Exploring the Skin Mycobiome in Very Preterm babies during the early neonatal period in a Neonatal Intensive Care Unit of India

Introduction

Skin colonization in preterm babies commences in utero and continues after delivery. It is predominantly influenced by maternal genital flora, mode of delivery, type of feeding, level of sickness, and antibiotic exposure. Symbiosis between host and microbiome is critical for a stable environment in vivo. The role of bacterial colonization in neonates and the diversity of flora has being greatly studied over the past decade. ¹ Studies on cutaneous mycobiome in preterm babies are, however, sparse. Our prospective study was conducted to understand and bridge this gap regarding the mycobiome of preterm babies <32 weeks old in the early neonatal period.

Materials and methods

Skin swabs of 30 preterm neonates < 32 weeks of gestation admitted to a Level III NICU were collected in the first five days of life after admission following obtaining of consent over a six month period. The swab was taken from the skin of the neonate in a single sweep pattern starting from right axilla > right abdomen > groin > left abdomen > left axilla in order (Figure 1). Samples were sent to our genomics laboratory in sterile water for Next-generation sequencing (NGS). Detection was done using Credence Rapid Infection Detection (credence RIDTM) for Next Generation Sequencing after Polymerase Chain Reaction (PCR). ITS1 (internal transcribed spacer 1) of the 18S rRNA gene was used for the identification of fungal species. The reads obtained were then compared to standard library matching barcoded bacterial and fungal libraries that were multiplexed on a single chip on a 400 bp run to obtain sequencing data. Specimens were run in batches of 20 on an Ion 318TMv2 chip (Thermo Fisher Scientific). Data were analyzed using Credence Genomics proprietary bioinformatics pipeline for analysis of clinical isolates. Statistical analysis was done using IBM-SPSS v.23, New York, USA. As these were preliminary findings, a narrative description of the fungal mycobiome has been expressed as a graphical format and demographic description has been tabulated. Heat Map for demonstrating relative abundance at the species level has been carried out for the same. Ethical approval was obtained for the study from Institutional Ethics Committee (IEC- BMCRI/PS/254/2020-21)

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Figure 1.

Heat Map for demonstrating relative abundance at the species level. (X-axis : Sample Number, Y-axis: Right : Species type, Left : Relative abundance in %, Bottom right image inset: The swab was taken from the skin of the neonate in a single sweep pattern starting from right axilla > right abdomen > groin > left abdomen > left axilla in order).

Results

Out of 30 preterm babies, 13 (43.33%) were males with a median (IQR) gestation of 31(30–32) weeks and a mean (SD) birth weight of 1.34 (0.21) kg. Other neonatal and maternal baseline characteristics are mentioned in Table 1. Using the credence RIDTM, the NGS results were analyzed as percentages, i.e. relative abundances. The most abundant genera found in 22/30 samples were Candida followed by Bipolaris & Cladosporium. The hierarchical clustering based on species was carried out for the ten most common fungal abundances that were found. These include Candida albicans, Candida Africana, Candida tropicalis, Candida parapsilosis, Candida orthopsilosis, Cladosporium endophytica, Cladosporium halotolerans, Candida hyderabadensis, Bipolaris subramanianii, and Bipolaris variabilis. (Figure 1)

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Table 1.

Baseline characteristics y(mother /infant).

Baseline characteristic	(n = 30)
Maternal
Age (years), median (IQR)	24 (20–26)
Gravida(G), mode (range)	1 (1–6)
Para(P), mode(range)	0 (0–5)
Normal delivery	0 (0–2)
Gestational weeks, median (IQR)	31(30–32)
Antenatal steroids dose %(n)	80%(24)
Dichorionic Diamniotic % (n)	20 (6)
Monochorionic Monoamniotic % (n)	20 (6)
Singleton %(n)	60 (18)
Leak per vaginam (PV) present % (n)	30 (9)
PV discharge %(n)	10 (3)
Clear Liquor %(n)	96.6 (29)
Fever in last trimester %(n)	3.3 (1)
Antibiotics received % (n)	56.6 (17)
Birth Details
Birth weight (kg) mean (SD)	1.34 (0.21)
Gender
i) Male %(n)	43.3 (13)
ii) Female %(n)	56.7 (17)
Neonatal outcomes
1) Discharged %(n)	73.3 (22)
2) Left Against Medical Advice %(n)	23.3 (7)
3) Death %(n)	3.3 (1)

Discussion

Premature babies are at risk of skin colonization by various bacteria or fungi during the perinatal or neonatal period from maternal vaginal flora, NICU environment, or health care staff. Bacteria or fungal colonization predisposes these babies to invasive sepsis. ² In our study, we found colonization was mostly by commonly seen candida species and two uncommon species, Cladosporium and Bipolaris. Cladosporium, although ubiquitous, is rarely associated with invasive sepsis. ³ Bipolaris species are plant pathogens and detection of these species, although intriguing, could be due to environmental contamination. ⁴ A previous study ⁵ have found Malassezia as the most common cutaneous mycobiome in premature babies. This disparity could be explained by the geographical location of increased heat and humidity, population differences of low socio-economic status, high incidence of fungal sepsis in NICU, and a 56.6% antibiotic usage in the first week of life. No vaginal swab was done in any mother before delivery. Some 30% of mothers had a vaginal leak and 10% had a vaginal discharge before delivery. Colonization by Candida species in high abundance increases the risk of candidaemia according to previous studies, though none of our babies had any invasive fungal sepsis.^5,6 One baby died due to multi-organ dysfunction secondary to bacterial sepsis. The mere presence of these species in premature babies is alarming for the treating physician concerned about the possible hazards of invasive fungal sepsis, cross-infection, and outbreaks.

The strengths of our study included proper technique in taking the sample including transport and processing methods; therefore making the possibility of cross-contamination minimal.