The genus Streptococcus is 1 of the dominant bacterial groups in human milk, but the taxonomic identification of some species remains difficult.
Objective:
The objective of this study was to investigate the discriminatory ability of different methods to identify streptococcal species in order to perform an assessment of the streptococcal diversity of human milk microbiota as accurately as possible.
Methods:
The identification of 105 streptococcal strains from human milk was performed by 16S rRNA, tuf, and sodA gene sequencing, phylogenetic analysis, and Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry.
Results:
Streptococcus salivarius, Streptococcus mitis, and Streptococcus parasanguinis were the streptococcal dominant species in the human milk microbiota. Sequencing of housekeeping genes allowed the classification of 96.2% (16S rRNA), 84.8% (sodA), and 88.6% (tuf) of the isolates. Phylogenetic analysis showed 3 main streptococcal clusters corresponding with the mitis (73 isolates), salivarius (29), mutans (1)-pyogenic (2) groups, but many of the mitis group isolates (36) could not be assigned to any species. The application of the MALDI-TOF Bruker Biotyper system resulted in the identification of 56 isolates (53.33%) at the species level, but it could not discriminate between S pneumoniae and S mitis isolates, in contrast to the Vitek-MS system.
Conclusion:
There was a good agreement among the different methods assessed in this study to identify those isolates of the salivarius, mutans, and pyogenic groups, whereas unambiguous discrimination could not be achieved concerning some species of the mitis group (S mitis, S pneumoniae, S pseudopneumoniae, S oralis).
FernándezLLangaSMartínV. The human milk microbiota: origin and potential roles in health and disease. Pharmacol Res. 2013;69(1):1-10.
2.
MartínRHeiligHGZoetendalEG. Cultivation-independent assessment of the bacterial diversity of breast milk among healthy women. Res Microbiol. 2007;158(1):31-37.
3.
MarínMLArroyoRJiménezEGómezAFernándezLRodríguezJM. Cold storage of human milk: effect on its bacterial composition. J Pediatr Gastroenterol Nutr. 2009;49(3):343-348.
4.
HuntKMFosterJAForneyLJ. Characterization of the diversity and temporal stability of bacterial communities in human milk. PLoS ONE. 2011;6(6):e21313.
5.
JostTLacroixCBraeggerCChassardC. Assessment of bacterial diversity in breast milk using culture-dependent and culture-independent approaches. Br J Nutr. 2013;110(7):1253-1262.
6.
EuzebyJP. List of prokaryotic names with standing nomenclature (LPSN). Genus Streptococcus. www.bacterio.net/streptococcus.html. Accessed July 1, 2015.
7.
DoernCDBurnhamCA. It’s not easy being green: the viridans group streptococci, with a focus on pediatric clinical manifestations. J Clin Microbiol. 2010;48(11):3829-3835.
8.
CoykendallAL. Classification and identification of the viridans streptococci. Clin Microbiol Rev. 1989;2(3):315-328.
9.
FacklamR. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin Microbiol Rev. 2002;15(4):613-630.
10.
BishopCJAanensenDMJordanGEKilianMHanageWPSprattBG. Assigning strains to bacterial species via the internet. BMC Biol. 2009;7:3.
11.
KawamuraYHouXGSultanaFMiuraHEzakiT. Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol. 1995;45(2):406-408.
12.
ArbiqueJCPoyartCTrieu-CuotP. Accuracy of phenotypic and genotypic testing for identification of Streptococcus pneumoniae and description of Streptococcus pseudopneumoniae sp. nov. J Clin Microbiol. 2004;42(10):4686-4696.
13.
ItohYKawamuraYKasaiH. dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus. Syst Appl Microbiol. 2006;29(5):368-374.
14.
ZbindenAKöhlerNBloembergGV. recA-based PCR assay for accurate differentiation of Streptococcus pneumoniae from other viridans streptococci. J Clin Microbiol. 2011;49(2):523-527.
15.
GlazunovaOORaoultDRouxV. Partial recN gene sequencing: a new tool for identification and phylogeny within the genus Streptococcus. Int J Syst Evol Microbiol. 2010;60(9):2140-2148.
16.
PoyartCQuesneGCoulonSBerchePTrieu-CuotP. Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase. J Clin Microbiol. 1998;36(1):41-47.
17.
DrancourtMRouxVFournierPERaoultD. rpoB gene sequence-based identification of aerobic Gram-positive cocci of the genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella. J Clin Microbiol. 2004;42(2):497-504.
18.
PicardFJKeDBoudreauDK. Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species. J Clin Microbiol. 2004;42(8):3686-3695.
19.
FriedrichsCRodloffACChhatwalGSSchellenbergerWEschrichK. Rapid identification of viridans streptococci by mass spectrometric discrimination. J Clin Microbiol. 2007;45(8):2392-2397.
20.
HeikkiläMPSarisPEJ. Inhibition of Staphylococcus aureus by the commensal bacteria of human milk. J Appl Microbiol. 2003;95(3):471-478.
21.
DelgadoSArroyoRMartínRRodríguezJM. PCR-DGGE assessment of the bacterial diversity of breast milk in women with lactational infectious mastitis. BMC Infect Dis. 2008;8:51.
22.
JiménezEDelgadoSFernándezL. Assessment of the bacterial diversity of human colostrum and screening of staphylococcal and enterococcal populations for potential virulence factors. Res Microbiol. 2008;159(9-10):595-601.
23.
ArroyoRMartínVMaldonadoAJiménezEFernándezLRodríguezJM. Treatment of infectious mastitis during lactation: antibiotics versus oral administration of Lactobacilli isolated from breast milk. Clin Infect Dis. 2010;50(12):1551-1558.
24.
MartínVMañes-LázaroRRodríguezJMMaldonado-BarragánA. Streptococcus lactarius sp. nov., isolated from breast milk of healthy women. Int J Syst Evol Microbiol. 2011;61(5):1048-1052.
25.
Ruiz-BarbaJLMaldonadoAJiménez-DíazR. Small-scale total DNA extraction from bacteria and yeast for PCR applications. Anal Biochem. 2005;347(2):333-335.
26.
KullenMJSanozky-DawesRBCrowellDCKlaenhammerTR. Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex. J Appl Microbiol. 2000;89(3):511-516.
27.
ColladoMCDelgadoSMaldonadoARodríguezJM. Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR. Lett Appl Microbiol. 2009;48(5):523-528.
28.
TamuraKPetersonDPetersonNStecherGNeiMKumarS. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731-2739.
29.
MartínRLangaSReviriegoC. Human milk is a source of lactic acid bacteria for the infant gut. J Pediatr. 2003;143(6):754-758.
30.
WardTLHosidSIoshikhesIAltosaarI. Human milk metagenome: a functional capacity analysis. BMC Microbiol. 2013;13(1):116.
31.
IkryannikovaLNLapinKNMalakhovaMV. Misidentification of alpha-hemolytic streptococci by routine tests in clinical practice. Infect Genet Evol. 2011;11(7):1709-1715.
32.
TelesCSmithARamageGLangS. Identification of clinically relevant viridans group streptococci by phenotypic and genotypic analysis. Eur J Clin Microbiol Infect Dis. 2011;30(2):243-250.
33.
DaviesAPReidMHadfieldSJ. Identification of clinical isolates of α-hemolytic streptococci by 16S rRNA gene sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry using MALDI Biotyper, and conventional phenotypic methods: a comparison. J Clin Microbiol. 2012;50(12):4087-4090.
34.
ChiFNolteOBergmannCIpMHakenbeckR. Crossing the barrier: evolution spread of a major class of mosaic pbp2x in Streptococcus pneumoniae, S. mitis and S. oralis. Int J Med Microbiol. 2007;297(7-8):503-512.
35.
HakenbeckRBalmelleNWeberBGardesCKeckWde SaizieuA. Mosaic genes and mosaic chromosomes: intra- and interspecies genomic variation of Streptococcus pneumoniae. Infect Immun. 2001;69(4):2477-2486.
36.
KilianMPoulsenKBlomqvistT. Evolution of Streptococcus pneumoniae and its close commensal relatives. PLoS ONE. 2008;3(7):e2683.
37.
RupfSBreitungKSchellenbergerWMerteKKneistSEschrichK. Differentiation of mutans streptococci by intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Oral Microbiol Immunol. 2005;20(5):267-273.
38.
CherkaouiAEmonetSFernandezJSchorderetDSchrenzelJ. Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid identification of beta-hemolytic streptococci. J Clin Microbiol. 2011;49(8):3004-3005.
39.
StevensonLGDrakeSKMurrayPR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2010;48(2):444-447.
40.
SchulthessBBrodnerKBloembergGVZbindenRBottgerECHombachM. Identification of Gram-positive cocci by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry: comparison of different preparation methods and implementation of a practical algorithm for routine diagnostics. J Clin Microbiol. 2013;51(6):1834-1840.
41.
WernoAMChristnerMAndersonTPMurdochDR. Differentiation of Streptococcus pneumoniae from nonpneumococcal streptococci of the Streptococcus mitis group by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2012;50(9):2863-2867.
42.
IkryannikovaLNFilimonovaAVMalakhovaMV. Discrimination between Streptococcus pneumoniae and Streptococcus mitis based on sorting of their MALDI mass spectra. Clin Microbiol Infect. 2013;19(11):1066-1071.
43.
BrandaJAMarkhamRPGarnerCDRychertJAFerraroMJ. Performance of the Vitek MS v2.0 system in distinguishing Streptococcus pneumoniae from nonpneumococcal species of the Streptococcus mitis group. J Clin Microbiol. 2013;51(9):3079-3082.
44.
DuboisDSegondsCPrereMFMartyNOswaldE. Identification of clinical Streptococcus pneumoniae isolates among other alpha and nonhemolytic streptococci by use of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system. J Clin Microbiol. 2013;51(6):1861-1867.
45.
López RoaPSánchez CarrilloCMarínMRomeroFCercenadoEBouzaE. Value of matrix-assisted laser desorption ionization-time of flight for routine identification of viridans group streptococci causing bloodstream infections. Clin Microbiol Infect. 2013;19(5):438-444.
46.
KärpänojaPHarjuIRantakokko-JalavaKHaanperäMSarkkinenH. Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of viridans group streptococci. Eur J Clin Microbiol Infect Dis. 2013;33(5):779-788.
47.
DenapaiteDBrucknerRNuhnM. The genome of Streptococcus mitis B6—what is a commensal?PLoS ONE. 2010;5(2):e9426.
48.
MitchellJ. Streptococcus mitis: walking the line between commensalism and pathogenesis. Mol Oral Microbiol. 2011;26(2):89-98.
49.
KrzysciakWPluskwaKKJurczakAKoscielniakD. The pathogenicity of the Streptococcus genus. Eur J Clin Microbiol Infect Dis. 2013;32(11):1361-1376.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
