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Clinical Microbiology of Bacterial and Fungal Sepsis in Very-Low-Birth-Weight Infants

Department of Pediatrics, Division of Neonatology, University of Virginia Health System, Charlottesville, Virginia,¹ Department of Pediatrics, Division of Neonatology, University of Maryland School of Medicine, Baltimore, Maryland²

SUMMARY

INTRODUCTION

MICROORGANISMS AND VERY-LOW-BIRTH-WEIGHT INFANTS: SCOPE OF THE PROBLEM

DEFINITIONS OF SEPSIS AND FOCAL INFECTIONS IN VERY-LOW-BIRTH-WEIGHT INFANTS

DIAGNOSIS OF SEPSIS IN VERY-LOW-BIRTH-WEIGHT INFANTS

Use of Adjunct Laboratory Tests To Predict Septicemia

Nonculture Microbiological Methods for Predicting Bloodstream Infection

Antigen detection.

Molecular diagnostics.

Routes of Infection

Susceptibility of Very-Low-Birth-Weight Infants to Infection

BACTERIA

Gram-Positive Organisms

Group B Streptococcus

Staphylococcus aureus.

Coagulase-negative staphylococci.

Enterococcus species.

Group A, C, D, and G Streptococcus species.

Listeria monocytogenes.

Gram-Negative Organisms

Enterobacteriaceae. (i) Escherichia coli

(ii) Enterobacter, Klebsiella, and Serratia species.

(iii) Citrobacter species.

Members of other families. (i) Pseudomonas species.

(ii) Haemophilus influenzae.

Anaerobic Bacteremia

Necrotizing Enterocolitis and Infection

Focal Intestinal Perforation

Concurrent Bacteremia and Fungemia

Antibiotic Resistance in Gram-Negative Pathogens

Mortality and Morbidity Associated with Bacterial Sepsis

Treatment of Bacterial Sepsis

FUNGAL ORGANISMS

Host Defense against Fungal Infection

Fungal Colonization

Risk factors.

Incidence.

(i) Skin.

(ii) Gastrointestinal tract.

(iii) Respiratory tract.

Fungal Infections

Congenital candidiasis.

Mucocutaneous candidiasis.

Fungal sepsis.

(i) Risk factors.

(ii) Candida species causing fungal sepsis.

(iii) Timing.

(iv) Clinical presentation.

(v) End-organ dissemination.

Outcome of invasive fungal infection. (i) Neurodevelopmental outcome.

(ii) Mortality.

Treatment of Candida sepsis. (i) Antifungals.

(ii) Central vascular catheter removal.

(iii) Empirical treatment

Malassezia, Trichosporin, Aspergillus, and Zygomycetes

PREVENTION OF NOSOCOMIAL SEPSIS IN VERY-LOW-BIRTH-WEIGHT INFANTS

Bacterial Infections

Necrotizing Enterocolitis

Invasive Fungal Infections

Oral antifungal prophylaxis.

Fluconazole prophylaxis.

Azole resistance.

Mortality associated with fluconazole prophylaxis.

CONCLUSION

REFERENCES

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INTRODUCTION

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Immature host defense mechanisms and invasive life support systems make the premature neonate particularly susceptible to overwhelming infection. Approximately 20% of very-low-birth-weight (VLBW) (birth weight <1,500 g) preterm infants experience a serious systemic infection during their initial hospital stay (441, 454, 457). While advances in neonatal intensive care have resulted in improved survival of preterm infants, mortality is as much as threefold higher for VLBW infants who develop sepsis than for those without sepsis (136, 454). In fact, sepsis accounts for approximately half of all deaths beyond the second week of life in VLBW infants (452). While the past decade has been marked by a significant decline in early-onset group B streptococcal (GBS) sepsis in both term and preterm neonates, the overall incidence of early-onset sepsis has not decreased in many centers, and several studies have found an increase in sepsis due to gram-negative organisms (95, 283, 454, 473). Infections with multidrug-resistant organisms (62, 231, 454, 473) and Candida (454) are also increasing in incidence. This review focuses on the bacterial and fungal organisms causing perinatally acquired and nosocomial sepsis in VLBW neonates and the various efforts to prevent infection in this vulnerable population. In addition, we discuss nonculture methods of predicting or detecting infection that may in the future enable clinicians caring for these infants to limit the use of empiric antibiotics and facilitate earlier detection of life-threatening infections.

MICROORGANISMS AND VERY-LOW-BIRTH-WEIGHT INFANTS: SCOPE OF THE PROBLEM

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Recent data indicate that VLBW infants account for approximately 1.4% of all live births in the United States, or about 56,270 infants per year, and about one-third of this group are extremely low birth weight (ELBW) (birth weight <1,000 g) (18). While it is well known that the incidence of neonatal sepsis is inversely proportional to birth weight and gestational age, it is only recently that studies of neonatal sepsis have addressed VLBW and ELBW infants separately from other preterm and term infants. It is important to bear this in mind when interpreting the literature on neonatal sepsis, since the incidence of sepsis in term neonates is around 0.1%, compared to the incidence among all VLBW neonates of approximately 20% (454). With decreasing birth weight comes increasing risk of sepsis, since only 10% of infants with birth weights between 1,000 and 1,500 g develop sepsis compared with 35% of infants with birth weights of <1,000 g and 50% of infants with birth weights of <750 g. Some studies group infants according to gestational age, a more accurate determinant of immune function but a more subjective criterion than birth weight. While the two are usually directly related, factors such as intrauterine growth restriction may lead to a small-for-gestational-age (SGA) VLBW infant whose immune competence and risk for infection are more in line with gestational age rather than with birth weight. Immune competence and other sepsis risk factors vary widely between, for example, a 24-week 600-g infant, a 30-week 1,250-g infant, and a 37-week 1,200-g SGA infant, all of whom by definition are VLBW infants. Immature host defenses (Table 1) appear to play a larger role in risk of infection in infants of lower birth weight (<750 g) and gestational age (<28 weeks), whereas for more mature infants, other risk factors such as abdominal surgery or the presence of a central venous catheter or endotracheal tube are more important in identifying high-risk patients. Hence, studies that examine sepsis by categories of 100-g increments of birth weight or by narrow ranges of gestational age contribute the most to our understanding of sepsis in the VLBW infant.

Recent data on the epidemiology of sepsis in preterm neonates in the United States may be obtained from several clinical consortia. The National Institute of Child Health and Human Development (NICHD) Neonatal Research Network, composed of approximately 15 neonatal intensive care units (NICUs), prospectively collects epidemiologic data on EONS and LONS sepsis in VLBW neonates (Table 2) (453, 454). In the most recent NICHD surveys, from 1998 to 2000, 1.5% of VLBW infants had EONS, defined as a positive blood culture with clinical symptoms (453). Given the widespread use of intrapartum antibiotics with preterm labor, the incidence of clinical sepsis (symptoms and abnormal laboratory findings with no growth from blood culture) is significantly higher than the incidence of culture-proven sepsis. LONS is over 10 times more common than EONS in VLBW infants (Fig. 1). The NICHD reported a 21% incidence of blood culture-proven LONS among VLBW infants (454). The incidence is higher among infants of <25 weeks gestation, with 46% of these infants suffering from LONS (454). Similar rates of sepsis in VLBW infants have been reported by other clinical consortia, with a range of 11 to 30% (61, 85, 451); (Vermont Oxford Network 2001 Database Summary). The Vermont Oxford Network reported that, among over 30,000 patients with birth weight of 501 to 1,500 g admitted to their hospitals in 2001, the incidence of EONS and LONS was 2 and 21%, respectively (Vermont Oxford Network 2001 Database Summary). The Centers for Disease Control and Prevention collect data on neonatal nosocomial infections through the Pediatric Prevention Network and the National Nosocomial Infections Surveillance (NNIS) System. In a point prevalence survey of 29 hospitals in 1999, the Pediatric Prevention Network found that the prevalence of NICU-acquired bloodstream infection was 11% among VLBW neonates (172). Relating infection to particular high-risk interventions, such as the presence of central vascular catheters, can provide useful information for comparisons of interinstitutional variations in sepsis rates. An NNIS survey of 138 high-risk nurseries, conducted between 1995 and 2001, reported the incidence of umbilical and central line-associated bloodstream infections to be 11.3 per 1,000 catheter-days for neonates with birth weight of <1,000 g and 6.9 per 1,000 catheter-days for infants with birth weight between 1,000 and 1,500 g (335a). Similar numbers were recently reported by the Canadian Neonatal Network (85).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Timing of bacterial and fungal sepsis in VLBW infants. Percentages indicate the approximate number of VLBW infants with septicemia. EONS usually occurs via ascent of organisms from the birth canal to the amniotic fluid, with or without rupture of amniotic membranes. LONS occurs with vertical and horizontal spread of organisms. While the vast majority of cases of sepsis in VLBW infants occur in the first 30 days of life, VLBW infants requiring prolonged intensive care are at risk for VLONS beyond 2 months of age.

While the majority of VLBW infants have only one episode of culture-proven sepsis during their NICU hospitalization, 20% have two events, 6% have three, and 2% have four (454). Multiple sepsis episodes are more common in the lowest-birth-weight categories, with almost 40% of infants with birth weights of <750 g having two or more episodes. Also striking is that only one of every five evaluations for sepsis with a blood culture yielded a microorganism. This underscores the finding, that 80% of the time, empiric antibiotics will be given when no organism is isolated from culture (454).

DEFINITIONS OF SEPSIS AND FOCAL INFECTIONS IN VERY-LOW-BIRTH-WEIGHT INFANTS

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Isolation of an organism from a blood culture of a neonate with clinical symptoms of infection constitutes the common definition of sepsis. Due to technical constraints, often only a single peripheral blood culture is obtained from a septic-appearing neonate, and in most studies the isolation of an organism from one blood culture is considered evidence of sepsis. In the case of coagulase-negative Staphylococcus (CoNS), which is both a common cause of sepsis and a frequent blood culture contaminant, many recent studies require either isolation from two blood cultures or a single positive blood culture with other laboratory evidence of sepsis, such as an elevated serum C-reactive protein level (CRP).

Many neonates with strong clinical indicators of sepsis, including severe apnea, lethargy, and hypotension (136), and laboratory abnormalities such as neutropenia, bandemia, and elevated CRP levels, have a negative blood culture. For this reason, some published studies of neonates include patients with the loosely defined entities "clinical sepsis" or "probable sepsis," either as a separate group or together with culture-proven sepsis. In these patients, the blood culture may be falsely negative or the patient may be experiencing a systemic inflammatory response due to a viral infection or noninfectious process. An official classification scheme has been adopted for pediatric and adult patients, with four categories of sepsis: systemic inflammatory response syndrome, sepsis, septic shock, and severe sepsis (284). However, no similar classification scheme is in routine use for neonates. Development of a "neonatal sepsis scale" including sepsis with shock and a more rigorous definition of "clinical sepsis" in neonates will allow better correlation with outcomes and a more consistent interpretation of epidemiologic and clinical research studies and should be a priority for those performing research in the field of neonatal sepsis.

Focal infections of the skin, urinary tract, lungs, central nervous system (CNS), and intestinal tract are also common in preterm infants and may occur with or without a positive blood culture. While a comprehensive discussion of focal infections in preterm infants is beyond the scope of this review, definitions and brief descriptions of their association with sepsis are warranted.

Urinary tract infection (UTI) is generally defined as growth of at least 100 CFU per ml from a specimen obtained by suprapubic bladder aspiration or at least 10,000 CFU per ml from a sterile bladder catheterization (40, 124). Lower colony counts may be indicative of infection in preterm infants, particularly if the isolate is a gram-negative organism or Candida, and treatment should be considered due to the high risk of ascending infection and sepsis. Urine culture is not indicated in the evaluation of EONS since the urine is sterile in the first 48 to 72 h of life (464). However, urine culture should be obtained in all evaluations of sepsis beyond day 3 of life, since the clinical presentations of UTI and sepsis are similar. In a study evaluating VLBW infants with suspected sepsis, the incidence of UTI was 12.2% in ELBW infants and 5.7% in infants with birth weights of 1,000 to 1,500 g, for a combined incidence of 8.1% in all VLBW infants (124). In this study, vesicoureteral reflux was present in 14% of VLBW infants with UTI (124); therefore, a voiding cystourethrogram and renal ultrasound should be considered for preterm infants with a UTI.

Any isolation of a microorganism from a cerebrospinal fluid (CSF) culture is generally considered evidence of meningitis in preterm infants, regardless of the CSF cell count or chemistries. Diagnosis of meningitis is problematic in VLBW infants since the CSF white blood cell count is an unreliable indicator of meningitis in these infants and the lumbar puncture is often delayed until after antimicrobials have been administered, when the patient is judged to be more stable (114). There are no data in preterm infants examining trends in CSF pleocytosis after antibiotics have been administered. Some clinicians do not routinely perform lumbar puncture in preterm infants with suspected sepsis unless there is growth of an organism on blood culture (142, 497). Since meningitis may occur without septicemia and since blood cultures may be falsely negative, this places VLBW infants at risk of being undertreated for meningitis (454a).

Pneumonia remains the most difficult infection to diagnose in preterm infants (17, 34, 94). In intubated VLBW infants, bronchopulmonary dysplasia and chronic lung disease exhibit similar clinical signs and symptoms, timing, and radiographic changes compared with pneumonia. Organisms cultured from the endotracheal tube often represent colonization rather than pneumonia. Quantitative cultures from endotracheal tube aspirates may reveal a large number of organisms, and Gram stain may demonstrate many white blood cells at times when there are no radiographic or clinical symptoms of pneumonia (123, 232). Better methods of assessing the microorganisms in the lungs are needed because this is probably a very common site of infection in intubated VLBW infants (34, 94).

Necrotizing enterocolitis (NEC), while multifactorial in etiology, is frequently associated with either clinical or culture-proven sepsis in VLBW infants. NEC is commonly defined using Bell's criteria (41), which are based in large part on radiographic findings. However, several studies have found that radiography and ultrasonography have a lower sensitivity than expected in detecting pneumatosis intestinalis, portal gas, and intestinal perforation in VLBW infants with NEC (263, 293, 463). The more extremely preterm infants, as well as some VLBW infants, particularly those who have never been fed, may present with a gasless abdomen, and clinical symptoms and laboratory data may be just as vital as imaging studies in determining the medical or surgical management of these infants.

DIAGNOSIS OF SEPSIS IN VERY-LOW-BIRTH-WEIGHT INFANTS

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The diagnosis of neonatal sepsis begins with clinical suspicion (136). The challenge for the neonatal practitioner is to decide which babies need empiric antibiotic therapy and for how long, a decision which is complicated by the common occurrence and nonspecific nature of sepsis-like symptoms in preterm infants. The prevalence and predictive value of common signs and symptoms of sepsis were described in a study by the NICHD Neonatal Network (136). The most common presenting symptoms in VLBW infants undergoing evaluation for suspected sepsis were increased apnea (55%), gastrointestinal problems (46%), increased need for oxygen or ventilatory support (36%), and lethargy/hypotonia (23%). The positive predictive value of these signs was low for culture-proven septicemia, ranging from 14 to 20%. The strongest predictor of septicemia in this study was hypotension, present in fewer than 5% of infants, which had only a 31% positive predictive value.

The "gold standard" for diagnosing neonatal sepsis remains the blood culture, even though, in many cases, blood cultures are negative in the face of strong clinical indicators of septicemia and even in autopsy-proven disseminated bacterial or fungal infection. In a 1999 autopsy study of ELBW infants, infection was deemed the primary cause of death by pathologists in the majority of infants (56 of 111), while sepsis was not diagnosed prior to death for 61% of these neonates (38). Maternal antibiotics given in the majority of preterm deliveries may suppress the growth of bacteria in culture, yet the neonate may have clinical symptoms and laboratory findings consistent with a diagnosis of sepsis. False-negative blood cultures in apparently septic neonates may also result from insufficient sample size. One study estimated that as many as 60% of blood cultures would be falsely negative for common neonatal pathogens if only 0.5 ml of blood is sampled in low-colony-count (<4 CFU/ml) sepsis (251). While neonates are commonly thought to have high-colony-count bacteremia compared with adults, as many as half of the neonates in one study were found to have low-colony-count bacteremia (251). Furthermore, in a prospective study of nearly 300 blood cultures drawn from critically ill neonates, 55% of culture vials contained less than 0.5 ml of blood (337). Technical difficulties associated with phlebotomy in small, sick preterm neonates often limit the volume of blood obtained and thus decrease the sensitivity of blood culture for diagnosing sepsis in this population.

In recent years, neonatal studies have reported that abnormal heart rate characteristics (HRC), defined as lack of heart rate variability and transient decelerations, occur 12 to 24 h prior to the onset of other clinical signs in infants with sepsis and sepsis-like syndrome (169, 170). There is speculation that cytokines, whose levels are elevated as much as 48 h before onset of sepsis symptoms, may be responsible for these changes in HRC (272). Griffin and coworkers have suggested that continuous monitoring of HRC may allow the earlier detection of infection and initiation of antibiotics, potentially improving outcomes. In a study of 633 NICU patients, an HRC index was developed and validated externally at another institution and was able to identify infants with a five- to six-fold-increased risk for developing sepsis or a sepsis-like illness in the following 24 h (169, 170).

Molecular diagnostics. PCR has proved to be a valuable adjunct for detection of neonatal viral infections such as human immunodeficiency virus, herpes simplex virus, and hepatitis C virus when used in conjunction with other diagnostic testing such as serologic testing or culture. However, the use of PCR to detect bacteremia and fungemia is more challenging and thus is still under investigation. Detection of bacterial DNA in the blood has been accomplished by PCR amplification of the gene for 16S rRNA, a gene universally present in bacteria but absent in humans. Detection of as few as 10 organisms per ml of whole blood has been reported, and research into how to enhance the sensitivity and automate the PCR is under way (314). A major concern about bacterial PCR is possible contamination due to the widespread presence of bacterial DNA in the environment, which may be a major stumbling block to clinical applications. However, several studies with neonates have shown promising results. In the largest study to date of PCR for detection of neonatal bacteremia, Jordan and colleagues collected 548 paired blood specimens from patients admitted to the NICU with suspected sepsis. Of 25 specimens with positive blood cultures, 24 were positive by 16S rRNA PCR. PCR results were available within 9 h of sample acquisition, and subsequent DNA hybridization was able to distinguish gram-positive from gram-negative organisms. Only 3 of 548 samples were positive by PCR and negative by blood culture. The sensitivity, specificity, positive predictive value, and negative predictive value of this PCR were 96, 99.4, 88.9, and 99.8%, respectively (230). In this study, blood samples were cultured for 4 to 6 h prior to hydribization and PCR to increase the sensitivity of the assay. Two smaller studies of neonates have shown similar high sensitivity of whole-blood 16S rRNA PCR for detection of culture-proven bacteremia (273, 431). While these are encouraging results, further large clinical studies are necessary to determine whether PCR will be a useful adjunctive test to rapidly detect the presence or absence of bacteremia in high-risk neonates. If the high negative predictive value of PCR is substantiated in larger trials, this test may be particularly useful for avoiding the overuse of empiric antibiotics in patients with nonspecific symptoms common to both septic and nonseptic preterm neonates.

PCR detection of fungemia is another important area of investigation. Preterm infants are at high-risk of morbidity and mortality from invasive Candida disease, and earlier treatment of Candida sepsis, including central vascular catheter removal, may be associated with an improved outcome (146). PCR assays to detect a variety of genes present in multiple species of fungi have been developed and tested both in vitro and in blood samples from patients at risk for fungal sepsis. Einsele et al. used PCR for the 18S rRNA gene, which is present in nearly all clinically relevant fungal species, to test over 600 blood samples from adult cancer patients with febrile neutropenia and healthy controls (122). Compared to blood culture, PCR was 100% sensitive and 98% specific for detection of invasive fungal infection. Two studies have reported the use of PCR for detecting fungemia in pediatric patients. Jordan and colleagues reported the detection of fungal DNA in 26 of 27 pediatric blood samples that were culture positive for Candida, using PCR amplification of the chitin synthase gene involved in fungal cell wall formation (229). Our group recently reported that, in NICU and pediatric ICU patients with suspected sepsis, PCR for the panfungal 18S rRNA gene was positive in all nine paired blood samples from which blood cultures yielded Candida species (468). Furthermore, all 44 samples negative for fungal DNA by PCR were associated with a negative blood culture for Candida. We also identified three patients with positive fungal PCR and negative blood culture who had other evidence of invasive fungal disease, including two NICU patients with candiduria and signs of sepsis and one patient with NEC, intestinal perforation, and Candida peritonitis. While animal studies suggest that PCR may be more sensitive than blood culture for the detection of fungemia (480), further studies are necessary to determine whether this may be the case in humans as well. Positive fungal PCR results may indicate true invasive disease, transient fungemia that will clear without therapy, presence of dead organisms in the bloodstream, or specimen contamination during collection or processing. Jordan found that blood samples from 29 neonates at high risk for Candida sepsis, but with no clinical evidence of disease, as well as blood from individuals with mucosal colonization, were negative by fungal PCR. In contrast, our study identified seven patients with negative blood culture and no other evidence of invasive fungal disease whose blood samples were positive for fungal DNA by 18S rRNA PCR. Further refinements of PCR methods, including the use of newer methods such as quantitative real-time PCR, together with large clinical trials, are necessary to determine whether PCR will be a useful tool to rapidly predict the presence or absence of disseminated fungal or bacterial infection in high-risk patients with sepsis-like symptoms.

Another promising approach to molecular diagnosis of sepsis involves the use of DNA microarray technology (343a). Devices are being developed which incorporate automated DNA analyzers with integrated sample preparation and biosensing elements which could allow rapid and sensitive detection of small numbers of organisms in minute volumes of blood. In the near future, microchip detection of bloodstream infection may allow practitioners to tailor antibiotic therapy toward specific pathogens and avoid unnecessary antibiotic therapy and its negative consequences.

LONS most commonly occurs via horizontal or nosocomial transmission, but it may occur via vertical transmission at birth, leading to colonization and, later, to infection. Skin or mucosal colonization with potential pathogens may be acquired from the hands of health care workers or family members, from water used in incubator or ventilator humidification systems, or from contaminated fomites such as stethoscopes, which may carry organisms directly from one patient to another. Colonizing organisms may enter the bloodstream through breaks in the skin or mucosa or by gastrointestinal translocation or may be introduced through invasive devices such as vascular catheters, endotracheal tubes, or feeding tubes. Alternately, nosocomial infection may result from infusion of contaminated intravenous solutions (especially lipid-based or high-glucose solutions) or from contaminated formula or breast milk.

In addition to the compromised skin barrier, the respiratory and gastrointestinal tracts of the VLBW infant provide surfaces for microbial colonization and invasion. These mucosal surfaces are both colonized and injured by nasogastric and endotracheal tubes and suction catheters. Mucous membrane defenses such as secretory immunoglobulin A (IgA), mucin, and defensins have been shown in some studies to be deficient in VLBW neonates (306, 387). Feeding tubes may also serve as reservoirs for pathogens. In a study of 50 preterm infants in whom orogastric feeding tubes were changed once a week, bacteria were isolated from 117 of 125 weekly feeding-tube cultures (320). The most common isolates were Staphylococcus species and members of the Enterobacteriaceae, whereas Candida was rarely isolated. All seven patients whose orogastric tube cultures yielded >100,000 CFU/ml developed NEC. More frequent changing of enteral feeding tubes may reduce colonization and risk of infection in VLBW neonates.

Once organisms have breached the mucous membranes, immaturities in both innate and adaptive immunity make preterm infants particularly vulnerable to rapid spread of infection (reviewed in references 127, 272, 286 and 376a). Low levels of serum complement, fibronectin, and defensins, as well as abnormalities in cytokine production, have been demonstrated in newborns, with preterm infants exhibiting a greater degree and longer duration of these abnormalities compared with term newborns. Most maternal-fetal transfer of IgG occurs in the third trimester of pregnancy; therefore, infants born prior to 32 weeks' gestation have low levels of passively acquired antibody, in addition to limited production of type-specific antibody in response to invading pathogens (420, 446). Cellular deficiencies of chemotaxis, phagocytosis, and microbial killing further contribute to the the vulnerability of preterm neonates to overwhelming infection (reviewed in reference 127, 272, 286, and 376a).

It is well established that the incidence of sepsis is inversely proportional to birth weight and gestational age (453, 454). Infants who are SGA have been found to be at decreased risk for EONS (454) but at higher risk for LONS than their appropriately grown gestational age-matched peers (82, 437). This may be because SGA infants are susceptible to neutropenia associated with maternal preeclampsia or because they may require central venous catheters and parenteral nutrition for long periods due to a predisposition to feeding intolerance and NEC. Another factor that may contribute to sepsis risk is gender, with male infants showing a disadvantage in some studies. In a 1988 to 1991 survey of 2416 VLBW infants, the NICHD Neonatal Network found that males were nearly 50% more likely to develop LONS than were females (odds ratio [OR], 1.48; 95% confidence interval [CI], 1.17 to 1.88) (136). Of note, however, the more recent Neonatal Network surveys did not find a preponderance of males among VLBW infants with either EONS or LONS. Male gender is a risk for sepsis in adults, attributed at least in part to the immunostimulatory properties of estrogen and the immunosuppressive properties of testosterone (15). Neonates have low levels of sex hormones; therefore, any predisposition to sepsis among male preterm infants it is likely to be attributable to other factors. Adult females generally exhibit a more robust humoral and cell-mediated immune response, leading to increased risk of autoimmune disease and possibly providing some advantage against sepsis compared with males (445), although this has not been thoroughly studied in infants. Some have postulated that genes on the X chromosome regulating thymic development and antibody production may lead to a sepsis gender gap (416). The absence of a sepsis gender gap in more recent studies of VLBW infants may reflect the fact that with increasing survival of the smaller, more vulnerable preterm infants, other risk factors such as prolonged invasive life support systems have overshadowed the male gender risk.

In addition to endogenous risk factors, exogenous factors associated with intensive care, such as parenteral nutrition and medications, further contribute to the vulnerability of preterm infants to infection. Several in vitro studies have shown that exposure to lipid emulsion solutions impairs the chemotaxis of neonatal leukocytes (129, 264, 477), although ex vivo studies of serum or cells from neonates given infusions of lipid emulsion compared to nonexposed patients showed no differences in various immune functions (376a). Lipid solutions support the growth of microorganisms (481), and a number of epidemiologic surveys have found an association between lipid infusions and CoNS bacteremia (21, 148, 311) and fungemia (215, 402) (particularly with Malassezia furfur) (294, 384, 460). Although severe sepsis is associated with altered lipid metabolism, it is not known whether discontinuation of intravenous lipid therapy for 1 to 2 days (except for Malassezia infections [see below]) at the onset of sepsis will decrease the duration of septicemia or otherwise improve the immune response or outcome in VLBW neonates.

A number of pharmacologic agents commonly used in NICU patients have been implicated as risk factors for sepsis. A single course of maternal antenatal corticosteroids, which reduces pulmonary and neurologic morbidity in preterm neonates, has been associated with a decreased risk of EONS, even with premature rupture of membranes (OR, 0.52; 95% CI, 0.31 to 0.88) (507). The effect of antenatal steroids on LONS is less clear. While the NICHD Neonatal Network found an increased risk for LONS in infants exposed to antepartum corticosteroids (OR, 1.29; CI, 1.10 to 1.51) (507), other studies have not found this association (212, 499). In any case, the benefits of antenatal steroids, including decreased mortality, intraventricular hemorrhage, respiratory distress syndrome, and EONS, strongly outweight the risk for LONS. Postnatal systemic steroid treatment has been clearly associated with a higher risk of sepsis in preterm infants, with the incidence of LONS increasing to 33% in steroid-treated VLBW infants (228, 455, 511). While the use of dexamethasone for acute or chronic lung disease has declined due to concerns about adverse neurodevelopmental effects, hydrocortisone use in the most immature VLBW infants with pressor-resistant hypotension and adrenal insufficiency has increased. Practitioners must balance the potential benefits of glucocorticoids with the increased risk of infection in this population.

Preterm infants are commonly treated with histamine type 2 receptor (H₂) antagonists for prevention or treatment of stress-induced gastritis or for treatment of gastroesophageal reflux, yet these agents have immunomodulatory properties as well. H₂ antagonists impair neutrophil rolling in vitro (510), while other studies have shown the presence of immunostimulatory properties of H₂ antagonists (280, 343, 448). In preterm neonates, effects of H₂ antagonists and proton pump inhibitors on neutrophil function or cytokine production are probably overshadowed by their effect on pH and gastrointestinal tract colonization with potential pathogens. Gastric alkalinization increases bacterial and fungal proliferation in the small bowel (39, 455), and administration of H₂ antagonists has been associated with increased microbial growth in enteral feeding tubes (320) and hence with an increased risk of sepsis and NEC (402) in preterm neonates.

Another pharmacologic agent that may affect neutrophil function is indomethacin, which is commonly used in preterm neonates for closure of a patent ductus arteriosus and for prevention of intraventricular hemorrhage (IVH). Indomethacin has been associated with impaired neutrophil motility and chemotaxis, particularly in preterm infants (236). Although one prospective study found a temporal association of indomethacin treatment and sepsis (200), a more recent meta-analysis (144) and multicenter study found no association (144, 417). Vitamin E therapy has also been shown in a meta-analysis to decrease the risk of severe IVH and rationopathy of prematurity, but may be associated with an increased risk of sepsis in VLBW infants (60).

Finally, the frequent use of broad-spectrum antibiotics, particularly third-generation cephalosporins, in preterm infants alters the intestinal milieu, favoring colonization and infection with multidrug-resistant bacteria and fungi (11, 401, 439). The microflora of the skin and gastrointestinal tract limits fungal proliferation through its direct antifungal properties as well as through competition for adhesion sites and micronutrients (218). Efforts to minimize exposure to broad-spectrum antibiotics in preterm infants are warranted.

BACTERIA

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GBS sepsis was the first neonatal infection to be defined as EONS, LONS, and LLOS. The majority of LLOS GBS sepsis occurs in preterm infants at an age when the immune system is more mature; thus, mortality due to LLOS GBS sepsis is much lower than that presenting at earlier ages (220, 509). Infants may have persistent colonization from birth or may acquire the organism through nosocomial routes. Transmission of GBS from breast milk, patient-to-patient spread, and colonized nursery personnel has been reported (119, 350). Increased adherence of GBS to buccal epithelial cells from preterm compared to term infants may also be an contributing factor (97). Recurrent GBS sepsis after appropriate antibiotic therapy has also been documented, since treatment fails to eradicate colonization in up to 50% of infants, infants can be repeatedly exposed through breast milk or horizontal contact, and systemic infection does not stimulate the production of protective levels of type-specific antibodies, particularly in preterm infants (166).

In 1996, consensus guidelines aimed at reducing the incidence of GBS EONS were issued. Intrapartum antibiotic prophylaxis of women with GBS rectovaginal colonization or with specific risk factors for sepsis significantly decreased the incidence of GBS EONS among both term and preterm neonates. The Neonatal Network found that the incidence of GBS EONS in VLBW infants declined from 5.9 per 1,000 in 1991-1993 to 1.7 per 1,000 in 1998-2000 (450, 453). A recent study has shown that the screening-based approach is more effective than the risk-factor-based approach in preventing GBS EONS (419), and in 2002 the Centers for Disease Control and Prevention released new recommendations for universal GBS screening of pregnant women and intrapartum penicillin prophylaxis for carriers (419). While this approach is estimated to prevent over 85% of cases of GBS EONS, a number of concerns regarding widespread intrapartum antibiotic prophylaxis have been raised. With over 25% of all pregnant women and over half of those delivering preterm receiving intrapartum antibiotics, the incidence of infections with gram-negative bacteria and antibiotic resistance among gram-negative pathogens has increased in some centers (62, 231, 473). Emergence of penicillin resistance among GBS has not become a problem in the United States; however, approximately 20 to 40% of GBS isolates in some centers are resistant to clindamycin or erythromycin (particularly type V strains) (261, 307). A recent report from Japan describes clinical GBS isolates with intermediate sensitivity and with resistance to penicillin (329), but the clinical significance of this finding is unclear. Because of the risk of emerging resistance and the failure of intrapartum antibiotics to prevent GBS LONS as well as some cases of EONS, efforts to develop a multivalent GBS vaccine are ongoing (24).

Another research priority in the field of GBS disease prevention is the development of a rapid, sensitive, and inexpensive test to detect GBS colonization in women who present to the hospital in labor. A number of commercially developed assays for GBS antigen have been tested, including a recently developed optical immunoassay that appears to have higher sensitivity than enzyme immunoassays. While these tests have high sensitivity for detecting heavy GBS colonization, the overall sensitivity is much lower than that of selective broth culture (23, 403). Since approximately 15% of cases of neonatal GBS sepsis occur when mothers have only light GBS colonization, immunoassays do not currently have adequate sensitivity to be clinically useful. Molecular biology-based assays such as fluorescence in situ hybridization and PCR for rapid GBS screening are under development (20, 250).

Staphylococcus aureus. Staphylococcus aureus is a much less common cause of neonatal sepsis in recent decades than at its peak incidence in the 1950s through the 1970s. However, it can be a highly virulent pathogen in immunocompromised patients such as premature neonates. Extensive research has focused on the pathogenesis of S. aureus infection and is the subject of a recent review (205). Although S. aureus is more commonly associated with nosocomial sepsis, maternal-fetal infections have been reported. In a case series spanning 3 years from a single institution, seven preterm infants with congenital S. aureus infection were identified, including one with methicillin-resistant S. aureus (MRSA) (14). In all seven cases, amniotic fluid culture as well as initial blood culture of a sample from the infant yielded S. aureus, and in three cases, antenatal invasive procedures (amniocentesis or amnioinfusion) performed within a day of delivery were presumed to have contributed to infection of the fetus. Late-onset S. aureus infections in neonates include skin and deep-seated tissue abscesses, bacteremia/sepsis, endocarditis, septic arthritis, osteomyelitis, pneumonia, and meningitis (19, 224, 334, 365, 368). In addition, S. aureus is one of the most common etiologic agents of ventriculoperitoneal shunt infections in preterm infants with posthemorrhagic hydrocephalus (63, 106). Compared with other neonatal pathogens, S. aureus is associated with a relatively high incidence of deep-seated infection and suppurative complications. Of the pathogens responsible for pneumonia in preterm infants, S. aureus is the most likely to cause pneumatoceles and empyema, sometimes requiring chest tube drainage. S. aureus meningitis may be associated with brain abscesses, and neuroimaging is recommended to determine the duration of therapy. S. aureus endocarditis may occur in the absence of clinical signs such as a heart murmur, and some researchers have advocated routine echocardiography in preterm infants with S. aureus bacteremia, particularly those with a central venous catheter in or near the heart and those with two or more positive blood cultures.

S. aureus toxin-associated diseases have been reported in preterm neonates. Staphylococcal scalded skin syndrome (SSSS), in which S. aureus exfoliative toxins A or B split the granular layer of the skin, resulting in sloughing and erythema, has been found in NICU patients (301, 400). Although SSSS is not associated with severe systemic illness or bacteremia, nosocomial spread among NICU patients has been reported (400), and strict infection control measures should be implemented when a case is suspected. In contrast to the relatively benign nature of SSSS, toxic shock syndrome (TSS) due to S. aureus enterotoxins presents a more fulminant clinical picture. Criteria for diagnosing TSS include fever, hypotension, multiorgan system dysfunction, a diffuse macular rash leading to desquamation, and evidence against an alternative diagnosis. Two cases of mother-infant transmission of a TSS-like illness associated with S. aureus surface colonization have been reported (87, 167), although neither case met the strict definition of TSS. A Japanese group reported 20 infants with exanthema and thrombocytopenia in the first week of life, all of whom were colonized with TSS toxin 1 (TSST-1)-producing strains of MRSA, and they named this disease entity neonatal toxic shock syndrome-like exanthematous disease (NTED) (462). Further studies revealed that seven infants colonized with MRSA, but without NTED, had high titers of anti-TSST-1 IgG, compared with negligible titers in all four patients with NTED. These titers were measured during the acute phase of disease, suggesting that maternal anti-TSST-1 antibodies may be protective against TSS-like disease in neonates. Preterm infants born before transplacental transfer of maternal antibody may thus be at higher risk of acquiring S. aureus toxin-associated disease.

The vast majority of S. aureus clinical isolates produce ß-lactamases and are resistant to penicillin. Most S. aureus strains causing colonization and infection in NICUs have remained sensitive to extended-spectrum penicillins, and treatment with oxacillin or nafcillin usually eradicates infection. Persistent or deep-seated infections may require the addition of an aminoglycoside or rifampin for effective clearance (465). Methicillin resistance among S. aureus strains in NICUs has been reported and is commonly associated with episodic outbreaks from a single clone (486). Epidemics of MRSA infection have been associated with understaffing, overcrowding, improper cleaning of equipment and hands, and mixing of patients in the NICU (13, 182). Successful eradication of MRSA outbreaks has been accomplished by scrupulous attention to infection control measures as well as by intranasal mupiricin treatment of colonized patients and health care workers (22, 184, 202). Hexacholoraphene hand washing has also been used to control an MRSA outbreak in a NICU (383).

Vancomycin- or glycopeptide-intermediate S. aureus isolates with drug MICs in the 8-µg/ml range, have been detected since the late 1980s in adults (151, 291, 424); since 2002, vancomycin-resistant S. aureus strains have also been found (766). To date, there are no published reports of vancomycin-intermediate or vancomycin-resistant S. aureus isolated in neonates. With increasing use of vancomycin for treating documented MRSA and CoNS infection and as empiric therapy for suspected LONS, it is possible that vancomycin resistance among S. aureus strains will evolve in the NICU. One approach to preventing the emergence of resistant S. aureus strains is prevention of infection through active and passive immunization of susceptible hosts. Vaccination with S. aureus capsular polysaccharides 5 and 8, which account for up to 90% of infections (128, 295), has proven efficacious in adults (434), and hyperimmune intravenous immune globulin (IVIG) preparations against staphylococcal surface proteins are under investigation for use in VLBW infants.

Coagulase-negative staphylococci. CoNS are the etiologic agents of the majority of nosocomial infections in premature neonates. Although CoNS are common commensal organisms with little pathogenicity in immunocompetent hosts, premature neonates are particularly susceptible to invasive infection. The first step in the pathogenicity of CoNS involves adherence of the bacteria to skin, mucosal surfaces, or indwelling artificial devices, such as intravascular catheters and CNS shunts, which are commonly used in preterm infants. Adherence of CoNS is facilitated by a capsular polysaccharide adhesin consisting of poly- N-succinyl glucosamine (435, 469). Once adherence and colonization have been established, some CoNS produce an exopolysaccharide "slime," which allows the organisms to form a biofilm and evade host defense mechanisms and antibiotic activity. The major component of slime is polysaccharide intercellular adhesin (PIA), a linear homoglycan composed of N-acetylglucosamine residues (163). In one study of 179 strains of S. epidermidis, 51% produced PIA and most of these strains formed a biofilm (299). Genes encoding PIA are located in a gene cluster termed the ica (for "InterCellular Adhesion") operon.

The ability of CoNS to produce slime and biofilms has been linked to increased virulence in preterm infants. One longitudinal study of colonizing strains of CoNS found in 18 preterm neonates demonstrated slime production in 68% of strains on day 1 of life, 89% on day 4, and 95% on day 7 and later (104). In contrast, another study found an overall incidence of slime production of 50% in 105 nasopharyngeal colonizing strains of CoNS from 28 VLBW infants (187). This study did not find an increase in slime production over time in infants hospitalized from 4 to 15 weeks. Another study of 180 clinical isolates of CoNS from NICU patients found that although the ica operon was present in all isolates, quantitative biofilm production was greater among isolates from patients with sepsis than among those associated only with colonization (111). Mixed-species biofilms of S. epidermidis and Candida albicans may be particularly pathogenic to preterm infants. A recent report demonstrated that the slime produced by S. epidermidis inhibited the penetration of fluconazole into mixed fungal and bacterial biofilms and, conversely, that C. albicans protected staphylococci from the action of vancomycin (6). This may play a role in the large number of concomitant CoNS and Candida infections in VLBW infants, with one microbe facilitating colonization and infection with the other (see below).

Although slime and other virulence factors are important to the pathogenicity of CoNS, several studies did not find evidence of hypervirulent clones of CoNS causing disease in neonates (111, 335). However, in a study of 97 blood isolates of CoNS (29 considered sepsis isolates and 68 considered contaminants) from a single center over a 15-year period, sepsis isolates were phenotypically and genotypically more homogeneous than contaminating isolates, suggesting that disease-causing strains of CoNS may have a higher invasive capacity (53).

Of the 31 species of CoNS and the 13 known to colonize human skin, species reported to cause disease in infants include S. epidermidis, S. haemolyticus, S. hominis, S. warneri, S. saprophyticus, S. cohnii, and S. capitis. The major species involved in neonatal infection is S. epidermidis, which accounts for approximately 50 to 80% of CoNS colonization (52, 185, 407) and 60 to 93% of CoNS bloodstream infection (104). S. epidermidis colonization rates of 86 to 100% have been reported among NICU patients (104, 138, 187, 228). Occasionally CoNS is acquired from the mother at birth. Hall et al. showed that 51% of pregnant women were colonized with CoNS, and the proportion of slime-positive strains increased from 40 to 68% from the first to the third trimester of pregnancy (186). In this study, although 30% of neonates were colonized with CoNS at birth, in only three of these cases were the maternal and infant species identical based on biotype, antibiotic sensitivity pattern, slime production, phage type, and plasmid pattern profile. The majority of CoNS colonization is acquired nosocomially, predominantly from the hands of health care workers. In a survey using multiple molecular typing techniques, 62% of NICU nurses were colonized with methicillin-resistant CoNS, with similar species distribution to that of bacteremic strains in the unit (364).

Identification of intraspecies strains is important in investigating outbreaks of CoNS sepsis in the NICU, and a number of epidemiologic methods have been used in the past for strain identification, including antibiotic susceptibility testing, phage typing, and analysis of slime production. Newer studies have employed molecular typing of chromosomal or plasmid DNA using techniques such as restriction endonuclease fingerprinting, random amplification of polymorphic DNA, and pulsed field gel electrophoresis (10, 100, 339, 380). Since none of these tests has proven to have sufficient discriminatory ability when used alone, most epidemiologic studies have used a combination of two to four tests to identify related strains. For the most part, multiple strains of CoNS are found in nurseries and in individual patients at one time and over time (235, 338). However, endemic strains of CoNS can persist in NICUs for many years (217, 298, 440, 485, 486). One study found a higher proportion of distinct chromosomal patterns for S. epidermidis in blood cultures (7 of 11) than in mucocutaneous cultures (6 of 18), suggesting that the more invasive strains are more likely to become endemic (51). This study also found that S. haemolyticus strains were genotypically much more homogeneous than S. epidermidis strains, with 4 of 4 blood cultures and 15 of 15 surface cultures falling into two distinct chromosomal patterns.

While reports of S. epidermidis bacteremia on the first day of life suggest that the organism may be perinatally acquired (7, 449), it is more commonly a nosocomial pathogen. Neonates with intravascular catheters, particularly those with central vascular catheters which remain in place for prolonged periods, are at high risk for CoNS bacteremia. Another significant risk factor for CoNS septicemia is the administration of intravenous lipid infusions, which provide a growth medium for the organism (21, 148). Sepsis with CoNS is often indolent rather than fulminant, although fatalities have been reported (240). Neonates with CoNS bacteremia generally present with nonspecific symptoms such as decreased activity, increased apnea, and feeding intolerance. Since these symptoms are common even in nonseptic preterm neonates, a positive blood culture for CoNS may represent either contamination or true bacteremia, and for this reason, many studies require more than one positive blood culture (458) or other laboratory evidence of infection, such as an elevated CRP level (454), to distinguish CoNS bacteremia from contamination. In one prospective study of positive blood cultures for CoNS in infants younger than 6 months, only 25 of 59 episodes were considered to represent true infection, and hematologic indices were not found to be helpful in distinguishing between infected and uninfected neonates (335). A more recent study compared the use of two versus one peripheral blood culture for the diagnosis of CoNS sepsis in 100 neonates with suspected sepsis (458). While CoNS was isolated from 26 patients, in only 16 cases were cultures from two sites positive, and the other 10 cases were considered to represent contamination. Of note, three patients had only one of the two cultures positive for other organisms (S. aureus, Serratia marcescens, and E. coli), highlighting the challenge in isolating microorganisms from blood culture. This study, requiring two positive cultures for a diagnosis of CoNS sepsis, resulted in an 8.2% reduction in vancomycin usage, but 20 babies underwent a second blood collection and culture for every "false-positive" case of CoNS detected. In addition, the authors noted that due to the added time, expense, and discomfort involved in obtaining two peripheral-blood samples for culture, on completion of the study they reverted to the practice of using a single blood culture for preterm neonates with suspected sepsis.

In addition to being the most common cause of nosocomial bacteremia in NICU patients, CoNS may cause focal infections. The organism has been isolated from the CSF of septic preterm neonates, sometimes in absence of CSF pleocytosis (174). CoNS is among the common causes of ventriculoperitoneal shunt infection in VLBW infants (63, 106). An association of CoNS infection and NEC has been reported by some groups (174, 412), while other studies have not found this association (179, 392). Likewise, studies of CoNS delta-like toxin in neonates with NEC have yielded conflicting results (179, 413). Another nidus of infection with CoNS is the endocardium. Right-sided endocarditis due to S. epidermidis may be associated with placement of an umbilical venous catheter in the right atrium (347).

Resistance to antibiotics such as penicillin, semisynthetic penicillins, and gentamicin is common among clinical isolates of CoNS. One NICU study found an increase in multiple antibiotic resistance among colonizing CoNS strains from 32% at birth to 82% after the first week of life (104). Similarly high rates were reported by another group (187). Of particular concern is the emergence of strains of CoNS with decreased sensitivity to vancomycin (424). While this has not yet been described for neonates, heteroresistance to vancomycin has been reported. A recent report described a preterm neonate with persistent S. capitis bacteremia for 3 weeks despite treatment with vancomycin and replacement of all central venous catheters (479). The authors screened the patient's strain and 218 other strains of CoNS isolated from neonates over the previous 4 years. Using population analysis, the patient strain and 47 others proved to be heteroresistant to vancomycin. It is possible that heteroresistance to vancomycin is more common in the NICU than is currently realized, since standard susceptibility testing fails to detect subpopulations of the organism for which the MICs are higher (486). Development of vancomycin resistance has been linked to prolonged antibiotic pressure. In an adult oncology patient, for example, a colonizing strain of S. haemolyticus which was initially susceptible to vancomycin (MIC, 1 to 2 µg/ml) developed increased resistance (MIC, 8 to 32 µg/ml) and caused bacteremia after 34 days of vancomycin therapy (482). Given the potential severe sequelae associated with the emergence of vancomycin resistance, efforts should be made to limit vancomycin use in the NICU. Several studies have shown that initial therapy of suspected LONS with nafcillin or oxacillin and an aminoglycoside, rather than vancomycin and broad-spectrum cephalosporin, are not associated with increased morbidity or mortality, even in units in which the majority of CoNS are oxacillin resistant (240, 268, 312).

Enterococcus species. Although accounting for only a small proportion of neonatal sepsis, Enterococcus species deserve special mention because of the increasing incidence of neonatal enterococcal sepsis in several studies (89, 317) and the emergence of vancomycin resistance among enterococci. Both Enterococcus fecalis and E. faecium cause sepsis in preterm neonates, with E. fecalis accounting for over 80% of cases. McNeely et al. reviewed all cases of enterococcal bacteremia in NICU patients in a large metropolitan teaching hospital and found a three-fold increase in cases in 1983 to 1993 compared with the previous decade (318). In 100 cases reviewed, the mean age of onset was approximately 45 days and the mean gestational age was 31 weeks. Of note, 64% of patients had other organisms isolated concurrently from blood culture. Another study also found a significant proportion of polymicrobial bacteremia associated with enterococcal sepsis in 83 pediatric patients, including 16 neonates (89). This may be due to the common association with central vascular catheters or NEC, found in 77 and 33% of cases, respectively.

For reasons that are unclear at present, vancomycin resistance among enterococci has not become a significant problem in most NICUs (470), yet several studies have reported endemic or epidemic vancomycin-resistant enterococci (VRE) among hospitalized neonates. In the review by McNeely et al., six neonates had bacteremia with VRE, and one died of the infection (317). Interestingly, none of the six had been given prior therapy with vancomycin, although they all had prolonged NICU stays prior to the VRE infection (mean age, 100 days). Other studies have found an association of prior antibiotic use and colonization or infection with VRE. In one study, 68% of pediatric patients infected with VRE had been treated with vancomycin within 90 days of detection of the organism (317). A recent case report describes a 4-month old ELBW infant with a central venous catheter who, after three 10-day courses of vancomycin for various infections including CoNS and E. faecalis bacteremia, developed endocarditis with vancomycin-resistant E. faecium. The infection was successfully cleared by intravenous followed by oral linezolid therapy. Limited studies of neonates suggest that linezolid is well tolerated and as effective as vancomycin for the treatment of infections with resistant gram-positive bacteria (112, 233). Rare resistance to linezolid has been observed with prolonged therapy, and its use in neonates is still under investigation (237).

Rapid spread of VRE among NICU patients was documented by Malik et al., who reported the spread of related strains of VRE to 40% of the NICU patient population (305). Two preterm i