ABSTRACTObjective

We aimed to determine the association between newborn bacterial colonization and infant respiratory morbidity, in the first six months of life.

Methods

This prospective study included healthy newborn infants. Nasopharyngeal swabs performed within 72hrs of delivery were analyzed via polymerase chain reaction. We assessed cumulative respiratory morbidity of infants at 6-months-old.

Results

Four hundred and twenty-six mother-infant pairs were recruited. In 53.3% (n=225) of newborns, S. pneumoniae (46%) and S. aureus (7.3%) was isolated. None had H. influenzae nor M. catarrhalis. At 6-months-old, 50.7% had experienced respiratory symptoms, 25% had unscheduled doctor visits, and 10% were treated with nebulizers. Colonization with S.pneumoniae was associated with reduced risk of any respiratory symptom (aOR 0.39[95% CI 0.16,0.50]), unscheduled doctor visits (aOR 0.35 [95% CI 0.18,0.67]) and nebulizer treatment (aOR 0.23 [95% CI 0.07,0.72]) at 6 months. Pregnancy-induced hypertension was also associated with increased need for nebulizer treatment (aOR 9.11 [95% CI 1.43,58.1]).

Conclusion

Colonization of the newborn respiratory tract occurred in 53% of infants. Streptococcus pneumoniae was the most common organism, and this was associated with a reduced risk for respiratory morbidity at six months of life.

Keywords

INTRODUCTION

Historically the lung was believed to have been a sterile environment. However, this is a myth, and 2000 bacterial genomes per cm2 (Hilty et al., 2010),(Segal et al., 2014) indeed occupy the lung, and the upper respiratory tract is colonized by a vast amount of aerobic and anaerobic bacteria.(Charlson et al., 2011; Segal et al., 2014)There is a homogenous microbiota and reducing biomass from the nose to the lung, thus, implying that the lung microbiota may originate from the upper airway.(Charlson et al., 2011)

As for the infant, mode of delivery was thought to play an important role in new-born colonisation. However recent evidence suggests that the placenta, amniotic fluid, and membranes, do in fact harbour a unique low biomass with low abundance microbiome. Hence infant colonisation may happen in utero rather than at delivery.(Aagaard, 2020; Liu et al., 2019; Yassour et al., 2016)

While much is known about intestinal microbiota, what is the role of organisms in the airway and lung? Knowledge on how respiratory bacterial colonization can modulate the immune system and, in turn, the respiratory outcome from infancy is still lacking.(Bisgaard et al., 2007; Bosch et al., 2017; Gallacher & Kotecha, 2016; Yap et al., 2018) Unlike intestinal microbiota, the lung does not shape the adaptive and innate immune system.(Gallacher & Kotecha, 2016) However, it has been the ability to dampen down the inflammatory response to infections or allergens.(Wang et al., 2013) This may be explained by different organisms evoking different types of T cell response, e.g., Moraxella catarrhalis and Haemophilus influenzae have been shown to induce a mixed T cell response, which results in a reduced Th1 response.(Frati et al., 2018) This, in turn, leads to an inability to clear microorganisms resulting in chronic inflammation.(Folsgaard et al., 2013) Other studies have shown that a stable microbiota is associated with a reduced risk of colonization with pathobionts, while the disappearance of initial colonizers is related to the emergence of bacteria causing disease.(Esposito & Principi, 2017) Therefore, altering the lung microbiota may have a therapeutic effect both in treating and preventing diseases.(Gallacher & Kotecha, 2016)

We hypothesize that neonatal bacterial colonization occurs early and is associated with the risk of developing respiratory tract illness later on in life. Hence this study aimed to determine (a) the prevalence and type of bacterial colonization of the respiratory tract of healthy infants at birth, (b) the association between antenatal factors and the type of bacterial colonization, and (c) the association between bacterial colonization birth and the incidence of acute respiratory tract symptoms in the first six months of life.

METHODS

Study design and subjects

This is a single-centre, prospective study in University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia, from 1st March 2019 to 17th March 2020. The obstetric unit in UMMC has about 1200 deliveries per year.

We included healthy mother-baby dyads from the postnatal ward within three days post-delivery to ensure the respiratory tract sampling would represent airway colonization at birth.(Mourani et al., 2011) We excluded mothers with underlying chronic medical illness, psychiatric disease, or malignancies, or those who refused consent. Babies born premature (<36 weeks gestation) or with underlying medical diseases or significant perinatal illness were also excluded.

The sample size was calculated based on the study by Bisgaard et al., who looked at the association between prevalence of asthma in children with bacterial colonization of the respiratory tract during infancy.(Bisgaard et al., 2007) The sample size required was 400 subjects to achieve a confidence interval of 95% and 80% power of detection, with an odds ratio of 3.0 between the two comparable groups.

Social demographics and clinical data

Initial data collection on social demographics was done face-to-face with an interviewer-administered questionnaire. Information on the living environment was obtained: smoke exposure at home, air pollution in the neighbourhood (exposure from construction sites, factories, or near to the main road within 200 metres), mould in the house, level of crowding at home (number of children and the total number of people in the house), pets or other animal exposure. We also enquired about family history of asthma and atopy (eczema, allergic rhinitis, allergic conjunctivitis, and food allergy). Socioeconomic status was determined by the family's total monthly income, parental highest educational level and their occupation. Vaccination against pneumococcal was also obtained as this was not part of the National Vaccination Programme until January 2021.

We also collected data regarding antenatal, perinatal, and postnatal history. Information collected included: smoking behaviour or environmental tobacco smoke (ETS) exposure during pregnancy, maternal intake of folic acid, antibiotic exposure either during the antenatal or intrapartum period, mode of delivery, complications during pregnancy (e.g., gestational diabetes mellitus (GDM), pregnancy-induced hypertension (PIH), anaemia during pregnancy and urinary tract infection (UTI).

New-born Nasopharyngeal swab collection and preparation

We took written consent before the nasopharyngeal swab was performed. We chose Copan Diagnostics Flexible Minitip Size Nylon ® Flocked Swabs (FLOQSwab®) with 100mm Breakpoint in Peel Pouch for this study. Each swab was individually packaged and in sterile condition before use.

The infant was held with the head supported by hands and the body swaddled in a blanket. The swab was moistened with sterile 0.9% normal saline and subsequently inserted straight back (not up) the nose until some resistance was met (at least 1 cm). Contact between the swab and nasal mucosa was made and the swab was rotated gently for at least 5 seconds. Both nostrils were swabbed using the same swab to increase the yield.

The swab was then immediately placed into Trypticase Soy Broth (Soybean-Casein Digest Medium), a liquid enrichment medium used to cultivate aerobic organisms, particularly pathogenic bacteria that are not excessively fastidious (i.e., high nutritional requirements). The swab specimens were then immediately sent to the microbiology lab for the identification of organisms. The identified organisms are best-known commensals in the upper respiratory tract (URT) of children: Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis.(Bisgaard et al., 2007)

The samples were analyzed via bacterial DNA isolation and polymerase chain reaction (PCR). Samples were frozen at -80 deg C and analyzed in batches. For bacterial DNA isolation, rapid purification of total DNA was done using DNeasy® Blood & Tissue Kit (Qiagen, Germany). Specimens were first lysed using proteinase K. Buffering conditions were adjusted to provide optimal DNA binding conditions, and the lysate was loaded onto the DNeasy Mini spin column. During centrifugation, DNA was selectively bound to the DNeasy membrane as contaminants pass through. The remaining contaminants and enzyme inhibitors were removed in two efficient wash steps. DNA was then eluted in buffer and ready for use. Purified DNA had A260/A280 ratios of 1.7–1.9, and absorbance scans showed an asymmetric peak at 260 nm, confirming high purity. DNA was then stored at -20°C until further usage. PCR was performed as previously described to detect S. pneumoniae(Zbinden et al., 2011), H. influenzae,(Meats et al., 2003) S. aureus,(Baron et al., 2004) and M. catarrhalis.(Post et al., 1996)

Respiratory morbidity and outcomes

We solicited the following questions regarding both upper and lower respiratory tract symptoms experienced by their infants: any respiratory symptom (cough, runny nose, fever, wheezing, noisy breathing, difficulty breathing), need for unscheduled doctor visits due to a respiratory problem, hospital admission, and need for nebulizer use. These questions were based on common symptoms experienced by infants. We considered the need for unscheduled doctor visits, nebulizer treatment or hospitalization as significant respiratory illnesses as opposed to complaints of "any respiratory symptom".

All infants were followed up at the 1st, 3rd, and 6th months of age via a structured data online Google form. Other social factors were also obtained, i.e., ETS exposure, details regarding carers, and vaccination status (including pneumococcal). Mothers were asked if they breastfed their children exclusively. The questionnaire was prepared in both English and Malay language versions after forward and backward translation. The questions were pilot tested on ten mothers and responses harmonized to ensure correct understanding before using them in the study. Mothers were contacted at 1-, 3- and 6 months to ensure accurate recording of respiratory morbidity. Mothers who did not respond to the questionnaire were contacted via telephone calls at least 5 times before they were considered defaults. Their data was not analyzed.

Statistical analyses

Statistical analyses were performed using SPSS 24.0 (SPSS Inc., Chicago, IL, USA). For demographic information, quantitative variables were expressed as mean ± standard deviation or median (interquartile range), while categorical variables were expressed as numbers and percentages. Missing data of infant outcome at 6 months was not analysed. Differences between categorical variables were examined using the chi-square (χ2) test or Fisher test, while independent t-test or ANOVA were used to investigate the differences between numerical variables, where appropriate. Differences between categorical data were also presented using odds ratio (OR) and 95% confidence interval (95% CI), wherever possible. For non-parametric numerical variables, we performed Kruskal Wallis or Mann Whitney tests to examine the differences between them. Logistic regression (LR) was used to adjust significant associations with the infants' risk of respiratory tract symptoms. Factors that were significant in univariate analysis (p<0.10) or which were considered clinically significant were entered into LR, and adjusted OR (aOR), 95% CI and p values were tabulated. Statistical significance was considered as p< 0.05.

RESULTS

Patients were recruited from 1st March 2019 until 17th March 2020. There were 1850 mother-infant pairs who fulfilled the inclusion criteria. After excluding those who fulfilled the exclusion criteria, a total of 426 mother-infant pairs were recruited (Figure 1).

Figure 1Study flow showing recruitment of mothers as well as number of respondents at 1 month, 3-months and 6-months of age.

Responses to the respiratory morbidity questionnaire were as follows: 363 (85.2%) at 1-month-old, 334 (78.4%) at 3-months-old and 259 (60.7%) at 6-months-old. Data from non-respondents were excluded from data analysis.

Maternal social demographics and clinical characteristics (Supplemental Table 1)

The mothers' median (range) age was 31.4 (20-42)-years old. Ethnic distribution was very similar to the National Statistics on the ethnic composition in Malaysia, where Malays represent 67.4%, Chinese represent 24.6%, and Indians represent 7.3% of the total population. More than 2/3rds of the families had a monthly household income in the middle to the high-income range. A family history of asthma was present in 35.8% of mothers. Nearly half (44.2%) were exposed to ETS during pregnancy, mostly from husbands. Only one mother actively smoked during pregnancy.

Complications during pregnancy were present in more than two-thirds of the mothers (68.8%), with the most common problems being anaemia (23.8%) and GDM (26.7%).

Infant demographics at 1-, 3- and 6-months-old (Supplemental Table 2)

The exclusive breastfeeding rate of the infants decreased over time, with only half (54.4%) of the infants exclusively breastfed at 6-months-old. At 6-months-old, about half (53.3%) of the infants were placed in a day care facility and around one-third (28.6%) had exposure to ETS.

Respiratory morbidity of infants (Table 1)

Table 1Respiratory morbidity among the infants at 1, 3 and 6-months old

Half of the respondents (50.6%) at 6-months-old had experienced respiratory tract symptoms. The most common respiratory symptoms reported were noisy breathing, runny nose, and cough. Finally, 25.2% of infants had at least one unscheduled doctor visit while 10% received nebulizers.

Bacterial colonization of the respiratory tract of infants (Table 2)

Table 2Respiratory Tract Colonization of infants

Organisms were isolated in 53.3% (n=225) children. Streptococcus pneumoniae was isolated in 46.0% of infants while Staphylococcus aureus in 7.3%. Haemophilus influenzae and Moraxella catarrhalis were not isolated in any infant.

Factors associated with infant respiratory tract colonization (Table 3)

Table 3Association between Antenatal Maternal Factors with Infant Respiratory Tract Microbiome Colonization

We analysed antenatal factors (family history of asthma, antenatal maternal exposure to ETS, maternal complications, i.e., GDM, UTI, PIH) and peripartum factors (maternal antibiotics, mode of delivery) to determine if there was an association with presence and type of bacteria isolated. There was no significant association between mode of delivery (caesarean section versus vaginal delivery) and colonisation of organisms. The use of maternal antibiotics (either during the antenatal or intrapartum period) was associated with an increased risk for neonatal colonization of S. pneumoniae (OR 1.67[95% CI 1.04,2.67], p=0.03). The most common antibiotics prescribed were ampicillin (n=38), cefuroxime (n=17), cephalexin (n=8) and ampicillin-clavulanic acid (n=7).

Association between maternal antenatal factors, breastfeeding, day care attendance, and environmental tobacco smoke exposure and respiratory morbidity of infants in the first six months of life (Table 4)

Table 4Association between antenatal factors, perinatal factors, breastfeeding, day care attendance and environmental smoke exposure with respiratory morbidity of infants during first six months of age

Exclusive breastfeeding was associated with reduced respiratory symptoms among infants (OR 0.43[95% CI 0.23,0.77], p=0.004) but not unscheduled doctor visits (OR 0.53[95%CI 0.28,1.01], p=0.05) nor need for nebulizer treatment (OR 1.01[95% CI9.39,2.79], p=0.99). Pneumococcal vaccination was associated with reduced respiratory symptoms (OR 0.55[95% CI 0.32,0.92], p=0.02) and reduced nebuliser treatment (OR 0.03[95% CI 0.09,0.910], p=0.03) but not unscheduled doctor visits (OR 0.67[95% CI 0.36,1.24], p=0.21).

Association between bacterial colonization during birth and respiratory morbidity at six months of life (Table 5)

Table 5Association between Infant Respiratory Tract Colonization and Incidence of Respiratory Morbidity

Colonization with S. aureus was associated with increased risk of any respiratory symptoms (OR 4.33[95%CI 1.45,8.99], p=0.004), increased risk of unscheduled doctor visits (OR 2.76 [95%CI 1.23,6.13], p=0.02) but not increased need for nebulizer treatment (OR 2.71[95%CI 0.91,7.30], p=0.07).

Colonization with S. pneumoniae was associated with reduced respiratory symptoms (OR 0.25 [95%CI 0.12,0.53], p<0.001), reduced unscheduled doctor visits (OR 0.32[95%CI 0.17,0.59], p<0.001) and reduced nebulizer use (OR 0.18[95%CI 0.06,0.55], p=0.001).

Multivariate analysis of significant factors associated with respiratory morbidity (Table 6)

Table 6Logistic Regression of Significant Factors associated With Respiratory Morbidity At 6 Months

Significant factors (p<0.10) identified in univariate analysis were analysed with logistic regression. At 6-months, colonization with S. pneumoniae was associated with a reduced risk for respiratory morbidity: any respiratory symptom (aOR 0.29 [95% CI 0.16-0.50], p=<0.001), unscheduled doctor visits (aOR 0.35 [95% CI 0.18-0.67], p=0.001) and need for nebuliser treatment (aOR 0.23 [95% CI 0.07-0.72], p=0.01). Interestingly, PIH was also associated with need for nebuliser treatment (aOR 9.12 [95% CI 1.43-58.1], p=0.02).

DISCUSSION

This study aimed to investigate the association between organisms colonized in the newborn respiratory tract and respiratory disease in the first six months of life. In the nasopharyngeal swabs of 426 newborns, 1 out of 2 infants was positive for either Streptococcus pneumoniae or Staphylococcus aureus. None had isolated Moraxella catarrhalis nor Haemophilus influenzae. Antibiotic use, either during the antenatal or perinatal period, was the only maternal factor associated with increased neonatal colonization of S. pneumoniae. Interestingly, neonatal isolation of S. pneumoniae was associated with a reduced incidence of any respiratory tract symptom, unscheduled doctor visits and need for nebulizer treatment at 6-month-old. S. aureus was associated with an increased incidence of respiratory tract symptoms in univariate analysis, although this did not achieve statistical significance in logistic regression. Antenatal history of PIH was also associated with an increased need for nebulizer treatment.

We investigated the incidence of both upper and lower respiratory tract infections in this study. Half of the infants (50.6%) had any respiratory symptom (which included runny nose or cough or noisy breathing or breathing difficulty) in the first six months of life. This is high, and maybe an overestimate as not all mothers consulted the doctor with these complaints. However, 25% had unscheduled doctor visits and 10% required nebulizer treatment, representing more serious illness.

This study found that infants were colonized with organisms: S. pneumoniae and S. aureus. In the COPSAC study, which investigated bacteria colonization of 321 healthy infants at 1-month-old, 9 % were colonized with S. pneumoniae or Haemophilus influenzae, 8% with Moraxella catarrhalis and 61% with S. aureus, while Strep. pyogenes was isolated in < 1%. (Bisgaard et al., 2007; Vissing et al., 2013) A more recent study by Bosch AA et al., who looked at nasopharyngeal microbiota of infants at birth via 16S rRNA, found an initial expansion of Streptococcus species at Day 1 of life. Although, this was replaced at one week of life with predominantly Staphylococcus.(Bosch et al., 2017) Our results indeed are similar to Bosch et al,(Bosch et al., 2017) but differ from the COPSAC.(Vissing et al., 2013) This is probably because of the different techniques used to identify the organisms, with the COPSAC using culture methods and ours using PCR; as well as the timing of obtaining the nasal microbiota, which was done later at one month in the COPSAC study versus right after birth as in this study and that of Bosch AA et al.

The other interesting finding in this study was the association between colonization with S. pneumoniae and a reduced risk for respiratory morbidity at 6-months-old: any respiratory symptom, unscheduled doctor visits and need for nebulizer use . This has not been described before as, few authors have looked at the association between organisms colonised early at birth and risk of respiratory tract infections early in life. Resident respiratory tract microbiota has a role as the gate keeper, that either facilitates or provides resistance to colonization by respiratory virus.(Beigelman & Bacharier, 2016; Lynch et al., 2017) We know that Streptococcus spp is a known colonizer of the maternal vaginal tract.(Mendling, 2016) However, its exact role is unsure. We hypothesize that its early presence is protective i.e., having an anti-inflammatory role by evoking differential T cell responses, as seen with other bacteria.(Frati et al., 2018) .

There was no significant association between mode of delivery and detection of pathogens in this study. Infants born via LSCS usually have microbiota reflective of skin commensals (Staphylococcus and Corynebacterium(Grice & Segre, 2011)) and more pathogenic bacteria. In contrast, those born via vaginal delivery had commensals reflective of the maternal vaginal tract (Staphylococcus, Streptococcus, and Dolosigranulum(Mendling, 2016)) and thus more protective microbiota.(Dominguez-Bello et al., 2010) However, there is emerging data suggesting that new-born colonization may not be affected by Caesarean section per se but by many other factors like parity, maternal diet, stress, use of antibiotics, and presence of maternal comorbidities.(Liu et al., 2019; Singh & Mittal, 2020) These factors may affect placenta amniotic fluid microorganism colonization and in turn new-born colonisation.(Liu et al., 2019)

None of the patients in this study had Moraxella catarrhalis nor Haemophilus influenzae isolated. Bosch AA et al. found that early enrichment with Moraxella and reduced Corynebacterium and Dolosigranulum, and more abundant and prolonged presence of oral types of bacteria in the nasopharynx, including Neisseria and Prevotella spp, were associated with an increased risk of respiratory tract infections.(Bosch et al., 2017) We attribute our lack of Moraxella due to the early sampling of the infants, as Moraxella tends to inhabit the respiratory tract of children at 2-3 months of life.(Bosch et al., 2017)

Maternal antibiotic exposure was associated with neonatal colonisation with S. pneumoniae in univariate analysis. This has been shown before as antibiotics can significantly alter the microbiota, with lower amounts of Alloiococcus and Corynebacterium and a greater abundance of Haemophilus spp, Streptococcus spp, and Moraxella spp.(Teo et al., 2015; Wong et al., 2020)

We also found a significant association between antenatal history of PIH and increased need for nebulizer treatment. It is presumed that PIH may affect foetal lung function.(Shaheen et al., 2016) Interestingly, a pooled analysis from 14 European birth cohorts have also reported that hypertensive disorders during pregnancy, especially pre-eclampsia, was associated with an increased risk of developing recurrent wheezing episodes among infants at 12-24 months of age (aRR 1.09 [95% CI: 1.01–1.18]).(Zugna et al., 2015)

The strengths of this study are that this data is from a middle-income country, where environmental exposures would be different from developed countries, where most of the microbiome studies have been done.(Biesbroek et al., 2014; Bosch et al., 2017; Vissing et al., 2013) Furthermore, this longitudinal study adds to the limited existing data looking at the association between neonatal microbiome and risk for respiratory tract disease in early life. Many have investigated colonization of the respiratory tract in premature and sick infants.(Sakwinska et al., 2017) We also enquired about relevant environmental exposures during follow-up to consider confounding factors during statistical analyses, adding robustness to our significant findings,

However, limitations in this study are also recognized. We were not able to recruit all eligible mothers due to time constraints. However, as these mothers were not systematically excluded, this should not introduce significant reporting bias. The identification of airway microbiota is not comprehensive as other methods like 16S rRNA which will more accurately detect the respiratory microbiome. This explains the high negative microorganism finding of 46.7% in this study too. This study did not investigate the succession pattern of respiratory microbiota in infants, which has been shown to impact the respiratory morbidity.(Biesbroek et al., 2014) The high dropout rate of mothers may introduce bias in symptom reporting, and may have resulted in an increased incidence of respiratory morbidity. Finally, as this study involved infants during the COVID pandemic, the incidence of respiratory symptoms may be markedly reduced in patients recruited from March 2021; however, this only involved a small number of children (n=39).

In conclusion, this longitudinal study from a middle-income country found that more than half of the neonates had their upper airway colonized with S. pneumoniae or S. aureus. Colonization with S. pneumoniae was significantly associated with a lower risk of any respiratory symptom, unscheduled doctor visits and need for nebulizer treatment in the first six months of life. PIH was associated with an increased need for nebulizer treatment.

FUNDING

This study received financial support from the Malaysian Thoracic Society, Malaysia, in 2018 (grant: MTS 2018 grant) as well as the University of Malaya Partnership Grant (RK-003-2020).The funders were not involved in the conception of this study, writing, editing, or analyses of this study.

AUTHOR CONTRIBUTIONS

Anna Marie Nathan: Conceptualization; funding acquisition; data curation; formal analysis; methodology; supervision; visualization; writing-original draft; writing-review & editing. Kai Ning Chong: Conceptualization; data curation; formal analysis; methodology; supervision; visualization; writing-original draft; writing-review & editing. Cindy Shuan Ju Teh: Funding acquisition; data curation; formal analysis; methodology; supervision; visualization; writing-original draft; writing-review & editing. Jessie Anne de Bruyne: Conceptualization; funding acquisition; methodology; supervision; visualization; writing-original draft; writing-review & editing. Anis Najwa Muhamad: Data curation; formal analysis; visualization; writing-original draft; writing-review & editing. Quraisiah Adam: Data curation; formal analysis; methodology; writing-original draft; writing-review & editing. Rafdzah Ahmad Zaki: Conceptualization; funding acquisition; data curation; formal analysis; methodology; supervision; visualization; writing-original draft; writing-review & editing. Shih Ying Hng: Conceptualization; data curation; formal analysis; methodology; supervision; visualization; writing-original draft; writing-review & editing. Kah Peng Eg: Visualization; writing-original draft; writing-review & editing. Nuguelis Razali: Funding acquisition; data curation; formal analysis; methodology; supervision; visualization; writing-original draft; writing-review & editing.

DISCLOSURE STATEMENT

The authors do not have any conflict of interest.

HUMAN ETHICS APPROVAL DECLARATION

Ethics approval was obtained before commencement of study from the Medical Research Ethics Committee in UMMC (MREC ID NO: 2018116-5961), and signed informed consent from all participants was obtained before we undertook any procedure.

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Received: June 3, 2022

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