Exposure to antibiotics before birth may impair lung development in premature infants

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New research in mice suggests that exposure to antibiotics before birth may impair lung development in premature infants.

The study, the first to explore the gut-lung axis in prematurity, is published ahead of print in the American Journal of Physiology—Lung Cellular and Molecular Physiology and was chosen as an APSselect article for December.

Premature infants, particularly those who receive oxygen treatment soon after birth, are at high risk of developing lung problems characterized by scarring (fibrosis) and inflammation.

Emerging research suggests that the communities of bacteria in the digestive tract (gut microbiome) can impair development of the immune system and may also affect inflammation, which in turn plays an important role in lung disease.

The interaction among these body systems is called the gut-lung axis.

Antibiotics are known to change the makeup of the gut microbiome and are linked to an increased risk of lung injury.

Antibiotic treatment is common in premature babies, but how the gut is involved is not clear.

Looking at how antibiotics affect offspring – even before birth – may help researchers better understand the gut-lung axis.

In a new study, researchers exposed one group of pregnant mice to the widely used antibiotic penicillin in their drinking water (“treated dams”). Another group of pregnant mice received plain water (“control dams”).

After the babies were born, the research team used a cross-over experimental model to assess the effects of antibiotics on the offspring in combination with oxygen treatment. They looked at four groups:

  • One group was exposed to penicillin before birth and was fed by treated dams.
  • One group was exposed to penicillin before birth and was fed by control dams.
  • One group was not exposed before birth and was fed by treated dams.
  • One group was not exposed before birth and was fed by control dams.

The research team examined lung structure, including thickness of the walls, size of capillaries and scar tissue – together all of these affect breathing ability.

They also looked at the inflammation-causing cells and proteins in the lungs of all the animals.

Mice that were exposed to antibiotics before birth and fed by control dams had more fibrosis with oxygen treatment than mice exposed to antibiotics only after birth.

Offspring exposed to penicillin in the womb also had lower body mass and reduced capillary size compared with those not exposed before birth.

Prenatal exposure also altered levels of proteins that promote inflammation and immune function as well as those that affect microbial signaling in the lungs.

“Our study provides valuable experimental evidence that manipulation of the gut microbiota by antibiotic exposure influences the progression of lung injury,” the researchers wrote. These findings “may assist in the interpretation of future observational studies in human newborns examining the role of the gut-lung axis in [bronchopulmonary dysplasia].”


Neonatal sepsis remains a significant cause of morbidity and mortality, particularly among preterm and low birth-weight neonates (Thaver and Zaidi, 2009Shane et al., 2017). It is estimated that within 28 days of birth, 13% of neonatal mortality worldwide is caused by sepsis or meningitis (Liu et al., 2012). Neonatal sepsis is commonly divided into early-onset sepsis (EOS) and late-onset sepsis (LOS). EOS (≤3 days of birth) is generally associated with organisms transferred to the neonate from the mother while LOS (>3 days of birth) is usually associated with nosocomial or community acquired infections (Dong and Speer, 2015).

Diagnosing neonatal sepsis can be enigmatic—there is no consensus among clinicians or researchers. The current “gold standard” for diagnosis is a positive blood culture (Wynn et al., 2014).

Blood culture-confirmed sepsis is only one facet of the disease; suspected sepsis in culture-negative cases is still considered clinical sepsis (Lukacs and Schrag, 2012Wynn et al., 2014). Current recommendations call for empiric use of antibiotics before culture confirmation if sepsis is suspected, and possible continued antibiotic treatment if signs of sepsis persist when the culture is negative (Camacho-Gonzalez et al., 2013Shane et al., 2017).

Because of this approach, antibiotics are frequently administered in neonatal intensive care units (NICUs). In a study of 253,651 NICU neonatal records within the Pediatrix Medical Group database, the two most commonly administered drugs were ampicillin (69%) and gentamicin (58%); additionally, 18% of neonates were exposed to cefotaxime and 10% to vancomycin (Clark et al., 2006). With an increasing emphasis on antibiotic stewardship, it is essential to understand how neonatal sepsis is confirmed and treated, and to identify opportunities for reducing antibiotic use.

A retrospective analysis was conducted using data obtained from The University of Utah Health system, comprising four hospitals and 12 community clinics. Our objective was to thoroughly describe the blood culture confirmation and antibiotic treatment of neonatal sepsis within this hospital system. Antibiotics used to treat culture confirmed and unconfirmed sepsis are compared for both early and late onset sepsis over a 12-year period.

Methods

Collection of medical records for analysis was approved by the University of Utah Institutional Review Board (IRB_00091312). Records were collected from neonates born and/or admitted to the University of Utah Hospitals within 28 days of birth between January 1, 2006, and December 31, 2017, with ICD9 billing code 771.81 or ICD10 code P36 (newborn sepsis). Neonates with major birth defects such as spina bifida, gastroschisis, congenital heart defects (excluding patent ductus arteriosus and patent foramen ovale), Down syndrome, encephalocele, and microcephaly or neonates born from mothers with a family history of congenital immune disorders (x-linked agammaglobulinemia, x-linked neutropenia/myelodysplasia, common variable immunodeficiency, severe combined immunodeficiency, severe congenital neutropenia, Shwachman-Diamond syndrome, and Kostmann syndrome) were excluded from data collection.

We categorized these patients as suspected early-onset sepsis (EOS) when any blood culture was drawn and antibiotics were initiated ≤3 days of birth and suspected late-onset sepsis (LOS) when any blood culture was drawn and antibiotics were administered >3 but ≤28 days of birth and ≥7 days after a positive blood culture associated with confirmed EOS.

Neonates with suspected late or early-onset sepsis are not mutually exclusive, that is, a single neonate can be categorized as both suspected early and late-onset sepsis if it meets the criteria for both. Neonates suspected of having sepsis were further divided into two categories, confirmed sepsis and unconfirmed sepsis. These categories are mutually exclusive (i.e., a single neonate suspected of having LOS must be either confirmed or unconfirmed LOS).

Only blood cultures were used to confirm neonatal sepsis. Centers for Disease Control and Prevention (CDC) bloodstream infection criteria were adapted for use to confirm sepsis with some modifications (Horan et al., 2008).

According to these guidelines, if there was a recognized pathogen in a blood culture, sepsis was confirmed. If a common skin contaminant was cultured, and the organism was cultured again in the following 48 h, sepsis was also confirmed. If a common skin contaminant was cultured, and the organism was not cultured again in the following 48 h, then these cultures were possibly contaminated and these patients were excluded from analysis as ambiguous cases.

The following microorganisms were considered common skin contaminants: diphtheroids, Bacillus spp., Micrococcus spp., non-pathogenic Neisseria spp., gram-positive rods, coagulase negative staphylococci (CoNS), and non-pathogenic streptococcal species (e.g., Streptococcus viridans) (Hall and Lyman, 2006). We also analyzed cerebrospinal fluid (CSF) cultures.

Antibiotic exposure was measured by days of treatment (DOT) and length of treatment (LOT) (Ibrahim and Polk, 2014). DOT is measured by multiplying the number of doses by the dosing interval in days for each antibiotic administered. DOT is normalized to per 1,000 patient days (PD) which is the mean length of stay multiplied by 1,000. LOT is measured by the number of calendar days a patient received any antibiotic. LOT is normalized to per admission and per treatment course. LOT/course is equivalent to the mean number of days that neonates are treated for an episode of sepsis. Antibiotics administered topically were not included.

Treatment courses were calculated as continuous periods of antibiotic exposure with no gap in administration greater than or equal to two calendar days to accommodate for extended intervals of aminoglycoside dosing in extremely low birthweight newborns. In some cases, there was continuous administration of antibiotics from within three days of birth to well beyond three days after birth where there was an obvious switch from treatment of unconfirmed EOS to treatment of suspected LOS.

For example, in one neonate, a negative culture was drawn on day one of life and ampicillin and gentamicin were administered as treatment for EOS. At day five of life, more cultures were drawn, ampicillin administration ceased, and vancomycin administration began as treatment for LOS.

In these cases, we considered treatment courses ending for EOS and treatment courses beginning for LOS when there was a blood culture drawn after three days of birth, then a change in antibiotic regimen on or after the same calendar day.

Severity of illness and risk of mortality were measured using All Patient Refined Diagnosis Related Groups (APR-DRG). Univariate comparisons were evaluated using Student’s t-test for continuous variables and chi-squared test or Fisher’s exact test for categorical. Median values are reported with an interquartile range (IQR) in brackets and mean values are reported with standard deviation (SD) in parenthesis. T

rends over time were analyzed using simple linear regression for continuous variables, logistic regression for dichotomous variables, or one-way ANOVA for severity of illness and risk of mortality. Statistical tests were considered significant when p < 0.05. All analyses were undertaken with R version 3.5.3 (R Core Team, 2019) in conjunction with the dplyr version 0.8.0 R package.

Discussion

Confirmation of Neonatal Sepsis

Establishing blood culture-confirmed neonatal sepsis is complicated by the presence of false negative culture results and culture contamination. False negative cultures are commonly attributed to low volume of blood or low pathogen concentrations, which is an inherent problem in the mostly low body weight newborns with neonatal sepsis (Kellogg et al., 2000Dien Bard and McElvania TeKippe, 2016).

Additionally, it is believed by some that exposure to intrapartum antibiotics when treating chorioamnionitis or other maternal illnesses can reduce sensitivity of blood cultures (Peralta-Carcelen et al., 1996). To overcome these limitations, it is recommended that ≥ ≥1 ml of blood is collected for culturing (Polin, 2012Dien Bard and McElvania TeKippe, 2016).

Using 1 ml of blood for a blood culture has been demonstrated to provide 98% sensitivity even when pathogen concentrations are low (4 colony forming units per ml) (Schelonka et al., 1996). The volume of cultured blood was not available in this study, therefore the effect of blood volume on false negative results in this hospital system are difficult to infer.

Even though a blood culture may provide a sensitive diagnostic tool, the results may take several days. In the past, attempts have been made to detect true cases of sepsis earlier than a blood culture, (Philip and Hewitt, 1980) but there is no consensus on how to incorporate laboratory tests in diagnosing neonatal sepsis (Wynn et al., 2014). New biomarkers such as bacterial surface antigens and emerging technologies such as genetic sequencing based tools may be used more frequently in neonatal sepsis diagnostics in the future (Chauhan et al., 2017Iroh Tam and Bendel, 2017). Automated multiplex PCR systems can provide pathogen specific identification of common microorganisms including many organisms found in neonates such as E.coli and S. aureus in as little as an hour, (Salimnia et al., 2016) but it is unknown whether all pathogens are adequately detectable.

Regardless of the method used to detect microorganisms in a blood sample, sample contamination can mislead diagnosis. To complicate the issue, when a central venous catheter is used to draw blood, positive cultures may be due to catheter colonization (Hall and Lyman, 2006). Some organisms, such as Escherichia coliStaphylococcus aureus, and Streptococcus pneumoniae, are almost always associated with a true infection, while others are commonly contaminants, such as CoNS and Streptococcus viridans (Hall and Lyman, 2006). When a common contaminant is cultured, misdiagnosis is reduced by obtaining and comparing multiple cultures; and when blood is drawn from a catheter, a second sample should be drawn percutaneously (Schifman et al., 1998Hall and Lyman, 2006).

CoNS are the most common blood culture contaminants, (Hall and Lyman, 2006) but they are also a prominent causative agent of LOS (Dong and Speer, 2015).

In this study, approximately 54% of cultures positive for CoNS were identified as true positives, 46% were identified as potentially contaminated. Additionally, at least 15% of CoNS-positive cultures were from catheter-drawn blood samples. Distinguishing between contaminants and pathogens is imperative in these cases. CoNS cultures cannot simply be completely excluded from, or completely included in, culture-confirmed cases.

Clinical signs and laboratory results indicating sepsis beyond blood cultures are commonly used to classify confirmed cases of sepsis (Bizzarro et al., 2005Haque, 2005Wynn et al., 2014Zea-Vera and Ochoa, 2015). Incorporating any of these methods into confirming sepsis retrospectively has shortcomings; in particular, data are sometimes sparse.

It is important to realize that when laboratory results or clinical signs remain abnormal but blood cultures are negative, sepsis may still be suspected and the use of antibiotics may be prolonged. To allow comparisons with other studies and since blood cultures are the gold standard for the diagnosis of neonatal sepsis, the criteria for retrospectively confirming neonatal sepsis via a modified version of CDC guidelines (Horan et al., 2008) within this study used only culture results.

Treatments for Neonatal Sepsis

Of neonates treated for EOS in the University of Utah Hospital system, only 2.3% had blood culture confirmation and there was a high rate of empiric use of ampicillin and gentamicin. This type of empiric treatment is common practice in neonatal intensive care units (NICUs) within the United States. Oliver et al. reported that within the Pediatric Health Information System, 92.5% of neonates in a NICU that were administered antibiotics within three days of birth received ampicillin and/or gentamicin on the first day of treatment (Oliver et al., 2017). Cefotaxime appeared to be a preferred choice in therapy for confirmed EOS with gram-negative infections for the University of Utah Hospital system.

Empiric treatment for LOS seems to vary more than empiric treatment for EOS, but within the University of Utah Hospital system, vancomycin and/or gentamicin were the most common treatments. In a 2002 survey of 278 neonatology clinicians at 35 hospitals with NICUs, at least 60% prescribed vancomycin as part of empiric therapy for unconfirmed LOS, commonly combined with an aminoglycoside (Rubin et al., 2002). Prudent use of vancomycin has been advised for decades due to the evolution of vancomycin resistant strains of pathogens (The Hospital Infection Control Practices Advisory Committee, 1995).

There is some evidence that empiric vancomycin does not improve short-term mortality or length of stay when neonates are infected with CoNS (Ericson et al., 2015). Within the University of Utah Hospital system, there are signs of improvement in the antibiotic stewardship of vancomycin where the DOT/1,000 PD for vancomycin use was 37.3% lower in unconfirmed compared to confirmed cases of LOS with a significant reduction in empiric use over time (Table 4).

Rifampin was often combined with vancomycin to treat persistent CoNS infections within the University of Utah Hospital system—there is some limited evidence that this strategy is effective (Acar et al., 1983Shama et al., 2002van der Lugt et al., 2010Rodriguez-Guerineau et al., 2013).

Neonates with unconfirmed sepsis within the University of Utah Hospital system were treated for a median of 7 days LOT IQR (Clark et al., 2006Liu et al., 2012), which is similar to, or longer than, treatment lengths for unconfirmed sepsis in similar studies within the United States and Europe (Patel et al., 2009Cantey et al., 2015Fjalstad et al., 2016). Antibiotic administration may be prolonged even when cultures are negative if blood cell counts, C reactive protein levels, or other diagnostic tests remain abnormal. Continued administration of antibiotics when cultures are negative appears to be a common practice.

This is in spite of evidence that prolonged empirical antibiotic administration within neonates (≥5 days) is associated with adverse outcomes such as necrotizing enterocolitis, prolonged hospital stay, and death (Kuppala et al., 2011Ting et al., 2016). Recent studies on the incubation time for blood cultures in cases of neonatal sepsis suggest that antibiotic treatment can be ceased after 48 h if cultures remain negative (Jardine et al., 2006Durrani et al., 2019) Cantey et al. demonstrated that when empiric neonatal antibiotic therapy was set to automatically discontinue after 48 h and continued therapy was limited to 5 days when cultures were negative, there was no difference in safety outcomes compared to prior practices where antibiotics were regularly administered ≥6 days when cultures were negative (Cantey et al., 2016).

Since the gestational age, birth weight, severity of illness, risk of mortality, and the ratio of culture confirmed to unconfirmed cases of neonatal sepsis remained fairly consistent throughout the study period, it was unexpected that the neonatal sepsis mortality rate within the University of Utah Hospital system increased over the study period. In 2006, Clark et al. showed that the concurrent use of ampicillin and cefotaxime within 3 days of birth rather than ampicillin with gentamicin had an increased risk of mortality (Clark et al., 2006).

Logistic modeling showed that the use of cefotaxime within 28 days of birth may be a cause of the increased neonatal sepsis mortality within the University of Utah hospital system, even after adjusting for potential confounders.

This conclusion is limited by the retrospective nature of this study: cefotaxime administration may be a surrogate for some other unknown factor. Further analysis of the causes of death and the motivations for prescribing cefotaxime may reveal other factors that may be responsible for the increased mortality rate.

One potential limitation of this study is that antibiotic therapy may have been prolonged for neonates with infections other than sepsis such as pneumonia or meningitis. In our patient cohorts, there were no neonates that had both negative blood cultures and positive CSF culture which would suggest neonatal meningitis.

When neonates with any other positive cultures were removed from the unconfirmed cohorts (see Table 1), the LOT/course for courses starting ≤3 days of birth reduced from 6.1 to 6.0 for unconfirmed EOS and the LOT/course for courses starting >3–28 days of birth reduced from 7.8 to 7.5 for unconfirmed LOS. Both of these differences were insignificant (p > 0.05); therefore, we believe that the main findings of this study are still supported. This study has other limitations.

Comorbidities and mother’s characteristics were not accounted for and may affect suspicion of sepsis and choices in antibiotic treatments. The results of this study cannot be generalized to other hospitals since epidemiology and practices are likely to differ from region to region.

Twelve-Year Overview

Over the past 12 years, the level of empirical treatment remained high. The major limitations to reduce empirical treatment are highly variable clinical presentation of sepsis in neonates and low levels of confirmed sepsis.

The development or improvement of diagnostic tests capable of accurately confirming sepsis within 1–2 h after blood draw is a key factor that will reduce empirical treatment of neonatal sepsis. One of the possible risk factors for the development of sepsis in neonates is an underdeveloped immune defences (Gervassi and Horton, 2014). Recently, it has been proposed that immune responses in neonates are suppressed by afterbirth residual myeloid derived suppressor cells (MDSCs) (Gervassi and Horton, 2014). MDSCs are a heterogeneous population of immature myeloid cells capable of expanding following pathologic conditions such as infections and cancer. MDSCs have been detected in the blood of septic patients (Cuenca et al., 2011).

The ontogenetic destiny of MDSCs is to suppress the fetal immune response against the mother. MDSCs possess the remarkable ability to suppress T, B, and NK cell functions and by this mean impair protective immune responses to infectious agents (Rieber et al., 2013Gervassi et al., 2014).

Presence of these cells in cord blood can potentially serve as early biomarker of neonatal sepsis. If MDSCs indeed suppress anti-microbial immunity of neonates, the immune system can be restored and sepsis development prevented by blocking MDSCs activity or by differentiation of MDSCs into mature myeloid cells lacking suppressive activities. This approach may reduce the rate of neonatal sepsis, empirical use of antibiotics, and increase survival of preterm babies.Go to:

Conclusions

In conclusion, the large proportion of unconfirmed to confirmed neonatal sepsis combined with a high empiric antibiotic administration rate and high level of exposure indicates an opportunity to reduce unnecessary antibiotic use in the neonatal population. This may be accomplished by implementing new and improved methods for more sensitive and timely diagnosis of neonatal sepsis and reevaluating guidelines for appropriate antibiotic use.


More information: Kent A Willis et al. Perinatal maternal antibiotic exposure augments lung injury in offspring in experimental bronchopulmonary dysplasia, American Journal of Physiology-Lung Cellular and Molecular Physiology (2019). DOI: 10.1152/ajplung.00561.2018

Journal information: American Journal of Physiology
Provided by American Physiological Society

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