The Shifting Environment of Neonatal Sepsis. Lessons After Comparison of Two Different Periods

1. Introduction

Neonatal sepsis is one of the leading causes of neonatal morbidity and mortality in neonatal intensive care units (NICUs) worldwide [1]. Sepsis disproportionally affects the most preterm and low birth weight neonates, owing to their immature immune system, comorbidities, increased need for invasive procedures, and prolonged hospitalization [2,3]. The prevalence of neonatal sepsis in the United States (US) is reported to be 1 per 1000 live births, with a significantly higher incidence observed among preterm neonates, approximately tenfold higher than that of term neonates [4]. A significant variation in the incidence of neonatal sepsis is observed between different geographic regions. It has been estimated that the global pooled incidence of neonatal sepsis is 22 per 1000 live births, with a mortality rate of 11-19%. The prevalence of sepsis is 40 times higher, and the mortality rates are twice as high in low-and middle-income countries (LMICs) compared to high-income countries (HICs) [5].

Depending on the age of onset, sepsis can be categorized as early-onset sepsis (EOS) or late-onset sepsis (LOS). EOS is defined as sepsis occurring during the first 72 hours from birth in hospitalized neonates or the first seven days after birth in nonhospitalized neonates, and LOS, sepsis that occurs thereafter [1]. The acquisition of pathogens in EOS cases is typically vertical, in contrast to the implication of nosocomial or community-acquired pathogens in LOS cases [3].

Neonatal sepsis is associated with significant morbidity and mortality, and prompt diagnosis and antibiotic treatment are critical for the prognosis. However, the non-specific clinical presentation of neonatal sepsis and the low predictive value of the biomarkers result in the overuse of antibiotics in NICUs [1,6]. Overprescription of antibiotics, prolonged antibiotic courses, and broad-spectrum antibiotics are associated with the emergence of resistant pathogens, which represent a growing global threat [6].

The increasing prevalence of multidrug-resistant pathogens in NICUs worldwide poses a significant challenge to the management of neonatal sepsis [7]. Antimicrobial resistance has evolved over the last few decades. Pathogens that were considered emerging during the early 2000s are now recognized as endemic in many NICUs [7]. In the 2000s, extended-spectrum beta-lactamase (ESBL) Enterobacterales, vancomycin-resistant (VRE) Enterococci, and methicillin-resistant Staphylococcus aureus (MRSA) emerged in NICUs and were implicated in outbreaks [8,9,10]. At present, these pathogens continue to pose significant threats in NICUs, while other multidrug-resistant pathogens, such as carbapenem-resistant Enterobacterales (CRE) and multidrug-resistant Acinetobacter baumannii, have emerged [7,11] .

The management of multidrug-resistant sepsis is challenging due to the limited antimicrobial agents available, representing a major global health concern. Antimicrobial agents authorized for use in neonates are even more restricted [12].

In response to the increasing prevalence of antimicrobial resistance, optimization of antibiotic use in NICUs through antibiotic stewardship programs is a critical strategy to mitigate the spread of resistance. Multidisciplinary team approach and the use of advanced molecular techniques to detect pathogens and understand transmission dynamics are the cornerstones of antibiotic stewardship [6,13].

The aim of this narrative review is to compare the epidemiology and resistance patterns of neonatal sepsis in NICUs between two periods, 2000-2005 and 2020-2025. In addition to the evolution of antimicrobial resistance, changes in diagnostic techniques and management of neonatal sepsis will be reviewed, highlighting the major changes that have occurred in the last two decades. By comparing these two periods, this review aims to highlight key lessons and discuss future priorities to limit the emergence and dissemination of antimicrobial resistance in NICUs.

2. Methods

A structured and comprehensive search was conducted using the online databases PubMed, Scopus, and Google Scholar to identify relevant studies published during the periods 2000-2005 and 2020-2025. The following keywords were used: neonatal sepsis; neonatal intensive care unit; antibiotic resistance; multidrug-resistant pathogens; extended-spectrum beta-lactamases; vancomycin-resistant Enterococcus; methicillin-resistant Staphylococcus aureus; coagulase-negative Staphylococcus; carbapenem-resistant Enterobacterales; Acinetobacter baumannii; antibiotic stewardship; molecular diagnostics. Only full-text, peer-reviewed studies written in English were included. Studies not related to antibiotic resistance in neonates or involving non-bacterial pathogens were excluded. The reference lists of the retrieved articles were reviewed to assess for the presence of relevant articles that may not have been detected in the initial search. The article selection, which involved screening titles and abstracts, followed by full-text evaluation and data extraction, was performed independently by two authors. In cases of uncertainty, the decision-making process involved discussion with the co-authors.

3. Common Pathogens in Neonatal Sepsis

3.1. 2000-2005

During the early 2000s, a shift was observed in the causative pathogens implicated in EOS in the neonatal population. Large cohorts of term and preterm neonates reported Group B Streptococcus (GBS) as the most prevalent cause of EOS, followed by E. coli. In preterm neonates, non-GBS infections were more common. However, these studies were conducted prior to the implementation of antenatal GBS screening. The authors hypothesized that 66% of GBS infections could have been prevented through screening and intrapartum prophylaxis [14]. The 2002 revision of the Centers for Disease Control and Prevention (CDC) guidelines recommended universal GBS screening for all pregnancies at 35-37 weeks of gestation and intrapartum antibiotic prophylaxis for all colonized women [15]. A significant decline in the incidence of GBS EOS was documented following the implementation of intrapartum prophylaxis, from 2.79 to 0.21 cases per 1000 live births [16]. Despite this decline, GBS remained one of the most common causes of EOS, alongside E. coli, accounting for 60-80% of cases [17,18]. Less frequently isolated pathogens included Klebsiella pneumoniae, other Enterobacterales, Listeria monocytogenes, and Enterococcus species [17].

In very low birth weight (VLBW) neonates, data from the National Institute of Child Health and Human Development (NICHD) Neonatal Research Network indicated a shift toward an increased prevalence of gram-negative pathogens causing EOS compared with the past decade, during which gram-positive pathogens predominated [19,20]. E. coli was the most prevalent organism isolated (41.2%) [20]. The rate of GBS infections was low (11.8%), which is likely attributable to the widespread use of intrapartum prophylaxis. The most common gram-positive pathogen was coagulase-negative Staphylococcus (CoNS), accounting for 14.7% of EOS.

LOS are nosocomial infections caused by pathogens acquired during NICU hospitalization. Staphylococcus aureus, CoNS, K. pneumoniae, and Pseudomonas aeruginosa were the most frequent pathogens. In many institutions, CoNS was the most prevalent cause of LOS, particularly affecting ELBW neonates [17,21,22]. According to the NICHD, 21% of VLBW neonates had one or more episodes of LOS. Gram-positive organisms were identified in 70% of LOS episodes, with nearly half attributable to CoNS. Of the gram-negative organisms, E. coli, K. pneumoniae, P. aeruginosa, and Enterobacter spp. were identified as the most prevalent pathogens. Fungal infections, predominantly due to Candida species, accounted for approximately 12% of LOS episodes [23].

3.2. 2020-2025

The most common pathogens implicated in neonatal sepsis have remained largely unchanged over the past few decades. Despite the considerable reduction in the prevalence of GBS sepsis following the implementation of routine intrapartum screening and antibiotic prophylaxis, GBS remains the most prevalent cause of EOS, followed by E. coli. It is estimated that approximately two-thirds of EOS cases are attributable to these two pathogens [1,3,7,24].

As in previous decades, E. coli is the most prevalent cause of EOS in the preterm population, accounting for approximately 50% of cases, whereas GBS is the second most common causative pathogen, accounting for approximately 20% of EOS episodes [24,25]. Stoll et al., in a large prospective surveillance study, reported an increased prevalence of E. coli infections in VLBW neonates compared with the previous decade (8.68 vs. 5.07 per 1000 live births; P=0.008) [26]. The remaining one-third of EOS cases are attributable to other gram-positive pathogens, including Staphylococcus aureus, Enterococcus spp., and Listeria monocytogenes, as well as gram-negative pathogens, such as Klebsiella, Enterobacter, and Haemophilus species [24,25].

Although the burden of LOS, particularly in preterm neonates, remains substantial, a decline in the incidence has been observed compared to previous decades [27,28]. Gram-positive pathogens remain the most prevalent, predominantly CoNS, and less frequently, Staphylococcus aureus, Enterococcus spp., and GBS [1,24,29]. Gram-negative pathogens account for 20-30% of LOS, with E. coli and K. pneumoniae being the most prevalent, and, to a lesser extent, Serratia marcescens, Enterobacter spp., and P. aeruginosa [3,24,29]. Approximately 5% of LOS episodes in extremely preterm neonates involve Candida spp. [28]. However, significant variation exists in the pathogens most commonly implicated in LOS across different geographical regions and even within the same region. Gram-positive pathogens are more frequently implicated in LOS in HICs, while gram-negative pathogens predominate in MLICs [29,30].

4. Antimicrobial Resistance

4.1. 2000-2005

During the early 2000s, an increasing prevalence of antimicrobial resistance in NICUs was observed, particularly among gram-negative organisms. Increased resistance to third-generation cephalosporins and broad-spectrum penicillins was observed during this period. Moreover, resistance to aminoglycosides was documented, while resistance to carbapenems and quinolones remained sporadic [31].

The evolution of pathogen resistance depends on the antibiotic prescribing practices [31]. The most widely used empirical antibiotic regimen was ampicillin plus an aminoglycoside or third-generation cephalosporin [32]. Ampicillin and third-generation cephalosporins are associated with the selection of gram-negative pathogens that produce ESBLs, rendering them resistant to a variety of antibiotics, including beta-lactams [33].

The impact of antibiotic selection on resistance patterns was highlighted in the study by De Man et al., which demonstrated that using a narrower-spectrum antibiotic was associated with reduced potency in selecting resistant pathogens. The authors compared the prevalence of colonization with resistant bacteria among neonates admitted to two identical NICUs, each using a different empiric antibiotic regimen: amoxicillin plus cefotaxime and penicillin plus tobramycin. An 18-fold relative risk of colonization with pathogens resistant to the empirical antibiotic regimen was reported in the NICU that used amoxicillin plus cefotaxime [32].

An increasing prevalence of pathogens resistant to the widely used EOS regimen, ampicillin, and gentamicin, was documented. High rates of ampicillin resistance among E. coli isolates had been reported since the early 2000s, likely attributable to the high rates of ampicillin intrapartum prophylaxis and the widespread use of ampicillin as an empiric regimen in neonates with suspected sepsis [16,20,34,35,36]. The 2005 NICHD report on EOS in VLBW neonates documented 77% ampicillin resistance among E. coli isolates [20]. Furthermore, Alarcon et al. reported an increased prevalence of ampicillin-resistant E. coli isolates causing EOS in preterm neonates over a 10-year period (25% vs. 91%), however, no association with the administration of intrapartum prophylaxis could be established. Interestingly, during the same period, the authors reported no increase in the prevalence of ampicillin-resistant E. coli isolates causing EOS in term neonates [16]. This is in accordance with the results of other studies [34].

Resistance to aminoglycosides was reported less frequently; however, concerns regarding the efficacy of standard empirical regimens were raised. Gentamicin resistance among E. coli isolates was documented in approximately 8-12% of cases [16,20].

The selective pressure exerted by commonly used antibiotics in NICUs was concerning. Moreover, the emergence of specific resistance mechanisms, including ESBLs in gram-negative pathogens and vancomycin resistance among enterococci, as well as MRSA, posed significant challenges to the management of infections in NICUs.

4.2. 2020-2025

The recommended empirical coverage for EOS, ampicillin and gentamicin, has remained unchanged over the last decades. However, high levels of ampicillin resistance, documented since the 2000s among E. coli isolates, persist, with increasing reports of gentamicin resistance [1]. Flannery et al. reported an 76.6% resistance to ampicillin of gram-negative pathogens implicated in EOS, 8.5% to gentamicin, and 7.3% to both agents. All gram-positive pathogens were susceptible to ampicillin and/or gentamicin [37]. A large multicenter US study documented 70% resistance to ampicillin and 16.8% to aminoglycosides in E. coli isolates from neonates admitted to NICUs. Among E. coli isolates associated with EOS, resistance to both ampicillin and gentamicin was observed in 10% of cases [38]. Stoll et al. reported that ampicillin-resistant E. coli was more frequent among preterm compared to term neonates (83.1% vs. 37.5%, P < 0.001) [26]. In light of increasing antimicrobial resistance, pediatric infectious disease specialists have suggested that while ampicillin and gentamicin remain reasonable empirical regimens for term and most preterm neonates, we are approaching a point at which we should consider alternative empirical regimens at the local or national level [38,39]. However, in the case of critically ill, particularly VLBW neonates, at higher risk for EOS, the use of broader-spectrum antibiotics should be considered [26,38,39].

A higher level of heterogeneity is reported among antibiotics used to treat LOS [40]. Vancomycin in combination with an aminoglycoside or a third-generation cephalosporin is the most commonly employed antibiotic regimen, while beta-lactams/beta-lactamase inhibitors, carbapenems, and linezolid are also used [29,40]. The widespread use of vancomycin, third and fourth-generation cephalosporins, and carbapenems has led to the emergence of multidrug-resistant pathogens worldwide [30].

The prevalence of antibiotic-resistant strains has increased globally compared to previous decades. ESBL Enterobacterales have been recognized as a serious threat, and CRE and multidrug-resistant (MDR) Pseudomonas aeruginosa as urgent threats according to the CDC 2019 report [41]. Compared with the early 2000s, when ESBL-producing pathogens and VRE were emerging, the current era is characterized by the widespread dissemination of multidrug-resistant pathogens, including CRE, ESBL, VRE, and MRSA, representing a major challenge in NICUs due to the outbreak potential and limited therapeutic options.

5. Multidrug-Resistant Pathogens

5.1. Extended-Spectrum Beta-Lactamases (ESBL)

5.1.1. 2000-2005

Until the 1980s, resistance to beta-lactam antibiotics was observed only in pathogens harboring chromosomal beta-lactamase genes, a form of resistance that is not transmissible. In 1983, Knothe et al. described strains of nosocomial Enterobacterales demonstrating transmissible, plasmid-mediated resistance to cephalosporins [42]. In the following years, ESBLs emerged among Enterobacterales, particularly Klebsiella spp. and E. coli, as a global concern [43]. Outbreaks in intensive care units caused by ESBL-producing pathogens were increasingly reported [44].

During the early 2000s, the prevalence of ESBL-producing bacteria varied significantly across geographic regions and within centers within the same region [45]. In the US, according to the CDC National Nosocomial Infections Surveillance, the national average for ESBL-producing Enterobacterales was low, at approximately 3%, although rates varied widely between centers (0-25%) [45]. In European countries, significant heterogeneity was also observed. In the Netherlands, very low prevalence (<1%) was reported, whereas substantially higher rates were documented in other countries, such as France, where up to 40% of K. pneumoniae isolates were ceftazidime-resistant, and 30% of Enterobacter aerogenes isolates were ESBL-producing [46,47,48]. These variations across European countries were highlighted in a large-scale study involving 100 intensive care units, in which the prevalence of ESBL-producing Klebsiella spp. ranged from 3% in Sweden and 34% in Portugal [49]. Similarly, in Asia, prevalence ranged from negligible levels in Japan to approximately 12% in China [50,51]. Of particular concern was the evidence of the rapid increase in ESBL infections over a short period. For instance, data from Taiwan demonstrated a substantial increase in the rate of ESBL Enterobacterales infections in pediatric intensive care units (PICUs) and NICUs, from 11% of nosocomial infections in 1999 to 44% in 2001 [52].

Although data specific to NICUs during this period were relatively limited, studies consistently demonstrated a substantial burden of ESBL-producing pathogens in both colonization and infection. In a US NICU, 41% of neonates who developed antimicrobial-non-susceptible Enterobacterales sepsis were infected with ESBL-producing strains. Of particular note, 91% of Klebsiella spp. and 73% of Enterobacter aerogenes were ESBL producers [53]. A Chinese study involving a two-year observation period in a NICU reported that 56.4% of infections caused by K. pneumoniae and E. coli were due to ESBL-producing strains [54]. Colonization rates among neonates ranged from approximately 20% to over 50% with K. pneumoniae spp. predominating. In a Malaysian NICU, 21.7% of admitted neonates were colonized with ESBL K. pneumoniae at a median age of nine days [55]. Pessoa-Silva et al. observed that 53.8% of 383 neonates admitted to a level II/III NICU in Brazil were colonized with ESBL-producing K. pneumoniae [56]. A study from Turkey examined the resistance of the fecal flora of 118 neonates, including those admitted to the NICU and the neonatal ward, as well as healthy term neonates, and reported a 33.7% ESBL colonization rate. Of the K. pneumoniae and E. coli isolates evaluated, 44.8% and 45.1%, respectively, were identified as ESBL-producing. Interestingly, no difference in the ESBL colonization rate was observed between hospitalized and healthy neonates [57].

In 2005, more than 200 ESBLs had been identified [58]. Whereas TEM- and SHV-producing ESBLs were predominant in previous decades, CTX-M-producing ESBLs emerged as the most frequent genotype in the early 2000s across various geographic regions [59,60]. However, molecular data on ESBL genotypes in NICUs were scarce. Consistent with the broader epidemiology of ESBLs during that period, Wu et al. analyzed 88 ESBL-producing isolates using restriction site PCR and reported CTX-M-3 as the predominant genotype among E. coli and K. pneumoniae. In all E. cloacae isolated, SHV-12 was identified.

In view of the increased prevalence of ESBLs in NICUs, several investigators examined potential risk factors for neonatal colonization and infection. The risk of colonization increased by 26% for each additional day of hospitalization, and with the combined use of a cephalosporin and an aminoglycoside. Previous colonization and the use of a central venous catheter were independent risk factors for infection, observed in 3.4% of neonates [56]. Boo et al. also reported hospital stay duration as an independent risk factor for ESBL K. pneumoniae colonization, with a 1.3-fold increase in risk per additional day of hospitalization [55]. Huang et al. identified low birth weight, prior use of third-generation cephalosporins, and prolonged mechanical ventilation as risk factors for ESBL-producing bacterial infection [54]. The role of transmission via healthcare workers was highlighted in a case-control study by Gupta et al. during an outbreak of ESBL-producing K. pneumoniae in a US NICU, in which 19 neonates were colonized or infected. The acquisition of the outbreak strain was associated with both length of stay and exposure to a healthcare worker wearing artificial fingernails [61].

While some consistency across institutions in antimicrobial susceptibility of ESBL-producing pathogens was noted, variability was also observed. Carbapenems exhibited high levels of activity against ESBL-producing Enterobacterales and were considered the most effective therapeutic option [52,54]. Although susceptibility to quinolones was demonstrated in most instances, emerging resistance was reported in several studies [10,52,54]. Moreover, ESBL isolates that exhibited increased resistance to beta-lactam/beta-lactamase inhibitor combinations, including amoxicillin/clavulanic acid, cefoperazone/sulbactam, and ticarcillin/clavulanic acid, were reported and demonstrated to be associated with high-level ESBL production [54,62].

5.1.2. 2020-2025

During 2020-2025 and to the present, ESBL-producing pathogens remain a significant threat in NICUs worldwide. The prevalence of ESBL colonization and infection varies significantly across regions, with higher rates in LMICs [11]. Reports of NICU outbreaks are increasing [63,64,65,66,67]. The endemic presence of ESBL in numerous NICUs has become a notable concern [11].

Recent surveillance studies have highlighted the substantial burden of ESBL colonization in neonatal populations. In a Moroccan NICU, approximately 55% of admitted neonates were colonized by ESBL K. pneumoniae [68]. Similarly, a recent Iranian systematic review and meta-analysis reported a 57% third-generation cephalosporin resistance among gram-negative isolates implicated in neonatal sepsis [69]. A recent meta-analysis of studies conducted in LMICs reported a pooled prevalence of third-generation cephalosporin-resistant Enterobacterales colonization of 30.2%, rising to 48.2% when including only hospitalized neonates [70]. In contrast, substantially lower colonization rates have been reported in several HICs. In an Irish NICU, 9% of very preterm neonates were found to be colonized with ESBL-producing isolates, whereas the prevalence of colonization remained low in countries such as the US and Japan [7,71,72].

The CTX-M genotype, which emerged in the early 2000s, has become the predominant type in recent years, particularly CTX-M-15, followed by SHV and TEM genotypes [7,24,63,65,71].

ESBL-producing pathogens are typically resistant to a broad range of antibiotics, including penicillins and cephalosporins. Although ESBLs typically do not inactivate non-beta-lactam antibiotics, pathogens often harbor additional genes or mutations that confer resistance to various agents, including fluoroquinolones and aminoglycosides. Carbapenems are generally effective against ESBL-producing Enterobacterales and are the treatment of choice for invasive infections [7,73,74]. The widespread use of carbapenems, driven by the rising prevalence of ESBL-producing pathogens, has raised concerns about selection pressure and the subsequent emergence of carbapenem-resistant Enterobacterales in NICUs. Newer beta-lactam/beta-lactamases combination, such as ceftazidime/avibactam and ceftolozane/tazobactam are approved for neonatal use and effective against ESBLs, preferentially reserved for carbapenem-resistant isolates [74,75].

5.2. Carbapenem-Resistant Enterobacterales (CRE)

5.2.1. 2000-2005

During the early 2000s, Enterobacterales resistance to carbapenems was rare and only sporadically reported [76,77]. However, the increasing prevalence of ESBL-producing Enterobacterales was associated with increased carbapenem use during this period. The increased selective pressure raised concerns that carbapenem-resistant strains could emerge in NICUs [54].

5.2.2. 2020-2025

The steadily increasing prevalence of ESBL-producing and multidrug-resistant pathogens has led to an increased use of carbapenems, more commonly meropenem and imipenem, in NICUs, thereby contributing to the emergence of carbapenem-resistant pathogens [11]. Carbapenem resistance may be mediated by the production of carbapenemases, such as Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), and oxacillinase OXA-48-like enzymes, or production of ESBL or AmpC beta-lactamase combined with structural mutation leading to impaired membrane permeability [7,74,78]. Carbapenem resistance may occur in various Enterobacterales species; Klebsiella pneumoniae is the most common [7,70].

Recent studies have demonstrated increasing prevalence of CRE colonization and infections in NICUs, particularly in LMICs (Table 1). A Chinese multicenter study reported that 13% of sepsis cases caused by Enterobacterales in very preterm neonates were attributable to carbapenem-resistant strains [79]. According to a recent systematic review, the pooled global prevalence of carbapenem-resistant K. pneumoniae sepsis in neonates admitted to the NICU is 0.3%. Notably, the analysis included 23 studies, all of which were conducted in LMICs [80].

High colonization rates have also been reported in several regions, especially in developing countries. Prematurity, invasive procedures, prolonged antibiotic courses, and prolonged hospitalization have been identified as risk factors for colonization. Moreover, a correlation has been demonstrated between prior carbapenem use and colonization with carbapenemase-resistant strains [81,82]. Colonization has been demonstrated to serve as a reservoir for invasive infections and transmission, with the potential to result in outbreaks within intensive care units [82,83]. Studies conducted in African countries, Morocco and Tanzania, have reported a colonization rate of 30% [84]. High colonization rates of 25% have also been documented in a Serbian NICU [81]. In India, colonization rates have been reported to be 5-9% among hospitalized neonates [85,86]. A recent meta-analysis of 26 studies conducted in LMICs demonstrated a pooled prevalence of 6.3% for CRE colonization in hospitalized neonates [70]. Data on CRE colonization in HICs are limited, however, evidence suggests a significantly lower prevalence than in LMICs [87,88].

Molecular studies have demonstrated the predominance of carbapenemase-producing strains in isolates from NICUs [89,90]. Geographic variations exist in the prevalence of different carbapenemase genes [80,89]. In Saudi Arabia, 71.2% of CRE K. pneumoniae isolates encoded OXA-48 type carbapenemases, followed by NDM-1 (20.5%) [91]. The predominance of the NDM gene has been demonstrated in studies conducted in China [82,90]. KPC and VIM carbapenemase were most common in a Portuguese NICU, whereas NDM was most prevalent in Italy [88,92]. A global systematic review reported that approximately 65% of carbapenem-resistant Klebsiella pneumoniae isolates implicated in neonatal sepsis encode NDM [80]. Notably, the presence of co-harbored carbapenemase genes has been frequently described [89,93].

The global emergence of carbapenem-resistant strains is a significant concern, primarily due to the limited therapeutic options. According to the World Health Organization (WHO), carbapenem-resistant pathogens are classified as high priority for antimicrobial research [78]. In neonates, therapeutic options are considerably more limited [94]. Older drugs such as polymyxins and newer drugs such as ceftazidime/avibactam have been used in the treatment of CRE [95].

Table 1. Epidemiological and molecular characteristics of carbapenem-resistant Enterobacterales in neonatal intensive care units.

Author	Country	Population	CRE %	CRE species	CRE genotyping
Labi, 2020 [96]	Ghana	228	7.9	Klebsiella pneumoniae 18/18	OXA-181: 100%
Yin, 2021 [97]	China	1230	11.7	Klebsiella pneumoniae 110/144 Escherichia coli 29/144 Enterobacter cloacae 5/144	NDM-1: 82.7% OXA-23: 46.5% NDM-5: 15.5% KPC: 1.7%
Almeida, 2021 [88]	Portugal	173	5.8	Klebsiella pneumoniae 7/10 Enterobacter cloacae 3/10	KPC: 40% VIM: 40% OXA-48: 20%
Wang, 2022 [98]	China	1650	14.2	Klebsiella pneumoniae Escherichia coli	NDM: 94.4%
Agosta, 2023 [92]	Italy	230	7.4	Escherichia coli 12/20 Klebsiella pneumoniae 8/20	NDM: 100%
Demir, 2023 [89]	Turkey	206	24.3	Klebsiella pneumoniae 44/50 Klebsiella oxytoca 6/50	NDM: 42% OXA-48: 16% VIM: 2% NDM+OXA-48: 36% No genes detected: 4%
Edwards, 2023 [99]	Nigeria, Kenya	42	62.4	ND	NDM: 95.2% OXA-48: 4.8%
Mussa, 2023 [93]	Morocco	339	22.1	Klebsiella pneumoniae	OXA-48: 100% NDM: 30.8% VIM: 9.9% KPC: 2.2%
Mijac, 2023 [81]	Serbia	350	25.1	Klebsiella pneumoniae 87/88 Escherichia coli 1/88	KCP: 51.1% OXA-48: 47.7% NDM: 1.2%
Guan, 2025 [100]	China	1108	7.3	Klebsiella pneumoniae	KCP-2: 45.7% NDM-1: 40.7% NDM-5: 13.6%
Naburi, 2025 [84]	Tanzania	51	29.4	Klebsiella pneumoniae 10/15 Escherichia coli 4/15 Klebsiella oxytoca 1/15	NDM-5: 100% OXA-181: 53%
Dihn, 2026 [101]	Vietnam	36	38.9	Klebsiella pneumoniae 6/17 Enterobacter cloacae 6/17 Escherichia coli 4/17

KPC: Klebsiella pneumoniae carbapenemase; NDM: New Delhi metallo-beta-lactamase; OXA: oxacillinase ; VIM: Verona integron-encoded metallo-beta-lactamase.

Table 1. Epidemiological and molecular characteristics of carbapenem-resistant Enterobacterales in neonatal intensive care units.

Author	Country	Population	CRE %	CRE species	CRE genotyping
Labi, 2020 [96]	Ghana	228	7.9	Klebsiella pneumoniae 18/18	OXA-181: 100%
Yin, 2021 [97]	China	1230	11.7	Klebsiella pneumoniae 110/144 Escherichia coli 29/144 Enterobacter cloacae 5/144	NDM-1: 82.7% OXA-23: 46.5% NDM-5: 15.5% KPC: 1.7%
Almeida, 2021 [88]	Portugal	173	5.8	Klebsiella pneumoniae 7/10 Enterobacter cloacae 3/10	KPC: 40% VIM: 40% OXA-48: 20%
Wang, 2022 [98]	China	1650	14.2	Klebsiella pneumoniae Escherichia coli	NDM: 94.4%
Agosta, 2023 [92]	Italy	230	7.4	Escherichia coli 12/20 Klebsiella pneumoniae 8/20	NDM: 100%
Demir, 2023 [89]	Turkey	206	24.3	Klebsiella pneumoniae 44/50 Klebsiella oxytoca 6/50	NDM: 42% OXA-48: 16% VIM: 2% NDM+OXA-48: 36% No genes detected: 4%
Edwards, 2023 [99]	Nigeria, Kenya	42	62.4	ND	NDM: 95.2% OXA-48: 4.8%
Mussa, 2023 [93]	Morocco	339	22.1	Klebsiella pneumoniae	OXA-48: 100% NDM: 30.8% VIM: 9.9% KPC: 2.2%
Mijac, 2023 [81]	Serbia	350	25.1	Klebsiella pneumoniae 87/88 Escherichia coli 1/88	KCP: 51.1% OXA-48: 47.7% NDM: 1.2%
Guan, 2025 [100]	China	1108	7.3	Klebsiella pneumoniae	KCP-2: 45.7% NDM-1: 40.7% NDM-5: 13.6%
Naburi, 2025 [84]	Tanzania	51	29.4	Klebsiella pneumoniae 10/15 Escherichia coli 4/15 Klebsiella oxytoca 1/15	NDM-5: 100% OXA-181: 53%
Dihn, 2026 [101]	Vietnam	36	38.9	Klebsiella pneumoniae 6/17 Enterobacter cloacae 6/17 Escherichia coli 4/17

KPC: Klebsiella pneumoniae carbapenemase; NDM: New Delhi metallo-beta-lactamase; OXA: oxacillinase ; VIM: Verona integron-encoded metallo-beta-lactamase.

5.3. Vancomycin-Resistant Enterococcus (VRE)

5.3.1. 2000-2005

In the early 2000s, a further emerging threat was identified in NICUs, concerning the increasing prevalence of VRE. The first isolated strains of VRE were reported in Europe in 1986 [102]. Over the following years, a significant increase in prevalence was reported worldwide, rendering VRE strains as important nosocomial pathogens, particularly affecting intensive care units [103]. According to the CDC’s National Nosocomial Infections Surveillance System report, in 2004, 28.5% of enterococcal infections in intensive care units were attributable to VRE-producing strains [104].

During this period, outbreaks of VRE in intensive care units were increasingly reported [105]. Data on the burden of VRE in NICUs remained limited, focusing primarily on colonization rather than infection. However, it is well recognized that colonized neonates and environmental VRE contamination serve as reservoirs for pathogen transmission via healthcare workers’ hands and contaminated equipment [106]. Evidence of healthcare-associated spread was provided by Khan et al., who reported the first 10 VRE isolates of the same clone in a tertiary hospital in Pakistan. Four of the isolates were sourced from the NICU, while six were obtained from the intensive care unit. It should be noted that these two units were not located in the same facility; however, they shared a number of personnel [107]. Moreover, Yuce et al. collected rectal swabs from neonates admitted to the NICU and from healthy neonates in the obstetric ward to evaluate the incidence and risk factors for VRE colonization. None of the healthy neonates demonstrated VRE colonization, whereas 8/110 hospitalized neonates did. Prolonged hospitalization and prior use of broad-spectrum antibiotics were identified as risk factors for colonization [105].

Several studies highlighted the potential for VRE outbreaks in NICUs (Table 2). Borgmann et al. described two outbreaks caused by at least two distinct clones of VRE E. faecium in a German NICU [108]. In a US NICU, VRE-producing strains were isolated from 65 out of the 1,820 admitted neonates over a three-year period. Five patients developed infections, including bloodstream infections, meningitis, and urinary tract infections. The infection-to-colonization ratio was 1:12 [106]. In contrast, in a prospective longitudinal study of a NICU in Israel, Toledano et al. detected no cases of VRE colonization among hospitalized neonates, although 61% were colonized with vancomycin-sensitive enterococci during their NICU stay. Notably, in other wards of the same hospital, 14.7% of inpatients were colonized with VRE isolates [109].

Given the emergence of VRE infections as a significant threat to NICUs, there was particular interest in developing strategies to reduce their spread and prevent outbreaks. The significance of targeted surveillance cultures was emphasized in numerous studies, which demonstrated that even a single positive culture may be associated with a substantial number of colonized infants who serve as reservoirs for VRE [110]. Indeed, as evidenced by several studies, active surveillance and cohorting, in combination with enhanced infection control measures, were highly effective in controlling NICU outbreaks [106,108,111]. Golan et al. emphasized the significant role of environmental contamination as a reservoir for VRE transmission, as evidenced by the persistence of VRE transmission facilitated by contaminated incubators despite patient cohorting and infection control measures [112].

Although E. faecalis is the most common strain associated with human disease, E. faecium was the most common strain implicated in VRE infections [103,113]. Most outbreaks in NICUs involved VanA-type VRE strains, which are associated with high-level resistance to vancomycin and teicoplanin [107,113,114]. The VanB-type is associated only with high or low-level vancomycin resistance [105]. An additional concern with the VanA phenotype is the potential for transmission of this gene and vancomycin resistance to other pathogens, such as Staphylococcus aureus and Listeria monocytogenes [9,107]. The therapeutic management of VRE infections is particularly challenging, as vancomycin resistance often occurs with resistance to other antibiotics, including ampicillin, tetracycline, and aminoglycosides [107]. Chloramphenicol was a commonly used agent, whereas linezolid and quinupristin-dalfopristin represented newer agents that were increasingly used, despite the limited experience in neonatal use [103,107,113,114].

Table 2. Epidemiological, molecular and infection control measures of vancomycin-resistant enterococci in neonatal intensive care units.

Author	Country	Population	Infection/ Colonization	Site of isolation	Genotype	Clonality	Intervention
Yuce, 2001 [105]	Turkey	8	0/8	Rectal swabs	E. faecalis (5) E. faecium E. gallinarum	ND	ND
Rupp, 2001 [111]	US	28	0/28	Rectal, oropharyngeal swabs	E. faecium (VanB (25), VanA)	27/28 one clone	Cohorting, active surveillance cultures, environmental decontamination, infection control measures, reduced vancomycin use
Khan, 2002 [107]	Pakistan	4	1/3	Blood/ rectal swabs	E. faecium, (VanA)	One clone	Cohorting, discontinuation of sharing personnel with ICU, monitoring adherence to infection control measures
Borgmann, 2004 [108]	Germany	24	1/23	Stool samples	E. faecium (VanA)	Two clones	Cohorting, active surveillance cultures
Singh, 2005 [106]	US	65	5/60	Blood, urine, CSF, sputum/ rectal swabs	E. faecium (63) E. gallinarum (2)	Multiclonal	Cohorting, active surveillance cultures, infection control measures
Golan, 2005 [112]	England	14	0/14	Rectal swabs	ND	One clone	ND

US: United States; ICU: intensive care unit; CSF: cerebrospinal fluid; ND: no data.

Table 2. Epidemiological, molecular and infection control measures of vancomycin-resistant enterococci in neonatal intensive care units.

Author	Country	Population	Infection/ Colonization	Site of isolation	Genotype	Clonality	Intervention
Yuce, 2001 [105]	Turkey	8	0/8	Rectal swabs	E. faecalis (5) E. faecium E. gallinarum	ND	ND
Rupp, 2001 [111]	US	28	0/28	Rectal, oropharyngeal swabs	E. faecium (VanB (25), VanA)	27/28 one clone	Cohorting, active surveillance cultures, environmental decontamination, infection control measures, reduced vancomycin use
Khan, 2002 [107]	Pakistan	4	1/3	Blood/ rectal swabs	E. faecium, (VanA)	One clone	Cohorting, discontinuation of sharing personnel with ICU, monitoring adherence to infection control measures
Borgmann, 2004 [108]	Germany	24	1/23	Stool samples	E. faecium (VanA)	Two clones	Cohorting, active surveillance cultures
Singh, 2005 [106]	US	65	5/60	Blood, urine, CSF, sputum/ rectal swabs	E. faecium (63) E. gallinarum (2)	Multiclonal	Cohorting, active surveillance cultures, infection control measures
Golan, 2005 [112]	England	14	0/14	Rectal swabs	ND	One clone	ND

US: United States; ICU: intensive care unit; CSF: cerebrospinal fluid; ND: no data.

5.3.2. 2020-2025

VRE infections remain a significant concern among hospitalized patients, and a substantial increase in the prevalence has been reported compared to previous decades. In Asia, the pooled prevalence of VRE infections has been estimated at 9.1% in a systematic review, compared with 6.4% during 2000-2010 [115]. Significant variations have been observed among European countries. However, a significant increase was reported in 2021 compared to 2017, from 13.4% to 17.2% [116].

Data specific to neonatal populations remains limited and mostly focuses on colonization rather than infections. Recent studies continue to document VRE colonization among hospitalized neonates, with prevalence varying widely across geographic regions and institutions, from approximately 2% to more than 50% in outbreak settings [117,118,119]. A recent report in an Australian NICU documented an outbreak of vancomycin-resistant E. faecium, with a colonization rate of 15.3% among hospitalized neonates [120]. During an outbreak in a NICU in Israel, 55.2% of neonates were colonized [118].

Invasive infection is uncommon compared with colonization, and an estimated 0-10% of colonized neonates will develop invasive infection [117,118,120]. Potential increased risk of necrotizing enterocolitis has been reported among colonized neonates [119,121]. Despite the common characterization of these strains as less virulent than other multidrug-resistant pathogens, the limited therapeutic options available for neonates, along with the immaturity and comorbidities of these patients, are a cause for concern in NICUs [117,120,122]. Linezolid is the most commonly used agent in VRE infections, although daptomycin is also used in selected cases [12]. The increasing use of linezolid raises concerns about the emergence of linezolid-resistant isolates. However, to date, such isolates have been reported only sporadically [122,123].

Molecular testing, including PCR-based methods and whole-genome sequencing (WGS), is increasingly used in hospital outbreaks, as they offer rapid and accurate results, enabling prompt implementation of infection control measures [124]. Indeed, Saliba et al. demonstrated that PCR was associated with a median reduction of six days in turnaround time compared with rectal swab culture [125].

E. faecium is the most prevalent vancomycin-resistant strain of enterococci worldwide and is responsible for most outbreaks in nosocomial settings [115]. VanA type remains the most common VRE genotype. However, significant geographic variation exists, and an increased prevalence of vanB E. faecium has been reported in Australia and in several European countries since 2010 [126,127]. Regarding NICUs, molecular data are limited, but the existing literature reports the vanA genotype as the most frequently reported in outbreaks [118,119,120].

5.4. Methicillin-Resistant Staphylococcus Aureus (MRSA)

5.4.1. 2000-2005

The first documented isolate of MRSA was identified in Europe in 1962, and by the 1970s, global dissemination had occurred [128]. In 2000, over 55% of Staphylococcus aureus strains causing nosocomial infections in intensive care units were methicillin-resistant [129]. Although reports of MRSA infections and outbreaks in neonates had been documented in the literature since the 1970s, it was not until the 1990s that there was an increased prevalence of reports of outbreaks in NICUs worldwide [130]. By the 2000s, MRSA had already been recognized as a significant threat in NICUs [8,129,131,132,133,134,135].

The increased prevalence of MRSA led to the use of several antibiotic classes, exerting additional selective pressure and driving resistance to various non-beta-lactam antibiotic classes. Consequently, multidrug-resistant strains evolved, often retaining susceptibility to only a limited number of agents, such as vancomycin, rifampicin, mupirocin, and chloramphenicol [130,136]. Vancomycin resistance was rarely reported, yet this potential threat was already recognized [137].

The resistance of MRSA to beta-lactams is attributable to the mecA gene, which is located in the staphylococcal cassette chromosome mec (SCCmec). The SCCmec is distinguished by seven variants, type I to VII. Until the 1990s, hospital-associated MRSA clones were implicated in nosocomial infections [128,138]. However, in the early 2000s, new, more virulent, community-associated MRSA clones emerged and were identified as causative agents in nosocomial outbreaks [128,139,140]. These clones have a different SCCmec, often carry genes encoding toxins such as Panton-Valentine leukocidin, and are susceptible to more antibiotic classes than multidrug-resistant hospital-associated MRSA clones, including several non-beta-lactam antibiotics [128,139]. The increasing number of reports of community-associated MRSA as the causative agent of outbreaks in NICUs indicated a shift in MRSA epidemiology and that these strains had become endemic in NICUs [139,140,141].

In view of the increasing reports of MRSA outbreaks in NICUs, there was a growing interest in evaluating the most effective methods of transmission prevention and MRSA eradication. Molecular techniques, such as pulsed-field electrophoresis, had revealed that several outbreaks were caused by a single clone, highlighting the central role of horizontal transmission in the NICU [8,133,134]. Active surveillance cultures, nasal and/or umbilical, cohorting, adherence to infection control measures, and decolonization of neonates and healthcare workers were recognized as effective measures for outbreak control [134,142,143,144]. Various approaches were used in different institutions for MRSA eradication, including intranasal mupirocin administration to colonized neonates and healthcare workers or unselectively in the NICU population, methylprosaniline chloride, hexachlorophene, or dilute povidone iodine baths with encouraging outcomes [142,143,144].

5.4.2. 2020-2025

Although the prevalence of MRSA infections has decreased in several geographic regions, MRSA has become an endemic pathogen in NICUs worldwide, associated with significant morbidity and mortality [145,146,147]. Community-associated MRSA clones, initially recognized in the early 2000s, are now well established in healthcare settings [148]. The cumulative incidence of neonatal MRSA colonization in NICUs was reported to be 7.2% in a systematic review comprising 62 studies. Significant geographic variations were detected, with the highest incidence reported in Taiwan and the lowest in Brazil [146].

Although vertical transmission may occur, horizontal transmission is the most probable factor underlying MRSA endemicity in NICUs [146,149]. Healthcare workers have two to three times higher MRSA colonization rates than the general population [148]. A recent systematic review, including mostly studies from HICs, reported a pooled prevalence of MRSA carriage among healthcare workers of 9.5% [150]. Quan et al. reported that control of a prolonged outbreak was achieved only when persistently colonized healthcare workers were decolonized successfully [151].

Advances in molecular technologies have improved our understanding of MRSA transmission dynamics in the NICU. These technologies have been used in clinical practice, aiding the prompt recognition and implementation of infection control measures to control outbreaks [152]. Recent studies have documented the successful control of MRSA outbreaks in NICUs following screening with WGS and nanopore sequencing [152,153,154].

According to the CDC’s latest guidelines, active surveillance cultures for MRSA should be performed in outbreak settings. The recommended site for testing is the anterior nares. Targeted decolonization of colonized neonates alongside infection control measures should be implemented. However, no recommendation regarding the optimal decolonization agents could be made [155]. Intranasal mupirocin and chlorhexidine baths are the most common decolonization strategy in NICUs [148,151]. Nevertheless, the safety and efficacy in the neonatal population, particularly in VLBW neonates, have not been established [147,155].

Methicillin resistance may be isolated; however, combined resistance to other antimicrobial classes, such as fluoroquinolones, rifampicin, and clindamycin, is commonly observed [145,148]. Vancomycin is the most commonly used first-line agent for treating MRSA infections, often in combination with a beta-lactam [156]. However, the emergence of vancomycin-resistant Staphylococcus aureus (VRSA) and vancomycin-intermediate Staphylococcus aureus (VISA) is a significant concern [157]. Linezolid represents a potentially effective agent against VISA and VRSA, and resistance to linezolid is rarely observed [148].

MRSA remains an important endemic pathogen in NICUs worldwide. Advancements in molecular technologies have improved outbreak detection and control, while active surveillance and decolonization remain the primary strategies for mitigating transmission. However, the emergence of multidrug-resistant strains and strains with reduced susceptibility to glycopeptides poses significant challenges in NICUs.

5.5. Coagulase-Negative Staphylococcus (CoNS)

5.5.1. 2000-2005

CoNS represented the most prevalent pathogens causing LOS in the NICU, particularly affecting VLBW neonates, accounting for approximately half of all LOS episodes and associated with significant morbidity [158]. As demonstrated in several studies using molecular techniques, specific CoNS clones exhibited prolonged persistence in NICUs [159,160,161]. Antibiotic resistance was considered the primary selective force for these persistent CoNS clones, alongside virulence characteristics such as adhesion and biofilm production [160]. Multidrug resistance and biofilm formation on indwelling medical devices, such as central venous catheters, pose significant challenges to the effective treatment of invasive infections.

A significant proportion of CoNS strains resistant to commonly used antibiotics, including methicillin, were reported in NICUs [162,163]. Jain et al. reported 94% penicillin resistance and 66% methicillin resistance among 100 CoNS isolates derived from neonates with LOS [163]. A significant proportion of methicillin-resistant CoNS isolates, up to 92%, as determined by the presence of the mecA gene, were reported in NICUs worldwide [160,164,165]. It is noteworthy that combinations of cephalothin or oxacillin with gentamicin were reported to be efficacious in the treatment of CoNS neonatal sepsis in NICUs with a high prevalence of mecA gene-carrying CoNS isolates [164,166]. Aminoglycoside resistance was reported in 19-50% of isolates [163,165]. However, beta-lactam and aminoglycoside resistance was reported to be more common in isolates implicated in LOS compared to contaminants, potentially suggesting an association between antimicrobial resistance and pathogenicity [162].

A significant concern was the emergence of glycopeptide resistance among CoNS isolates [167,168]. Center et al. reported that 3.9% of CoNS isolates from colonized neonates admitted to the NICU demonstrated decreased susceptibility to vancomycin. Prolonged hospitalization, exposure to vancomycin, and colonization with Staphylococcus warneri strains were recognized as risk factors [168]. Although glycopeptide resistance was uncommon during this period, the widespread empirical use of vancomycin was recognized as associated with an increased risk of the emergence of glycopeptide-resistant strains.

5.5.2. Coagulase-Negative Staphylococcus (CoNS)

CoNS remains the leading cause of LOS in NICUs, particularly affecting VLBW neonates [1,28]. Staphylococcus epidermidis still represents the most prevalent strain associated with neonatal sepsis [169,170,171]. In addition to their ability to form biofilms, the increasing antimicrobial resistance among CoNS isolates is a major concern (306). Resistance to beta-lactams is widespread; methicillin resistance has been observed in up to 90% of CoNS isolates [172,173].

Glycopeptides, particularly vancomycin, are considered the first-line treatment for CoNS sepsis. However, decreased vancomycin susceptibility with increased minimum inhibitory concentration (MIC), particularly in methicillin-resistant isolates, has been observed [169,170]. A recent meta-analysis reported a 41.1% glycopeptide heteroresistance among CoNS isolates, raising concerns about the efficacy of glycopeptides as first-line agents [174].

5.6. Acinetobacter Baumannii

5.6.1. 2000-2005

A. baumannii, a gram-negative coccobacillus, was increasingly recognized as an important nosocomial pathogen in the early 2000s, particularly in intensive care units [175]. Outbreaks in NICUs were sporadically reported [176,177,178]. Although resistance to carbapenems, aminoglycosides, and fluoroquinolones was rarely reported, concerns had already been raised about the emergence of multidrug-resistant strains due to the selective pressure exerted by the widespread use of these broad-spectrum antibiotics [175,178].

5.6.2. 2020-2025

Multidrug-resistant Acinetobacter baumannii has emerged as an important cause of nosocomial infections and outbreaks in NICUs, particularly in LMICs [7,179,180]. A. baumannii has the capacity to survive in the hospital environment for a prolonged time and the potential for clonal spread [180,181]. Moreover, it has acquired resistance to various antibiotic classes, including cephalosporins, aminoglycosides, fluoroquinolones, and carbapenems [180]. Of particular concern is the increasing prevalence of carbapenem-resistant isolates, mediated by oxacillinase genes, more commonly OXA-23 [179,182]. The treatment options for MDR A. baumannii are limited, and colistin has been widely used. However, there is a growing concern regarding the emergence of colistin-resistant A. baumannii strains [183,184].

A. baumannii is estimated to be implicated in 1-6% of neonatal sepsis cases, geographic variations are observed [179]. According to a large observational study across 11 countries, primarily in Africa and Asia, Acinetobacter was identified as the third most prevalent causative agent of neonatal sepsis, accounting for 12.8% of cases. More than 70% of the isolates were resistant to meropenem [185]. In a NICU in South Africa, 13% of culture-confirmed sepsis cases were attributed to A. baumannii. In this study, 17% of the isolates were identified as extremely drug-resistant, displaying susceptibility exclusively to colistin [180]. In Ethiopia, all A. baumannii isolates associated with neonatal sepsis demonstrated multidrug resistance [186].

6. Evolution of Diagnostic Technologies in Neonatal Sepsis

Blood and/or other sterile-fluid cultures and conventional susceptibility testing were the main diagnostic tools in the early 2000s. Whilst culture from a sterile body site remains the gold standard for sepsis diagnosis, it has significant limitations. These include low sensitivity, contingent on the blood volume obtained, and a long turnaround time of 24-72 hours [187,188]. The delay in detecting pathogens may lead to unnecessary broad-spectrum antibiotic administration and prolonged courses, which can contribute to antimicrobial resistance [1].

Although molecular techniques were already employed in the early 2000s, their use was limited, primarily for research purposes, such as identifying specific resistance genes and investigating outbreaks [43,108,114,132,138,189]. Molecular epidemiologic studies primarily relied on pulse-field gel electrophoresis (PFGE), which was regarded as the most accurate typing technique for assessing the genetic relatedness of isolates and determining clonal transmission during outbreaks [111,128,189].

Over the past two decades, significant advances have been made with the development of novel, more accurate and rapid molecular methods, including multiplex polymerase chain reaction (PCR) assays, T2 Magnetic Resonance (T2MR) technology, matrix-aided laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), and WGS have significantly improved pathogen identification and characterization and outbreak surveillance [1,152,153,154,187,188]. Utilization of these techniques can guide clinical decisions and limit the prolonged empirical antibiotic use. However, the increased cost and the limited availability of these techniques significantly reduce their widespread use [1]. Optimization of molecular methods, the increased availability of these techniques for a wider range of institutions, and the development of new methods to assess virulent and pathogen susceptibility are future goals to improve sepsis diagnosis and outbreak control [187].

7. Evolution of Antimicrobial Therapy

Over the past two decades, there have been no significant changes in the empirical antibiotic treatment regimens employed in NICUs for both EOS and LOS cases. However, the increasing antimicrobial resistance led to important changes, including the introduction of newer agents and the implementation of antimicrobial stewardship programs.

Despite the introduction of novel antimicrobial agents against multidrug-resistant pathogens, treating multidrug-resistant infections in NICUs remains challenging due to the limited therapeutic options approved for neonates. Novel beta-lactam/beta-lactamase inhibitors, effective against multidrug-resistant gram-negative pathogens, ceftazidime/avibactam and ceftolozane/tazobactam, have been recently approved for use in neonates [75]. Older drugs, such as colistin and fosfomycin, have been reintroduced in clinical practice to combat resistant pathogens [184].

Although the association between broad-spectrum antibiotic use, prolonged antibiotic courses, and the emergence of antibiotic resistance had already been recognized in the early 2000s, the systematic implementation of antimicrobial stewardship programs in NICUs has largely occurred in recent years [190]. Antimicrobial stewardship programs aim to optimize antibiotic use and reduce unnecessary antibiotic exposure by implementing a series of strategies [13]. Minimization of treatment duration in confirmed sepsis, de-escalation of therapy based on susceptibility tests, empirical treatment selection based on local susceptibility patterns, and early cessation of antibiotics in unconfirmed sepsis are the cornerstones of these strategies [6].

Evidence from studies on the implementation of antimicrobial stewardship programs in the NICU is encouraging. Kahn et al. reported a 34.1% reduction in antibiotic use at NICU admission and a 45.3% reduction in antibiotic use beyond the 72 hours following the implementation of a stewardship program [191]. Several studies have demonstrated that a reduction in the duration of antibiotic treatment can be achieved through stewardship measures [191,192,193,194,195]. A recent meta-analysis of 70 studies reported a significant reduction in antibiotic initiation, duration of therapy, and antibiotic administration for more than five days. Overall, a 20% reduction in antibiotic days and a two-day reduction in treatment length were observed [190].

8. Discussion

Antimicrobial resistance is not a recently emergent phenomenon; rather, it is a persistent and increasing threat. As early as 1945, Alexander Fleming predicted that the inappropriate use of penicillin could lead to the emergence of resistant species. He was confirmed in the early 1950s, when penicillin-resistant Staphylococcus aureus had emerged and disseminated globally [196].

The comparison between the two periods, separated by two decades, highlights that antimicrobial resistance is an evolutionary phenomenon, dependent on the selective pressure exerted by the antibiotic prescribing patterns. The evolution of resistance can be evidenced by the emergence of ESBLs, a consequence of the widespread use of ampicillin and third-generation cephalosporins, and by the subsequent emergence of carbapenem-resistant strains due to increased carbapenem use. Similarly, the extensive use of vancomycin to treat MRSA has led to the recent emergence of VISA and VRSA strains. This represents the most important lesson from this comparison: the extensive use of an effective drug or even the development of a new agent does not prevent the emergence of resistance. Instead, preserving the effectiveness of antibiotics through rational use remains the most effective strategy to limit the emergence of resistant pathogens [33,190]. It is evident that the consequences of irrational antibiotic use are becoming increasingly apparent. Pathogens regarded as emerging two decades ago are now considered endemic, and we are confronted with multidrug-resistant species.

Although the consequences of irrational antibiotic use have been recognized since the 2000s, in the last decade, there has been considerable effort by NICUs worldwide to implement antibiotic stewardship programs and limit unnecessary antibiotic use. However, the effective implementation of antimicrobial stewardship in NICUs is primarily challenged by the nonspecific clinical presentation of neonatal sepsis and the lack of specific biomarkers in daily clinical practice [197,198]. Because prompt administration of antibiotic therapy is critical for survival and reducing morbidity, empirical antibiotic therapy is routinely administered in suspected cases of sepsis, and prolonged antibiotic courses are also frequently given despite negative cultures [196,197,199,200]. Despite efforts over the past decades, the ideal biomarker with high sensitivity and specificity has not yet been identified and should be prioritized in future studies, as it is a prerequisite for targeted antibiotic use [29].

A further challenge to antimicrobial stewardship in neonates is the limited antibiotics approved for neonatal use against multidrug-resistant pathogens. Although several novel agents have been approved against multidrug-resistant sepsis for adult patients, with few exceptions, these agents are not labeled for use in neonates [196]. Despite the progress made over the past two decades and the expansion of therapeutic options for neonates, there remains a pressing need for further research and development. It is urgent that clinical trials are conducted in neonates to provide a basis for safe and effective use of these agents. Thus, providing clinicians with the means to combat resistant pathogens.

Infection prevention and the rational use of antibiotics are key to disrupting the vicious cycle of resistance. As early as 2000, the CDC recognized that preventing antibiotic resistance comprises four components: infection prevention, effective diagnosis and treatment, appropriate antibiotic use, and prevention of transmission [196]. Although these principles have been established for decades, antimicrobial resistance has continued to emerge and disseminate, highlighting implementation gaps. It is therefore imperative to strengthen adherence to infection prevention and antibiotic stewardship to effectively limit the future burden of resistance.

9. Conclusion

Over the past few decades, antimicrobial resistance has significantly challenged the management of neonatal sepsis. Broad-spectrum antibiotics and prolonged antibiotic courses are established factors predisposing to the emergence of multidrug-resistant pathogens.

Antimicrobial resistance is not static but rather an evolutionary process, driven by the selective pressure exerted by widely used antibiotics in a particular era. In the early 2000s, although resistance to aminoglycosides was uncommon, concerns were raised about the efficacy of standard empirical regimens for neonatal sepsis. The emergence of resistant pathogens in NICUs, including ESBL-producing Enterobacterales, VRE, MRSA, and CoNS, was associated with increasingly reported outbreaks in institutions worldwide. The significance of horizontal pathogen transmission in the outbreak context was established, and prevention measures were implemented in clinical practice, including active surveillance cultures, cohorting and infection control measures.

The increasing prevalence of antibiotic-resistant strains was associated with widespread use of vancomycin, third and fourth-generation cephalosporins, and carbapenems, further driving the selection and emergence of resistant strains. In the 2020s, the widespread dissemination of multidrug-resistant pathogens has been documented. MRSA and ESBL-producing Enterobacterales have become endemic in many NICUs. The increased use of carbapenems in NICUs has contributed to the emergence of carbapenem-resistant pathogens. MRSA and CoNS are associated with increased vancomycin use, contributing to the increasing prevalence of VRE and the emergence of VRSA and VISA.

Despite novel antimicrobial agents and repurposing of older drugs, the management of multidrug-resistant sepsis in neonates remains challenging, as the therapeutic options are limited and multidrug-resistant sepsis is associated with significant morbidity and mortality. Reducing inappropriate and unnecessary antibiotic use is considered the most effective strategy for mitigating antimicrobial resistance. In recent years, antimicrobial stewardship programs have been implemented in NICUs worldwide and combined with advances in molecular diagnostic techniques that enable rapid pathogen identification, have been recognized as key strategies to limit antibiotic selective pressure and improve outbreak control in NICUs.

The evolution of antimicrobial resistance over the past two decades indicates that future efforts should focus not only on developing new antimicrobial agents but also on preserving available antibiotics. Thus, priority should be given to antibiotic stewardship, the identification of reliable biomarkers, wider access to rapid molecular diagnostics, and the development of new agents effective against multidrug-resistant pathogens. A coordinated strategy that combines enhanced surveillance, infection prevention, antibiotic stewardship, and research focused on the diagnosis and management of multidrug-resistant neonatal sepsis is warranted to limit the spread of multidrug-resistant pathogens and the emergence of new resistant strains.