DIARRHEA CAUSING ENTEROBACTERIACEAE AND THEIR ANTIBIOTIC RESISTANCE PATTERNS AMONG DIARRHEA PATIENTS FROM SANA’A CITY, YEMEN

 Gamil Taher Ahmed AbdulMughni1image, Khaled Abdulkarim Al-Moyed1,2image, Eshrak Adulmalik Al-gunaid3image, Huda Zaid Al-Shami2image,  Naif Mohammed Al-Haidary2image, Anowar  Gasem Al-Madhaji2image, Hassan Abdulwahab Al-Shamahy1,2image 

1University of 21 September for Medical and Applied Sciences, Sana’a, Yemen.

2Medical Microbiology and Clinical Immunology Department, Faculty of Medicine and Health Sciences, Sana’a University. 

3The National Center of Public Health Laboratories (NCPHL), Sana'a, Yemen.

4Basic Sciences, Faculty of Dentistry, Sana’a University, Republic of Yemen.

 

Background and aims: The rise of antibiotic-resistant strains of the bacteria causing diarrhea remains a major worldwide health concern, especially in children under five. The purpose of this surveillance study was to ascertain the prevalence and antibiotic susceptibility profile of Enterobacteriaceae, which cause diarrhea.

Materials and Methods: The National Center for Public Health Laboratories (NCPHL) in Sana'a city was the site of this cross-sectional investigation. Patient stool samples exhibiting diarrhea were gathered and cultivated using standard differential/selective medium. Standard biochemical assays and serological investigations were employed to identify the isolates. With the aid of Kirby-Bauer disk diffusion, the antibiogram was identified. 

Results: Enterobacteriaceae isolates were obtained from 972 stool samples collected; 15.3% (149/972) were diarrheagenic Enterobacteriaceae bacteria, including E. coli, Salmonella, and Shigella. There were 8.3% (81/972) Salmonella spp., 5.7% (56/972) Shigella spp., and 1.2% (12/972) E. coli. E. coli strains were grouped, with the most prominent being serogroups O153 (3, 2%) and O25 (2, 1.4%). We detected high resistance of Salmonella isolates to tetracycline (29/81, 35.8%) but relatively low resistance to ciprofloxacin (7/81, 8.6%) and ceftriaxone (5/81, 6.3%). 

Conclusion: The many Enterobacteriaceae linked to instances of diarrhea in Sana'a, Yemen, were demonstrated in this study, along with the necessity of differential diagnosis for improved treatment results. Salmonella, Shigella, and Escherichia coli have all been linked to episodes of diarrhea in Sana'a city. To stop Yemen's antimicrobial resistance from spreading, regular antimicrobial susceptibility testing, efficient monitoring, and national surveillance of AMR bacteria should be put into place.

Keywords: Antibiotics, bacteria, diarrhea, Enterobacteriaceae, Yemen.

 

INTRODUCTION

 

Though largely treatable and preventive, diarrheal disease ranks second globally in terms of causes of death for children under five, taking the lives of approximately 525,000 children each year1. The World Health Organization defines diarrhea as the excessive and frequent evacuation of watery stools, which typically indicates a gastrointestinal illness or condition lasting three to seven days1. Numerous viral, bacterial, and parasite pathogens are the primary cause of diarrheal disorders, and their etiology varies based on a number of factors, including host characteristics, socioeconomic circumstances, and geographic and climatic settings2. Children under the age of two are known to suffer from diarrhea primarily from viral diseases such rotavirus and norovirus, with the latter resulting in the greatest death rate among children3. It has also been documented that parasites such Giardia lamblia, Entamoeba histolytica, and Cryptosporidium parvum can induce episodes of severe diarrhea4-6.

Escherichia coli is the most prevalent bacterium responsible for diarrhea in children in developing nations and is an emerging enteropathogen that is resistant to antibiotics in industrialized nations, according to research7. However, Vibrio cholerae has been identified as the primary bacteria pathogen responsible for diarrhea-associated disorders; certain strains, such Ogawa, have the potential to spread epidemics if left unchecked8-11. This is mostly due to unhygienic conditions like feces entering rivers and streams that serve as drinking water for people and cattle, a shortage of potable water, and incorrect handling of household food, which can result in an increase in diarrhea cases12-14.

There have been documented cholera epidemics and outbreaks in Yemen in the years 2016, 2017, 2018, 2019, and 2024. All parts of the nation were impacted by the 2016–2019 pandemic, which resulted in roughly 1,28,9220 cases overall and 3243 deaths8-10. 

Nonetheless, it has been demonstrated that bacteria other than V. cholerae can also cause diarrhea, such as Campylobacter species, Salmonella species, Shigella species, and pathogenic E. coli O15:H7 (though this issue is still open), all of which can be just as deadly if left untreated15,16.

As advised by the WHO, zinc and oral rehydration solutions (ORS) are commonly effective treatments for infections caused by these bacteria. Nevertheless, in cases of severe, bloody, or chronic diarrhea, certain individuals may also benefit from antimicrobial medication17. Treatment of bacterial diarrhea would be difficult due to rising reports of increased antibiotic resistance among Yemeni enterobacteria18-20. There is serious worry over reports of over 40% of children receiving antibiotic treatment developing non-bacterial diarrhea, of easy access to medications in Yemen through unapproved ways, and of incorrect drug prescriptions21-23. Studies have linked bacteria, which have a 3% prevalence rate and a strong resistance to often overused medications like tetracycline, to instances of diarrhea in Yemen21-25. In patients presenting to the National Center for Public Health Laboratories (NCPHL) in Sana'a City, the objective of this study was to identify Enterobacteriaceae organisms linked to diarrhea and ascertain the isolates' profile of antibiotic susceptibility.

 

MATERIALS AND METHODS 

 

Study design and identification of microorganisms: This study used clinical samples of diarrheal stool that were obtained between January 1, 2021, and March 1, 2024 (data were complete and available for this period) at the National Center for Public Health Laboratories (NCPHL) in Sana'a, Yemen. The study was cross-sectional in nature. The National Center for Pathology Laboratory Health (NCPHL) serves as the nation's reference laboratory. Its microbiology section comprises six benches and employs twelve bacteriologists, each of whom processes six samples per day. Al-Gumhouri University Hospital and Al-Kuwait University Hospital, two significant Sana'a hospitals, contributed samples.

Bacteria isolation and characterization 

Patient stool samples exhibiting diarrhea were gathered and cultivated using standard differential/selective medium. Standard biochemical assays and serological investigations were employed to identify the isolates. Every stool sample was inoculated on three different media: MacConkey agar, which was used to isolate E. coli and non-lactose fermenters, and Selenite Faecal Broth, SFB, an enrichment medium used to isolate Salmonella and Shigella spp. The enteric bacteria were then isolated by aerobic incubation at 37°C for 18–24 hours. On Salmonella Shigella Agar (SS), a loopful of SFB and APW broth cultures were sub-cultured and incubated as before. Suspected colonies of E. coli, Shigella, and Salmonella spp. were identified and purified using standard biochemical techniques, the Gram staining response, and colonial features. Using commercially available serological assays, antigenically identified isolates of Salmonella, E. Coli, and Shigella were obtained.

Antimicrobial Susceptibility Testing 

Only isolates that were believed to be diarrheagenic bacteria were included in our study for antibiotic susceptibility testing. Following the guidelines set forth by the Clinical and Laboratory Standard Institute (CLSI)26, the susceptibility to antimicrobial medicines was ascertained using the Kirby Bauer disk diffusion method. The following were tested on isolates: 30 μg of ceftriaxone, 5 μg of norfloxacin, 30 μg of cefotaxime, 30 μg of amikacin, 30 μg of gentamycin, 50 μg of clotrimazole, 30 μg of nalidixic acid, 30 μg of tetracycline, 30 μg of ceftazidime, 30 μg of ciprofloxacin, 300 μg of nitrofurantoin, and 30 μg of cefazolin. On Mueller Hinton Agar plates, zones of inhibition were measured using the Kirby Bauer technique. Susceptibility, resistance, and intermediate susceptibility were ascertained using the CLSI breakpoints26.

Ethical Consideration: This cross-sectional study, which took place between January 2021 and March 2024, was approved by the Medical Ethics and Research Committee of Sana'a University's Faculty of Medicine and Health Sciences (approval number: 317; dated January 1, 2021). The ethical rules that were established by the review committee were consistently followed.

Statistical Analysis:  Epi-Info Statistics version 7 was used to screen information. A statistical study was conducted to calculate the frequency of different bacteria causing diarrhea, and the degree of drug resistance of the isolated bacteria.

 

RESULTS 

 

Table 1 show the sex and age distribution of diarrheal patients attending the National Laboratory Center for Stool Culture in Sana’a City, tested for anemia. The male patients counted 51.7%, and the female patients counted 48.3%. The mean age of our patients was 30.05 years, with an SD of 14.2 years, and their ages ranged from 3 to 68 years. Most of our patients were in the age group of 16–25 years (36.9%), followed by the group of 26–35 years (18.8%), while other age groups were less frequent. Enterobacteriaceae isolates were obtained from 972 stool samples collected; 15.3% (149/972) were diarrheagenic Enterobacteriaceae bacteria, including E. coli (from bloody stool as well as stool from children under five), and 2 other Enterobacteriaceae genera, namely: Salmonella and Shigella.

There were 8.3% (81/972) Salmonella spp., 5.7% (56/972) Shigella spp., and 1.2% (12/972) E. coli, from bloody or infant stool (infant <5 years) (Table 2).Upon serological analysis, E. coli strains were grouped, with the most prominent being serogroups O153 (3, 2%) and O25 (2, 1.4%), known to be enterotoxigenic E. coli (Table 2). Fifty-six (37.6%) Shigella species were identified as Shigella boydii type 2 and Shigella bodii type 17 were the most common Shigella isolated (11, 8.1% and 16, 10.7%, respectively), Shigella flexneri type II and Shigella flexneri type I (8, 5.3% and 4, 2.7%, respectively), Shigella dysenteriae type II (8, 5.3%), Shigella dysenteriae type I (6, 4%), and Shigella sonnei (21.4%). Among Salmonella isolates, serogroups O3.10 (16.1%), O4 (9.4%), and O7 (7.4%) were the most dominant. Twelve E. coli isolates were subjected to antimicrobial susceptibility testing; when these isolates were examined against all of the antimicrobials we had chosen, at least one antimicrobial resistance was found (Table 3). The isolates of E. coli exhibited the highest levels of resistance to cefazolin (7/12, 58.3%) and tetracycline (10/12, 83.3%). The isolates showed a high susceptibility to aminoglycosides that are given to treat E. coli infections, including gentamicin (11/12, 91.7%), amikacin (8/12, 66.7%), and nitrofurantoin (10/12, 83.3%). For one fluoroquinolone, ciprofloxacin (4/12, 33.3%), we found high resistance. When tested against our chosen antimicrobials (Table 4), all 56 Shigella isolates were shown to be 100% tetracycline resistant. We found three isolates of Shigella that were resistant to multiple drugs. Six (10.7%) isolates were resistant to both norfloxacin and ciprofloxacin, while three (5.4%) isolates were resistant to just one of the three medications (norfloxacin, ciprofloxacin, and tetracycline). It was found that the Salmonella isolates had a significant tetracycline resistance (29/81, 35.8%), but a comparatively low ciprofloxacin (7/81, 8.6%) and ceftriaxone (5/81, 6.3%). When tested against gentamycin and norfloxacin, the isolates of Salmonella revealed complete susceptibility; nevertheless, no strains of the bacteria that were resistant to multiple drugs were discovered.

 

 

DISCUSSION 

 

Several significant discoveries have been made from our regular surveillance study of diarrhea from Sana'a city from January 2021 to March 2024. This study's isolates of Enterobacteriaceae were diverse and frequent, which is consistent with findings from China, Kenya, and India15,16,27. Shigella, Salmonella, and diarrheagenic E. coli frequencies are in line with earlier research conducted in African nations28,29. Lompo et al.'s results30 did not, however, identify Salmonella from stool samples that had diarrhea. Enterobacteri-aceae isolates were obtained from 972 stool samples collected; 15.3% (149/972) were diarrheagenic Enterobacteriaceae bacteria, including E. coli (from bloody stool as well as stool from children under five), and 2 other Enterobacteriaceae genera, namely: Salmonella and Shigella. There were 8.3% (81/972) Salmonella spp., 5.7% (56/972) Shigella spp., and 1.2% (12/972) E. coli, from bloody or infant stool (infant <5 years) (Table). When compared to other research (4%) the amount of diarrheagenic bacteria we identified in our study was noticeably higher15,28. The high incidence could potentially be attributed to the unhygienic living conditions and lack of potable drinking water at our sample site. As was previously mentioned, street food samples have been found to have diarrheagenic bacteria. This could lead to an increase in occurrences of bacterial diarrhea in places where living circumstances are unhygienic. This finding is crucial in a nation where the majority of diarrheal illnesses are believed to be exclusively caused by V. cholerae8-10.  

Shigella, Salmonella, and enterotoxigenic E. coli are common, which highlights the necessity of increasing laboratory-based surveillance and accurately determining the cause of sickness in order to treat diarrheal illnesses. It is quite concerning that strong medications used to treat Shigella and Vibrio, like ciprofloxacin, norfloxacin, and nalidixic acid, are showing signs of developing resistance. We discovered that all (100%) of the Shigella isolates were tetracycline resistant.Tetracycline has been widely used in Yemen throughout the years, and there have been numerous reports of antimicrobial resistance to these medications18,19,31,32. This could be the cause of this, due to easy access to the medication through pharmacies; there may also be some contribution from the careless use of the drug in the animal husbandry industry. In addition, the strains of Shigella were resistant to amikacin (10.7%), ciprofloxacin (28.8%), nalidixic acid (17.9%), and norfloxacin (5.4%), the latter two of which were once highly effective medications for treating adult cases of Shigella dysentery33. The findings that Shigella sp. exhibits strong resistance to tetracycline, amikacin, and nalidixic acid and high susceptibility to ceftriaxone are in line with the findings of Pourakbari et al.,34 and Basnet et al.,33. The emergence of high resistance to parenteral ceftriaxone is concerning because it is highly effective and advised for the treatment of hospitalized children with severe shigellosis35. E. coli strains were grouped based on serological analysis; the most prevalent serogroups were O153 (3, 2%) and O25 (2, 1.4%), which are recognized as enterotoxigenic E. coli strains.

Numerous virulent genes that can cause mild to severe infections have been found to be present in the enterotoxigenic Escherichia coli (ETEC) serotype O15336. This E. coli strain has been reported to have been isolated from patients during an outbreak, which may be a sign of its propensity to spread epidemics37. Public health may be seriously jeopardized by the spread of these aggressive strains of E. coli inside nearby populations. Antimicrobial resistance is becoming more common in populations, as evidenced by the study's detection of multi-drug resistant organisms.

The careless use of antibiotics as growth supplements in the farming and animal husbandry industries in Yemen is a significant issue since it reduces yield by preventing infection. This procedure accelerates the development of antibiotic resistance by pre-exposing medications to these microorganisms38,39.

In this surveillance project, a village with restricted access to pipe-borne water was selected as the sentinel site. Another issue in the neighborhood is the improper disposal of human waste. The increased incidence of diarrhea could be explained by open defecation into bushes and bodies of water as well as a shortage of drinkable water13. It is commonly established that some behaviors can stop the spread of bacterial germs that cause diarrhea, including washing your hands, preparing your food properly, and disposing of your trash and human waste properly.

Limitations of the study

We noted a few restrictions in the research. Compared to serological analysis alone, molecular techniques like PCR would have been more sensitive and provided a better knowledge of the diarrheagenic E. coli and ESBLs obtained. Since samples were only taken from patients who consented to be included in the study, we are unable to provide information on the overall number of people who present to medical institutions with diarrhea.

 

CONCLUSIONS 

 

This study has demonstrated the variety of bacteria (Salmonella, Shigella, and certain E. coli) linked to diarrheal illness and the importance of accurate differential diagnosis for improved therapeutic results. All isolates exhibited resistance, which raises concerns for public health because the medications used to treat Shigellosis, Salmonellosis, and other diarrheal illnesses demonstrated significant resistance. The high rates of resistance found in Shigella isolates serve as a warning and highlight the necessity of putting preventive measures in place to reduce transmission. To stop AMR microorganisms, regular antibiotic susceptibility testing, efficient monitoring, and national surveillance should be put into place.

 

ACKNOWLEDGEMENTS 

 

The authors express their gratitude to Yemen and the Sana'a University for their kind assistance.

 

AUTHOR’S CONTRIBUTION

 

Al-Shamahy HA: writing original draft, methodology, investigation. AbdulMughni GTA: formal analysis, data curation, conceptualization. Al-gunaid EA: writing, review and editing, methodology. Al-Shami HZ: editing, review. Al-Haidary NM: literature survey. Al-Madhaji AG: formal analysis, data curation. Al-Moyed KA: conceptualization. All authors have revised and approved the final version of the manuscript.

 

DATA AVILIABILITY

 

The data will be available to anyone upon request from the corresponding author.

 

CONFLICT OF INTEREST

 

None to declare.

 

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