Speciation and Antibiotic Susceptibility Pattern of Shigella Isolates in a Rural Tertiary Care Hospital, Kolar

Shigellosis is the most important cause of bacillary dysentery. It's a major public health problem. It is endemic in many developing countries resulting in a significant amount of mortality and morbidity. The incidence is 90 million with 6,00,000 deaths world over. In India, there is an emergence of resistance to Shigella resulting in limitation of treatment. To study the prevalence of Shigella species and their antibiogram in stool samples obtained from patients treated for dysentery at Rural Tertiary Care Hospital. A retrospective analysis of the Shigella isolates obtained from 400 stool samples from patients with dysentery , treated at Rural Tertiary Care Hospital from 2015 -2019 was conducted. The isolates were identified by conventional methods and antibiotic susceptibility testing was performed by Kirbybauers disk diffusion method and analysed. The percentage of isolation of Shigella species at our centre was 5% with Shigella flexneri being the predominant species isolated. A progressive pattern of resistance was observed. 100% strains of Shigella flexneri and Shigella sonnei were resistant to cotrimoxazole and nalidixic acid. 75% of Shigella flexneri were resistant to fluoroquinolones and 25% resistant to third generation cephalosporins. 100% isolates were sensitive to azithromycin. In view of rampant resistance observed among Shigella species, it is a need to test these isolates for antibiotic susceptibility and treat the patients based on the antibiogram.


iNtROduCtiON
Shigellosis is a gastrointestinal infection caused by genus Shigella. 1 One of the most common causes of morbidity and mortality among children in developing countries is acute gastroenteritis due to Shigella species.Shigella is a gram-negative bacilli that belongs to the family Enterobacteriaceae and is generically very similar to Escherichia coli. 2 Genus Shigella is divided into Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei. 3The mode of transmission is faeco-oral and the infective dose is as low as 10-to 100 bacilli.The pathogen colonizes and disrupts the intestinal epithelium resulting in fever, bloody diarrhoea and tenesmus. 4One of the most common causes of morbidity and mortality among children in developing countries is acute gastroenteritis due to Shigella species.It predominantly affects children below 5 years, elderly and immunocompromised.It accounts for 1.1 million deaths in developing countries, of which 60% is constituted by children below 5 years of age. 5 The mortality and morbidity due to Shigellosis is preventable by oral rehydration and appropriate antimicrobial agents.WHO reports of Multidrug-resistant (MDR) Shigella in India, and other developing countries has become a therapeutic challenge in the treatment of Shigellosis. 1,6So, this study is undertaken to know about the prevalence of Shigella species and their antibiogram in stool samples obtained from patients treated for dysentery at Rural Tertiary Care Hospital.
This study helps us understand the changing trend of antibiotic resistance among Shigella isolates in a rural background where antibiotic usage is comparatively low.

Objectives
To study the prevalence of Shigella species and their antibiogram in stool samples obtained from patients treated for dysentery.

MATeRIAlS ANd MeTHOdS
A retrospective analysis of the Shigella isolates from stool culture of patients with dysentery, treated at RL Jalappa Hospital from May 2015 -June 2019 was conducted.Ethical clearance was obtained from the institutional ethics committee.

Inclusion criteria
Shigella isolates from patients who presented with dysentery were included exclusion criteria Incomplete patient data.20 isolates of Shigella from patients who presented with dysentery were included in the study and were identified by conventional culture methods.The stool specimens were inoculated on Blood agar, Mac Conkey agar, Xylose lysine deoxycholate (XLD) agar directly.They were also inoculated into enrichment media, Selinite F broth and subcultured to MacConkey and XLD agar.Suspected colonies of Shigella was identified by standard biochemical tests and confirmed by specific antiserum (Denka -Seiken, Japan).Antibiotic susceptibility testing was done by Kirby Bauer disk diffusion technique as per CLSI (Clinical and Laboratory standard institute) guidelines using the following antibiotics, ampicillin (Amp)10µg, cotrimoxazole (Cot) 25µg, nalidixic acid (Na) 30µg, ciprofloxacin (Cip) 5µg, and ceftriaxone (Ctr) 30µg (Hi Media Laboratories, India).An additional drug azithromycin (Az) 15µg was tested as an investigational drug to find the usefulness of the drug as second line drug in case of pan drug-resistance.These isolates were further confirmed by NICED (National Institute of Cholera and Enteric Disease) Kolkotta.Further, their antibiotic susceptibility pattern was analysed as per CLSI (Clinical and Laboratory standard institute) guidelines.The data was retrieved from the Medical records department.

Statistical Analysis
A descriptive analysis of the data was done.

Results
A total number of 400 stool samples were cultured between May 2015-June 2019.20 (5%) isolates of Shigella species were obtained from patients with dysentery, treated at Rural Tertiary Care Hospital.Out of the 20 isolates, the predominant serotype was Shigella flexneri, 16 (80%) followed by Shigella sonnei, 4 (20%).A gradual increase in the number of isolates from 2015 to 2019 was observed as shown in (Table 1).The majority of the isolates 18 (80%) out of 20 isolates were from the adult population.All the 20 isolates showed 100% resistance to cotrimoxazole and nalidixic acid as shown in (Table 2). 4 (25%) isolates of Shigella flexneri showed resistance to ceftriaxone, 2 of the 4 were ESBL producers.All the 20 (100%) isolates were sensitive to azithromycin.

disCussiON
Global estimation of shigellosis is 164.7 million annually. 7One of the major public health problems in developing countries is shigellosis.It is transmitted through contaminated food and water and direct contact with infected individuals.In the present study, the rate of isolation was 5% which is higher than the rate quoted by Taneja et al. 8 The overall percentage of isolation of Shigella from different of our country ranges from 3 to 6%.It has been shown by many studies that shigellosis is prevalent among children less than 5 years. 9Contrary to the above observation made, the majority of the isolates 18 (80%) out of 20 were from the adult population in the present study.
The most common serotype isolated in our study is Shigella flexneri followed by Shigella sonnei.Madhavan et al. has reported Shigella sonnei as the predominant serotype in Kerala. 8higella sonnei 9 was found to be the predominant species in Thailand, Vietnam and Srilanka attributed to improved sanitary conditions.From various studies across the world, it is found that there is a shift from Shigella flexneri to Shigella sonnei in countries with improved sanitation. 9 The trend of antibiotic resistance was progressive in our study.An alarming increase in the resistance pattern of Shigella flexneri was observed.13 (81%) of the Shigella flexneri strains were MDR strains resistant to more than three classes of antibiotics.12 (75%) of the strains were resistant to ciprofloxacin.Fluoroquinolones were recommended by the WHO as drug of choice in 1990 but the emergence of resistance  to fluoroquinolone has limited its usage and has mandated the use of ceftriaxone.The emergence of fluoroquinolone resistance is probably due to the over use of these drugs for diarrhoea and urinary tract infections.5.9% of fluoroquinolone resistance was observed in 2004 which increased to 48.5% in 2007. 9Gururaj et al. proved that there was a uniform mutation in Gyr A among the fluoroquine resistant strains. 10Presently some of the strains have developed resistance to third-generation cephalosporins.Madhavan et al. 11 showed that 17% of Shigella isolates in his study were resistant to third-generation cephalosporin as compared to 25% in our study.Mandal J et al. showed 3% of cephalosporin resistance which was lesser in comparison to our study.ESBL production was observed in 2 (12.5%) isolates of Shigella flexneri as compared to 3% in the study conducted by Mandal J et al. 12 The greatest danger faced with MDR strains are the limited therapeutic options available to the treating physician. 13In a rural setup, the cost of procuring higher antibiotics results in delayed recovery and prolonged hospital stay, on the isolation of an MDR strain.
As CLSI recommends azithromycin to be tested as an investigational drug, azithromycin sensitivity was performed on all the isolates obtained in the year 2019 and 100% sensitivity was observed.Although there is a smaller sample size this work has gained success to fulfil the purpose of this work to achieve better treatment limitation of the Study The sample size is the limitation of the study.Extrapolation of the results could have been better with a larger sample size.

CONClusiON
Shigella flexneri has become increasingly resistant to antimicrobials.WHO in 2005 recommended ciprofloxacin as the first line treatment for shigellosis in children and ceftriaxone as a second line drug for children.Azithromycin as a second line drug for adults.Shigellosis should be treated with antibiotics as it shortens the duration of clinical symptoms and decreases fecal excretion of the organism and minimises transmission.Provision for improved sanitation and clean drinking water is needed to prevent shigellosis.There is a impending need for updation of the guidelines for treating shigellosis in the developing countries This work would ameliorate the treatment for dysentery, it exhibits a significance in the treatment among rural areas where in many cases are not diagnosed and treated effectively.Instead of the limitation due to the smaller sample size this work is very fruitful for the study of the prevalence of Shigella species and antibiogram which is very important for the proper treatment of patients with dysentery.

Table 1 .
Distribution of shigella isolates during 5 years period Note: A and B denotes the serotypes of Shigella.