.0%)###00 (0.0%)###00 (0.0%)

Co-trimoxazole (Co)###251(42.9%)###229 (39.2%)###109 (18.6%)###20 (3.4%)

Enaxacin (E)###00(0.0%)###00 (0.0%)###00 (0.0%)###00 (0.0%)

Erythromycin (Er)###294(50.3)###280 (47.9%)###155(26.5%)###56 (9.6%)

Gentamicin (G)###160(27.4%)###145 (24.8%)###43 (7.4%)###4 (0.7%)

Kanamycin (K)###253(43.3%)###232 (39.7%)###170 (29.1%)###46 (7.9%)

Nalidixic acid (Na)###171(29.3%)###151 (25.8%)###54 (9.2%)###16 (2.7%)

Penicillin (P)###380(65.1%)###353(60.4%)###266 (45.5%)###112 (19.2%)

Sulfamethoxazole-###244(41.8%)###210 (35.9%)###95 (16.3%)###19 (3.2%)

Trimethoprim (SxT)###182(31.2%)###159 (27.2%)###64 (10.9%)###17 (2.9%)

Streptomycin (S)###303(51.9%)###293(50.2%)###168 (28.8%)###60 (10.3%)

Tetracycline (T)

(C) 22.3% to cephalothin (Cl) and 19.5% to ceftriaxone (Cz). All S. flexneri isolates were sensitive to cefixime (Cfm) ciprofloxacin (CIP) and enoxacin (E).

Table I shows resistance of 584 strains of S flexneri against twenty antibiotics at four concentrations viz. 25 50 100 and 300g/ml. Generally the isolates showed the highest resistance against penicillin and the lowest resistance was against ceftriaxone at all the four levels of antibiotics screened. At 300g/ml the isolates showed considerable decrease in the resistance frequency of almost all the antibiotics tested.

MDR was observed in this study ranging from three to ten drugs. Out of 584 isolates screened for antibiotic resistance 36% were resistant at 25g/ml 32% were resistant at 50g/ml 10% were resistant at 100g/ml and 5% were resistant at 300g/ml to three or more antibiotics. The resistant isolates showed different patterns of antibiotics resistance. The most common pattern was PCaA at all the four levels shown in Table II.

Plasmid profile

A total of 225 isolates of S. flexneri werex processed for isolation of plasmids and only 74 (32.8%) carried plasmids. These isolates were resistant to three or more antibiotics used in this study. The number of plasmids varied from one to seven.

Table II.- Multiple antibiotic resistance patterns occurring in S. flexneri isolated from various clinical sources of Azad Kashmir.

###Percent of resistant

Antibiotics resistance patterns###isolates at (g/ml)

P Ca A###36###32###10###5

P A T###33###30###9###4

P Ca T###30###26###7###4

P Ca A T###24###22###7###3

P A T Er###21###18###5###3

P Ca A Er###17###31###5###2

P Ca A CXM###15###11###5###2

P Ca A T Er###12###10###4###1

P C A T CXM###9###6###4###1

P Ca T CXM K###7###5###3###1

P Ca A T K Co###5###5###2###1

P A T Er K Co###4###3###2###-

P Ca A T Er Co SxT Am###3###2###1###-

P A C Er K Co Am Ak S Na###2###1###-###-

P Ca A T Co Am Ak S Na G###1###1###-###-

In S. flexneri the analysis of plasmid DNA revealed that all the isolates contained a heterogeneous population of plasmids ranging between greater than 23.1 kb to less than 2.0 kb (Fig.1). The most dominant plasmids were 2.0 Kb (82.4%) 2.3 Kb (67.6%) 23.1 Kb (56.7%) 4.3 Kb (44.6%) 6.5 Kb (40.5%) greater than 23.1 Kb (36.5%) and 4.3 Kb (32.4%). Other plasmids were observed in lesser frequency. The frequency of 9.4 Kb plasmid was 25.7% for greater than 9.4 Kb it was 22.9% and for less than 2.0 Kb it was 8.1%.

Based on molecular weight the pattern of different plasmids was also very diverse. Depending upon the number of plasmids the 74 individual strains were grouped into nine different plasmid patterns designated P1-P9. Sixteen isolates (21.6%) had pattern P1 (5 plasmids) fifteen isolates (20.3%) had pattern P2 (3 plasmids) twelve isolates (16.2%) had pattern P3 (5 plasmids) nine isolates (12.2%) had pattern P4 (4 plasmids) seven isolates (9.4%) had P5 (5 plasmids) six isolates (8.1%) had P6 (5 plasmids) five isolates (6.7%) had P7 (5 plasmids) three isolates (4.0%) had P8 (4 plasmids) and the remaining one strain (1.3%) had pattern P9 (2 plasmids) (Table III).

Antimicrobial resistance determinants are plasmid borne

Plasmid curing

Representative MDR isolates of S. flexneri (BSf-475 BSf-4072 and BSf-4291) were selected for plasmid curing. Acridine orange was used as curing agent during this study for elimination of plasmids. Out of 100 colonies each from four treated cultures some had lost the resistance to one or the other antibiotic. Effect of plasmid curing on the drug resistance determinants of S. flexneri isolates is shown in Table IV.

Transformation

Of the 74 S. flexneri isolates the plasmids of 35 strains were used for transformation of E. coli

Table III.Plasmid profile analysis of 74 isolates of S. flexneri.

###Plasmid (Kb)

No of isolates###Plasmid pattern

###greater than 23.0###23.1###greater than 9.4###9.4###6.5###greater than 4.3###4.3###2.3###2.0###less than 2.0

###16###-###+###-###+###+###-###-###+###+###-###P1

###15###+###-###-###-###-###-###+###+###-###-###P2

###12###+###-###+###-###-###-###+###+###+###-###P3

###9###-###+###-###+###+###+###-###-###-###-###P4

###7###-###+###-###+###-###-###+###+###+###-###P5

###6###-###+###-###-###-###-###+###+###+###+###P6

###5###-###-###+###-###+###-###+###+###+###-###P7

###3###-###+###-###+###-###-###+###-###+###-###P8

###1###-###+###-###-###-###-###-###-###+###-###P9

Table IV.- Effect of acridine orange mediated plasmid curing on the antibiotic resistance pattern of S. flexneri isolates.

Isolate no.###Resistance pattern

###Pre-curing###Post-curing

BSf-475###PCaA###-

BSf-4072###PCATCXM###-

BSf-4290###PCAKCoSxTAmAk###Am

BSf-4291###PACErKCoAmAkSNa###AmS

HB101 separately ascertaining ampicillin (MIC-100 g/ml) chloramphenicol (MIC-100 g/ml) and sulfamethoxazole-trimethoprim (MIC-100 g/ml) sensitivity. Plasmids of 23 (65.7%) 19 (54.3%) and 17 (48.6%) isolates were used for transformation of E. coli HB101 and tested for ampicillin chloramphenicol and sulfamethoxazole trimethoprim resistance respectively. Of the 35 transformations 30 (85.7%) were successfully accomplished as E. coli HB101 acquired antibiotic resistance to ampicillin chloramphenicol and sulfamethoxazole-trimethoprim. Plasmids of three isolates (BSf-415 BSf-596 and BSf-4127) were successfully transferred to E. coli HB101 shown by the acquisition of resistance to ampicillin and plasmids of another three strains (no. BSf-418 BSf- 480 and BSf-4162) with chloramphenicol resistance were also successfully introduced into E. coli HB101.

Plasmids of 23 isolates resistant to ampicillin 19 isolates resistant to chloramphenicol and 17 isolates resistant to sulfamethoxazole- trimethoprim were also successfully introduced into E. coli HB101.

Table V.- Transformation of plasmids of S. flexneri into E. coli Hb101.

###Molecular

###weight of###Transformed

###No.###plasmids###plasmids that

Sample

###of###which were###conferred

no.

###plasmids###individually###antibiotic

###transferred to###resistance.

###E. coli HB101.

475###5###greater than 23.1Kb###greater than 23.1Kb

###greater than 9.4Kb 4.3Kb

###2.3Kb 2.0Kb.

4072###5###23.1Kb 4.3Kb###23.1Kb

###2.3Kb 2.0Kb

###less than 2.0Kb.

2091###2###23.1Kb 2.0Kb###23.1Kb

In some multiple plasmid isolates (no. BSf- 475 BSf-4072 and BSf-4291) all the DNA bands of different molecular sizes were cut out of the gel extracted purified and then successfully transferred to E. coli HB101 individually. The plasmids (greater than 23.1 Kb and 23.1 Kb) could only confer ampicillin chloramphenicol and sulfamethoxazole- trimethoprim resistance to the competent cells of E. coli HB101 (Table V).

DISCUSSION

Prevalence vs age group

Shigellosis is primarily a childhood disease in both developed and developing countries including Pakistan whereas epidemic shigellosis affects all age groups (Keusch and Bennish 1991; Ahmed and Shakoori 1996). The information about the etiology and drug sensitivity pattern of bacterial strains is however lacking due to the lack of diagnostic facilities. In this study the prevalence S. flexneri decreased over the years from 66.2% in 2004 to 65.5% in 2008. The highest proportion of stool specimens infected with S. flexneri was observed in the age group of 6-10 years. Lowest infestation was observed in the age group 41-50 years (42.8%). Almost similar results were reported by earlier workers (Ahmed and Shakoori 2003). Khalil et al. (1998) reported slightly higher prevalence in the age groups of 11-20 and 21-30 years. The highest infestation of Shigella was observed in the age groups of 18-23 and 24-35 years.

Similarly Bhattacharya et al. (2005) reported that the majority (79%) of Shigella species were isolated from children aged less than five years in Eastern Nepal. The high incidence of shigellosis in children is attributed to their poor resistance lack of previous exposure poor hygiene and higher exposure to contaminated environment due to play-related activities. In the present study the higher incidence of shigellosis in the age groups of 6-10 years is attributable to poor hygiene and fecal matter contaminated environment. In the areas where diarrhoeal disease is endemic S. flexneri is usually the most prevalent species (Henry 1991). Until recently at least 47 serotypes of Shigella were recognized of which 15 belonged to S. flexneri (Clemens et al. 1999).

Antibiotic resistance

The use of antimicrobial agents in the treatment of diarrhoea has greatly improved the quality of life among residents in and travelers to developing countries (Tjaniadiu et al. 2003). Development of resistance has however complicated the therapeutic activities. In the current study 65% S. flexneri isolates were resistant to penicillin followed by 56.3% to carbenicillin 55.6% to ampicillin 51.9% to tetracycline 50.5% to erythromycin 47.9% to ceftizoxime 43.3% to kanamycin 42.9% to co-trimoxazole 41.8% to piperacillin 39.0% to amoxicillin 37.8% to amikacin 31.2% to streptomycin 29.3% to nalidixic acid 27.4% to gentamicin 26.1% to chloramphenicol 22.3% to cephalothin and 19.5% to ceftriaxone. All S. flexneri isolates were sensitive to cefixime ciprofloxacin and enoxacin. These results are comparable with the results of a previous study in Iran (Nowroozi and Hakemivala 2006)

where it was reported that 56.7% of isolated S. flexneri strains had multi drug resistance to ampicillin tetracycline and trimethoprime- sulfamethoxazole but 100% of isolates were sensitive to ciprofloxacin. Similar results have been reported by Turner et al. (2003) where 100% of S. flexneri isolates were resistant to carbenicillin streptomycin chloramphenicol tetracycline ampicillin ticarcillin. However 39.3% isolates were resistant to trimethoprime-sulfamethoxazole and 6% were resistant to nalidixic acid. All the strains were susceptible to cephalotin colistin kanamycin amikacin ciprofloxacin cefoxitin and cefotaxime. Results of this study showed more resistance to tetracycline ampicillin and trimethoprime-sulfamethoxazole which might be due to geographic differences or indiscriminate use of these drugs.

In another study Kaisar et al. (2003) showed that 42% of isolates were resistant to three commonly used antibiotics ampicillin tetracycline and trimethoprime-sulfamethoxazole which was close to our result.

In their study Casalino et al. (1994) reported that all but three of the strains were resistant at least to ampicillin chloramphenicol spectinomycin and tetracycline. Of these resistant strains 41 were resistant to sulfonamide and streptomycin and 14 were resistant to trimethoprim or trimethoprim and kanamycin. Similarly in Bangladesh Talukder et al. (2003) reported that all S. flexneri serotype 1b were resistant to tetracycline 94% were resistant to ampicillin 72% were resistant to trimethoprim- sulfamethoxazole and 18% were resistant to nalidixic acid. None of the strains were resistant to mecillinam or ciprofloxacin. In a study in southeast Brazil Penatti et al. (2007) reported that the S. flexneri strains were resistant to ampicillin in 83.3% of cases chloramphenicol in 70.0% streptomycin in 86.7% sulfamethoxazole in 80.0% and tetracycline in 80.0% while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%).

In southren Ethiopia out of the ninety-nine S. flexneri group B strains isolated 95 were found to be susceptible to gentamicin and 90 to nalidixic acid. In addition 55.5% of S. flexneri strains were reported to be resistant to trimethoprim- sulfamethoxazole (Cobra and Sack 1996).

In the current study the isolates showed the highest frequency of resistance against penicillin at all concentrations of antibiotics used. The lowest resistance was against ceftriaxone followed by cephalothin and chloramphenicol. The resistance of S. flexneri to doses as high as 300g/ml is alarming because if S. flexneri become resistant to such high levels of antibiotics the treatment of disease with antibiotics would become quite difficult. In Pakistan Ahmed and Shakoori (1996) reported highest frequency of resistance against septran at 50 and 100g/ml whereas chloramphenicol resistance was 88.8%. In another study in Pakistan (Ahmed and Shakoori 2001) documented 50% resistance of Shigella strains and in Northern Areas of Pakistan Ahmed et al. (2003) reported 14.3% resistance of Shigella strains against chloramphenicol. MDR was observed in this study ranging from three to ten drugs.

The most common pattern was PCaA at all the four concentrations. Analogous results were reported by other investigators in many countries including Pakistan (Ahmed and Shakoori 2001; Ahmed et al. 2003).

Plasmid borne resistance factors

There are large numbers of antimicrobial agents such as penicillin cephalosporin tetracycline spectinomycin chloramphenicol fusidic acid sulfonamides heavy metal and others for which plasmid-mediated antibiotic resistance has been reported. The cause of the increase in R factor- carrying bacteria is due to the selective pressure caused by antibiotics and other chemotherapeutic agents. These drugs are currently being used not only in humans but also in animals cultured fish fruits vegetables rice plants and honey bees. It has been shown that the use of antibiotics in animal and fish culturing greatly increase the pool of R factor- carrying bacteria in the environment. It seems likely that the use of antibiotics for other non-medical purposes also helps the increase of the reservoir of R factors (Watanabe 1972). A high incidence of plasmid-mediated resistance has been observed in enterobacteria including Shigella in many countries.

The most common pattern was resistance to sulfonamide and streptomycin which were carried by a single plasmid. The strains of Shigella resistant to various commonly used antibiotics have been reported from various parts of the world including Pakistan (Ahmed and Shakoori 2001; Ahmed et al. 2003). Multiple drug resistance in Shigella has complicated the situation in recent years (Ghosh and Sehgal 1998). The genes for resistance to ampicillin chloramphenicol spectinomycin and tetracycline formed a linkage group located on the chromosome of the strains of all serotypes (Casalino et al. 1994).

Plasmid profile

Plasmid profile analysis is a useful tool in epidemiological studies dealing with enteric infections. In the present work the multiple drug resistant (MDR) strains of S. flexneri were processed for isolation of plasmids. This study revealed that (32.8%) strains of S. flexneri carried plasmids. These were found resistant to three or more antibiotics. The number of plasmids varied from one to seven. Shigella spp. is known to carry as many as 10 plasmids (Ling et al. 1993) these results indicate a high probability of identity between the R-plasmids found in S. flexneri which usually encoded resistance to ampicillin tetracycline chloramphenicol sulfamethoxazole- trimethoprim and co-trimoxazole. Genes for resistance to sulfonamide and streptomycin were located on a 63-kb plasmid in strains of S. flexneri serotypes lb 2a and 4 (Casalino et al. 1994).

The large plasmid was found to contain a gene conferring virulence (Lin and Chang 1992) the large plasmid was very unstable and easily lost only a small number of strains of Shigella species were found to have the large virulence plasmid (Vargas et al. 1999). Shigella with plasmidmediated multiple drug resistance including Pakistan (Ahmed and Shakoori 2001; Ahmed et al. 2003) have been well documented in India (Dutta et al. 2002) and in Bangladesh (Talukder et al. 2003; Iqbal et al. 2014). In the present study all the MDR isolates of S. flexneri contained a heterogeneous population of plasmids ranging between greater than 23.1 kb to less than 2.0 kb. Based on molecular weight the pattern of different plasmids was also very diverse. Depending on the number of plasmids individual strains were grouped into nine different plasmid patterns and were found among (MDR) S. flexneri strains.

Similar results are reported in a previous study in Iran (Nowroozi and Hakemvala 2006) where they reported that eleven distinct plasmid patterns were identical and plasmid size ranged from 0.564 kb to 21.226 kb. Plasmids of the same size were present in multiple strains for example most of strains (78.8%) harbored the 21.226 kb plasmid. Similarly the comparable results have been presented in a previous study Hoe et al. (2005) reported that the heterogeneous plasmid patterns were observed in all Shigella spp. There was a correlation between plasmid patterns and serotypes of S. flexneri S. dysenteriae and S. sonnei. Five common small plasmids (greater than 20.0 kb) were observed in S. flexneri 1b and 2a whereas six common small plasmids were found in serotype 3a. Some of these plasmids appeared to maintain their existence stably in each individual serotype. Plasmids of size 11.40 and 4.20 kb were present only in S. flexneri 2a isolates

Whereas the 4.40 kb plasmid was unique for serotype 3a. Large (greater than 150 kb) or mid-range plasmid (20.0150 kb) was not observed from any S. flexneri 1b isolates. Eighty-nine percent of S. flexneri of various serotypes harbored the plasmid of 3.20 kb. Similarly as reported in a previous study that the plasmid profiles of S. dysenteriae serotype 1 flexneri 2a and sonnei strains indicated presence of large plasmid (approx. 210 kb) and multiple copies (4-6 copies) of smaller plasmids in almost all strains. Each of the serotypes presented with unique plasmid profile hence this can be used as an epidemiological marker tool for strain differentiation.

Location of drug resistance determinant

The location (chromosomal or extra chromosomal) of drug resistance determinants was also confirmed by plasmid curing strategies. In this connection acridine orange mediated curing was performed. The loss of resistance to single or multiple antibiotics after acridine orange treatment of isolates point to the fact that observed antibiotic resistance was plasmid borne. In the present study the representatives multiple drugs resistant isolate of S. flexneri were selected for plasmid curing. Acridine orange was used as curing agent during this study for the elimination of plasmid. Out of 100 colonies each from treated cultures some had lost the resistance to one or the other antibiotic. Effects of plasmid curing on the drug resistance determinants of S. flexneri isolates are depicted in (Table V). Resultantly some of the resistance markers were stably lost (excluding amoxicillin and streptomycin in terms of the MDR S. flexneri strains;

Thereby showing the chromosomal location of these two markers). However a total loss to penicillin was found in the cultures. Similar studies were performed by earlier workers (Joan 1997; Rasool et al. 2003) where some of the representative isolates lost the antibiotic resistance after acridine orange mediated curing. The resistance markers were stably lost (excluding amoxicillin and streptomycin in terms of the MDR Klebsiella strains; there by showing the chromosomal location of these two markers.

In many cases resistance genes are found to reside on easily transferable R-plasmids and various studies report that between 45 and 73% of these plasmids are transferable (Ling et al. 1993; Hoe et al. 2005). In current study the plasmids of MDR S. flexneri strains were processed for transformation into E. coli HB101 separately for ampicillin (MIC- 100 g/ml) chloramphenicol (MIC-100 g/ml) and sulfamethoxazole-trimethoprim (MIC-100 g/ml. The transformations of (85.7%) were successfully accomplished as E. coli HB101 acquired antibiotic resistance to ampicillin chloramphenicol and sulfamethoxazole-trimethoprim. The plasmids (greater than 23.1 Kb and 23.1 Kb) could only confer ampicillin chloramphenicol and sulfamethoxazole- trimethoprim resistance to the competent cells of E. coli HB101. These results are comparable with results of a previous study in Somalia where Casalino et al. (1994) reported that the multiple drug resistance had become widely disseminated in the

S. flexneri population with a high rate (41%) of resistance to five or more drugs. The components of the resistance patterns mediated by plasmids were the resistance to SU and SM and the resistance to TP or TP and KM. Conjugative trimethoprim or trimethoprim and kanamycin resistance plasmids with lengths of 80 to 110 kb were present.

In conclusion the present study demonstrates that S. flexneri is the predominant species. There is a significant increase in resistance to several commonly-used antimicrobial agents. The results suggest reconsideration of the empiric use of these antimicrobial agents for the treatment of shigellosis. Our findings stress the need for distributing reliable information about antimicrobial resistance patterns and for ongoing drug resistance surveillance. Search for alternate new drugs should be continued because although newer antimicrobial drugs can offer hope for treatment of shigellosis emergence of resistance to the new drugs is also not far in the future. Thus intensive water and sanitation programs and vaccine development would seem to be critical. The preventive measures need to be directed not only at the prudent use of antimicrobial agents but also at risk factors such as preparation of food storage and treatment of water and interruption of person-to-person spread.

It is emphasized that hands should be washed before eating before feeding children after defecation and after disposal of children's excreta. These measures are further reinforced in epidemic situations when because of the very low infective dose of the organism and its potential for rapid spread stringent control measures need to be instituted through simple but effective health education messages to the common masses.

ACKNOWLEDGEMENT

The authors would like to express their sincere appreciation to the Deanship of Scientific Research at the King Saud University for funding of this research through the Research Group Project No. RGP- 341.

Conflict of interest statement The authors have no conflict of interest to declare.

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