Multiresistant Shigella spp. Isolated from Cases of Childhood Diarrhoea in Ile-Ife, Southwest Nigeria

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1 Journal of Microbiology Research and Reviews Vol. 2(3): 1929, May, 2014 ISSN: Multiresistant Shigella spp. Isolated from Cases of Childhood Diarrhoea in IleIfe, Southwest Nigeria Abdu B. Abdulrasheed 1*, Aboderin O. Aaron 2, Elusiyan B. Jerome 3, Kolawole O. Deboye 4, and Lamikanra Adebayo 5 1 Department of Medical Microbiology and Parasitology, Faculty of Basic Medical Sciences, College of Health Sciences, Niger Delta University, Wilberforce Island, BayelsaState, Nigeria. 2 Department of Medical Microbiology and Parasitology, Faculty of Basic Medical Sciences, College of Health Sciences, Obafemi Awolowo University, IleIfe, OsunState, Nigeria. 3 Department of Paediatrics, Faculty of Clinical Sciences, College of Health Sciences, Obafemi Awolowo University, Ile Ife, Nigeria. 4 Department of Microbiology, Faculty of Sciences, Obafemi Awolowo University, IleIfe, OsunState, Nigeria. 5 Department of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, IleIfe, OsunState, Nigeria. for Correspondence: abdulsoul@gmail.com; a.abdulrasheed@mail.ndu.edu.ng Abstract The study was carried out to determine the prevalence of antimicrobial resistance among Shigellae of different serogroups isolated from children up to sixty months of age with acute diarrhoea in IleIfe. Three hundred consecutive faecal samples were obtained from children who presented with acute diarrhoea at the Obafemi Awolowo University Teaching Hospital Complex (OAUTHC), IleIfe from September 2003September The shigellae isolates were identified biochemically, serologically, and subjected to antibiotic sensitivity tests. Of 102 Shigella isolates identified, all were resistant to Ampicillin and Tetracycline whilst more than 50% exhibited resistance to Trimethoprim (80.4%), Nalidixic acid (64.7%), Cephalothin (64.7%), Chloramphenicol (59.8%), Sulphonamide (56.9%), and Cefotaxime (52.0%). However, only five (4.9%) of the isolates were found to be resistant to Gentamicin, Tobramycin and Ciprofloxacin. Five of the isolates, all of them S. dysenteriae were also found to be resistant to all the antibiotics used in the test. The result of transconjugation experiments suggest that many of the resistances were carried on extrachromosomal genetic units and could therefore be transferred horizontally from one organism to another. Laws regulating the sale and use of antimicrobial agents must be strictly enforced and continual surveillance of the use of these antimicrobials and the dissemination of such information is also required in IleIfe. Keywords: Shigella spp., acute diarrhoea, antimicrobial resistance, IleIfe INTRODUCTION Over the last five decades, Shigella species have become progressively resistant to the most widelyused and inexpensive antimicrobials (Ashkenazi et al., 2003; Talukder et al., 2007), making it difficult to identify efficacious drugs of choice for the treatment of shigellosis. This difficulty is increased in developing countries, where shigellosis is endemic and indiscriminate use of antimicrobial agents is common (Shiferaw et al., 2000; WHO 2001; Dutta et al., 2003; Okeke et al., 2007). In Nigeria, strains that were resistant to many commonly used drugs have been reported in different parts of the country (Meremikwu et al., 1997; Iwalokun et al., 2001; Egah et al., 2003; Enabulele et al., 2006). In the aforementioned

2 20 Nigerian studies, the strains were found to be multiplyresistant to Ampicillin (> 90%), Chloramphenicol (>90%), Tetracycline (>70%), and Trimethoprim (>80%). Various reports have also documented strains that were multiply resistant in different parts of the globe (Niyogi and Pazhani 2003; Jeong et al., 2003; MoezArdalani et al., 2003; Fullá et al., 2005; Sivapalasingam et al., 2006). This study was carried out to determine the prevalence of antimicrobial resistance among Shigella serogroups isolated from 2003 to 2006 from children up to sixty months of age with acute diarrhoea to the most commonly used antibiotics in IleIfe, southwest Nigeria. MATERIALS AND METHODS Subjects and sample collection Sampling was performed during September 2003September 2006 and 300 stool samples were consecutively collected from children who presented with acute diarrhoea at the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), IleIfe. Bacteriological Procedures Rectal swabs or fresh stool samples were collected into Stuart (Oxoid, England) transport medium and processed within 2 hours of collection following standardised procedure as described by Chompook et al. (2005). The samples were simultaneously inoculated directly on MacConkey (Oxoid, England) agar and SalmonellaShigella agar, and were incubated overnight at 37 C. Growths were tested for the entire gamut of enteropathogens recovered by employing standard techniques (Bopp et al. 2003). Colonies morphologically resembling shigellae were further subcultured and identified by biochemical reactions on API 20E (Biomerieux, France). Serogroups were determined by using commercially available antisera kits (Sanofi Diagnostics, Pasteur, France and ProLab Diagnostics, England). Strains were subcultured on SalmonellaShigella agar (Oxoid, England) and serotyping was performed after 18 hours of incubation at 37ºC by using the slide agglutination tests. Antimicrobial Susceptibility Bacterial sensitivity to eleven antimicrobial agents was determined by using the disc diffusion method, as recommended by the National Committee for Clinical Laboratory Institute (NCCLI, 2004) with commercial antimicrobial discs (Oxoid, England and AB Biodisk, Sweden). The antibiotic used in this study and their disk concentrations were Ampicillin (AM, 10µg), Cefotaxime (CT, 30µg), Trimethoprim (TM, 5µg), Gentamicin (GM, 30µg), Sulphonamide (SU, 5µg), Nalidixic acid (NA, 30µg), Chloramphenicol (CL, 30µg), Tetracycline (TC, 30µg), Tobramycin (TO, 30µg), Cephalotin (CE, 30µg) and Ciprofloxacin (CI, 10µg). Zones of inhibition were measured in millimetres and interpretation was in accord with CLSI recommendations. E. coli NCTC and Staphylococcus aureus NCTC 6571 were used as controls for the susceptibility tests. Determination of minimum inhibitory concentration (MICs) The MICs of the isolates to Ampicillin, Chloramphenicol, Ciprofloxacin, Nalidixic acid and Trimethoprim were measured by using the agar dilution methods as described by NCCLS (2003). Each antimicrobial agent (Sigma, England) was serially diluted and tested at the following range of concentrations: μg/mL for each agent. The agar contained concentration ranges of the antibiotics prepared by twofold serial dilution. A Multipoint inoculator was used for dispensing 20.00mL of adjusted inoculum (10 7 cfu/ml) of each isolate onto the surface of the antibiotic plate to obtain a final inoculum size of cfu/spot. Antibioticfree plates were inoculated last and were used as negative controls. The positive controls were plates inoculated with reference strain E. coli NCTC The MIC of each antimicrobial agent against the Shigella serogroups were evaluated after incubating the plates, containing completely absorbed inocula, in an ambient air at 37 C for 18 h. In vitro transconjugation experiment Twenty isolates that showed resistance to Trimethoprim but were also Nalidixic acid sensitive were mated with a plasmidless E. coli C600 (recipient strain) in accordance to the protocol of Sundström et al. (1987). Individual colonies

3 21 Table 1. Age and sex distribution of subjects with Shigella serogroups isolated within the study period Age(Months) Isolates Sex Serogroups No % No. Of No. Of A B C D Untypeable Male Female TOTAL KEY. A: Shigella dysenteriae, B: Shigella flexneri, C: Shigella boydii, D: Shigella sonnei of the test (donor) organisms and the recipient organism (C600) were grown overnight in a 2.00mL of nutrient broth incubated at 37 C. 0.20mL proportions of the overnight cultures of the donor and the recipient were transferred using sterile pipettes onto the surface of over dried Isosensitest agar plates. The conjugation mixtures were incubated at 37 C for 24 hours. Growth on the plate was harvested into a sterile test tube and washed down with 3.00mL of fresh 0.85%W/V sodium chloride solution. The suspension was then streaked onto the recovery medium. The selecting plates were Isosensitest agar plates containing 40mg/L of Nalidixic acid (Sigma chemicals, USA) and 40ng/L, Trimethoprim. After 24 hours incubation at 37 C a few colonies showing growth on the recovery medium were transferred onto a fresh recovery plates to ensure that pure cultures of transconjugants were obtained. In the absence of growth on the original recovery plates, the plates were reincubated for a period of 4872 hours, before assuming that conjugation had not taken place. The transconjugants obtained were tested for their sensitivity to the antibiotics to which they were originally resistant to find out if such resistances had been cotransferred with Trimethoprim resistance. RESULTS Table 1 shows the age and sex distribution of subjects with Shigella serogroups isolated within the study period. Of the 300 diarrhoeal stool samples screened, a total of 102 (34.00%) yielded Shigellae serogroups with the frequency of occurrence being; S. flexneri, 45 (44.1%), S. dysenteriae 26 (25.5%), S. boydii, 19 (18.6%) and S. sonnei 6 (5.9%). Six (5.9%) of these isolates could not be typed under the conditions of the test. The in vitro antimicrobial susceptibility pattern of Shigella isolated in the course of this study is shown in Table 2, while Table 3 shows the rate of Shigella isolates with multidrug resistance. The highest rate of antimicrobial resistance (100%) observed was to Ampicillin, and Tetracycline and more than 50% of the strains demonstrated resistance to Trimethoprim (80.4%), Nalidixic acid (64.7%), Cephalothin (64.7%), Chloramphenicol (59.8%), Sulphonamide (56.9%), and Cefotaxime (52.0%). However, resistances to Gentamicin, Tobramycin and Ciprofloxacin were found to be infrequent with 5 (4.9%) of the isolates showing resistance to these antimicrobial agents. Ability to transfer antimicrobial resistant among isolated Shigella strains Nineteen (95.0%) of the twenty tested isolates were able to transfer their resistances. All the 17 isolates with MIC above 1000μg/mL to Trimethoprim were able to transfer their resistance during the transconjugation experiment. Two (66.7%) of the three isolates with MIC between μg/ml were also able to transfer their resistance. The ability of these isolates to cotransfer Trimethoprim resistance with Ampicillin (85.0%), Chloramphenicol (65.0%), Sulphonamide (100.0%) and Tetracycline (85.0%) is shown in Table 4.

4 22 Table 2. Comparison and Incidence of Antimicrobial Resistance to 11 antibiotics among 102 Shigella isolates and serogroups children with diarrhoea from IleIfe, Nigeria SSG No (%) AM CT TM GM SU TE TO CE CI NA CL A 26(25.49) 26(100) 17(65.4) 23(88.5) 5(19.2) 15(57.7) 26(100) 5(19.2) 18(69.2) 5(19.2) 20(76.9) 20(76.9) B 45(44.12) 45(100) 16(35.6) 40(88.9) 0(0) 31(68.9) 45(100) 0(0) 36(80.0) 0(0) 33(73.3) 30(66.7) C 19(18.63) 19(100) 3(15.8) 10(52.6) 0(0) 5(26.3) 19(100) 0(0) 5(26.3) 0(0) 4(21.1) 5(26.3) D 6(5.88) 6(100) 1(16.7) 3(50.0) 0(0) 1(16.7) 6(100) 0(0) 1(16.7) 0(0) 3(50.0) 0(0) Untypable 6(5.88) 6(100) 6(100.0) 6(100.0) 0(0) 6(100) 6(100) 0(0) 6(100) 0(0) 6(100) 6(100) TOTAL 102(100.00) 102(100) 53(52.0) 82(80.4) 5(4.9) 58(56.9) 102(100) 5(4.9) 66(64.7) 5(4.9) 66(64.7) 61(59.8) KEY: SSG: Shigella serogroup, No: Number of Isolates tested, Figures in parenthesis: Percentages, A: Shigella dysenteriae, B: Shigella flexneri, C: Shigella boydii, D: Shigella sonnei. AM: Ampicillin, CT: Cefotaxime, TM: trimethoprim, GM: gentamicin, SU: sulphonamide, NA: Nalidixic acid, CL: Chloramphenicol, TE: Tetracycline, TO: Tobramycin, CE: Cephalothin, CI: Ciprofloxacin. Table 3. Rates of Shigella isolates with multidrug resistance in IleIfe, September 2003 through September Antibiotic resistance pattern No. Of resistant strains S. dysentariae S. flexneri S. boydii S. sonnei Untypable Total (n=26) (n=45) (n=19) (n=6) (n=6) Amp Tet Amp Sul Tet Amp Tmp Tet Amp Tmp Tet Nal Amp Tmp Tet Nal Cef Amp Tmp Tet Chl Cep Amp Tmp Tet Nal Sul Amp Tmp Tet Cef Cep Chl Amp Tet Nal Sul Cep Chl Amp Tmp Tet Cef Cep Chl Amp Tmp Tet Nal Sul Cep Amp Tet Nal Sul Cef Cep Chl Amp Tmp Tet Nal Cef Cep Chl Amp Tmp Tet Nal Sul Cep Chl Cip Amp Tmp Tet Nal Sul Cef Cep Chl Cip Amp Tmp Tet Nal Sul Cef Cep Chl Cip Tob Amp Tmp Tet Nal Sul Cef Cep Chl Cip Gen Tob Total KEY.: Amp: Ampicillin; Cef: Cefotaxime; Cep: Cephalothin Chl: Chloramphenicol; Cip: Ciprofloxacin; Gen: Gentamicin; Nal: Nalidixic acid; Sul: Sulphonamide Tetracycline; Tob: Tobramycin; Tmp: Trimethoprim Tet:

5 23 Table 4. Incidence of Cotransfer of Antibiotics with Trimethoprim Resistance in Organisms from each of the Shigella Serotypes of the Sampled Population Serogroups A B C D untypable Amp (n=20) 3(15) 2(10) 2(10) Chl (n=20) 4(20) 2(10) NA 2(10) % cotransferred Sul (n=20) 4(20) 1(5) Tet (n=20) 3(15) 2(10) Total 17(85) 13(65) 20(100) 17(85) Table 5. Comparison of resistance rates of Shigella spp. to commonly used antimicrobial agents in different periods and different regions (Awassa, Lagos, Gondar, and present study), IleIfe. Antibiotic 2000 Awassa, Ethiopia (Belay et al.) Ampicillin Tetracycline Cotrimoxazole Trimethoprim Cephalothin Nalidixic acid Chloramphenicol Sulphonamide Cefotaxime Gentamicin Tobramycin Ciprofloxacin Lagos, Nigeria (Iwalokun et al.) Gondar (Gizachew et al.) Iran (MoezArdalan et al) Gondar (Tiruneh) Ghana (Opintan and Newman) IleIfe, Nigeria (Present study) DISCUSSION Shigellosis has become a significant public health problem due to development of multiple antimicrobial resistances, frequently resulting in treatment failure, leading in turn to health complications, increased costs of treatment (Hawkey and Jones, 2009) and mortality (Sur et al., 2004; CDC, 2013). The use of antibiotics in the treatment of shigellosis is important because there is evidence that antibiotics are capable of bringing about a marked improvement in exhibited symptoms as well as shorten the duration of illness

6 24 and in doing so reduce the rate of transmission of infection. A high prevalence of antibiotic resistant shigellae will therefore make it very difficult to control this infection. Previously, various antimicrobial agents have been found to be effective in the treatment of shigellosis, but options for treatment of this disease are now becoming increasingly limited due to the continuedemergence of multidrugresistant strains of Shigella as shown by Replogle et al. (2000), Lee et al. (2001), Mclver et al. (2002), Oh et al. (2003), and CDC (2013). As shown by the antimicrobial resistance patterns obtained in this study, many of the antibiotics which were once the mainstay of the treatment of shigellosis can no longer be relied upon to give effective treatment as they were found to be resistant to the broad spectrum of antimicrobial agents against which they were tested. For example, all the isolates were found to be resistant to Ampicillin and Tetracycline, even though Tetracycline is not recommended for any kind of infection in children and Ampicillin, was two decades ago regarded as the drug of choice for the treatment of shigellosis. In addition, more than 50% of the isolates were found to be resistant to Trimethoprim (80.4%), Nalidixic acid (64.7%), Cephalothin (64.7%), Sulphonamide (56.9%), Chloramphenicol (59.8%), and Cefotaxime (52.0%). However, the incidence of resistance to Ciprofloxacin, Gentamicin and Tobramycin was found to be below 5%.. This resistance pattern has been reported from lowincome countries such as Tanzania (Navia et al., 1999; 2005) and in several locations in Nigeria (Meremikwu et al., 1997; Iwalokun et al., 2001; Egah et al., 2003; Enabulele et al., 2006). However, when these figures are compared with others from earlier studies in Nigeria (Iwalokun et al., 2001) and other regions of Africa, such as Ethiopia (Belay et al., 2000; Gizachew et al., 2006; and Tiruneh, 2009) and Ghana (Opintan and Newman, 2007) these are the highest resistance rates so far recorded among Shigella species (Table 5). Prior to 1990, Ampicillin was the drug of choice for the treatment of shigellosis (CDC, 1986) and studies have shown that aside from the extensive use of Ampicillin for treatment, mobile genetic elements, including plasmids, transposons as well as cassettes in integrons, have been responsible for the spread of resistance determinants among Shigella isolates (Toro et al., 2005). The demonstrated ability of the isolates obtained in this study to transfer resistance to recipient bacteria during conjugation suggests that the high incidence of resistance observed is at least partly due to the presence of these mobile genetic elements. The persistence of Ampicillin resistance may be explained by the wide spread use of βlactams for the treatment of diverse infections in this environment, causing the selection of resistant strains and the propagation of βlactamaseresistant genes in the normal intestinal flora from where they can be transferred into enteropathogens (Blake et al., 2003). In India, investigations conducted by Pazhani et al. (2005) between 2001 and 2004 showed that all the Shigella isolates were resistant to Tetracycline, a figure of 95% was cited in the USA (Sivapalasingam et al., 2006), 88% in Brazil (Peirano et al., 2006), and 80% in Bangladesh (Rahman et al., 2004). Talukder et al. (2006) have earlier demonstrated that middlerange plasmids that are selftransmissible confer resistance to Tetracycline, and that these can cotransfer Ampicillin, carbenicillin, streptomycin, sulfisoxazone, and trimethoprimsulphamethoxazole resistances along with Tetracycline and tet (A, B, C, D, E and Y) genes have been identified for this mechanism (Pazhani et al., 2008). The majority (80.4%) of the shigellae isolates tested were found to be resistant to the Trimethoprim/Sulphonamide combination. Antimicrobial resistance to this agent varied slightly by the isolated strains. Notably, all the untypable isolates were resistant to this agent, while S. dysenteriae (89%) and S. flexneri (89%) were resistant to Trimethoprim/Sulphonamide combination respectively. With 80.4% of this study isolates resistant this agent Trimethoprim/Sulphonamide combination, the Shigella isolates obtained from children in this environment were found to resemble those reported from other parts of the globe. Conjugative plasmids encoding resistance to antibiotics have been detected in numerous studies on Shigella spp. (Talukder et al., 2006) and these have been implicated in the wide broadcast of antibiotic resistances. Chloramphenicol resistance was very common (59.8%) amongst the Shigella strains cultured from children with diarrhoea. Studies reported in various part of the world also showed high resistance rates to Chloramphenicol. For example, in Ghana (83% [Opintan and Newman 2007]), Chile (69% [Fullá et al. 2005]), Brazil (68% [Peirano et al. 2005]), Kenya (67% [Brooks et al. 2003]), USA (60% [Sivapalasingam et al. 2006]), Spain (56% [Navia et al. 2005]), and Ethiopia (53% [Yismaw et al. 2006]). Chloramphenicol resistance in this environment may be due to the indiscriminate use of this drug (Okeke et al. 2007) and these resistance are said to be associated with Chloramphenicol O acetyltransferase activity (Navia et al. 2005) which is borne on Chloramphenicol resistance gene cata1 (Pazhani et al. 2008) which has been documented to be easily transferable In contrast to our findings, other workers in some developing countries have documented a low proportion of resistance to Chloramphenicol among Shigella serotypes. For example, In Iran, Ranjbar et al. (2007) cited a 26% incidence of resistance, Thailand (15%) (Chompook et al., 2005) and Rio de Janeiro, Brazil (35%) (Peirano et al., 2006) Most of the Shigella strains recovered in this study were resistant to the cephalosporins: Cephalothin (66(64.5%)) and Cefotaxime (53(52.0%)). In late 1980 s and early 1990 s cephalosporins were reported to be effective against shigellosis and were among the drugs of choice (Neu, 1987; Shawar et al., 1989; Varsano et al., 1991; Prado et al., 1992; Khan et al.1997). However, resistance to cephalosporins due to the emergence of Extended Spectrum βlactamases (ESBLs) was reported shortly after the introduction of these agents (Bradford 2001). As observed by Radice

7 25 et al. (2001) among Shigella isolates, the isolates obtained in this study could have readily acquired resistance to cephalosporins along with Ampicillin through conjugative resistance plasmids carrying resistance cassettes to β lactamases. In contrast to high incidence of resistance observed in this study, Fullá et al. (2005) in Chile, Ranjbar et al. (2007) in Iran and Orret (2008) in Trinidad and Tobago reported a high susceptibility rate to the cephalosporinsceftriaxone and Cefotaxime among Shigella isolates. The reason adduced for this susceptibility was that ceftriaxone was an infrequently used drug and only used as alternative to other drugs in these areas. Nonetheless, higher incidences of resistant strains of Shigella comparable to that reported in this present study have been observed from other nations including France (Huang et al., 2005), Argentina (Fortineau et al., 2001Error! Bookmark not defined.), Korea (Pai et al., 2001), and Turkey (Acikgoz et al., 2003). As indicated in the result, the incidence of resistance in Shigella isolates to Nalidixic acid in this environment was 64.7%. The results of studies carried out in other parts of the world show however that Nalidixic acid resistance is even more widespread in those counties than in Nigeria. The reason for this has been attributed to the unrestricted use of this drug for the treatment of diarrhoea and other infectious diseases in Bangladesh (Naheed et al., 2004), India (Sarkar et al., 2003), Nepal (Talukder et al., 2004), and Canada (CCDR, 2005). Since, treatment for shigellosis is critical in persons who have severe disease, especially in children and those that are immunosuppressed, the evident increase in the proportion of these Shigella isolates that are resistant to Nalidixic acid suggests the loss of an important class of antimicrobial agents against Shigella. The high incidence of resistance to Nalidixic acid observed in the course of this study suggests the possibility of resistance to the chemically related Ciprofloxacin but results has shown that the isolates were yet to develop resistance to this drug as only five isolates were found to be resistant to it, probably because it had recently been introduced into clinical use in this environment at the time these isolates were collected. The continued effectiveness of this drug is particularly important because several investigators have shown that Ciprofloxacin offers advantages in the treatment of shigellosis (Leibovitz et al., 2000; Zimbasa, 2002; Sivapalasingam et al. 2006; Ranjbar et al. 2007; Pazhani et al. 2008) and is recommended for the treatment of shigellosis in children caused by multidrug resistant bacteria (Hampel et al., 1997; American Academy of Paediatrics, 2000; WHO 2005). In contrast to the results obtained in this study, high rates of resistance to Ciprofloxacin have been recorded among Shigella strains in areas where this drug is extensively used (Bhattachary et al., 2003; Pazhani et al., 2004; Taneja et al., 2004; CCDR, 2005; Enabulele et al., 2006; von Seidlein et al., 2006) suggesting that organisms in this environment are likely to develop resistance to the drug since the use of antibiotics are not controlled to any significant extent. Despite the development of new βlactams and fluoroquinolones, aminoglycosides are still extensively used for the treatment of severe infections caused by gramnegative bacteria. The aminoglycosides: Gentamicin and Tobramycin investigated in this study showed excellent in vitro activity against Shigella isolates and supports the recommendation of Yismaw et al. (2006), Ahmed et al. (2000), and Khan et al. (2004) that aminoglycosides should be incorporated as drugs of choice for the treatment of shigellosis.. The in vitro transconjugation experiment in this study has demonstrated that resistances to Ampicillin, Ciprofloxacin, Chloramphenicol, Sulphonamide and Tetracycline were transferred along with Trimethoprim resistance into plasmidless E. coli K12 C600. This result is in agreement with the report made by Seol et al. (2006), and Ahmed et al. (2000) and indicates a mechanism by which these resistances are broadcast within the study environment. A resistance borne on transferable genetic elements such as plasmids, transponsons and integrons which have been observed to move from normal flora into pathogens (Blake, et al., 2003; Saran, et al., 2013). The transfer of Ciprofloxacin resistance reported here is the first to be reported in this environment and shows that the continued effectiveness of Ciprofloxacin may not be sustained for very long. 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