Trend Analysis of Multidrug-Resistant Bacterial Pathogens Causing Neonatal Sepsis at University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia: A Retrospective Study

Background Neonatal sepsis is the most common cause of mortality and morbidity. It is a major global public health challenge, particularly in developing countries. Therefore, knowing the current status of bacterial isolates and their antimicrobial resistance profile is essential to physicians and health workers to implement appropriate intervention. The aims of this study was to assess a ten-year trend of bacterial prevalence isolates from blood culture among neonates (<1 month of age). Method A hospital-based retrospective study was conducted on 1854 neonatal patients who were admitted at University of Gondar Specialized Comprehensive Hospital between 2010 and 2020. Sociodemographical and laboratory data were collected from medical records. Quality of the data was assured through standard operational procedures. Data were entered and analysed using SPSS version 20. Bivariate analysis was employed to determine strings of association between the outcome variable and sociodemographic variables. A P value less than 0.05 will be considered to be statically significant. Results In a total of 1854 patients, 538 (29%) were culture positive. The overall neonatal sepsis infection rate was 287 (53.5%) for male and 249 (46.5%) for female. The highest proportion of neonatal sepsis infection rate was observed among the patients in the age range between 3 and 28 days and gestational at birth <37 weeks, 461(86%) and 278 (52%), respectively. Gestational at birth (P ≤ 0.001, AOR = 5.81, CI: 4.63–7.29) is significantly associated with bacterial isolates. The predominant pathogens were Staphylococcus aureus, 18 (76.6%), Klebsiella pneumoniae, 146 (38%), and E. coli, 45 (11.7%) among the age range less than one weak. Klebsiella spp, S. aureus, and E. coli showed a high level of resistance to most tested antimicrobials. Amikacin, ciprofloxacin and norfloxacin, and erythromycin were the most effective antibiotics whereas ampicillin, amoxicillin, and cotrimoxazole were the least effective antibiotics for isolates. Conclusion Neonatal sepsis infection is common in the 3–28 days of age range. S. aureus, E. coli, and K. pneumonia were the most common isolates. Most the bacterial pathogens were resistant to commonly prescribed antibiotics. Therefore, an antimicrobial sensitivity test for bacterial isolates is recommended to provide updated data for the physician in choosing the appropriate antibiotic for better patient treatment outcome.


Background
Neonatal sepsis is defined as a systemic infection with bacteria, virus, and fungi with/without signs and symptoms of infection during the first 28 days of life. Sepsis can be categorizing as early onset (prior 72 hr in the life of the neonate) and late onset if the diagnosis was carried out after this deadline. Usually, early-onset neonatal sepsis is associated with perinatal background and late-onset neonatal sepsis is mainly related to the medical and surgical invasive procedures required by neonates who already have the disease. Early-onset sepsis remains a common and serious problem for neonates, especially preterm infants [1,2].
Neonatal sepsis is one of the major public health challenges. Based on different studies of different concerns, 41% (3.6 million) of all deaths in children under 5 years of age is due to bacterial infection. e majority of these deaths occur in low-income countries. Furthermore, neonatal mortality for different African countries ranges from 68 per 1000 live births in Liberia to 11 per 1000 live births in South Africa [3][4][5].
Existing published data have suggested that neonates are at the highest risk for bacterial sepsis, with the prevalence at 1 to 10 per 1000 live births worldwide [5]. e main bacterial pathogens of neonatal sepsis are L. monocytogenes, Streptococcus, E. faecalis, E. faecium, group D streptococci, α-hemolytic streptococci and staphylococci, S. pneumoniae, and H. influenzae type B, which are recognized as the principal cause of early neonatal sepsis. Among Gramnegative enteric organisms, predominantly, E. coli, Klebsiella species, and N. meningitides and N. gonorrhea have been also reported as a cause of neonatal septicemia [6,7]. Bacteria such as staphylococci species and E.coli are predominantly caused late-onset sepsis [7]. Based on the current study, the prevalence of neonatal sepsis is increased during recent years, and it may be due to the development of resistant organisms [5]. Drug resistance of pathogens is a major problem in causing neonatal sepsis, especially in developing countries because of poor sanitary conditions, over-thecounter sale of antibiotics, and lack of legislation [5,7]. Especially, MDR Gram-negative organisms, methicillin-resistant S. aureus (MRSA), and extended-spectrum betalactamase-(ESBL-) producing bacteria represent the principal setbacks to fight against infections. Most Gram-negative bacteria are now resistant to ampicillin and cloxacillin, and many are becoming resistant to gentamicin [7,8]. e prevalence of bacterial organisms causing neonatal sepsis is constantly increasing, especially in developing and low-income countries, and the emergence of resistant bacteria has complicated the problem further. erefore, this study aimed to assess ten-year trend of bacterial pathogens and their antimicrobial susceptibility pattern in neonatal patients with sepsis.

Study Design, Period, and Area.
e study was conducted at University of Gondar Specialized Comprehensive Referral Hospital. It is found in the historical town of Gondar located 750 km far from the northwest of Addis Ababa, the capital city of Ethiopia, and in the North Gondar Zone of the Amhara National Regional State which is a metropolitan city in the Amhara National Regional State in Ethiopia. e hospital was purposely selected because that it is one of the biggest referral hospitals in the region. Currently, it provides services to more than five million people in Gondar town and the surrounding area. e hospital has an inpatient and outpatient department and laboratory service with more than 500 beds that provides health services such as surgery, internal medicine, pathology, TB/HIV, dermatology, antenatal care, delivery, postnatal care, laboratory, pharmacy, maternal and neonatal care, and other services for the population of Gondar town and surrounding areas [9].

Study Design, Period, and Data Collection.
A retrospective cross-sectional study to assess the prevalence and antimicrobial resistance profile of neonatal septicemia was conducted from 2010 to 2020 at University of Gondar Specialized Comprehensive Referral Hospital, Gondar. Data records of 1854 neonatal patients were collected and reviewed using a checklist from medical microbiology laboratory registration logbooks. Sociodemographical data, laboratory test results, and hospitalization status were collected using a data extraction checklist.

Sample Collection and
Processing. Two bottles of venous blood samples (1 ml for each bottle) from two different sites of the peripheral vein were collected aseptically by experienced nurses before any antibiotic use. e collected blood sample was inoculated into a Trypton soya broth blood culture medium pair of bottles (Becton, Dickinson, USA) under strict aseptic procedures [10]. All culture bottles were incubated aerobically at 37°C for seven days for the presence of visible microbial growth. Positive culture bottles were subcultured; subcultures were made during successive days on enriched and selective media including blood agar plate (Oxoid LTD), chocolate agar plate (incubated at 5% CO 2 atmosphere), MacConkey (Oxoid Ltd., Basingstoke, Hampshire, UK), and mannitol salt agar plates and examined for growth after 24-48 h of incubation. All positive blood cultures were identified using their cultural characteristics, appearance on their respective media, and Gram staining reaction. Bacterial isolates were confirmed by the pattern of standard biochemical reactions. Gram-negative bacteria were identified by an oxidase test, indole production, citrate utilization, motility, urease, oxidase, gas production, hydrogen sulfide production, carbohydrate fermentation, and lysine decarboxylases production tests [11]. Identification of Gram-positive bacteria isolates: coagulase, catalase test and mannitol salt agar, bacitracin (0.04 or 0.05 UI), and optochin (5 μg) susceptibility tests were conducted. Interpretation was made as sensitive if the zone diameter was at least 10 mm and resistant if the zone of diameter was less than 10 mm. Blood culture broth which shows no microbial growth within 7 days was reported as culture negative, only after results were subcultured on blood agar, Mac Conkey, and chocolate agar [11].

Antimicrobial Susceptibility
Tests. An antimicrobial susceptibility test was carried out for each identified bacterial isolates by using the disc diffusion method on Muller Hinton agar for nonfastidious organisms and Muller Hinton agar with 5% defibrinated sterile sheep blood for fastidious organisms [12]. ree to five pure colonies were emulsifies in 2 ml of nutrient broth. e bacterial suspensions' turbidity was matched and checked with 0.5 McFarland standards. en, a sterile cotton swab was dipped into the suspension and squeezed free from excess fluid against the side of test tube. e test organisms were uniformly seeded on the surface of Muller Hinton agar and exposed to a concentration gradient of antibiotic diffusing from the antibiotic-impregnated paper disc into the agar medium, and the medium was incubated at 35°C for 18-24 h [13]. e following antimicrobial disks were used

Quality Control.
All media were prepared according to the manufacturer's instruction and following the standard operational procedure. Culture media were checked for sterility by incubating 5% batch of the media at 37 o c for 24 hours, and performance of all prepared media was also checked by inoculating international standard-strains such as E.coli (ATCC 25922) for Gram-negative bacteria, S. aureus (ATCC 25923) for Gram-positive bacteria, and N. Gonorrhoeae (ATCC49226) for fastidious bacteria. To standardize the density of the inoculum of bacterial suspension for the susceptibility test, 0.5 McFarland standards were used.

Data Management and Statistical
Analysis. Data were entered and analysed by using SPSS version 20 software. e characteristics of the study population were summarized using frequencies, percentages, median, and standard deviation. Text descriptions, tables, and figures were used to describe or present the findings of the study. A chi-square test was used to compare the proportion of bacterial isolates with patients' age and year. A P value of <0.05 was considered statistically significant.

Demographic Characteristics of Study Participants.
Within the ten years, 1854 neonatal patients' data were collected and analysed at University of Gondar Specialized Comprehensive Referral Hospital. e majority, 995(53.7%), of the study participants were males. Out of the total, 1531(82.6%) of the study participants were 3-28 days old. e majority, 1425(76.9%), of neonates come from the NICU ward; of them, 27.8% were culture positive. e majority of bacterial isolates were observed between 2017 and 2019 (Table 1).

Antibiotic Susceptibility Patterns of Bacterial Isolates from
Neonatal Septicemia. Gram-positive bacterial species are also common causative agents for neonatal sepsis infection, significantly showing a high level of resistance to different antimicrobial agents. For instance, in this study, S. aureus and CoNs were the most predominantly isolated Grampositive bacteria and had a great extent of resistance to ampicillin (83%), amoxicillin (87%), trimethoprim-sulfamethoxazole (81%), and norfloxacin (80%). On the contrary, amikacin (100%) and vancomycin (86%) were active antimicrobial agents against S. aureus and CoNs. On other hand, S. pyogenes were highly resistant to amoxicillin (100%) ( Table 4).
Gram-negative bacterial species resistance to commonly used antimicrobials is alarmingly increasing. Klebsiella spp. and E. coli showed a high level of resistance to most tested antimicrobials. Ampicillin, amoxicillin, gentamycin, and International Journal of Microbiology ceftriaxone were among the tested antimicrobials resisted by K. pneumoniae with the resistance rate of 100%, 96%, 72%, and 90%, respectively. E. coli also showed a significant level of resistance to ampicillin (96%), and ceftriaxone (67%). However, antimicrobials such as kanamycin (89%) and norfloxacin (67%) had a good inhibition effect against K. pneumoniae (Table 5).

Multidrug-Resistant Isolates.
Out of the total numbers of bacterial isolates, 76% were resistant to three or more different classes of antibiotics. Among multidrug resistant (MDR), 103(25.4%), 69(17%), and 163(40%) isolates were resistant to three classes of antibiotics, greater than four and five antibiotics, respectively. Results of drug resistance patterns compared within species specific showed that 80.4% of S. aureus, 40% of K. pneumoniae, and 10.2% of E. coli were MDR isolates (Table 6).

Discussion
Neonatal septicemia, a life-threatening worldwide disease, has become a disease of neonates less than one month of age with a significant mortality rate. It has to be reviewed periodically because of specific microorganisms responsible for infection varying with time, geography, and patient age. Out of the total culture, positive rate of bacterial isolates identified from patients with symptoms of neonatal septicemia was 29%, and this prevalence rate is higher than that reported from Nepal (12.6% and 10.8%) and Botswana (9.8%) [14][15][16]. But, it is lower than that of a study conducted in Egypt (40.7%), Sudan (61.3%), Yemen (57%), and in the Oromia region, southeast Ethiopia (34%) [17][18][19][20]. e variation could be due to fact that methodological variation, antibiotic administration before sample collection, control mechanisms in spread of bacterial infection, and difference in study setting might affect blood culture positivity rate.
In the present study, the prevalence of bacterial isolates was higher in males, 53.5%, than in females, 46.5%, which was comparable with the findings of previous studies where the prevalence of neonatal septicemia was higher in males (66%) than in females (33.9%) [16]. is might be due to the physiological and immunological makeup of nature and cultural practices in different societies. e prevalence of neonatal septicemia was higher in the preterm (<37 weeks) than in gestational age >37 weeks (term). Similarly, studies in Ethiopia, Mexico, and Indonesia have also showed that preterm (<37 week) had a significant association with neonatal sepsis [2,7,21,22]. is could be because a baby less than 37 weeks of age may have a limited capacity to increase neutrophil production in accordance to demand to overcome. In the current study, the predominant            Similarly, previous studies report that those isolates were also the major pathogens of neonatal septicemia [7,18,23]. In our study, amikacin, ciprofloxacin, and norfloxacin were the most effective drugs for Gram-negative isolates and vancomycin and ciprofloxacin were for Gram-positive isolates which correlates with the study conducted in Ethiopia [7]. S. aureus and CoNs showed the highest susceptibility towards vancomycin and ciprofloxacin. Amikacin showed 89% sensitivity towards K. pneumoniae isolates, similar to the findings of Muley et al. [24].
In this study, MDR was observed in S. aureus (80.4%), K. pneumoniae (40.5%), and E. coli (10.2%). Similarly, previous studies report that those MDR pathogens were the most common causative agents of neonatal septicemia [7,25]. S. aureus and K. pneumoniae were common pathogens that showed MDR due to genetic competence to acquire antibiotic resistance genes from other strains, intrinsic resistance mechanisms, and chromosomal and plasmid-encoded betalactam hydrolyzing enzymes [7,26,27].

Conclusions and Recommendations
e prevalence of septicemia among neonatal patients was 29%. Gestational age at birth <37 weeks (preterm) is a highrisk factor for neonatal septicemia than gestational age at birth >37 weeks (term), and it is significantly associated with bacterial isolates (P ≤ 0.001, AOR � 5.81, CI: 4.63-7.29). Also, most of the bacterial pathogens were resistant to commonly prescribed antibiotics. erefore, infection-preventive measures are required with a particular focus on neonatal patients. Further research is needed to explore the epidemiology and risk factors of neonatal septicemia.

Data Availability
Data and supporting materials associated with this study will be shared upon request.

Ethical Approval
e study was conducted after obtaining ethical clearance from the University of Gondar School of Biomedical and Laboratory Sciences ethical review committee. e collected data were used only for the study objective. e information obtained at any course of the study was confidential. Additionally, a permission letter from the hospital laboratory directorate was obtained before the data collection was performed.

Conflicts of Interest
All authors declare no conflicts of interest. International Journal of Microbiology 7