Neonatal sepsis is one of the leading causes of neonatal morbidity and mortality worldwide, particularly in developing countries, where access to timely diagnosis and optimal neonatal care remains limited [1]. Despite substantial improvements in perinatal care and advances in neonatal intensive care practices, sepsis continues to be a major contributor to neonatal deaths globally [2]. In India, neonatal sepsis represents a significant public health concern and accounts for a considerable proportion of neonatal mortality and long-term morbidity [3]. The burden of neonatal sepsis is particularly high in neonatal intensive care units (NICUs), where critically ill neonates are exposed to multiple invasive procedures, prolonged hospital stays, and broad-spectrum antibiotic therapy [4]. Over recent years, the emergence and rapid spread of multidrug-resistant (MDR) organisms have further complicated the management of neonatal sepsis, resulting in limited therapeutic options and poorer clinical outcomes [5]. Multidrug-resistant gram-negative organisms, in particular, have been associated with increased mortality, prolonged hospitalization, and higher healthcare costs [6]. Early diagnosis and prompt initiation of appropriate antimicrobial therapy remain the cornerstones of effective management of neonatal sepsis [7]. However, the choice of empirical antibiotics is often challenging due to changing microbial patterns and increasing antimicrobial resistance. The bacteriological profile of neonatal sepsis is known to vary widely across different geographical regions [8], between healthcare institutions [9], and even within the same center over time [10]. Knowledge of the local microbial flora and their antibiotic susceptibility patterns is therefore essential for formulating effective empirical antibiotic policies [11,12]. Regular surveillance of pathogens through NICU-specific antibiograms plays a crucial role in optimizing antibiotic use, preventing inappropriate therapy, and curbing the emergence of antimicrobial resistance [13,14].

The bacteriological profile and antibiotic susceptibility patterns of neonatal sepsis are known to differ across geographical regions and healthcare settings and continue to evolve over time. However, data from several regions of India, especially from certain parts of Rajasthan, remain limited or underreported. Generating region-specific data is essential, as empirical antibiotic policies based on data from other regions may not accurately reflect the local resistance patterns and may lead to inappropriate antibiotic use. In this context, the present study was conducted to determine the bacteriological profile and antibiotic susceptibility pattern among culture-proven neonatal sepsis cases admitted to the NICU of a tertiary care hospital.

MATERIALS AND METHODS

This prospective observational study was conducted in the Neonatal Intensive Care Unit (NICU) of a tertiary care hospital attached with Govt Medical College, Kota, Rajasthan, over a period of 12 months from July 2024 to June 2025. All neonates admitted to the NICU during the study period who exhibited clinical features suggestive of sepsis were assessed for eligibility. A total of 243 neonates with clinical suspicion of sepsis were enrolled in the study by consecutive sampling method. Neonates were included in the study if they had clinical signs and symptoms suggestive of sepsis and if appropriate culture samples were obtained prior to the initiation of antibiotic therapy. Neonates were excluded if informed consent was not obtained, antibiotics had been started before sample collection, had inborn errors of metabolism, discharged or expired before the availability of culture reports, or had major congenital malformations.

Approval from the Institutional Ethics Committee was obtained before the commencement of the study. Prior written informed consent was taken from the parents or legal guardians. Detailed demographic and clinical characteristics of each enrolled neonate, including gestational age, birth weight, sex and mode of delivery were recorded at the time of admission using a predesigned and structured proforma. Specimen collection was performed based on the clinical presentation and suspected focus of infection. Samples included blood cultures and, where clinically indicated, endotracheal tip or secretion samples, urine, cerebrospinal fluid (CSF), and umbilical tip specimens. Samples were processed using standard microbiological techniques (blood samples were inoculated into pediatric blood culture bottles and incubated in the BACTEC automated blood culture system and other samples were cultured on BLOOD AGAR and MACCONKEY AGAR. Organisms were identified based on standard microbiological techniques including colony morphology, Gram staining, and relevant biochemical tests and antibiotic susceptibility testing was performed using the Kirby–Bauer disk diffusion method, and results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) 2023 guidelines [13].

Clinical details, microbiological findings, and outcomes variables were entered into the predesigned proforma and subsequently compiled for analysis. Outcome variables were analyzed in terms of Discharged successfully, mortality and need of mechanical ventilation.  Data were analyzed using appropriate statistical software (SPSS 24). Categorical variables such as demographic characteristics, bacteriological profile, antibiotic susceptibility patterns, and clinical outcomes were described in number and percentages. Associations of outcome measures were analyzed using the Chi-square test. A p-value of less than 0.05 was considered statistically significant.

RESULTS

Total 243 suspected cases of sepsis were enrolled out of 8245 admitted neonates during the study. 186 cases (76.5%) had found culture positive sepsis. We found male predominance (141, 58%). 173 (71.1%) neonates had less than 2.5 kg weight while 157 cases (64.6 %) were preterm (Gestational age < 37 week). Demographic profile of the enrolled neonates is demonstrated in Table 1.

Table 1: Demographic profile of the enrolled neonates

Among the 186 culture-positive neonatal sepsis cases, Klebsiella pneumoniae was the most commonly isolated organism (60, 32.3%). This was followed by Acinetobacter species (39, 21.0%), and Escherichia coli (36, 19.4%). The detail of isolated organisms is illustrated in Table 2 and Figure 1.

Table 2: Bacteriological profile of isolated organisms

The antibiotic susceptibility pattern of the common isolates revealed a high level of resistance to frequently used antibiotics. Klebsiella pneumoniae showed resistance to ampicillin and cephalosporins, with variable sensitivity to aminoglycosides and reduced sensitivity to carbapenems. Acinetobacter species demonstrated sensitivity to ampicillin but were resistant to cephalosporins, exhibited variable susceptibility to aminoglycosides, and showed reduced sensitivity to carbapenems. Escherichia coli isolates were resistant to ampicillin and cephalosporins, showed variable sensitivity to aminoglycosides, and exhibited moderate sensitivity to carbapenems. Notably, all three major organisms—Klebsiella pneumoniae, Acinetobacter species, and Escherichia coli—remained uniformly sensitive to colistin, highlighting its role as an effective last-resort antibiotic against multidrug-resistant neonatal sepsis pathogens.

Figure 1: Distribution of bacterial isolates causing neonatal sepsis in NICU

Among the 186 culture-positive neonates, 138 (74.2%) were successfully discharged following treatment, while 48 (25.8%) succumbed to the illness. Culture-positive neonates had significantly higher mortality compared to culture-negative neonates (25.8% versus 15.8%, p = 0.01). The requirement for mechanical ventilation was higher among culture-positive cases than culture-negative cases ((55.9% versus 31.6%, p = 0.06). The data of outcome variables are shown in table 3.

Table 3: Comparison of outcome variables between culture-positive and culture-negative neonates

* Chi-square test

DISCUSSION

In the present study, the culture positivity rate among clinically suspected neonatal sepsis cases was 76.5%. This relatively high culture positivity rate is comparable to reports from other tertiary care NICUs in India and reflects the high burden of neonatal infections in referral centers that cater to critically ill and high-risk neonates [9].

A male predominance was observed among culture-positive neonates in this study. Similar findings have been consistently reported in earlier studies. This gender difference may be attributed to genetic and immunological factors, including X-linked immune regulatory mechanisms, which may predispose male neonates to a higher risk of infections [1]. This study demonstrated a clear predominance of gram-negative organisms as the causative agents of neonatal sepsis. Klebsiella pneumoniae was the most frequently isolated organism, followed by Acinetobacter species and Escherichia coli. This bacteriological pattern is consistent with several NICU-based studies from India and reflects a shift toward gram-negative predominance in neonatal sepsis in developing countries [15].

A major concern identified in this study was the high level of resistance to commonly used antibiotics, particularly third-generation cephalosporins, among Klebsiella and Acinetobacter isolates. The increasing prevalence of extended-spectrum beta-lactamase (ESBL)-producing gram-negative organisms causing neonatal sepsis has been widely reported and poses a significant therapeutic challenge [16]. In the present study, Colistin emerged as the most effective antibiotic against multidrug-resistant gram-negative organisms. This finding is in agreement with earlier reports that identify Colistin as a last-resort drug for the treatment of MDR neonatal infections, particularly in settings with high resistance to first-line and second-line antibiotics [17]. However, reliance on colistin raises concerns regarding toxicity and the potential emergence of colistin resistance. The rising resistance to first-line antibiotics underscores the urgent need for rational antibiotic use in NICUs. Regular monitoring of antimicrobial susceptibility patterns, strict adherence to antibiotic stewardship programs, and periodic revision of empirical antibiotic policies are essential strategies to limit the spread of antimicrobial resistance [18].

The mortality rate among culture-positive neonates in this study was 25.8%. High mortality associated with neonatal sepsis has been reported previously, particularly among preterm and low birth weight neonates. Infections caused by multidrug-resistant organisms further worsen outcomes by delaying effective therapy and limiting treatment options [3].

The present study has certain limitations that should be acknowledged. Being a single-center study conducted in a tertiary care NICU, the findings may not be fully generalizable to other healthcare settings or regions with different patient populations and microbial profiles. Although a reasonable number of neonates were included, the sample size was relatively limited, which may restrict the statistical power to detect significant associations between certain variables. In addition, the study focused only on aerobic bacterial pathogens; anaerobic and fungal cultures were not performed, which may have led to an underestimation of the complete spectrum of pathogens responsible for neonatal sepsis.

The present study highlights the predominance of multidrug-resistant gram-negative organisms as the major causative agents of neonatal sepsis in a tertiary care NICU. Klebsiella pneumoniae and Acinetobacter species were the most frequently isolated pathogens among culture-positive neonates A high level of resistance to commonly used antibiotics, particularly cephalosporins, was observed, while Colistin remained effective against most multidrug-resistant isolates. These findings emphasize the shrinking arsenal of effective antibiotics for neonatal sepsis and the growing dependence on last-line agents. Continuous surveillance of antimicrobial susceptibility patterns is essential to guide empirical antibiotic therapy in NICUs. Regular updating of unit-specific antibiograms, strict implementation of antibiotic stewardship principles, and rational antibiotic prescribing practices are crucial to reduce antimicrobial resistance, improve treatment outcomes, and ultimately decrease neonatal mortality.

ACKNOWLEDGEMENT

All authors were involved in work's conception, design, data acquisition, analysis, or interpretation, drafting or critically revising for intellectual content, final approval of the version to be published and taking the accountability for all work aspects, ensuring integrity and resolving questions. The authors acknowledge the support of all the NICU staff at Dept of Pediatrics, GMC, Kota and the patients’ parents throughout the study. The authors also wish to acknowledge Dr Shailendra Vashistha (Assistant Professor, Transplant Immunology Lab, Dept of IH&TM, GMC, Kota) and the VAssist Research team (www.thevassist.com) for their contribution in journal selection, manuscript editing, and submission process.

Declaration on competing / conflict of interest: None.

Source of funding: Nil.

REFERENCES

1. Shane AL, Sánchez PJ, Stoll BJ. Neonatal sepsis. Lancet. 2017;390(10104):1770–80.
2. Simonsen KA, Anderson-Berry AL, Delair SF, Davies HD. Early-onset neonatal sepsis. Clin Microbiol Rev. 2014;27(1):21–47.
3. Sankar MJ, Agarwal R, Deorari AK, Paul VK. Sepsis in the newborn. Indian J Pediatr. 2008;75(3):261–6.
4. Investigators of the Delhi Neonatal Infection Study (DeNIS) Collaboration. Characterisation and antimicrobial resistance of sepsis pathogens in neonates in India. Lancet Glob Health. 2016;4(10):e752–60.
5. Ballot DE, Nana T, Sriruttan C, et al. Bacterial bloodstream infections in neonates in a developing country. Int J Infect Dis. 2019;81:45–51.
6. Viswanathan R, Singh AK, Basu S, Chatterjee S, Sardar S, Isaacs D. Multidrug-resistant gram-negative bacilli causing early neonatal sepsis in India. Arch Dis Child Fetal Neonatal Ed. 2012;97(3):F182–7.
7. Agarwal R, Sankar MJ, Deorari AK, Paul VK. Sepsis in newborn. Indian J Pediatr. 2014;81(7):678–84.
8. World Health Organization. Managing possible serious bacterial infection in young infants. Geneva: WHO; 2015.
9. Roy I, Jain A, Kumar M, Agarwal SK. Bacteriology of neonatal septicemia in a tertiary care hospital of northern India. Indian J Med Microbiol. 2002;20(3):156–9.
10. Tsering DC, Chanchal L, Pal R, Kar S. Bacteriological profile of neonatal septicemia and antibiotic susceptibility pattern of isolates. Indian J Pediatr. 2011;78(7):1–6.
11. Kumar S, Bandyopadhyay S, Chakraborty R, Banerjee R. Changing trends of neonatal septicemia. J Neonatal Biol. 2017;6(2):1–5.
12. Agrawal P, Verma D, Meena M, Sharma A, Sharma S. An observational study from North India to evaluate microbial etiology, risk factors analysis, and antibiotic sensitivity pattern of surgical site infection in patients undergoing lower segment ceasarean section. CME J Geriatr Med. 2024 Jan;16(1):38-43.
13. National Neonatology Forum of India. Clinical Practice Guidelines: Neonatal Sepsis. New Delhi: NNF; 2021.
14. Tripathi N, Cotten CM, Smith PB. Antibiotic use and misuse in the neonatal intensive care unit. Clin Perinatol. 2012;39(1):61–8.
15. Investigators of the Delhi Neonatal Infection Study (DeNIS) collaboration. Characterisation and antimicrobial resistance of sepsis pathogens in neonates born in tertiary care centres in Delhi, India: A cohort study. Lancet Glob Health. 2016;4(10):e752-60. doi:10.1016/S2214-109X(16)30148-6.
16. Chandel DS, Johnson JA, Chaudhry R, et al. Extended-spectrum beta-lactamase–producing gram-negative bacteria causing neonatal sepsis. J Med Microbiol. 2011;60(Pt 4):541–5.
17. Viswanathan R, Singh AK, Basu S, Chatterjee S, Sardar S, Isaacs D. Multidrug-resistant gram-negative bacilli causing early neonatal sepsis in India. Arch Dis Child Fetal Neonatal Ed. 2012;97(3):F182–7.
18. Tripathi N, Cotten CM, Smith PB. Antibiotic use and misuse in the neonatal intensive care unit. Clin Perinatol. 2012;39(1):61–8.