Antimicrobial Resistance Profile and Associated Factors of Group B Streptococci Colonization among Pregnant Women Attending Antenatal Clinics in Jigjiga, Southeast Ethiopia

Background Streptococcus agalactiae (group B Streptococcus, GBS) is one of the causes of maternal and neonatal morbidity and mortality in many parts of the world. It is associated with severe maternal and neonatal outcomes. The colonization rate, associated factors, and antimicrobial sensitivity (AST) profile of GBS among pregnant women in Eastern Ethiopia is less studied. Methods A cross-sectional study was conducted from 1st  March to 30th May, 2021 in Jigjiga. A total of 182 pregnant women with a gestational period of ≥36 weeks were included. A structured questionnaire was used to collect data on the participants' demographic and clinical history. Vaginal-rectal samples were collected by brushing the lower vagina and rectum with a sterile cotton swab for bacteriological culture. An antimicrobial sensitivity test (AST) was performed using the Kary-Bauer disk diffusion method. Data were entered and analyzed using SPSS version 25. The logistic regression model was used to find out factors associated with GBS colonization. Results GBS colonization among pregnant women attending antenatal care was at 15.9% (29/182). The AST result showed that the majority of the isolates were sensitive to vancomycin (96.6%), chloramphenicol (96.6%), ampicillin (93.1%) azithromycin (89.7%), and penicillin (86.2%). In contrast, the isolates were found to be resistant to ceftriaxone, erythromycin, ciprofloxacin, clindamycin, and tetracycline at 17.2%, 20.7%, 27.6%, 27.6%, and 34.5%, respectively. Multidrug resistance (MDR) was noted in 4 isolates (13.79%). GBS colonization was significantly associated a with history of preterm labor (<37 weeks of gestation) (AOR = 3.87, 95% CI = 1.36–10.9) and a history of prolonged ruptured membrane (>18 hr.) (AOR = 3.44, 95% CI = 1.34–8.83). Conclusions The colonization rate of GBS was considerably high among pregnant women attending antenatal care in the present study area. The observed antimicrobial resistance for the common drugs and the reported MDR level calls for routine screening of pregnant women for GBS and actions to minimize antimicrobial resistance (AMR) should be strengthened.


Introduction
Streptococcus agalactiae, also called group B Streptococcus (GBS), is part of the normal flora of gastrointestinal and genital tracts [1]. e bacteria use numerous adhesins and stress response apparatuses and immune evasion schemes for vaginal colonization [2]. e lower intestinal tract is the most likely reservoir of the bacteria with a secondary spread to the genitourinary system. e risk of colonization of GBS is greater among infants who are born from mother with GBS colonization. GBS transmission is vertical, transmitted throughout labor, or in utero through the transmission of the bacteria from the vaginal or the mucosa that is anorectally colonized [3].
During the time of pregnancy, the colonization of GBS could be brief, chronic, or intermittent [4]. Colonization commonly does not show any symptoms. However, at some point in pregnancy, GBS multiplication around the vaginal area can lead to maternal morbidity as likely as neonatal complications [5]. About 10 to 30% of women of childbearing age lift GBS in the recto-vaginal area without showing any symptoms. When untreated, nearly 50-75% of neonates born from a GBS infected mothers could be potentially colonized [6]. In Ethiopia, studies revealed that maternal colonization of GBS ranges from 7.2% [7] to 20.9% [3].
Different factors are associated with GBS colonization. e history of premature rupture of membranes, gastrointestinal GBS colonization, increased maternal age, low vitamin D intake, poor personal hygiene, sexual intercourse, health care occupation, and illiteracy were reported as important associated factors with GBS vaginal colonization [8][9][10].
GBS is one of the leading cause of infections among neonates worldwide [11][12][13]. In 2017, the World Health Organization (WHO) stated that infection associated with GBS caused about 147,000 infant deaths worldwide, despite the use of intrapartum antibiotic prophylaxis (IAP). With 54% of estimated cases and 65% of stillbirths and infant deaths, Africa had the highest burden [14]. While majority of GBS infections could be detected during labor or delivery, women in their post-delivery period can also be at a greater risk for invasive GBS complications even in the absence of extra risk factors [15]. e early onset of GBS infection may cause severe neurological damages and other serious neonatal outcomes [16][17][18][19][20].
With regard to the antimicrobial sensitivity profile of GBS, it is regarded as uniformly susceptible to penicillin, the first-line antibiotic for Intrapartum Antibiotics Prophylaxis (IAP) [11]. Macrolides (e.g., erythromycin) and lincosamides (e.g., clindamycin) are used as alternative drugs [12]. However, the global spread of antimicrobial resistance (AMR) has led to increased GBS infections among pregnant women and newborns that are difficult to treat and poses significant health concerns. Increased use of antimicrobials for prophylaxis without proper bacteriological screening is one factor that raises this concern [21]. GBS is becoming resistant to different antibiotics, including macrolides and lincosamides, and recently resistance to penicillin and fluoroquinolone was also reported [22]. A systematic review and meta-analysis conducted in China showed that significant rates of resistance to erythromycin, clindamycin, and tetracycline were observed; 0-86%, 4-84%, and 23-96%, respectively [23]. Another meta-analysis that summarized findings from 21 Africa countries also revealed that antibiotic resistance for GBS is a major concern for the whole continent. Higher antimicrobial resistance was observed against tetracycline at 82.6% and penicillin at 33.6% [24].
A previous epidemiological study conducted in two hospitals in Jigjiga, Ethiopia, discovered neonatal sepsis (24.4%) and neonatal deaths (5.7%) as a major serious issue.
e study also indicates that 96% of deaths were early neonatal deaths that occurred in the first one week of life [25]. However, there is quite limited data on the colonization rate, antimicrobial susceptibility profile, and associated factors of GBS in Eastern Ethiopia and in the present study area in particular. erefore, our study was aimed at the description of colonization, antimicrobial susceptibility profile, and associated factors of GBS among pregnant women.

Study Design, Area, and
Period. An institution-based cross-sectional study was conducted at two selected health facilities of Jigjiga from 01 March 2021 to 30 May 2021. Jigjiga is located in the Southeastern part of Ethiopia, 621 km from the capital, Addis Ababa. e city is situated 1934 meters above the mean sea level. Based on the Ethiopian Central Statistical Agency report of the year 2015, the city had an estimated population of 304,000 of which 151,422 were men. ere were two hospitals and four health centers providing delivery services in the city during the time of data collection. For this study, Jigjiga University Sultan Sheik Hassen Yabere Referral Hospital (JJUSSHYRH) and Karamara General Hospital (KGH) were selected because these hospitals had the largest client flow for antenatal care and delivery services.

Study Population and Sampling.
Our study population were pregnant women with a gestational age of ≥36 weeks attending antenatal clinics at JJUSSHYRH and KGH during the time of data collection. e sample size was calculated based on the proportion of GBS colonization among pregnant women reported in Harar, Ethiopia, at 13.68% [26]. Using single population proportion formula, our calculated sample size was at 182. e sample size was proportionally allocated to each health facility based on their three months pregnancy visits. Accordingly, 123 and 52 participants were recruited from JJUSSHYRH and KGH, respectively.

Data Collection.
A pretested and structured questionnaire was used to collect data on the demographic, and clinical characteristics of the study participants. Demographic data such as maternal age, residence, marital status, occupation, educational status and clinical data such as gravidity, prenatal care, urinary tract infection, outcomes of the previous delivery, prolonged rupture of membrane, and gestational age were collected.

Specimen Collection, Transportation, and Bacteriological
Analysis. Vaginal-rectal swabs were collected during the antenatal care (ANC) follow-up by brushing the lower vagina and rectum with a sterile cotton swab by trained nurses following universal precautions. e swabs were immediately transported to the microbiology laboratory, JJUS-SHYRH, within 2-4 hours using Amies transporting media maintaining the temperature at 37. e bacteriological analysis was performed following the methods described in the center for disease control and prevention (CDC), and the American Society for Microbiology [27,28]. Samples were inoculated on Todd Hewitt broth media supplemented with gentamycin and nalidixic acid and incubated at 37°C for 24 hours, then subcultured on a blood agar plate at 37°C for 24 hrs. Colony characteristics, Gram-stain, and catalase test were used for presumptive identification. All Gram-positive cocci, beta-hemolytic, and catalase-negative isolates were further identified by Christie-Atkins-Munch-Petersen (CAMP) and bacitracin tests. e CAMP test was used to identify CAMP-positive GBS from other beta-hemolytic Streptococci. In brief, Staphylococcus aureus was inoculated onto a sheep blood agar plate by making a narrow streak down the center of the plate with a loop. en, the test organism (GBS) was streaked in a straight-line inoculum at right angles to the S. aureus within 2 mm. e plates were incubated at 35°C for 24°h. A positive CAMP test was indicated by an "arrowhead" shaped enhanced zone of beta-hemolysis in the area between the two cultures with the "arrow point" toward the S. aureus streak. No enhanced zone of beta-hemolysis was observed in a CAMP negative reaction. In addition, the bacitracin test was used to differentiate GBS from group A streptococcus which are both beta-hemolytic [29].

Antimicrobial SensitivityTesting (AST).
AST was performed using the Kirby-Bauer disk diffusion method. Bacterial suspension was prepared by adding 4-5 GBS colonies of the same morphology in 5 ml of sterile physiological saline equal to a 0.5 McFarland's standard that used as a reference to adjust the turbidity of the suspension. e suspension was inoculated on Mueller-Hinton agar (MHA) plates supplemented with 5% defibrinated sheep blood. en, antibiotic disks were placed and incubated at 35-37°C overnight. GBS were tested against antibiotics that are recommended by CLSI guideline. e zone of inhibition around antibiotic disks was measured by a calibrated ruler and interpreted as sensitive, intermediate, or resistant [30].
2.6. Data Quality Control. Standard operating procedures (SOPs) were followed during the sample collection, transportation, and bacteriological processing steps. Standard strains: Streptococcus agalactiae (American Type Cell Culture (ATCC) 27956), Staphylococcus aureus (ATCC 24923), Streptococcus pyogenes (ATCC 19615), and Escherichia coli (ATCC 25922) were used as quality control for culture and antimicrobial sensitivity testing.

Data
Analysis. Data were coded, cleaned, and entered into SPSS V.25 for analysis. Generated data were compiled by frequency tables and figures and other statistical summary measures. Bi-variable logistic regression analysis was carried out to identify the possible associated factors with GBS colonization. Variables with p-value ≤0.25 were further entered in to the multivariable logistic regression analysis. Finally, the results were presented using AOR with its 95% CI at p-value <0.05.

Antimicrobial Susceptibility Profile of GBS Isolates.
In this study, most of the GBS isolates were found to be sensitive to vancomycin and ampicillin. In contrary, they showed a moderate level of resistance for tetracycline 10 (34.5%), clindamycin 8 (27.6%), and ciprofloxacin 8 (27.6%) ( Table 3). Penicillin is the first choice for IAP and ampicillin as an alternative [5]. Studies conducted around the world, including reports in Ethiopia showed that these drugs have better action against GBS than other antibiotics [3,34,47,48]. In our study, the resistance of both penicillin and ampicillin was at 13.8% and 6.9%, respectively, which implies that these drugs are still good as a first choice to manage pregnant women colonized with GBS. Clindamycin and erythromycin could also be considered for patients who are allergic to penicillin once AST is performed [5]. Our finding is comparable with the studies conducted in Switzerland (8.2%) [49], Iran, Tehran (14.6%) [26], Egypt (22.6%) [50], Kenya (30.4%) [51], and Ethiopia (17.6-26.8%) [48,52].
We reported better sensitivity of GBS for chloramphenicol and azithromycin at 96.6% and 89.2%, respectively.
is result is concurring with other findings reported in Egypt [50] and similar studies in Ethiopia [52,53].
Vancomycin was also one of the commonly utilized drugs for patients who poorly responded for clindamycin and for the cases with a risk of high anaphylaxis. In this study, GBS isolates were found with 96.6% sensitivity for vancomycin which was similar with other studies conducted in Iran [26], Brazil [54], Cameron [33], Egypt [50] ,and Ethiopia [34,48] where GBS isolates were found 100% sensitive.
Multidrug resistance was observed in 4 isolates (13.79%) as presented in Table 4. is finding is similar with other studies in Ethiopia that reported 15.8%-43.9% MDR GBS isolates [34,52]. In general, the high level of AMR reported in our study might be associated with different factors including but not limited to the over and misuse of drugs in the study area where there is a weak drug regulatory practice and scarce bacteriological surveillance system because of lack of routine AST testing. Most of the antimicrobials listed above are available in the local market and individuals usually use these drugs without physician prescription.

Factors Associated with Colonization of GBS.
In the multivariable analysis, most of the variables tested in the regression model did not show statistical association with GBS colonization among pregnant women. Only two factors were found to be statistically significant; history of preterm labor (<37 weeks of gestation) (AOR = 3.87, 95% CI = 1.36-10.9, p-value = 0.01) and history of premature rupture of membrane (PROM) (>18 hr.) (AOR = 3.44, 95% CI = 1.34-8.83, p-value = 0.01). is finding is in line with other similar studies conducted in China [55], Nigeria [56], and Ethiopia [35,48,57]. According to different reports, GBS infection during pregnancy can cause complications like miscarriage, premature labor, or stillbirth. Likewise, PROM is also most often due to an infection in the uterus, cervix, or vagina, in this case; GBS might be an important component that should be ruled out.
As a limitation, neonatal outcomes are not included in our report since neonates born from the study participants were not included due to constraints.

Conclusions
e colonization rate of GBS was considerably high among pregnant women attending antenatal care in the present study area. In this study, most GBS isolates were found sensitive to vancomycin, chloramphenicol, ampicillin, azithromycin, and penicillin. However, the resistance to tetracycline, clindamycin, ciprofloxacin, erythromycin, and ceftriaxone was significant. GBS colonizationwas found significantly associated with history of preterm labor and premature rupture of membrane. erefore, stakeholders should consider routine culturebased bacteriological screening of pregnant women to help guide proper IAP. Actions to minimize AMR should be strengthened. Further large-scale studies that include neonatal outcome of GBS positive mothers should be considered. . Moreover, written informed consent was obtained from all the study participants and for those participants under the age of 16 years, written informed consent was secured from their parents or guardian. Client identifiers, including names and IDs, were also kept confidential, and any other special data security requirements were maintained and assured. Bacteriological results were communicated for health professionals attending the pregnant women.

Data Availability
All the generated data are included in this manuscript. e original data source can be shared up on request of the principal investigator.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
AT conceived the research idea. AT was also involved in the data collection and interpretation of the result. AD was involved in data analysis and evaluating the scientific content of the study. AM was involved in rationalizing the method section and manuscript preparation. All authors read and approved the final manuscript for submission.