María Marín

Is meconium from healthy newborns actually sterile

In a previous study, bacteria were able to be isolated from umbilical cord blood of healthy neonates and from murine amniotic fluid obtained by caesarean section. This suggested that term fetuses are not completely sterile and that a prenatal mother-to-child efflux of commensal bacteria may exist. Therefore, the presence of such bacteria in meconium of 21 healthy neonates was investigated. The identified isolates belonged predominantly to the genuses Enterococcus and Staphylococcus. Later, a group of pregnant mice were orally inoculated with a genetically labelled E. fecium strain previously isolated from breast milk of a healthy woman. The labelled strain could be isolated and PCR-detected from meconium of the inoculated animals obtained by caesarean section one day before the predicted date of labor. In contrast, it could not be detected in samples obtained from a non-inoculated control group.

Isolation of Commensal Bacteria from Umbilical Cord Blood of Healthy Neonates Born by Cesarean Section

In a previous study, lactic acid bacteria were isolated from meconium obtained from healthy neonates born by cesarean section. Such a finding suggested that term fetuses are not completely sterile, and that a mother-to-child efflux of commensal bacteria may exist. Therefore, presence of such bacteria in umbilical cord blood of healthy neonates born by elective cesarean section was investigated. The blood samples were submitted to an enrichment step and then inoculated onto agar plates. All the identified isolates belonged to the genus Enterococcus, Streptococcus, Staphylococcus, or Propionibacterium. Later, a group of pregnant mice were orally inoculated with a genetically labeled E. faecium strain previously isolated from breast milk of a healthy woman. The labeled strain could be isolated and polymerase chain reaction detected from the amniotic fluid of the inoculated animals. In contrast, it could not be detected in the samples obtained from a noninoculated control group.

Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR

ABSTRACT The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut.

Probiotic Potential of 3 Lactobacilli Strains Isolated From Breast Milk

Breast milk is an important factor in the initiation, development, and composition of the neonatal gut microbiota. In a previous study, the authors isolated lactic acid bacteria from milk of healthy mothers. Since some of the identified isolates belonged to the genus Lactobacillus, the objective of this work was to evaluate the probiotic potential of 2 Lactobacillus gasseri and 1 Lactobacillus fermentu strains. Different assays, including survival to conditions simulating those existing in the gastrointestinal tract, production of antimicrobial compounds, adherence to intestinal cells, production of biogenic amines, degradation of mucin, enzymatic profile, and pattern of antibiotic resistance, were performed. Globally, the results showed that the probiotic potential of lactobacilli isolated from milk of healthy mothers is, at least, similar to that of the strains commonly used in commercial probiotic products. This fact, together with the presence of prebiotic substances, indicates that breast milk is a natural synbiotic food. J Hum Lact. 21(1):8-17.

Staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics

BackgroundAlthough Staphylococcus aureus is considered the main etiological agent of infectious mastitis, recent studies have suggested that coagulase-negative staphylococci (CNS) may also play an important role in such infections. The aims of this work were to isolate staphylococci from milk of women with lactational mastitis, to select and characterize the CNS isolates, and to compare such properties with those displayed by CNS strains isolated from milk of healthy women.ResultsThe milk of 30 women was collected and bacterial growth was noted in 27 of them, of which Staphylococcus epidermidis was isolated from 26 patients and S. aureus from 8. Among the 270 staphylococcal isolates recovered from milk of women with mastitis, 200 were identified as Staphylococcus epidermidis by phenotypic assays, species-specific PCR and PCR sequencing. They were typified by pulsed field gel electrophoresis (PFGE) genotyping. The PFGE profiles of the S. epidermidis strains were compared with those of 105 isolates from milk of healthy women. A representative of the 76 different PFGE profiles was selected to study the incidence of virulence factors and antibiotic resistance. The number of strains that contained the biofilm-related icaD gene and that showed resistance to oxacillin, erythromycin, clindamycin and mupirocin was significantly higher among the strains isolated from mastitic milk.ConclusionS. epidermidis may be a frequent but largely underrated cause of infectious mastitis in lactating women. The resistance to diverse antibiotics and a higher ability to form biofilms found among the strains isolated from milk of women suffering mastitis may explain the chronic and/or recurrent nature of this infectious condition.

Cold Storage of Human Milk: Effect on Its Bacterial Composition

Objectives:In the last few years, it has been proved that human milk contains bacteria that constitute an important factor in the initiation and development of the neonatal gut microbiota. In this context, the objective of this study was to evaluate the effect of cold storage on the natural bacterial composition of breast milk. Materials and Methods:Breast milk samples provided by 34 healthy women and collected either by manual expression (n = 27) or breast pump (n = 7), were plated onto several culture media immediately after arrival at the laboratory (day 0) and after storage at −20°C for 6 weeks. A high number of isolates from 8 of the women were identified at the species level. Results:No statistically significant differences were observed between the counts obtained at both sampling times in those media in which growth was detected. In all of the culture media, bacterial counts in pump-collected samples were higher than in those obtained by manual expression. Staphylococci and streptococci were the predominant bacteria in both fresh and frozen samples, Staphylococcus epidermidis being the most abundant species at both sampling times. Lactic acid bacteria and bifidobacteria were also present in fresh and frozen breast milk samples, but among them, only 1 species (Lactobacillus gasseri) could be isolated at both sampling times. Conclusions:The results of this study suggest that cold storage of milk at −20°C for 6 weeks does not significantly affect either the quantitative or the qualitative bacterial composition of breast milk.

Lactic acid bacteria: hydrophobicity and strength of attachment to meat surfaces

The hydrophobicity and strength of a ttachment of several lactic acid bacteria with antimicrobial activity were studied. Hydrophobicity was determined by bacterial adherence to hydrocarbons (BATH; octane or xylene), adhesion to nitrocellulose filters (NCF), salt aggregation test (SAT) and adherence to phenyl–Sepharose beads (PSB). The relative hydrophobicity of lactic acid bacteria depended markedly on the method used. No correlation between either SAT or BATH (octane) and strength of attachment (Sr value) existed. However, a significant relationship between strength of attachment and BATH (xylene), NCF and PSB, respectively, was observed, showing the highest correlation coefficient (r = 0·778) for BATH (xylene).

A novel genetic label for detection of specific probiotic lactic acid bacteria

A novel method for genetic labelling of specific lactic acid bacteria strains was developed. The approach implied the transformation of the hosts with a plasmid containing a heterologous DNA fragment. The sequence of a DNA fragment that has been used to label a variety of genetically modified (GM) soya was used to design a forward primer and three reverse primers yielding PCR products recognisable by their sizes. Stability of the recombinant plasmid in the transformed strains was studied by PCR, and the results varied significantly depending on the strain. To test the usefulness of the DNA label to study in vivo properties of probiotic bacteria, such as viability after transit through the digestive tract, mice were orally inoculated with a genetically-labelled Enterococcus faecium strain. Later, their faeces were aseptically collected and the genetically-labelled strain was detected among the colonies that grew on MRS agar.

Virulence Factors-Pathogenicity Relationships for Aeromonas Species from Clinical and Food Isolates

The presence of virulence factors in 96Aeromonas strains isolated from food and clinical samples was studied. Neither cytotoxic activity and hydrophobicity, not the presence of pili or an extra surface layer made it possible to establish differences between food and clinical strains. Statistical studies showed that cytotoxin production was associated with a positive Voges—Proskauer reaction, inability to ferment arabinose and a positive lysine decarboxylation. Therefore, when comparing cytotoxic clinical and food strains with lysine decarboxylation phenotype, there was a significant difference (p<0.05) between the two groups. The association of a cytotoxin production and lysine decarboxylation character should thus be considered as a possible virulence marker.

Mutagenicity and cytotoxicity of fruits and vegetables evaluated by the Ames test and 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay / Mutagenicidad y citotoxicidad de frutas y vegetales evaluadas por el test de Ames y el ensayo del bromuro de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT)

The mutagenic and cytotoxic activity of the aqueous (H2O) and ethanolic (EtOH) extracts of fruits and vegetables were studied by the Ames test and the MTT assay. A significant mutagenic activity was found for three H2O extracts of broccoli (Brassica oleracea), carrot (Dacus carota) and licorice (Glycyrrhiza glabra) and for one EtOH extract of kiwi (Ananas sativus ) of the nine species analyzed. The frameshift tester strain TA98 was reverted by broccoli (2.4, 4.8 and 9.6 mg/mL; p < 0.01) and carrot (9.6 mg/mL; p < 0.001) aqueous extracts and by kiwi (9.6 mg/mL; p < 0.0001) EtOH extract, whereas strain TA100 was only sensitive to the mutagens of licorice H,O extract, within the nine fruits and vegetables tested. The mutagenic response of the extracts was not altered by the presence of S9 mix. Cytotoxicity was only found for three of the nine species tested. Percentage cytotoxic ac tivities at 4.8 mg/mL in pineapple (Actinidin diasinensis) and garlic (Allium sativum) H,O extracts were 77 and 91 %, respectively. Licorice EtOH extract was the only one that showed a cytotoxic activity at all of the concentrations used. The percentage of cytotoxic activity of licorice extract was 63% at 0.24 mg/mL and increased with increasing concentration of licorice up to 4.8 mg/mL. Thus, licorice (EtOH) extract was the most cytotoxic of the species tested.