Early Human Development 90S1 (2014) S84–S86 Late-onset neonatal group B streptococcal disease associated with breast milk transmission: molecular typing using RAPD-PCR Micaela Brandolini a, *, Marta Corbella a , Patrizia Cambieri a , Daniela Barbarini a , Davide Sassera b , Mauro Stronati c , Piero Marone a a Microbiology and Virology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy b Department of Veterinary Sciences and Public Health, Università degli Studi di Milano, Italy c Neonatology and Neonatal Intensive Care Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy ARTICLE INFO ABSTRACT Keywords: Group B streptococcal disease Random amplified polymorphic DNA (RAPD) Breast milk transmission Group B Streptococcus (GBS) is considered to be the major cause of neonatal sepsis and meningitis of bacterial origin. Late-onset GBS infection is infrequent and occurs between 1 week and 3 months of age. The transmis- sion of GBS through the ingestion of breast milk is reported in the literature, but only a few of these cases have been confirmed by molecular techniques. In this article we report five cases of late-onset GBS disease: transmission through maternal milk was confirmed in four cases, using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing assay. In addition, the RAPD-PCR assay showed that each of the isolated clones belonged to a different RAPD genotype, thus revealing that the late-onset GBS infections were not epidemiologically related. © 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Group B Streptococcus (GBS) is considered to be the major bacterial cause of neonatal sepsis and meningitis [1]. These severe infections are more common during the first week of life (early- onset disease), and generally vertically acquired, likely after the rupture of placental membranes, or during passage through the birth canal. On the other hand, late-onset infections are infrequent and occur between 1 week and 3 months of age. These infants usually have an identifiable focus (e.g. meningitis) in addition to the sepsis, and the infection is normally assumed to derive from human contacts, or from hospitals or community pathways [1,2]. Some cases of transmission of GBS through the ingestion of breast milk have been reported in the literature, but only a few of these have been confirmed by molecular techniques [1–3]. In this article we report five cases of late-onset GBS disease: transmission through maternal milk was confirmed in four of these cases, based on molecular typing using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) assay. 2.Methods We considered the cases of late-onset GBS infection that oc- curred in the Neonatology Unit of S. Matteo Hospital in Pavia from * Corresponding author: Dr. Micaela Brandolini, Microbiology and Virology, Unit, Fondazione IRCCS Policlinico San Matteo, via Taramelli 5, 27100 Pavia, Italy. Tel.: +39 0382 501089. E-mail address: m.brandolini@smatteo.pv.it (M. Brandolini). 0378-3782/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. August 2012 till February 2013. We analyzed positive GBS cultures obtained by neonatal blood culture, neonatal cerebrospinal fluid (CSF) and maternal breast milk (Table 1). All GBS isolates were culture-purified using standard procedures, antigen-group char- acterized by anti-sera agglutination (Prolex ™ , Latex Agglutination System–Pro-Lab Diagnostics, ON, Canada), subsequently analyzed by RAPD-PCR performed with primers NP3(5-GAAGCAGCCCGGTAG TAGGTTGAG-3), NP4(5-CTAATGCAGGAGTCGCATAAGGGAGA-3) and NP5(5-AGCGCTGTGAGAA AGATGA-3) and resolved by use of a 2% polyacrylamide gel, according to the procedure described in Telecco S. et al. (1999) [4]. In our samples, the primer NP5 exhibited the highest discriminatory efficiency, allowing to differentiate the highest number of samples (Fig. 1). 3. Results We present here five cases of GBS infections, in five infants admitted in Neonatology Unit of S. Matteo Hospital in Pavia, four in August–September 2012 and one in February 2013 (Table 1; the cases include two twins, patient no. 1 and 2 in Table 1). The characteristics of newborns and GBS isolates are shown in Table 1. All samples were collected at admission at the Neonatology Unit between days 12 and 57 after delivery (median days, 48; mean, 37.5). All patients were treated with double antibiotic therapy (ampicillin + gentamicin) for 21 days with resolution of late-onset infection (2 sepsis and meningitis, 3 sepsis). We identified only one GBS negative breast milk culture in one case of maternal positive screening for GBS vaginal-rectal colonization (Fig. 1, patient no. 4). RAPD-based genomic analysis of the isolated GBS (from five