ANTIBIOTIC SUSCEPTIBILITY PATTERN OF Shigella spp. ISOLATED FROM PATIENTS SUSPECTED OF ACUTE GASTROENTERITIS

Shigellosis, an intestinal infection caused by Shigella species, is manifested by bloody diarrhea. Due to the surge in multidrug-resistant (MDR) Shigella species, the control of shigellosis has been a big challenge. This study aims to determine the prevalence and assess the antibiotic susceptibility pattern of Shigella species. During our study period of five months from April 2014 to August 2014 at Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, a total of 653 stool samples were collected from the patients suspected of acute gastroenteritis. The standard microbiological procedure was followed for the isolation and identification of Shigella species. Assessment of antibiotic susceptibility pattern of the Shigella species was done by Kirby-Bauer disk diffusion method following CLSI guidelines. The study found 25(3.82%) cases were Shigella positive. Among them, 18(72%) were S. flexneri, 6(24%) were S. dysenteriae, and 1(4%) was S. sonnei. The patients in the age group 16-45 years were highly susceptible to infection as the higher proportion 16(64%) of Shigella species were isolated from this age group (p> 0.05). Shigella species were found to be highly susceptible to Cefotaxime (100%), a third-generation cephalosporin. Nalidixic acid, on the other hand, was the least effective antibiotic as 20(80%) of the Shigella isolates were resistant, followed by Ampicillin 18(72%), Cotrimoxazole 13(52%), and Ciprofloxacin 9(36%). A higher proportion of [10(40%)] of our study isolates were MDR. Our results show that Nalidixic acid, Ampicillin, Cotrimoxazole, Ciprofloxacin, and Ofloxacin cannot be used as empirical therapy for the treatment of Shigella infection as Shigella species were highly resistant to these antibiotics. So, for the MDR Shigella infection, we suggest third-generation cephalosporin as an option.


INTRODUCTION
Shigella, a food-borne pathogen, is responsible for acute invasive gastroenteritis and traveler's diarrhea (von Seidlein et al., 2006). It is a major global health problem as it is a prime cause of diarrhea, with 212,000 deaths every year worldwide (Arnold, 2021). It is a major cause of mortality and morbidity in children under the age of five in developing nations (Kotloff et al., 2018). It is manifested by bloody diarrhea and is caused by Shigella flexneri, S. sonnei, S. boydii, or S. dysenteriae (Heymann, 2008). S. flexneri is the major cause of diarrhea in the least developed and developing countries, whereas S. sonnei is the major species isolated in the developed nations (Kotloff et al., 2018;Bardsley et al., 2020). The organism is usually transmitted by the feco-oral route when contaminated food or water is ingested. The definitive diagnosis of Shigella infection is done by isolating the organism from the stool and serotyping the isolate (WHO, 2005).
The severity of shigellosis and potential complications can be avoided by the proper use of antimicrobials (Lima et al., 1995); however, antibiotic resistance to Shigella species and serotypes has emerged throughout the world (Taneja et al., 2012). Previously effective firstline drugs such as Sulphonamide-trimethoprim, Ampicillin, and Nalidixic acid are almost ineffective against the prevalent Shigella strains (Kotloff et al., 2018;Puzari et al., 2018). With the recent rise in fluoroquinolone-resistant strains, the choice of drugs for effective antimicrobial therapy has vastly reduced (Kotloff et al., 2018;Puzari et al., 2018). As an alternative antimicrobial therapy, WHO has recommended three antibiotics, namely Pivmecillinam, Ceftriaxone, and Azithromycin (WHO, 2005). However, resistance to these WHO-recommended drugs has been observed as suggested by the findings of various studies.
In Nepal, S. flexneri is the predominant species, followed by S. dysenteriae (Wilson et al., 2006;Khan et al., 2013). Studies conducted in Nepal have found that most patients who are vulnerable to infection and develop infection are under the age of ten (Bhattacharya et al., 2005;Khan et al., 2013). Nepal has experienced Shigella species resistance to multiple drugs over the past two decades. There are a few reasons for the emergence of antibiotic-resistant strains in Nepal, such as easy access to antibiotics in the pharmacies without a doctor's prescription, poor antibiotic policies, including poor laboratory diagnosis and antibiotic susceptibility tests (Dhital et al., 2017). Therefore, this study aims to provide more insight into the prevalence and the antibiotic susceptibility pattern of Shigella species isolated in a tertiary care hospital in Kathmandu.

MATERIALS AND METHODS
This cross-sectional study was done in Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, over five months from April to August 2014.

Patients and sample collection
A total of 653 stool samples were collected from the patients complaining of acute gastrointestinal disorder, i.e., patients with fever, fatigue, frequent defecation of painful stools containing blood and mucus defined by physicians as a probable case of bacillary dysentery. Therefore, the stool samples containing blood and mucus from patients with no antibiotics taken within three days were included as study samples, whereas those with firm consistency without blood and mucus were excluded from the study. One gram of stool samples was collected in clean screw-capped plastic containers. The samples were labeled with the patient's name, age, sex, and address. If there was a possible delay in the processing of the sample, it was stored in the refrigerator at 2-8 o C. The stool samples were further processed in the microbiology laboratory following standard operating procedures (McCartney et al., 1989).

Bacteriological analysis
A loopful of stool sample from each container was then inoculated in the Xylose lysine deoxycholate agar (XLD) and Mac-Conkey agar (MAC) plates and incubated at 37 o C for 24 hours for the isolation of Shigella species. Simultaneously, enrichment was done in Selenite-F broth and incubated at 37 o C for 24 hours. Subculture was then done in XLD agar and MAC and further incubated at 37 o C for 24 hours. The colony morphology resembling Shigella species was identified microscopically and biochemically. The non-lactose fermenting (NLF) colonies from both XLD and MAC were identified by the IMViC test, Urease test, Nitrate reduction test, and Triple sugar iron (TSI) test.

Serological identification
Differentiation of Shigella species was done by slide agglutination test using polyvalent antisera (Denka Seiken Co. Ltd. Japan). On the clean glass slide, milky suspension of the organism was made with saline. Shigella polyvalent antiserum was added starting from A to D polyvalent. The mixture was tilted to and fro for 30-60 seconds. While viewing under a good light against a dark background, distinct clumping within 60 seconds was a positive result. The suspension alone, on the other hand, as control was tested to exclude auto-agglutination. The appearance of agglutination (distinct clumping) by Group A polyvalent, Group B polyvalent, Group C polyvalent, and Group D polyvalent in kit were identified as S. dysenteriae, S. flexneri, S. boydii, and S. sonnei respectively.

Antibiotic susceptibility test
The antibiotic susceptibility test was done by the Kirby-Bauer disk diffusion method in Muller-Hinton Agar (MA) following 2014 CLSI guidelines (CLSI, 2014). The antibiotic susceptibility pattern of six antibiotics, namely Ampicillin (Amp, 10 µg), Cefotaxime (Ctx, 30 µg), Cotrimoxazole (TMP-SMX, 25 µg), Ciprofloxacin (Cip, 5 µg), Ofloxacin (Ofx, 15 µg), and Nalidixic acid (Nal, 30 µg) (Hi Media Laboratory Ltd. Mumbai, India) was assessed. The zone of inhibition of each antibiotic was measured, and they were interpreted as sensitive, intermediate sensitive, and resistant (CLSI, 2014). For the quality control purpose, strict aseptic conditions and techniques were followed according to the National Committee for Clinical Laboratory Standard (NCCLS) recommendation. Positive and negative control was always used during each testing with the standard organism. For the antibiotic susceptibility test, standard organism ATCC 25922 Escherichia coli was used for the quality control.

Statistical analysis
Data from the laboratory findings was analyzed using Microsoft Excel 365. The t-test was performed to compare the statistical significance. The relationship with a p-value greater than 0.05 was indicated as insignificant.

Ethics clearance
The written informed consent was taken prior to the study from the patients involved in the study. Our study was accepted by the Institutional Review Committee (IRC) at Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu.
The antibiotic susceptibility pattern of Shigella species suggests that Cefotaxime, the third-generation cephalosporin, was found to be the most effective (100% sensitive) against all the isolated Shigella species (Table 3). Among the tested antibiotics, Nalidixic acid was the least effective as 20(80%) of Shigella species were resistant to it ( Table 3). The second least effective antibiotic was Ampicillin, as 18(72%) of Shigella species were resistant to it (Table 3). This was followed by Cotrimoxazole 13(52%) and Ciprofloxacin 9(36%), respectively. Similarly, out of a total of 25 isolates, 10 isolates were found to be MDR, representing 40% of the total isolates (Fig. 2).

DISCUSSION
This study was conducted from April to August 2014 among the patients suspected of acute gastroenteritis visiting Sukraraj Tropical and Infectious Disease Hospital, Teku. Out of 653 studied cases, 25(3.82%) cases were Shigella positive. This isolation rate is comparable to a study done in Tribhuvan University Teaching Hospital and Kanti Hospital (2.1% culture positive) (Dhital et al., 2017). In contrast to our findings, two different studies conducted in Nepal reported a relatively higher prevalence rate (13.61% and 10.8%) (Wilson et al., 2006;Khan et al., 2014). The prevalence of food and water-borne infections like Shigellosis increases in the rainy season as the infection rate fluctuates according to the seasons. Our study shows that the patients in the age group 16-45 years were more susceptible to Shigella infection (16(64%)). However, there was no significant difference in the infection rate among all the age groups. In contrast to our study, the infection rate was higher in the age group <10 years in most studies (Kansakar et al., 2006;Wilson et al., 2006;Khan et al., 2014;Mengistu et al., 2014). This discrepancy is because the children usually visit the children's hospitals in Kathmandu rather than the tertiary care hospital where this study was carried out. This is also reflected in our study, as participants below the age of 10 were lower than other age groups.
S. flexneri [18(72%)] was found to be the predominant Shigella species in our study, followed by S. dysenteriae [6(28%)]. This finding is congruent to hospitalbased studies in Nepal (Wilson et al., 2006;Khan et al., 2014), whereas incongruent to a study done in eastern Nepal where the predominant isolate was S. dysenteriae (73.6%) followed by S. flexneri (23%) and S. boydii (4%) (Bhattacharya et al., 2005). In the studies in Nepal from 1995 to 2002, the prevalence of S. dysenteriae was higher than the S. flexneri, while in the studies done from 2003 to 2005, S. flexneri was found to be the predominant species (Kansakar et al., 2006). The recent studies conducted in India (Puzari et al., 2018), Nepal (Maharjan et al., 2017;Thapa et al., 2017), Iran (Nikfar et al., 2017), China (Chang et al., 2016), and Latin America (Sati et al., 2019) show that S. flexneri is the predominant species. On the other hand, nations such as England (Bardsley et al., 2020), Australia (O'Sullivan et al., 2002;Ingle et al., 2020), and the United States (Kozyreva et al., 2016) showed a higher predominance of S. sonnei. These findings support the claim that S. flexneri infection is associated with developing and underdeveloped countries, and S. sonnei infection is associated with developed countries (Kotloff et al., 2018;Bardsley et al., 2020). However, a rapid rise in the S. sonnei cases has been observed in developing countries in recent years (Anderson et al., 2016). This could be possibly associated with the climatic and environmental changes and the innate changing nature of Shigella serogroups and serotypes from time to time. Group of antibiotics 20% 40% 20% 10% 10% All the antibiotics used in our study were the routine antibiotics used in the diagnostic microbiology laboratory in Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu. Our study showed the increased resistance to Nalidixic acid (80%), Ampicillin (72%), Cotrimoxazole (52%), and Ciprofloxacin (36%) corresponding to various studies conducted in different parts of the world (Srinivasa et al., 2009;Bhattacharya et al., 2012;Gharibi et al., 2012). Although further confirmation is needed, our study suggests that Nalidixic acid, Cotrimoxazole, Ampicillin, and Ciprofloxacin can be used no longer for the empirical treatment of shigellosis. Similarly, Nalidixic acid is a drug of choice in developing countries, and Ciprofloxacin in developed countries (Gharibi et al., 2012). The increased resistance (80% resistance) of Nalidixic acid was observed in the present study and other studies conducted in Nepal (Bhattacharya et al., 2005;Khan et al., 2014;Dhital et al., 2017). This increased resistance to Nalidixic acid in Nepal can be attributed to the over-the-counter availability of drugs, self-prescription by patients, and poor antibiotic policies. In the early 1990s, Shigella isolates were susceptible to most prescribed antibiotics such as Gentamicin, Norfloxacin, and Nalidixic acid (Jesudason, 2002;Dutta et al., 2003;Rahbar et al., 2007). However, during the late 1990s, Shigella isolates were resistant to most of these antibiotics but susceptible to Ciprofloxacin (Sack et al., 1997). A study conducted in Bangladesh has shown the rising trend of Ciprofloxacin resistance, i.e., from 0% in 2004 to 48% in 2010 (Azmi et al., 2014). A similar study of the Shigella isolates between 2010 and 2015 revealed South-Asia as a reservoir for the global spread of Ciprofloxacin resistant S. sonnei (Chung The et al., 2016). Similarly, a study conducted from the Shigella isolates over the six years from 2011 to 2016 in Iran also suggested the higher resistance (76%) to Ciprofloxacin (Mamishi et al., 2019). These statistics show that the Ciprofloxacin-resistant Shigella species has increased considerably over time. Although WHO has recommended Ciprofloxacin as a drug of choice to treat shigellosis (WHO, 2005), our result suggests that Ciprofloxacin cannot be trusted blindly as the effective drug against shigellosis.
According to our findings, cefotaxime, a third-generation cephalosporin, was the most sensitive (100% of Shigella isolates were susceptible to it). This result is congruent to the studies performed by Dhital et al. (2017), where 93% of Shigella isolates were susceptible to Ceftriaxone, and by Kansakar et al. (2006), where 100% of Shigella isolates were susceptible. In similar settings, Maharjan et al. (2017) found 97% of Shigella species, and Thapa et al. (2017) found 71.4% of Shigella species susceptible to Ceftriaxone. Several studies across the world have also shown the increased resistance of Shigella species to third-generation cephalosporins (Radice et al., 2001;Vinh et al., 2009;Mandal et al., 2010). More recent research also consistently supports the fact that third-generation cephalosporins cannot be relied on treating Ciprofloxacin resistant Shigella species due to surge in third-generation cephalosporin-resistant Shigella species (Hosseini Nave et al., 2016;Nüesch-Inderbinen et al., 2016;Madhavan et al. 2018;Abbasi et al., 2019;Bian et al., 2019). Therefore, although our study has shown the high susceptibility of Shigella species to a third-generation cephalosporin (Cefotaxime), we still suggest using thirdgeneration cephalosporin with caution. Our study was conducted seven years ago, and recent studies across the world have shown increased resistance of Shigella species to third-generation cephalosporins. So, for further studies, we recommend carrying out the study in similar settings with few other third-generation cephalosporins included before concluding with certainty that thirdgeneration cephalosporins are the effective options for treating shigellosis.
Forty percent (40%) of the Shigella-positive isolates were MDR. This figure corresponds to a study conducted in the hospital settings in Pokhara (74.7%) and Kathmandu (86%) (Wilson et al., 2006;Dhital et al., 2017). MDR isolates in our study were resistant to Nalidixic acid, Ciprofloxacin, Cotrimoxazole, and Ampicillin, and Ofloxacin. The emergence of MDR Shigella isolates is of great concern in South Asian countries, including Nepal (Rahman et al., 2004). The high resistance level in Shigella can be attributed to the transfer of resistant determinants such as plasmids, integrons and transposons (Barrantes & Achí, 2016). Due to the rise in resistance to thirdgeneration cephalosporins (Abbasi et al., 2019), a recent study has suggested using Azithromycin and Piperacillin tazobactam as an alternative (Madhavan et al., 2018). However, resistance to Azithromycin has also been reported (Nüesch-Inderbinen et al., 2016). Therefore, we can see that the MDR Shigella species is increasing continuously. Continuous monitoring of the MDR strains and an effective antibiotic prescription policy in Nepal could help mitigate increased drug resistance seen in Shigella species.

CONCLUSIONS
Our study found the predominant Shigella serovar to be Shigella flexneri. Shigella species were highly resistant to commonly prescribed antibiotics in Nepal, such as Nalidixic acid, Ampicillin, Cotrimoxazole, and Ciprofloxacin, whereas susceptible to a third-generation cephalosporin (Cefotaxime). We found a higher prevalence of MDR Shigella, and we suggest using thirdgeneration cephalosporins to treat MDR Shigella infection.
provided by the teaching staff in the Department of Microbiology, Kirtipur.

AUTHOR CONTRIBUTIONS
BB contributed to the study design, laboratory work, and manuscript writing. DN performed data analysis and manuscript writing. JA and SB contributed to the conceptualization and study design.