Antimicrobial Resistance Patterns in Patients with Vaginal Discharge: A 2019-2022 Analysis at the National Health Laboratory in Eritrea

Background Antimicrobial resistance poses a significant global threat to the treatment of bacterial infections, particularly in low- and middle-income regions such as Africa. This study is aimed at analyzing antimicrobial resistance patterns in vaginal swab samples from patients at the National Health Laboratory from 2019 to 2022. Methods This retrospective study examined patient records from vaginal swab analyses performed at the National Health Laboratory between January 1, 2019, and December 31, 2022. Ethical approval was obtained from the Ministry of Health Research Ethical Approval and Clearance Committee on 15/02/2023. Results Of the 622 samples, 83% underwent microbial isolation and identification. Citrobacter spp. exhibited high resistance (>43%) to antibiotics such as cephalexin, ceftazidime, nalidixic acid, ampicillin, gentamicin, and tetracycline. E. coli showed resistance rates of more than 50% to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Klebsiella spp. and Proteus spp. exhibited resistance rates that exceeded 47% to specific antibiotics. Gram-positive bacteria have resistance rates of more than 49% with ampicillin, trimethoprim-sulfamethoxazole, tetracycline, oxacillin, vancomycin, and penicillin G. In particular, S. aureus demonstrated no resistance to rifampicin or clindamycin, while Streptococcus spp. showed 100% resistance to rifampicin and vancomycin. Several species, including Proteus species, Streptococcus spp., S. aureus, and Klebsiella spp. exhibited multidrug resistance. Conclusion Most gram-negative bacteria displayed higher resistance of >45% to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Among gram-positive bacteria, a higher resistance rate with ampicillin, trimethoprim-sulfamethoxazole, tetracycline, oxacillin, vancomycin, and penicillin G was recorded. S. aureus showed no resistance to rifampicin and clindamycin, and Strep. spp. indicated 100% resistance to rifampicin and vancomycin. This study highlights critical gaps and areas for further exploration. Expanding the spectrum of antibiotics tested and investigating underlying multidrug resistance mechanisms would provide a more comprehensive understanding of resistance patterns.


Introduction
The vagina is a complex ecosystem containing a variety of microorganisms [1].This unique environment undergoes significant changes throughout life, from birth to puberty and menopause [2].Women are more prone to urinary and vaginal infections due to their anatomical and functional proximity to their anal canal and due to the short urethra [3,4].The vaginal area is considered a complex microbial environment that harbors a multitude of microbial species [5].A common cause of vaginal discharge in women is bacterial vaginosis.Various rod-shaped gram-positive and gram-negative bacteria, including E. coli, Klebsiella spp., Enterococcus spp., Enterobacter spp., and Staphylococcus spp., contribute significantly to bacterial vaginosis [6,7].
Vaginal discharge is a common symptom seen by clinicians.It may be physiological or pathological.Normal physiological discharge changes with the menstrual cycle.It is thick and sticky for most of the cycle but becomes clearer, wetter, and stretchy for a short period around the time of ovulation.However, abnormal vaginal discharge is characterized by a change in color, consistency, volume, or odor and may be associated with symptoms such as itch, soreness, dysuria, pelvic pain, or intermenstrual or postcoital bleeding [8].
Pathologic vaginal discharges are caused by a variety of infectious and noninfectious causes.Discharge may be caused by infections of the vagina itself, but infections or inflammation of the cervix also leads to increased vaginal discharge [9].About 75% of women have the risk of vaginal infections at least once during their lifetime, and the vaginal tract can be infected by common pathogens, including Enterobacteriaceae spp., Enterococcus sp., and Streptococcus sp., Staphylococcus sp., Lactobacillus sp., and Candida albicans [5].
Antimicrobial resistance (AMR) is defined as the inherited or acquired ability of a microorganism to stop the antimicrobial drug from working against it to the extent that it cannot be used any longer [10].In the last two decades, multi-drug-resistant bacteria have increased and the number of pharmaceutical companies developing new antimicrobial agents has declined [11].Antibiotic susceptibility testing remains the standard diagnostic method for detecting bacterial resistance and guides clinicians in the appropriate and timely treatment of bacterial infections [12].The growing multidrug resistance of gonococci and the absence of an antibiotic regimen that is shown to be optimal in terms of safety and effectiveness are a challenge for almost every health system [13].
Pathogenic bacteria colonize the birth canal primarily after fecal contamination [14] and are then sometimes transmitted to the baby during labor and delivery [15].This transmission is probably one of the main sources of neonatal bacterial infection within the first week of life, particularly if there was prolonged/blocked labor or premature rupture of the membranes [16][17][18].Vaginal swabs were obtained from 50 individuals with symptoms of vaginal discharge.Bacterial isolates include 20 (43.4%) of E. coli, 8 (17.3%) of Klebsiella spp., and 8 (17.3%) of Staphylococcus spp.The E. coli, Staph-ylococcus spp., Enterobacter spp., E. faecalis, and R. ornithinolytica isolates were found resistant to several antibiotics and considered multidrug resistance (MDR) [6].The most common pathogens that cause vaginal discharge are Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium [19,20].
There is no data repository on AMR-related research on humans, animals, food, plants, and environment isolates in Eritrea, and, as such, it is difficult to estimate the national health and economic impact of AMR [22].The availability of updated epidemiological data on antimicrobial resistance in frequently encountered bacterial pathogens will be useful not only for deciding on treatment strategies but also for designing an effective antimicrobial stewardship program in hospitals.Therefore, this study was carried out to evaluate the bacterial pathogens involved in vaginal discharge and their antimicrobial susceptibility pattern in patients referred to the National Health Laboratory (NHL) Microbiology Department from 2019 to 2022.The study included the drugs listed on the Eritrean National List of Medicines (ENLM) that are available in the country according to the government policy corresponding to the Clinical Laboratory Standard Institute (CLSI) guidelines, in order to provide baseline information on sensitivity to these drugs.Furthermore, this study is expected to make a great contribution as it could be a baseline for further research and will help access the common cause of vaginal discharge and its pattern of antibiotic resistance, which may help to design a strategy for effective and proper use of antimicrobial drug use.To ensure a representative dataset, we used a census sampling approach, which included all available laboratory records of patients with complete data within the study period.This methodology was chosen to minimize selection bias and provide a comprehensive overview of antimicrobial resistance patterns in the study population.

Inclusion and Exclusion
Criteria.The inclusion criteria comprised all laboratory records of patients who underwent vaginal swab analysis during the study period and who had complete and nonduplicated information.Patients with incomplete or duplicate records were systematically excluded from the study.These criteria were established to maintain the integrity of the data and ensure the validity of the analysis.

Data Collection.
A specific data extraction tool was meticulously designed to retrieve essential information from the laboratory record.Skilled laboratory personnel, specifically trained for this study, were responsible for data collection to ensure data accuracy and reliability.Before full-scale data extraction, a pilot study was conducted to validate the data collection tool.This pilot study not only confirmed the effectiveness but also provided information on its refinement.Adjustments were made to the context and objectives of the study findings based on the pilot study.The data collection tool included critical variables, including the patient's age, the year of analysis, the isolated pathogen, and the resistance pattern exhibited by the isolated organism.Resistance patterns were classified according to the established criteria to ensure consistency in data interpretation.
2.6.Ethical Considerations.Ethical approval for this retrospective study was obtained from the Ministry of Health, State of Eritrea, Research Ethics Approval and Clearance Committee (no.: 15/02/2023).Throughout the data collection process, strict measures were implemented to protect patient confidentiality and informed consent procedures were followed as required by ethical guidelines.The genital specimen was inoculated on chocolate agar, MacConkey agar, mannitol salt, and Thayer-Martin and Sabouraud chloramphenicol agar plates.Gram staining was done on the original samples.The chocolate and Thayer-Martin plates were incubated in a jar with a CO 2 generation kit, and the MacConkey, mannitol salt, and Sabouraud chloramphenicol agar plates were incubated aerobically at 35-37 °C for 18-24 hours.Plates were examined after incubation based on their morphology, size, color, consistency, and number of colonies.Gram staining was performed to verify the predominant organisms and subgroup the colonies into gram-negative and gram-positive groups.
The appropriate biochemical tests were then performed to help identify the suspected bacteria, and the appropriate sensitivity tests were performed.Finally, with the help of the growth characteristic of each plate and/or with the result of biochemical tests (catalase tests, deoxyribonuclease (DNase test), triple sugar iron (TSI), Simmons citrate, urease test, tryptophan deamination (TDA), methyl red (MR), indole, amino acid decarboxylation tests, and carbohydrate fermentation tests) and with the help of the numerical identification of the reference book using the API 20E system [23], the possible identification results and the sensitivity result were stated and documented.2.7.2. Candida albicans and Yeast Detection.Candida albicans and yeast cells were obtained by cultivating suspected samples on Sabouraud chloramphenicol agar (SCA) and subsequently confirmed morphologically using filamentation test methods.
2.8.Quality Control.The strains E. coli ATCC 25922, S. aureus ATCC 25923, and E. faecalis ATCC 29212 were used as control organisms to check the performance of the media and discs.They were tested for quality control after each microbiological procedure, such as staining, antimicrobial susceptibility tests, and biochemical identification procedures once a month and before the use of a new batch of reagents and antimicrobials.
2.9.Data Analysis.The completeness of the collected data was further checked for completeness prior to data entry.Finally, the data were entered in MS Excel and further exported to SPSS version 25 for analysis.The frequency and percentage were determined.After collecting the results, some of the bacteria were grouped into their species for analysis as follows: Citrobacter spp.( was informed, and permission was sought from the authorities.Since these were secondary data, informed consent was not sought, but confidentiality of the patient's laboratory records was maintained secure and the patient's personal identification was coded and analyzed as aggregates.

Characteristics of Microbial Isolates from Vaginal
Discharge in National Health Laboratory.During the study period, 622 vaginal samples were cultured in the NHL, and 515/622 were infected with microorganisms, a positivity rate of 83%.Most of the patients were between 18 and 35 years old, 431 (61.9%) and 11 of the patients were under 6 years old, and 36% of the cases were investigated in 2022 (Table 1).

Antimicrobial Sensitivity Based on Specific Bacterial
Isolates from Vaginal Swab in National Health Laboratory (Gram-Negative Species).A total of 12 antibiotics including amikacin, cephalexin, ceftazidime, ceftriaxone, nalidixic acid, ampicillin, chloramphenicol, ciprofloxacin, trimethoprim-sulfamethoxazole, gentamicin, nitrofurantoin, and tetracycline were tested in gram-negative bacteria.The result showed that no Citrobacter spp. was resistant to nitrofurantoin and also no other gram-negative bacteria were resistant to amikacin.Citrobacter spp.recorded the highest resistance rate (>43%) with cephalexin, ceftazidime, nalidixic acid, ampicillin, gentamicin, and tetracycline.For E. coli, the highest resistance rates were recorded (>50%) with ampicillin, trimethoprim-sulfamethoxazole, and tetracycline.Furthermore, the lowest resistance rates (<9%) were recorded with amikacin, ceftazidime, chloramphenicol, and nitrofurantoin.In Klebsiella spp., the highest resistance rates (>47%) were recorded with ampicillin, nitrofurantoin, and tetracycline and the lowest resistance rates (<11%) for this strain were recorded with amikacin and ciprofloxacin.For Proteus spp., the resistance rates were more than 50% for almost all antibiotics tested.In the other gram-negative bacteria, high resistance rates (>57%) were recorded with cephalexin and ampicillin and the lowest resistance rates (<14%) were recorded with ceftriaxone, nalidixic acid, ciprofloxacin, and gentamicin.Of all isolated gram-negative bacteria, the highest resistance rates (>46%) were recorded with ampicillin and tetracycline.However, the lowest resistance rates (15%) with these bacteria were recorded with amikacin, ceftazidime, and ceftriaxone (Table 3 and Figure 1).

Antimicrobial Sensitivities of Vaginal Swab in National
Health Laboratory (Gram-Positive Species).A total of 13

Discussion
Combating antimicrobial resistance is crucial, especially in developing countries like Eritrea.This can add a burden to different preexisting challenges such as misuse of medications, availability, and higher level of substandard medications.Another study revealed that in developing countries, the high proportion of life-threatening bacterial infections, exacerbated by inadequate awareness, laboratory facilities, and human resources for health, is expected to worsen the impact of AMR.Similarly, different studies reported that self-medication, empirical therapy, misuse, and overuse of antimicrobials increase antimicrobial resistance and lead to prolonged illness, disability, increased health care costs, and death [24].Enhancing community awareness and health professionals can stop the growth of antimicrobial resistance.The microbial growth rate was 83%, and the bacteria that dominated the most were E. coli (51.2%) and S. aureus (15.9%).This was consistent with the previous study in which E. coli (43.4%),Klebsiella spp.(17.3%), and Staphylococcus spp.(17.3%) were the most common isolates [6].Another study showed that the most prevalent pathogen was E. coli, Klebsiella pneumoniae, Staphylococcus aureus, and Citrobacter spp.[25].Furthermore, a study conducted in Eritrea showed that the most common isolates included Citrobacter spp., Klebsiella spp., E. coli, Proteus spp., S. aureus, CONS, and Streptococcus viridans, although the sample and frequency order were different [21].However, it was inconsistent with a study that 22.7% of isolates were G. vaginalis, and the predominant aerobic bacteria were S. aureus (25.4%), S. agalactiae (14.1%), and E. coli (13.5%) [11].This study revealed that 11 of the patients were under six years old and six of these samples showed bacterial growth and antimicrobial susceptibility was done.Another study reported that a total of 99 samples were collected from children, of which 78 (30.4%) were culture positive [26].Clinically, different articles in the literature reported that vaginal discharge is not a common presentation in this age group and bacterial growth is not commonly expected [26].These children could have any predisposing factors such as immune suppressive diseases and malnutrition and need further investigation to determine the primary cause.
The antimicrobial susceptibility assay revealed a variable resistance pattern to penicillin G, ampicillin, trimethoprimsulfamethoxazole, tetracycline, and oxacillin.And most were 6 BioMed Research International sensitive to ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, nitrofurantoin, and rifampicin.This was inconsistent with a study in which clindamycin (40%), ampicillin (27%), vancomycin (26%), ciprofloxacin (18%), and nitrofurantoin (12%) showed the highest resistance to surgical site infections in Eritrea [21].This could be mainly due to the type of sample, and commonly used medications can differ for the indication of treating vaginal discharge, which can result in various patterns of drug resistance.Gram-negative bacteria such as Citrobacter spp., E. coli, Klebsiella spp., and Proteus spp.were resistant to ampicillin.Similarly, another study showed that E. coli was resistant to trimethoprim/sulfamethoxazole and ceftriaxone but susceptible to ciprofloxacin, gentamicin, and nitrofurantoin [11].Ampicillin is one of the antibiotics commonly prescribed for gram-positive and gram-negative bacteria that can develop resistance, but it is not among the medications commonly prescribed for patients with vaginal discharge.
Citrobacter spp.were resistant to ampicillin and cephalexin, which coincides with other studies showing that Citrobacter spp.isolates had >70% resistance to various agents, including tetracycline, cephalexin, ceftazidime, and ceftriaxone [21].This group of gram-negative bacteria is not among the common causes of vaginal discharge but was isolated in a previous study from surgical site infections in Eritrea [21].Klebsiella spp.were resistant to ampicillin, tetracycline, nitrofurantoin, and trimethoprim-sulfamethoxazole.Similarly, studies reported that Klebsiella spp.exhibited high resistance to ampicillin and trimethoprim-sulfamethoxazole [6] and isolates exhibited high resistance (>60%) to nitrofurantoin, cephalexin, ceftazidime, and ceftriaxone [21].This could be mainly due to the fact that these antibiotics are among the antibiotics commonly prescribed for these gram-negative bacteria for different diseases.
The Proteus species were resistant to tetracycline, ampicillin, trimethoprim-sulfamethoxazole, cephalexin, ceftazidime, nalidixic acid, nitrofurantoin, gentamicin, chloramphenicol, and ciprofloxacin.Proteus vulgaris showed a sensitivity to ciprofloxacin (87.0%).Diphtheroids showed sensitivity to ampicillin (95.7%) and ceftriaxone (91.3%) [27].These bacteria showed a high degree of antimicrobial resistance to most medications that could be a threat to the community and health professionals.
This study reported that most gram-positive bacteria were resistant to trimethoprim-sulfamethoxazole, tetracycline, ampicillin, oxacillin, vancomycin, and penicillin G. Similarly, another study reported that most gram-positive bacteria were resistant to erythromycin, trimethoprim/sulfamethoxazole, and amoxicillin [11].This high level of antimicrobial resistance of gram-positive bacteria will have an effect on the use of these antibiotics for the use of other infections.

BioMed Research International
Staphylococcus aureus was 100% sensitive to rifampicin and clindamycin and was highly resistant to penicillin G, trimethoprim-sulfamethoxazole, oxacillin, and tetracycline.This was consistent with other studies that S. aureus was highly resistant to tetracycline and trimethoprim-sulfamethoxazole [24] and S. aureus was resistant to erythromycin, trimethoprim/sulfamethoxazole, and amoxicillin.Furthermore, all S. aureus isolates were resistant to penicillin in surgical site infection samples [21], and Staphylococcus has a high level of resis-tance to oxacillin, benzylpenicillin, levofloxacin, nitrofurantoin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, and tetracycline [6].Streptococcus spp.showed 100% resistance to rifampicin and vancomycin.Furthermore, there is greater resistance to ampicillin, ciprofloxacin, chloramphenicol, tetracycline, clindamycin, erythromycin, and oxacillin.Although these gram-positive bacteria were not among the common causes of vaginal discharge, they had a high level of resistance that could reduce their use for other indications.[21,28,29].In our study, most species (Proteus species, Streptococcus spp., S. aureus, and Klebsiella spp.) show multidrug resistance.This result coincides with another study conducted on vaginal swabs in which E. coli, Staphylococcus spp., and Enterobacter spp.isolates were found to be resistant to several antibiotics and considered multidrug resistant [6].Similarly, another study conducted in Eritrea on surgical site infections revealed that E. coli, Klebsiella spp., and Citrobacter spp.isolates had multiple antimicrobial resistances [21].This higher rate of commonly used broad-spectrum antibiotics will be a threat to the community, physicians, and patients.
The study was not without limitations.This study showed that there were no isolates of common causes of vaginal discharge such as C. trachomatis and N. gonorrhoeae.This could be mainly due to the fact that these pathogens are fastidious and need special media, a specific collection, transport, and storage system; thus, they may not show growth in the culture medium.Furthermore, since it was a retrospective study, the background characteristics of the patients were not determined, which may show a specific association with antimicrobial resistance.Furthermore, culture and sensitivity for some bacteria (CONS) were not performed, and poor sample collection may also impact the growth of some pathogens.As this was a retrospective study, we did not follow the outcome of the patients.Furthermore,   9 BioMed Research International this result does not generalize antimicrobial resistance for the general population, and self-medication and empirical therapy could contribute to antimicrobial resistance.

Conclusion
Most isolates were sensitive to ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, nitrofurantoin, and rifampicin and resistant to ampicillin and penicillin.Most gramnegative bacteria such as Citrobacter spp., E. coli, Klebsiella spp., and Proteus spp.were resistant to ampicillin.Additionally, most Proteus spp.were resistant to cephalexin, ampicillin, trimethoprim-sulfamethoxazole, tetracycline, and nitrofurantoin.Furthermore, most of S. aureus were sensitive to clindamycin and rifampicin and resistant to penicillin G, while all species of Streptococcus were resistant to rifampicin and vancomycin.Streptococcus spp., Proteus species, Klebsiella spp., and Staphylococcus aureus showed multiple antibiotic resistances.

Recommendations
Enhancing community awareness of the misuse of medications, substandard drugs, and the burden of antimicrobial resistance is crucial to solve this growing concern.Furthermore, the wise use of medications and the prescription of antibiotic-based culture and sensitivity are cornerstones for the life use of antibiotics.Further prospective studies based on vaginal, urine, and blood cultures are highly recommended in conjunction with the Eritrean national action plan to combat antimicrobial resistance through the "One Health" approach, 2021-2025.

1 .
Collecting Clinical Samples and Identification of Pathogens.
COT GEN F TET CD ERY ox Rif Van PEN Resistant strain (%) Antibiotics S. aureus Strep spp.

Table 1 :
Characteristics of microbial isolates from vaginal discharge in the National Health Laboratory.

Table 2 :
Antimicrobial sensitivities to different antibiotics from vaginal swab isolates in the National Health Laboratory.

Table 3 :
Antimicrobial sensitivity of vaginal swab in National Health Laboratory (gram-negative species).

Table 4 :
Antimicrobial sensitivities of vaginal swab in National Health Laboratory (gram-positive species).

Table 5 :
Multidrug resistance of gram-negative and gram-positive isolates from vaginal discharge.