PCR-DGGE assessment of the bacterial diversity of breast milk in women with lactational infectious mastitis

Susana Delgado, Rebeca Arroyo, Rocío Martín, Juan M Rodríguez, Susana Delgado, Rebeca Arroyo, Rocío Martín, Juan M Rodríguez

Abstract

Background: Infectious mastitis is a common condition during lactation and in fact, represents one of the main causes leading to a precocious weaning. The number of studies dealing with lactational mastitis is low and, up to now, the etiological diagnosis is frequently made on the basis of unspecific clinical signs. The aim of this study was to investigate the microbial diversity of breast milk in 20 women with lactational mastitis employing culture-dependent and culture-independent (PCR-DGGE) approaches.

Methods: Breast milk samples were cultured in different media to investigate the presence of bacteria and/or yeasts, and a total of 149 representative isolates were identified to the species level by 16S rRNA gene PCR sequencing. The microorganisms recovered were compared with those found by PCR-DGGE analysis. To identify the DGGE profiles two reference markers of different microbial species were constructed. Sequence analysis of unknown bands was also performed.

Results: Staphylococci were the dominant bacterial group and Staphylococcus epidermidis was the dominant species. In a lower number of samples, other bacteria (mainly streptococci and a few gram-negative species) were also identified. Globally, PCR-DGGE results showed a good correlation with those obtained by culture-based methods. However, although DNA bands corresponding to different lactic acid bacteria were detected, such bacteria could not be isolated from the milk samples.

Conclusion: Staphylococci seem to be the main etiological agents of human lactational mastitis. The combined use of culture and molecular techniques allowed a better characterization of the bacterial diversity in milk from women suffering from infectious mastitis. Our results suggest that this condition could be the result of a disbiotic process where some of the bacterial species usually present in human milk outgrow (staphylococci) while others disappear (lactobacilli or lactococci).

Figures

Figure 1
Figure 1
DGGE profiles of the 16S rDNA amplicons used to construct the DGGE markers. Lanes: a, Staphylococcus epidermidis; b, Staphylococcus aureus; c, Klebsiella oxytoca; d, Pseudomonas fluorescens; e, Streptococcus mitis; f, Streptococcus salivarius; g, Streptococcus oralis; h, Streptococcus parasanguis; i, Rothia mucilaginosa; j, Lactobacillus salivarius; k, Enterococcus faecalis; l, Enterococcus faecium; m, Lactobacillus reuteri; n, Lactobacillus johnsonii; o, Lactobacillus gasseri; p, Lactobacillus fermentum.
Figure 2
Figure 2
DGGE analysis of the bacterial diversity in the milk samples. Lanes: I and II, DGGE identification markers. DGGE marker I comprises the species listed from (a) to (i) in the Figure 1 legend while DGGE marker II contains the species listed from (j) to (p). M1 to M20, DGGE profiles of samples M1 to M20. Bands identified by gel excision and PCR sequencing are indicated by letters from (q) to (z) and from (α) to (γ), and correspond to the following bacteria: q, Gemella haemolysans [EMBL: AM774574]; r, Acinetobacter sp. [EMBL: AM774586]; s, Staphylococcus capitis [EMBL: AM774573]; t, Arthrobacter sp. [EMBL: AM774575]; u, Streptococcus suis [EMBL: AM774576]; v, Streptococcus cristatus [EMBL: AM774577]; w, Streptococcus pneumoniae [EMBL: AM774579]; x, Streptococcus anginosus [EMBL: AM774580]; y,Lactobacillus animalis [EMBL: AM774581]; z, Lactococcus lactis [EMBL: AM774582]; α, Lactococcus garvieae [EMBL: AM774583]; β, Propionibacterium acnes [EMBL: AM774584]; γ, Neisseria weaveri [EMBL: AM774585]. The arrows indicate the bands that matched with sequences identified as" uncultured bacterium DGGE band 16S ribosomal RNA gene" in the database.

References

    1. Foxman B, D'Arcy H, Gillespie B, Bobo JK, Schwartz K. Lactation mastitis: occurrence and medical management among 946 breastfeeding women in the United States. Am J Epidemiol. 2002;155:103–114. doi: 10.1093/aje/155.2.103.
    1. World Health Organization (WHO) Mastitis: Causes and Management. Geneva, Switzerland: Dept. of child and adolescent health and development WHO/FCH/CAH/00.13; 2000.
    1. Osterman KL, Rham V. Lactation mastitis: bacterial cultivation of breast milk, symptoms, treatment, and outcome. J Hum Lact. 2000;16:297–302. doi: 10.1177/089033440001600405.
    1. Michie C, Lockie F, Lynn W. The challenge of mastitis. Arch Dis Child. 2003;88:818–821. doi: 10.1136/adc.88.9.818.
    1. Carmichael AR, Dixon JM. Is lactation mastitis and shooting breast pain experienced by women during lactation caused by Candida albicans? Breast. 2002;11:88–90. doi: 10.1054/brst.2001.0313.
    1. Francis-Morrill J, Heinig MJ, Pappagianis D, Dewey KG. Diagnostic value of signs and symptoms of mammary candidosis among lactating women. J Human Lact. 2004;20:288–295. doi: 10.1177/0890334404267226.
    1. Heikkila MP, Saris PE. Inhibition of Staphylococcus aureus by the commensal bacteria of human milk. J Appl Microbiol. 2003;95:471–478. doi: 10.1046/j.1365-2672.2003.02002.x.
    1. Martín R, Langa S, Reviriego C, Jiménez E, Marín ML, Xaus J, Fernández L, Rodríguez JM. Human milk is a source of lactic acid bacteria for the infant gut. J Pediatr. 2003;143:754–758. doi: 10.1016/j.jpeds.2003.09.028.
    1. Martín R, Langa S, Reviriego C, Jiménez E, Marín ML, Olivares M, Bouza J, Fernández L, Xaus J, Rodríguez JM. The commensal microflora of human milk: new perspectives for food bacteriotherapy and probiotics. Trends Food Sci Tech. 2004;15:121–127. doi: 10.1016/j.tifs.2003.09.010.
    1. Perez PF, Doré J, Leclerc M, Levenez F, Benyacoub J, Serrant P, Segura-Roggero I, Schiffrin EJ, Donnet-Hughes A. Bacterial imprinting of the neonatal immune system: lessons from maternal cells? Pediatrics. 2007;119:e724–732. doi: 10.1542/peds.2006-1649.
    1. Kullen MJ, Sanozky-Dawes RB, Crowell DC, Klaenhammer TR. 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;89:511–516. doi: 10.1046/j.1365-2672.2000.01146.x.
    1. Martín R, Heilig HG, Zoetendal EG, Jiménez E, Fernández L, Smidt H, Rodríguez JM. Cultivation-independent assessment of the bacterial diversity of breast milk among healthy women. Research Microbiol. 2007;158:31–37. doi: 10.1016/j.resmic.2006.11.004.
    1. Muyzer G, de Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 1993;59:695–700.
    1. Sanguinetti CJ, Dias Neto E, Simpson AJ. Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. BioTechniques. 1994;17:914–921.
    1. dos Santos Nascimento J, Fagundes PC, de Paiva Brito MA, dos Santos KR, do Carmo de Freire Bastos M. Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis. Vet Microbiol. 2005;106:61–71. doi: 10.1016/j.vetmic.2004.10.014.
    1. Soltys J, Quinn MT. Selective recruitment of T-cell subsets to the udder during staphylococcal and streptococcal mastitis: analysis of lymphocyte subsets and adhesion molecule expression. Infect Immun. 1999;67:6293–6302.
    1. Thorberg BM, Kuhn I, Aarestrup FM, Brandstrom B, Jonsson P, Danielsson-Tham ML. Pheno- and genotyping of Staphylococcus epidermidis isolated from bovine milk and human skin. Vet Microbiol. 2006;115:163–72. doi: 10.1016/j.vetmic.2006.01.013.
    1. Zhang S, Maddox CW. Cytotoxic activity of coagulase-negative staphylococci in bovine mastitis. Infect Immun. 2000;68:1102–1108. doi: 10.1128/IAI.68.3.1102-1108.2000.
    1. Thomsen AC, Mogensen SC, Love Jepsen F. Experimental mastitis in mice induced by coagulase-negative staphylococci isolated from cases of mastitis in nursing women. Acta Obstet Gynecol Scand. 1985;64:163–166.
    1. Gill SR, Fouts DE, Archer GL, Mongodin EF, Deboy RT, Ravel J, Paulsen IT, Kolonay JF, Brinkac L, Beanan M, Dodson RJ, Daugherty SC, Madupu R, Angiuoli SV, Durkin AS, Haft DH, Vamathevan J, Khouri H, Utterback T, Lee C, Dimitrov G, Jiang L, Qin H, Weidman J, Tran K, Kang K, Hance IR, Nelson KE, Fraser CM. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacteriol. 2005;187:2426–2438. doi: 10.1128/JB.187.7.2426-2438.2005.
    1. Vuong C, Otto M. Staphylococcus epidermidis infections. Microbes Infect. 2002;4:481–489. doi: 10.1016/S1286-4579(02)01563-0.
    1. Ziebuhr W, Hennig S, Eckart M, Kranzler H, Batzilla C, Kozitskaya S. Nosocomial infections by Staphylococcus epidermidis : how a commensal bacterium turns into a pathogen. Int J Antimicrob Agents. 2006;28:S14–S20. doi: 10.1016/j.ijantimicag.2006.05.012.
    1. Martín R, Olivares M, Marín ML, Fernández L, Xaus J, Rodríguez JM. Probiotic potential of 3 Lactobacilli strains isolated from breast milk. J Hum Lact. 2005;21:8–17. doi: 10.1177/0890334404272393.
    1. Martín R, Jiménez E, Olivares M, Marín ML, Fernández L, Xaus J, Rodríguez JM. Lactobacillus salivarius CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother-child pair. Int J Food Microbiol. 2006;112:35–43. doi: 10.1016/j.ijfoodmicro.2006.06.011.
    1. Beasley SS, Saris PE. Nisin-producing Lactococcus lactis strains isolated from human milk. Appl Environ Microbiol. 2004;70:5051–5053. doi: 10.1128/AEM.70.8.5051-5053.2004.

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