Microbiological Characteristics and Resistance Patterns in a Neonatal Intensive Care Unit: A Retrospective Surveillance Study

Background and objective: This study aims to assess the prevalence and antimicrobial susceptibility patterns of bacterial infections associated with both early-onset sepsis (EOS) and late-onset sepsis (LOS). Methodology: This descriptive retrospective surveillance research was conducted on all neonates admitted to the neonatal ICU with bacterial sepsis, where positive cultures were isolated from sterile sites (either cerebrospinal fluid or blood) at Tawam Hospital, Al Ain, Emirate of Abu Dhabi, UAE, from January 2012 and December 2021. Antimicrobial susceptibility analysis was performed. Results: The incidence of LOS (94.43%) was higher compared to EOS (5.56%). The most prevalent isolates (59.2%) were gram-positive bacteria, with gram-negative bacteria accounting for 40.8%. The leading isolates included coagulase-negative Staphylococci (CONS, 40.98%), Klebsiella (16.04%), Staphylococcus aureus (8.46%), Escherichia coli (8.24%), Pseudomonas (7.57%), and Group B Streptococcus (GBS, 5.12%). CONS were predominant in LOS cases (42.9%), while GBS was the main pathogen in EOS cases (44%). Conclusions: We observed reduced resistance levels of CONS against ampicillin, benzylpenicillin, clindamycin, erythromycin, fusidic acid, gentamicin, oxacillin, rifampicin, and trimethoprim/sulfa. S. aureus exhibited increased resistance to erythromycin, fusidic acid, gentamicin, and levofloxacin, while E. coli demonstrated decreased resistance against cephalothin, gentamicin, and trimethoprim/sulfa. The antibiotics currently employed empirically appear to provide adequate coverage against the most prevalent bacteria causing early- and late-onset neonatal infections.


Introduction
Globally, neonatal sepsis is a significant contributor to both morbidity and mortality in neonatal intensive care units (NICUs) [1].Neonatal sepsis comprises a cluster of clinical presentations that arise from inflammatory reactions triggered by systemic infections, including but not limited to meningitis, septicemia, pneumonia, and bone-related diseases [2].About 30%-50% of neonatal deaths in developing nations are caused by sepsis.Despite recent advancements in healthcare, the increasing number of neonatal deaths continues to be mostly attributed to delayed diagnosis and inappropriate treatment.Nevertheless, these issues can be prevented with the use of enhanced adjuvant care and proper antimicrobial selection [3].Despite blood culture being widely regarded as the definitive method for neonatal sepsis, the relatively low frequencies of positive results pose a significant management obstacle [4].
Neonatal sepsis is categorized based on the time at which it manifests: early-onset sepsis (EOS) occurs before 72 hours of age, and late-onset sepsis (LOS) occurs after 72 hours of age.This classification has effects on the suspected organism, potential risk factors, and proposed empirical therapy [5].Neonatal sepsis is predominantly induced by various species of gram-positive and gram-negative bacteria, with sporadic instances including fungi such as Candida species [6].
As a first-line therapy for the management of suspected neonatal infections, the World Health Organization (WHO) guidelines advise empirical treatment with a combination of ampicillin and gentamicin.In cases where drug-susceptibility testing of bacterial isolates indicates resistance to first-line therapy or when nonresponders develop resistance to first-line therapy, a third-generation cephalosporin is recommended as a second-line therapy.In addition, empirical treatment of EOS with a combination of ampicillin and gentamicin is advised as the initial line of therapy.Diverse empirical therapy suggestions have been put forth for LOS, involving vancomycin paired with gentamicin for nosocomial LOS, ampicillin combined with gentamicin for LOS, and piperacillin-tazobactam for both EOS and LOS [7].
Conversely, the expansion of multi-drug-resistant organisms (MDROs) impedes the development of effective treatments and diminishes the range of adequate antibiotics; thus, institutional guidelines based on the prevalence of local microbes and their sensitivity patterns to antibiotics are required [8].The scope and resistance of virulent microorganisms have changed year after year due to the broad application of antimicrobial medications [9].A global threat has emerged in the form of antibiotic resistance.In developing nations, there are increased reports, especially in ICUs, about multi-drug-resistant (MDR) bacteria causing neonatal sepsis.Antibiotic misuse and unnecessary usage, especially of broad-spectrum antibiotics, have been well-established as major contributors to the expansion of drug-resistant strains [10].
Therefore, this study aimed to evaluate the prevalence and antimicrobial-susceptibility patterns of bacterial infections associated with EOS and LOS in our NICU.

Materials And Methods
This retrospective surveillance study was conducted in the NICU at Tawam Hospital.The ethical committee of the institutional review board approved the study.Data were acquired from the microbiological database, and detailed records of all newborns admitted to our unit from January 2012 and December 2021 who developed bacterial sepsis with a positive culture isolated from a sterile site (either blood or cerebrospinal fluid [CSF]), with antimicrobial susceptibility analysis performed.Neonates with an isolated positive culture from a nonsterile site were excluded from this study.For patients with persistent positive cultures, we considered only the first positive culture in our research.Neonatal sepsis was classified into two groups, namely EOS, which occurs 72 hours after birth, and LOS, which takes place after 72 hours of birth [11].

Diagnostic and microbiological criteria
Upon admission to the NICU, newborns underwent peripheral blood cultures if there were indications of suspected sepsis based on their clinical condition or medical history.When a blood culture using a yellow top (pediatric aerobic) tube produced a positive result, the cultures were repeated until achieving a negative result.CSF cultures were obtained if the patient's condition indicated the potential presence of meningitis.
A bacteremia/meningitis episode was defined by the presence of a single positive blood culture and/or CSF culture, according to criteria modified by the Centers for Disease Control and Prevention.However, this requirement was waived in the case of known commensal bacteria, in which case at least two positive cultures were needed, spaced no more than one calendar day apart [12].Positive cultures of the same bacteria that were repeated within a time frame of less than 14 days were considered to be a single episode.A positive CSF culture was regarded as indicative of an episode, irrespective of a negative blood culture, except for the bacteria mentioned beforehand and in cases involving ventriculostomy or open neural tube malformations.
Our unit's empirical antibiotic treatment regimens were Penicillin G and gentamicin for early-onset bacteremia or flucloxacillin and gentamicin for late-onset bacteremia.Vancomycin was administered in cases where bacterial sensitivity recommended its use when clusters of gram-positive cocci were seen before the definitive diagnosis of the organism or where there was an elevated likelihood of central line infection.In cases when there is a high level of suspicion or confirmation of meningitis, a third-generation cephalosporin was provided to ensure adequate coverage.
Regarding the antibiotic sensitivity test, the antibiotics used for gram-negative bacilli included amikacin, augmentin, aztreonam, cefepime, cefotaxime/ceftazidime, cefuroxime, cephalothin, ciprofloxacin, gentamicin, imipenem/meropenem, levofloxacin, piperacillin/tazobactam, tobramycin, and trimethoprim/sulfamethoxazole.The gram-positive bacteria antibiotics used included ampicillin, benzylpenicillin, clindamycin, erythromycin, fusidic acid, gentamicin, levofloxacin, linezolid, oxacillin, rifampicin, tobramycin, trimethoprim/sulfamethoxazole, and vancomycin.All discs were obtained from Oxoid, England.Interpretation of the inhibition zones associated with different antibiotics was conducted in accordance with Clinical and Laboratory Standards Institute (CLSI) recommendations [2].MDR bacteria can be identified as bacteria that exhibit resistance to a minimum of one agent from three or more of the evaluated antimicrobial classes [13].

Statistical analysis
Statistical analysis was done using IBM SPSS Statistics for Windows, Version 28 (IBM Corp., Armonk, NY).Qualitative variables were presented as frequency and percentage (%) and analyzed using the chi-square test or Fisher's exact test when appropriate.A two-tailed P-value < 0.05 was considered statistically significant.

Results
A total of 449 septic episodes with positive blood or CSF cultures fulfilled our inclusion criteria during the specified period, among which 25 (5.56%) were EOS episodes and 424 (94.43%) were LOS episodes.Table 1 shows the incidence of EOS and LOS episodes throughout the 10-year period, where we found that the incidence of LOS was higher compared to EOS.The antibiotic resistance patterns of the predominant gram-positive bacteria from 2012 to 2021 are outlined in Table 3. CONS exhibited high resistance levels to ampicillin, penicillin, oxacillin, gentamicin, erythromycin, and fusidic acid, and relatively lower resistance to vancomycin, rifampicin, and cotrimoxazole.S. aureus demonstrated the highest resistance to penicillin and ampicillin, followed by erythromycin, with no observed resistance to vancomycin and lower resistance to rifampicin and clindamycin.Enterococci displayed the highest resistance to erythromycin, followed by gentamicin, with no resistance to ampicillin, and lower resistance to vancomycin and penicillin.GBS showed the highest resistance to erythromycin, lower resistance to penicillin, and no resistance to vancomycin and ampicillin.Other streptococci did not exhibit resistance to any of the antibiotics used.Data were presented as frequency (%).

GBS, Group B Streptococcus; CONS, coagulase-negative staphylococci
The most prevalent gram-positive isolates during the study period, spanning from 2012 to 2021, were CONS and S. aureus.We observed a significant decrease in resistance levels of CONS against ampicillin, benzylpenicillin, clindamycin, erythromycin, fusidic acid, gentamicin, oxacillin, rifampicin, and trimethoprim/sulfamethoxazole (P < 0.05).Furthermore, CONS did not develop resistance to levofloxacin, linezolid, tobramycin, and vancomycin during the same period.
In contrast, S. aureus exhibited a significant increase in resistance levels against erythromycin, fusidic acid, gentamicin, and levofloxacin (P < 0.05).Additionally, there were insignificant increases in resistance against ampicillin, benzylpenicillin, clindamycin, and oxacillin, with constant resistance against trimethoprim/sulfamethoxazole observed from 2012 to 2021.Notably, S. aureus did not develop resistance to linezolid, rifampicin, tobramycin, and vancomycin during the same period (Table 4).Data were presented as frequency (%).

GBS, Group B streptococcus; CONS, coagulase-negative staphylococci
Table 5 illustrates the antibiotic resistance patterns of the predominant gram-negative bacteria from 2012 to 2021.E. coli displayed a high level of resistance against co-trimoxazole, followed by cefuroxime, cefotaxime/ceftazidime, cefepime, and gentamicin, with a lower level of resistance against piperacillin/tazobactam and no resistance against meropenem.Klebsiella exhibited the highest resistance to cefuroxime, followed by cefotaxime/ceftazidime, with lower resistance observed against gentamicin and piperacillin/tazobactam, and no resistance against meropenem.Pseudomonas demonstrated a low level of resistance to the antibiotics used, with no resistance against gentamicin and meropenem.Acinetobacter showed the highest level of resistance against cefuroxime only, with no resistance against other antibiotics.Data were presented as frequency (%).
Less common gram-negative rods, such as Haemophilus influenzae (which developed resistance against cotrimoxazole only), Morganella morganii, and Serratia, exhibited high resistance against cefotaxime/ceftazidime, cefepime, and cefuroxime.Enterobacter displayed a high level of resistance against amoxicillin/clavulanate, and cefuroxime, followed by cefotaxime/ceftazidime and cefepime.Citrobacter showed resistance against cefuroxime only.In conclusion, meropenem was identified as the most effective antibiotic against the isolated gram-negative rods.
The most prevalent gram-negative isolates during the study period from 2012 to 2021 were Klebsiella, E. coli, and Pseudomonas, respectively.We observed that E. coli exhibited significantly decreased levels of resistance against cephalothin, gentamicin, and trimethoprim/sulfamethoxazole (P = 0.010, 0.003, and 0.001, respectively).Additionally, E. coli showed insignificant changes in resistance levels against augmentin, cefepime, cefotaxime/ceftazidime, and cefuroxime and did not develop resistance to amikacin, aztreonam, ciprofloxacin, imipenem/meropenem, levofloxacin, piperacillin/tazobactam, and tobramycin.

Discussion
Neonates, being immunocompromised individuals, are susceptible to infections that can result in significant morbidity and mortality.The American Academy of Pediatrics recommends the initial treatment of neonatal sepsis and meningitis with a combination of ampicillin and gentamicin.Our study revealed that 13% of early-onset gram-negative organisms were resistant to gentamicin, compared to 15% resistance to cefotaxime, 7% to amikacin, and 10% to meropenem [14].Due to the rapid evolution of bacterial resistance to β-lactam antibiotics, meropenem is typically reserved for treating the most severe and resistant infections [15].The most common organism causing early-onset disease was GBS (29.2%, 0.38/1,000 live births), while CONS were the most prevalent in late-onset disease (51%) [14].
Regarding the resistance of gram-positive cocci organisms to antibiotics, we found that CONS showed a high level of resistance against ampicillin, penicillin, oxacillin, gentamicin, erythromycin, and fusidic acid and less resistance against vancomycin, rifampicin, and co-trimoxazole.S. aureus showed the highest resistance to penicillin, followed by ampicillin and erythromycin.E. coli exhibited a high level of resistance against cotrimoxazole, followed by cefuroxime, cefotaxime/ceftazidime, cefepime, and gentamicin, with less resistance against piperacillin/tazobactam.Klebsiella displayed the highest resistance to cefuroxime, followed by cefotaxime/ceftazidime, with less resistance observed against gentamicin and piperacillin/tazobactam. Pseudomonas showed a low level of resistance to the used antibiotics, with no resistance against gentamicin and meropenem.In conclusion, meropenem was identified as the most effective antibiotic against the isolated gram-negative rods.
Sorsa et al. [21] reported the identification of bacteria in 88 (29.4%) of the investigated blood cultures.The most frequently found bacteria were S. aureus (16, 18%), CONS (22, 25%), and E. coli (E.Coli) 18 (20.5%).The respective resistance rates of CONS and S. aureus to ampicillin were 20 (91%) and 11 (69%).The prevalence of resistance among E. coli to gentamycin and ampicillin was 10 (55.6%) and 12 (66.7%),respectively.In contrast, the resistance rates of Klebsiella spp. to these two first-line antibiotics were considerably higher (10, 91%, and 9, 82%, respectively).Similarly, gram-negative and gram-positive bacterial isolates exhibited significant resistance to third-generation cephalosporins, with 63 (72%) of the isolated bacteria demonstrating resistance to multiple drugs.On the other hand, the susceptibility patterns of gram-positive bacteria isolates to third-line antibiotics such as clindamycin, ciprofloxacin, and vancomycin were superior to those of gram-negative isolates to amikacin and ciprofloxacin.
Enterobacter, as studied by Almohammady et al. [2], exhibited 100% susceptibility to levofloxacin, 67% susceptibility to amikacin and ciprofloxacin, and 100% resistance to imipenem.Sharma et al. [22] reported 100% susceptibility of Enterobacter to levofloxacin, gentamicin, and imipenem, with 25% susceptibility to amikacin and ciprofloxacin.Al-Matary et al. [23] found Enterobacter to be 100% sensitive to amikacin and 83% sensitive to gentamycin and cefepime.These variations among studies may stem from differences in antibiotic preferences in different communities, influencing distinct patterns of antibiotic resistance development.
Bromiker et al. [14] demonstrated resistance patterns of staphylococci and gram-negative organisms to key antibiotics.CONS showed 73% resistance to methicillin, with no resistance to vancomycin.Most grampositive organisms, excluding staphylococci, were susceptible to ampicillin, and all were susceptible to vancomycin.For gram-negative isolates, 74% were resistant to ampicillin.E. coli exhibited a low susceptibility (38%) to ampicillin, while 83% of isolates were susceptible to gentamicin.Pseudomonas species showed resistance to gentamicin (36%), mezlocillin, and meropenem, with lower resistance to ceftazidime (18%) and amikacin (9%).Overall, there was a trend of increasing resistance of nosocomially acquired gram-negative organisms to β-lactam and aminoglycoside antibiotics compared to vertically acquired ones, with statistical significance observed only for ampicillin and mezlocillin (P < 0.05 and P < 0.01, respectively).Fahmey [24] reported that of 673 screened neonates, 138 (20.5%) had positive blood cultures, confirming early-onset neonatal sepsis (EONS).Among recovered isolates, 86.2% were gramnegative pathogens, with Klebsiella pneumoniae (42.8%),Enterobacter cloacae (22.5%), and E. coli (13.8%) being the most common.The most frequent gram-positive microorganism was S. aureus, constituting only 8.7% of isolates.All Klebsiella isolates and 93% of Enterobacter isolates were resistant to ampicillin.Gramnegative pathogens displayed maximum overall sensitivity to imipenem, cefepime, and ciprofloxacin, while gram-positive isolates were most susceptible to vancomycin, imipenem, and piperacillin.Species were generally resistant to ampicillin.Shahian et al. [25] found that approximately 33% of Klebsiella isolates were sensitive to ampicillin, while high susceptibility to imipenem (98%) and ciprofloxacin (96%) was reported by Macharashvili et al. [26].Due to the rapid evolution of bacterial resistance to β-lactam antibiotics, imipenem is often reserved for treating the most severe and resistant infections [14].Moreover, prior use of antibacterial drugs, particularly cephalosporins, ampicillin, and gentamicin, along with prolonged exposure, is associated with a high prevalence of MDR bacteria [27].MDR bacteria are not confined to hospitals; they are also prevalent in the community environment, particularly in the Middle East, due to the excessive use and over-the-counter availability of antibiotics [28].
We observed that CONS exhibited significantly decreased levels of resistance against ampicillin, benzylpenicillin, clindamycin, erythromycin, fusidic acid, gentamicin, oxacillin, rifampicin, and trimethoprim/sulfa.Conversely, S. aureus displayed significantly increased levels of resistance against erythromycin, fusidic acid, gentamicin, and levofloxacin.E. coli demonstrated significantly decreased levels of resistance against cephalothin, gentamicin, and trimethoprim/sulfa.Both Klebsiella and Pseudomonas showed insignificant changes in resistance levels against all tested antibiotics from 2018 to 2021 compared to 2012-2017.
In a study by Tessema et al. [29], E. coli, the predominant gram-negative bacteria, exhibited the highest overall proportion of resistance to ampicillin, followed by ampicillin-sulbactam and piperacillin during the study period.Furthermore, E. coli displayed rising trends of resistance against ampicillin, ampicillinsulbactam, piperacillin, and cefuroxime during the study period.However, it is noteworthy that E. coli demonstrated significantly declining trends of resistance to ciprofloxacin and levofloxacin over the years.Additionally, all E. coli isolates were susceptible to gentamicin, amikacin, colistin, fosfomycin, imipenem, piperacillin-tazobactam, and tobramycin, consistent with previous research [30].
an interest in the submitted work.Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.

Table 7
depicts the trend of resistant microorganisms over the 10 years from 2012 to 2021.The detection of methicillin-resistant Staphylococcus aureus (MRSA) decreased in the last three years, with four identifications in 2019 and only one in 2021.Pseudomonas with MDROs was not isolated in the previous four years of our study.There was a significant increase in the isolation of Enterobacteriaceae resistant to third-generation cephalosporin or extended-spectrum beta-lactamase (ESBL) in the last year, 2021, accounting for seven cases (15.91%).Enterobacteriaceae resistant to carbapenem were not reported throughout the study period.