Antimicrobial Susceptibility Patterns of Shigella Species among Children under Five Years of Age with Diarrhea in Selected Health Centers, Addis Ababa, Ethiopia

Background Shigella and parasitic infections are common public health problems throughout the world. Shigellosis is an acute gastroenteritis infection and one of Ethiopia's most common causes of morbidity and mortality, especially in children under five. High resistance rates to commonly used antibiotic agents have been documented in different locations in Ethiopia. Objective This study aimed to characterize the antimicrobial features of the Shigella species isolated from children under five years of age with acute diarrhea in Addis Ababa, Ethiopia. Methods Using a cross-sectional study, freshly passed fecal specimens were collected for intestinal parasite and bacterial isolation. Fecal samples for bacterial identification were placed immediately in Cary–Blair media and transported to the Ethiopian Public Health Institution (EPHI) laboratory. Antimicrobial susceptibility testing (AMST) was conducted using the disk diffusion method. Data were described using descriptive statistical tools. The association of independent and dependent variables was evaluated with logistic regression. A P value ≤0.05 was considered statistically significant. Results The prevalence of intestinal parasites was 8.2% with seven different species. Among the 534 stool-cultured specimens, 47 (8.8%) were positive for Shigella species. Antimicrobial susceptibility testing (AMST) showed that 100%, 93.6%, 80.9%, 72.3%, and 57.5% were susceptible to norfloxacin, nalidixic acid, ciprofloxacin, gentamicin, and cefoxitin, respectively. However, 100% of the isolates were resistant to amoxicillin and erythromycin. More than 50% of the isolates were resistant to three and above antibiotics, while none of them were susceptible to all the antibiotics tested. All risk factors assessed did not show a statistically significant association with Shigella infection. Conclusion The high levels of antibiotic resistance observed among the commonly prescribed antibiotics are alarming. The emerging resistance to ciprofloxacin and nalidixic acid signals a severe public health threat in the management of shigellosis. Raising awareness about resistance and educating health professionals, policymakers, and the public can help improve the quality of patient care and rational antibiotic use.


Introduction
Te burden of diarrheal disease is highest in developing countries associated with parasite and Shigella infection where there is poor sanitation, inadequate hygiene, unsafe drinking water, and poorer overall health and nutritional status [1]. Shigellosis is caused by the ingestion of bacteria of the genus Shigella. It is gram-negative enteropathogenic bacteria responsible for human diarrhea and dysentery. Tere are four species of Shigella based on serological and biochemical characteristics: Shigella dysenteriae (S. dysenteriae), S. fexneri, S. boydii, and S. sonnei [2]. Species or serogroups are further subdivided into serotypes based on "the O antigen of a lipopolysaccharide outer cell wall membrane." It is a germ that is extremely infectious; only 10 bacilli are required to cause infection [3]. Serogroup A (S. dysenteriae) has 15 serotypes and 2 provisional serotypes [4]. Serogroup B (S. fexneri) has 6 serotypes and 16 subserotypes. Serogroup C (S. boydii) has 20 serotypes, and serogroup D (S. sonnei) has only 1 serotype [5]. Morbidity and mortality result from inadequate hand washing after defecation or nappy changing or from person-to-person directly via the fecal-oral route [6]. Serogroup D (S. sonnei) is the most physiologically distinct and can be distinguished by key biochemical and genetic characteristics, including lactose fermentation and diferences in the utilization of a range of substrates. Shigella sonnei (S. sonnei) can be divided into fve biotypes (a, b, e, f, and g) based on biochemical properties [4].
Disease usually begins with fever, fatigue, anorexia, and malaise. Some patients have mild watery diarrhea, whereas others experience severe dysentery with systemic complications such as electrolyte imbalance, seizures, and hemolytic uremic syndrome [7]. Shigellosis is the most common cause of epidemic dysentery and is found all over the world. In developing countries, it is the leading cause of infant diarrhea and mortality [2]. Te burden of Shigella in children under fve years of age was variable from region to region. Te prevalence of shigellosis in Ethiopia was reported to be high in children [8]. Shigella fexneri (S. fexneri) is dominant in Africa and Asia, although S. sonnei, the most dominant species in South America, was the predominant isolate in one study in Ethiopia [9]. Tis may give clues to the scientifc world about the migration and movement of strains from one region to another. In Ethiopia, it is difcult to evaluate the burden of Shigella infection because the diagnosis is on clinical grounds and there is a lack of coordinated epidemiological surveillance systems. In addition, underreporting of cases and the prevalence of other diarrhea illnesses regarded as high priorities may have obscured the shigellosis issue. Since Shigella species are not frequently grown and their antibiotic resistance cannot be tested in Addis Ababa health centers, more research is needed to address the true status of their antibiotic resistance. Antimicrobial resistance (AMR) patterns vary from region to region and even within a single region [4]. Te majority of diarrheal illnesses in Ethiopia are treated clinically without laboratory confrmation, mainly in health centers.
Increasing prevalence of multidrug resistance (MDR) to Shigella species is a serious threat, especially in Ethiopia with health and nutritional problems. Tere was identifed MDR among several serotypes of Shigella species isolated from acute diarrheal patients [10]. Irrespective of the serogroup/ serotype, most of the strains carried similar genes encoding resistance to specifc antimicrobials. Tis emergency of drug resistance calls for the rational use of efective drugs and underscores the need for alternative drugs to treat infections caused by resistant strains. Resistance to commonly used antibiotics is high; therefore, it is important to look at the susceptibility pattern before starting treatment. Te occurrences of invasive bacterial infections from numerous sites were presented, revealing not only the presence of Shigella species infection but also signifcant continent-wide variation in estimated disease prevalence and antimicrobial susceptibility patterns. Terefore, this study aimed to determine the prevalence and antimicrobial sensitivity pattern of Shigella species infections among under-fve children in selected health centers in Addis Ababa, Ethiopia.

Study Sites.
Te investigation was carried out in Addis Ababa, Ethiopia, in the outpatient pediatric departments of the chosen medical facilities (Wereda 1 health center, Wereda 2 health center, Arada health center, and Shegole health center) from June 2021 to April 2022. Addis Ababa is the capital of Ethiopia and one of the largest cities on the African continent, with an altitude of 2,355 meters above sea level and an annual temperature ranging from 18°C to 25°C. Addis Ababa has a total population of 3,602,000 as of the 2019 projection based on the 2007 census, consisting of 1,134,150 children under the age of fve [11].

Study Design.
A laboratory-based cross-sectional study design was conducted in order to obtain stool specimens and related information from children with acute diarrhea (within 7 days) in Addis Ababa, Ethiopia.

Study Population and the Sampling Process.
First, the sampling technique was carried out by listing the subcities of Addis Ababa. Second, by the simple random method, three subcities (Gulele, Kolfe Keranio, Arada, and Nefassilk Lafto) were selected. Ten, four health centers were also selected by the simple random method. 534 under-fve children with diarrhea who met the inclusion criteria during the study period were chosen using the systemic sample method based on population allocation of the four health centers utilizing the case interval. By using a lottery, the frst case of the interval was chosen. Te study included all under fveyear-old children with acute diarrheal disease (144 children from Wereda 1 health center and 390 children from the other three health centers (130 children from each)). Children whose parents/guardians were not voluntary to participate, newborn, inpatients, and those who had persistent diarrhea and had taken antimicrobial treatment 1 week prior to and at the time of data collection were excluded from the study.

Data and Sample Collection.
Te study was conducted in four public health facilities. Data related to the sociodemographic profle of the children and associated factors were collected using a pretested questionnaire adapted from the relevant literature; data were collected by face-to-face interviews [12][13][14]. Data were collected by four clinical nurses and four medical laboratories. After the physicians established that the children had acute diarrhea, data collectors interviewed the parents or guardians on behalf of the children at the health facility. Te age of the children information was verifed from birth certifcates. Prior to collecting data, parents and guardians were fully informed of the study's objectives and the advantages of participation. Participation was on voluntarily basis, and they were informed that it is within their right to withdraw from the study at any time during the course of the study. Diarrheasuspected children that were in the pediatrics department during the frst day of the study period and registered from the case interval were the frst subject of the study. After proper instruction using their identifcation card (ID), each caregiver was asked to bring the stool specimen from their children in clean, dry, leak-proof disposable stool cups. Approximately, 1 g of stool was immediately transferred into the Cary-Blair transporting medium (Oxoid Ltd., Basingstoke, UK), labeled, and transported within 1-2 hours of collection in an ice-packed cold box (4°C) to the Ethiopian Public Health Institute (EPHI) of Bacteriology and Mycology Laboratory for bacterial identifcation.

Culture and Identifcation.
Isolation and characterization of Shigella species were performed based on the practical guideline and handbook of clinical microbiology procedures [15,16]. In order to enrich the bacteria, a mixture of a feces sample (1 mL) was added to a tube containing 9 mL of Selenite F broth (Oxoid Ltd., UK) and incubated at 37°C for 24 hours. Ten, an inoculum was transferred from Selenite F broth into MacConkey agar (MAC) and xylose lysine deoxycholate (XLD) agar plates. Shigella growth was distinguished from other lactose-negative suspected colonies on MAC by their distinctive colony morphology on XLD agar after subculture and overnight incubation at 37°C. Suspected Shigella colonies' further identifcation was performed biochemically using oxidase, Kligler iron agar (KIA), urea, indole, citrate, SIM media, and lysine iron agar (lysine decarboxylase (LDC)) tests. After incubation for 18-24 h at 37°C, the test media were read for characteristic Shigella species reactions.

Intestinal Parasite Identifcation.
After interviewing, respondents were asked to give a fresh stool specimen in a sterile, clean, wide-mouthed plastic container by using a clean wooden applicator stick, which was transported to the microbiology laboratory for analysis. Two stool samples were collected; one stool sample was immediately emulsifed using saline (0.85% NaCl) for parasitological examination of trophozoite, cyst, larva, and ova stages by direct wet mount preparation in normal saline and iodine solution, and the second stool was used for parasites identifcation using formol-ether concentration sedimentation techniques.

Antimicrobial Susceptibility
Testing. According to the standard operating procedure (SOP) adopted from the Clinical Laboratory Standards Institute (CLSI) [17], the antimicrobial susceptibility test (AMST) was carried out using the Kirby-Bauer single disk difusion method on Muller-Hinton agar (MHA) (Oxoid, Hampshire, UK) plates. To produce a homogeneous suspension according to 0.5 McFarland densitometer standards, three to fve pure colonies were selected using a sterile loop and mixed with sterile normal saline (0.85 percent NaCl). Using a sterile cotton swab, the suspension was evenly distributed onto an MHA plate. For 3-5 minutes, the plate was dried at room temperature. Antimicrobial disks used in the study were ampicillin (10 μg), chloramphenicol (30 μg), cotrimoxazole (25 μg), gentamycin (10 μg), amoxicillin (10 μg), doxycycline (30 μg), tetracycline (30 μg), erythromycin (15 μg), cefoxitin (10 μg), ciprofoxacin (5 μg), nalidixic acid (30 μg), and norfoxacin (10 μg). Based on the zone of inhibition, the disks were measured using a ruler to the nearest whole millimeters after the plates (3-4 antibiotic disks on a 100 mm plate) were incubated at 37°C for 24 hours. By comparing the diameter of the standard with the current inhibition zone, the result was classifed as susceptible or sensitive (S), intermediate (I), and resistant (R). Te technique was closely adhering to the SOP for the preparation and standardization in order to standardize the inoculum density for a susceptibility test. Te term "multiple drug resistance" (MDR) was defned as the resistance of bacterial isolates to two and more antibiotics in more than two antimicrobial categories tested [18][19][20].

Data Quality Control.
Training was given to data collectors. Te English version of the questionnaire was translated to local language (Amharic). Control strains were used during the microbiological investigation of AMST and checking the efciency of culture media. Te standard operating procedure (SOP) and CLSI were used throughout the procedures. In addition, sterility of the culture media was checked frequently by incubating the prepared culture media at 37°C overnight and checked for growth. American Type Culture Collection (ATCC) reference strains were used to test the performance of each culture medium and antimicrobial disks before use. 2.9. Data Analysis. EpiData version 3.1 (EpiData Association, Odense, Denmark) was used to input the data, and STATA version 14 software program (Stata Corporation, Lakeway Drive, College Station, Texas) was used to export it for analysis. Te frequency, percent, mean, and standard deviation of the data were used to explain the data using descriptive statistical techniques. Te outcome variables' predictors were found using independent variables such as Canadian Journal of Infectious Diseases and Medical Microbiology 3 sociodemographic and clinical characteristics. Te rate of Shigella isolates was examined using descriptive statistics. To determine the adjusted odd ratio (AOR) and P value, logistic regression was used to assess the relationship between the independent and dependent factors in the binary outcome statistics. In the logistic regression analysis with a 95% confdence interval (CI), P ≤ 0.05 was considered statistically signifcant.

Clinical Characteristics.
Tere was no history of antibiotics use by children in the past two weeks prior to and at the time of data collection. Te most common clinical complaints recorded were mixed illness (diarrhea vomiting, abdominal pain, and fever) (40.3%), followed by watery diarrhea (36.9%) ( Table 1).

Prevalence of Shigella Species and Associated Risk Factors.
As shown in Table 1, 47 (8.8%) of the stool specimens tested positive for Shigella species. Shigella species isolated from male (n � 34; 6.4%) had a higher frequency than female (n � 13; 2.4%) children. Te age range between 1-and 2.11year-old had the largest percentage of infections (5.4%), whereas no pathogen was isolated from less than 1-year-old. Children with two days of diarrhea, four family sizes, occupational (housewife), monthly income (3001-4000EB), and one child in the household were more likely to be exposed to Shigella infection although the diference was not statistically signifcant (P > 0.05). All the study participants visited the health center 1-5 days after the onset of diarrhea.
In general, all risk factors assessed did not show a statistically signifcant association with the presence of Shigella infection.

Prevalence and Types of Intestinal Parasites.
Te overall prevalence of intestinal parasites was 8.2% with seven different species. Entamoeba histolytica was the leading parasite isolated (3.0%), followed by Ascaris lumbricoides (1.9%), and least parasites isolated were Giardia lambelia (0.4%). No mixed parasite infection was isolated from the study participants (Table 2).

Antimicrobial Susceptibility Pattern of the Isolated Shigella Species.
Te antimicrobial susceptibility patterns of Shigella isolates were tested against 12 selected antibiotics, and generally, more than half of the isolates showed resistance to tested antibiotics. With respect to susceptibility, 100%, 93.6%, 80.9%, 72.3%, and 57.5% of the isolates were susceptible to norfoxacin, nalidixic acid, ciprofoxacin, gentamicin, and cefoxitin, respectively. However, all isolates were 100% resistant to amoxicillin and erythromycin ( Figure 1). Out of the 47 Shigella isolates, 40 (85.1%) were identifed as MDR. More than 50% of the isolates were resistant to three and more antibiotics, while none of them were susceptible to all the antibiotics tested. High levels of resistances were observed for erythromycin, amoxicillin, and ampicillin. Nearly, 55.3% of the Shigella isolates have developed resistance to four antibiotics, followed by 36.2% resistant to fve antibiotics, 12.8% to six antibiotics, and 4.3% of isolates were resistant to seven antibiotics (Table 3).

Discussion
As one of the most prevalent causes of epidemic dysentery, shigellosis is virtually universal. It is the main contributor to newborn mortality and diarrhea, primarily in developing nations [2]. In Ethiopia, Shigella species are not regularly cultured for routine patient diagnosis, and their antibiotic resistance cannot be verifed. Antimicrobial resistance (AMR) patterns vary from region to region and even within a single region [4].
In the current investigation, 47 (8.8%) children under the age of fve had positive stool cultures for Shigella species. Shigella poses a serious threat to the public's health because only a small number of cells (10-100 bacilli) are required to generate bacillary dysentery, which can result in shigellosis [3]. Comparable to this fnding, the reported isolation rates in the previous research conducted in Ethiopia were 8.3% in Hossana [18], 9.1% in Addis Ababa [21], 9.5% in Adama [22], and 6.9% in Mekelle [23]. Te outcome is, however, less than those from Iran (12.6%) [24] and Kenya ((14.8%) [25] and (23.6%)) [26]. Te possible explanation for this variance could be attributed to variations in the sample, regional variation, and socioeconomic situations. In addition, the diference in prevalence may be caused by an improved standard of living, increased understanding of sanitation and hygiene, and the use of preventative measures to control infectious diseases. Terefore, greater community awareness, particularly among mothers, about personal and environmental hygiene directly afects the prevalence of Shigella species in children. Tis study showed a higher rate of Shigella species isolation compared to reports from Juba, South Sudan (5.2%) [27], Arba Minch, Ethiopia (4.8%) [12], Robe and Goba, Ethiopia (4.3%) [28], Adigrat, Ethiopia (3.7%) [29], Ambo, Ethiopia (2.5%) [14], Brazil (2.2%) [30], Debre Markos, Ethiopia (2.3%) [31], Nepal (2.1%) [32], and Jimma, Ethiopia (1.1%) [33]. Tis might be because of variations in methodology, study participant types, sample sizes, and geographic locations. Te prevalence of Shigella species reports varied in diferent regions and time [7]. Children under the age of fve are naturally taking contaminated soils, food, and water into their mouth and may acquire disease, causing microbes including Shigella species [28]. According to a review study conducted in Ethiopia [8], the pooled prevalence of shigellosis in children was 7.0%, whereas it was 2.2% in the adult population. Tis reveals that newborns and young children are more susceptible to diarrheal morbidity due to Shigella than adults. Similar to the current study, earlier research conducted on children under the age of fve found no statistically signifcant link between Shigella infection and sociodemographic characteristics [12,18]. In the current study, infants were not associated with Shigella infection. Te most plausible explanation for this outcome is that the mother's immunity, which can shield the infant from infection for about six months, or that the parents provide their child adequate medical care. In the present investigation, children aged 1-2.11 years had a greater prevalence of Shigella species though this was not signifcant. Tis result was consistent with multiple investigations [12,18,28] which showed that children with a lower immunological state and those whose parents had lower educational backgrounds are more prone to Shigella infection. Tis study reported a more isolation rate of Shigella species among those with low educational background in connection to educational status and the frequency of isolation (1-8 grade). Tis result is analogous with the past research in south Ethiopia [34]. To control other factors that cause this disease and to raise community awareness of the mechanisms for managing infectious diarrhea, education is essential. Poor environmental  sanitation, malnutrition, inadequate water supply, poverty, and limited education are the major factors implicated in the occurrence, spread, and severity of diarrheal disease [35]. One to fve days following the commencement of diarrhea, all study participants went to the health centers. In this investigation, the most common symptoms of Shigella cases with a positive culture were fever, vomiting, and abdominal pain. Tis is caused by the bacteria's capacity to enter and reproduce in the cells lining the colon and rectum. Similar results have been reported from Asia [36]. In northern Ethiopia, more Shigella isolates were found in bloody and mixed (blood and mucus) samples [23]. In the current study, patients with watery diarrhea and mixed (mucus and blood) samples were more susceptible to Shigella species. Tis contrary outcome may be due to variations in the unidentifed strains connected to the patient or to the fact that more patients with watery diarrhea than bloody diarrhea participated in this investigation.
In the current study, Entamoeba histolytica was the leading parasite isolated (3.0%), followed by Ascaris lumbricoides (1.9%) and least parasites isolated were Giardia lamblia (0.4%). A similar fnding has been reported in the previous study conducted in Addis Ababa [37]. Te predominant intestinal parasite reported in Aksum, Ethiopia (5%) [38], Iran (3.7%) [39], and Saudi Arabia (9%) [40] was Giardia lamblia; however, Ascaris lumbricoides was reported in Dilla, Ethiopia (11.4%) [41]. Te high prevalence of those parasites might be due to low personal hygiene practice with easy transmission of the parasite which is usually found in unsafe food, water, soil, or contaminated surface.
In developing nations like Ethiopia, where infectious illnesses are prevalent, AMR is a major global public health concern. Shigella species were tested in this study against 12 antibiotics, and the results showed a variety of sensitivity and resistance patterns. In the current and earlier research in Ethiopia, both patterns have shown diferences. By 2050, a person will die every three seconds if antibiotic resistance is not addressed [42]. In the current investigation, Shigella species showed a high frequency of isolates resistant to antimicrobial drugs, although some of the isolates were also sensitive to norfoxacin, nalidixic acid, ciprofoxacin, cefoxitin, and gentamicin. Tis may be linked to the widespread usage of basic antibiotics and their simplicity of availability. In Ethiopia, it is simple for anyone to visit a pharmacy or a drug vendor and get the most common antibiotics without a prescription [14,43]. Te high resistance rates seen could be due to this and other malpractices such as the use of lower dosages, improper care of antibiotics for instance selling antibiotics in open markets in rural areas of the country, and low efcacy of imported drugs [44]. Te resistance patterns of Shigella against antimicrobial drugs in the present study range from 100% (for amoxicillin and erythromycin) to 6.4% (for nalidixic acid); however, norfoxacin was 100% susceptible to all isolates, which is in agreement with the previous report from Ethiopia [7]. Te three most crucial medications used to treat shigellosis in Ethiopia are amoxicillin, cotrimoxazole, and ciprofoxacin. In the treatment of shigellosis, the emergence of resistance to ciprofoxacin and nalidixic acid raises serious public health concerns. Te Clinical and Laboratory Standards Institute (CLSI) on performance standards for AMST stated that the result of ampicillin can be used to predict the result for amoxicillin and tetracycline for doxycycline regardless of the fnding [17]. Ampicillin and doxycycline resistance patterns in the current investigation were 93.6% and 83%, respectively. Te resistance pattern of amoxicillin (100%) in this study was similar to that of studies carried out in other regions of Ethiopia [28,45,46] and also showed comparable resistance to tetracycline (91.5%) with earlier studies in Bahir Dar (93.8%) [47] and Gonder (89.7%) [48]. According to Tosisa et al. [14], amoxicillin resistance was present in 50% of Shigella species in Ambo, Ethiopia, which is contrary to the fndings of this study. Tis can be because the study site sources and laboratory standards employed difered. Te current data showed complete resistance against erythromycin, which is consistent with a research conducted in Debre Markos, Ethiopia [49]. Te accessibility and prolonged use of the drug could be the reasons for such levels of resistance. Shigella isolates create R plasmids, which can confer several antibiotic resistances and contain numerous resistance genes [50]. Similar analyses of the genes responsible for antibiotic resistance in Shigella isolates from southeastern Africa that caused diarrhea in children under the age of fve revealed the presence of oxa-1-like-lactamases for ampicillin, dfrA1 genes for trimethoprim-sulfamethoxazole/cotrimoxazole, tetB genes for tetracycline, and Chl acetyltrans [51]. Limiting the fndings to Shigella species made identifying other common causes of diarrhea more difcult. Tis study included only children with acute diarrhea and was limited to public health facilities. Despite these limitations, the fndings of this study may be useful in informing clinicians, public health ofcials, and researchers about Shigella-associated diarrhea, its contributing causes, and the antibiotic susceptibility pattern.

Conclusion
In conclusion, 8.8% of the children under the age of fve tested positive for Shigella species in the study, which involved all study participants visiting the health facility between one and fve days after the onset of diarrhea. Te high prevalence of Shigella species with antibiotic resistance, combined with MDR among commonly prescribed antibiotics such as ampicillin, cotrimoxazole, tetracycline, erythromycin, and chloramphenicol, necessitates immediate strengthening of the monitoring mechanism on the prudent use of antibiotics as well as a broad-based behavioral change.
Te emerging resistance to ciprofoxacin and nalidixic acid signals a serious public health threat in the management of shigellosis. Raising awareness about resistance and educating health professionals, policymakers, and the public can help improve the quality of patient care and the rational antibiotic use.

Data Availability
Te datasets supporting the conclusions of this article are included in the article.

Ethical Approval
Te Jimma University Institute of Health Institutional Review Board granted ethical approval (IHRP6/1092/2021). A letter was received from the Addis Ababa Health Bureau, and the relevant medical facilities also granted approval for the study to be carried out.

Consent
Parents or guardians gave their permission. Information about the objective of the study and procedures was given before the participants enrolled to the study. Oral assent and written informed consent were obtained from each mother or caregiver of the children selected for the study. All information obtained during this study was kept confdential and used merely for the research purpose.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
BA participated in the design of the study, conducted data collection, analyzed the data, and drafted the paper. ZM, TST, and GB participated in the design of the study and analysis of the data and revised subsequent drafts of the paper. EA and ET participated in data collection and laboratory analysis. All the authors read and approved the fnal manuscript and agree to be accountable for all aspects of the work.