Effects of High and Low Dose Iron-Containing Micronutrient Powders for In-Home Fortification of Complementary Foods on the Gut Microbiome and Gut Inflammation in Kenyan Infants.

Objectives: Primary outcome was change in composition of gut microbiome, after 3 weeks and 4 months. Secondary outcomes were changes in faecal calprotectin, treated diarrhoea, anaemia, iron status and systemic inflammation. Methods: We performed two randomized controlled trials in 6-month-old Kenyan infants consuming home-fortified maize porridge daily for four months. 1) infants received an MNP containing 2.5 mg iron as NaFeEDTA (+2.5 mgFeMNP) or the identical MNP without iron (-2.5 mgFeMNP). 2) a different MNP containing 12.5 mg iron as ferrous fumarate (+12.5 mgFeMNP) or the identical MNP without iron (-12.5 mgFeMNP). Results: We enrolled 117 infants, and 101 infants completed the studies between March 2010 and September 2012. Baseline prevalence of anaemia and systemic inflammation were 67.3% and 29.7%, respectively. At baseline, 63% of the total microbial 16S rRNA could be assigned to Bifidobacteriaceae; using qPCR, Salmonella was detected in 22.8% of infants, B. cereus in 38.6%, S. aureus in 71.3%, C. difficile in 53.5%, and C. perfringens in 86.1%. Body iron stores increased in the +12.5 mgFeMNP (p=0.001), but not in the +2.5 mgFeMNP. Using pyrosequencing, +FeMNPs increased enterobacteria, especially Escherichia/Shigella (p=0.048), the enterobacteria/ bifidobacteria ratio (p=0.020), and Clostridium (p=0.03) compared to -FeMNPs; +FeMNPs also increased faecal calprotectin (p=0.002). Most of these effects were confirmed using qPCR, and many were statistically stronger in ±12.5 mgFeMNP study than in ±2.5 mgFeMNP study. During the trial, 27.3% of infants in the +12.5 mgFeMNP group required treatment for diarrhoea vs. 8.3% in the -12.5 mgFeMNP group (p=0.092). Conclusions: In rural Africa where infectious disease burden is high, provision of iron-containing MNPs to infants increases gut inflammation and modifies the gut microbiome toward a potentially more pathogenic profile.

Phenotypic and Genotypic Antibiotic Resistance Patterns of Staphylococcus aureus From Raw and Spontaneously Fermented Camel Milk.

Aims: To pheno- and genotypically characterise Staphylococcus aureus isolated from raw and fermented camel milk from Kenya and Somali for their antibiotic resistance. Methodology: Microdilution assays to determine minimal inhibitory concentrations (MICs) were done using to 20 different antibiotics. Further tests with selected antibiotics were done using disk diffusion test. Genotypic antibiotic resistance was tested using by microarray hybridization with selected isolates and consequent screening of antibiotic resistance genes by PCR. Results: Prevalence of antibiotic resistance among the 47 S. aureus tested were ampicillin 26% (12), gentamicin 26% (12), streptomycin 11% (5), tetracycline 13% (6), trimethoprim 6% (3) and fusidic acid 2% (1). Multi-resistance was detected with three isolates resistant to two antibiotics, six to three antibiotics and six to four or more antibiotics. Three multi-resistant S. aureus isolates were positive for the β-lactamase resistant genes (blaZ), the tetracycline resistance gene tet38 and the Panton-Valentine leukocidin gene pvl according to microarray hybridization assays. Two of the three isolates harbored additionally streptomycin resistance gene ant(6)-Ia. The tetracycline resistance gene tet(K) was also detected by microarray in four isolates. PCR detected tet(K) and blaZ in 2 and 7 additional isolates respectively. Conclusion: Controlled antibiotic therapy in camels should be introduced to prevent the increase of AB resistant bacteria for this and similar milk and hygienic situations in similar production environment. Detection of the Panton-Valentine leukocidin gene pvl by microarray hybridization calls for further research on possibility of community-acquired methicillin-resistant S. aureus (CA-MRSA) in the milk as CA-MRSA with high virulence potential has been associated with the gene lukF-PV (pvl).

Biodiversity and Enterotoxigenic Potential of Staphylococci Isolated from Raw and Spontaneously Fermented Camel Milk.

Aims: To characterise the diversity, genotypic and phenotypic properties of coagulase negative and coagulase positive staphylococci from camel milk. Place and Duration of Study: Laboratory of Food Biotechnology, Department of Health Sciences and Technology (D-HEST), Swiss Federal Institute of Technology, Zurich, Switzerland, between July 2009 and June 2011. Methodology: Staphylococci isolated from 59 raw and spontaneously fermented camel milk (suusac) samples from Kenya and Somalia were identified, pheno- and genotypically characterized. Preliminary screening of colonies was done by catalase test, Gram staining reactions, clumping factor/protein A and microscopy. Further identification was done by 23S rDNA species PCR, thermostable nuclease gene (nuc) PCR and rep-PCR followed by staphylococcal genus ID32 Staph system and coagulase negative species specific PCR. PCR amplification of the genes encoding capsular polysaccharides cap5 and cap8, and staphylococcal enterotoxins SEA to SEE and SEG to SEJ was also carried out. Results: From a total of 235 BP medium isolates, staphylococci were 146 (62 %) of which, 66 (45 %) were Staphylococcus aureus. S. epidermidis accounted for 43 % of the coagulase negative staphylococci (CNS). The rest of the CNS were 25 % S. simulans, 16.3 % S. saprophyticus, 2.5 % S. haemolyticus, 2.5% S. hyicus, 2.5 % S. xylosus, 2.5 % S. lentus, 1.3 % S. carnosus and 1.3 % S. microti. Capsular polysaccharide gene cap5 was present in 15 % and cap8 in 23 % of the S. aureus isolates. Enterotoxin genes were detected in 47 % of the staphylococci with sej in 33 %, seb in 6 %, sed in 5 % and seg in 3 % of the isolates. Within the species enterotoxin genes were detected in 100 %, 64.7 %, 38.5 % and 22.7 % of the S. simulans, S. epidermidis, S. sapropyticus and S. aureus respectively. Conclusion: The diversity of CNS is remarkable and the prevalence of enterotoxin genes amongst CNS and CPS further informs generalizations for other milk and hygienic situations in similar production environment.

Fermentation technologies for the production of exopolysaccharide-synthesizing Lactobacillus rhamnosus concentrated cultures

The exopolysaccharide (EPS)-producing cultures such as Lactobacillus rhamnosus RW-9595M present a challenge for the culture producers because the high viscosity of the fermented growth medium makes it difficult to recover the cells by centrifugation or filtration. This study examined four approaches to reduce viscosity of the medium while producing high cell densities: incubation temperature, extended incubation in the stationary growth phase, production in alginate gel beads and fed-batch fermentation technology. Automated spectrophotometry (AS) was used to study the effects of temperature, pH and lactate level on growth of the strain. In AS assays, there was no significant difference in final maximal biomass production at temperatures ranging between 34 ºC to 44 ºC, but lower yields were noted at 46 C. A pH below 6.0 and a lactate concentration higher than 4 percent almost completely prevented growth. Under batch fermentation conditions, the viscosity of the medium obtained at 37 C was two fold higher than for 44 ºC. For cultures produced at 37 ºC, centrifugation at 10000 g during 5 min did not allow complete recovery of cells, in contrast to cultures grown at 44 ºC. An extended period of incubation (5 hrs) in the stationary growth phase did not reduce the final viscosity of the growth medium. For similar biomass levels, the glucose-based fed-batch fermentation allowed a 40 percent reduction in viscosity of the fermented medium in comparison to traditional batch cultures. High-density cell populations (3 x 10(10) CFU/g) were obtained when L. rhamnosus RW-9595M was grown in alginate beads. However, overall biomass yields in the immobilized cell bioreactor were half of those obtained in free-cell fermentations. Therefore three methods of producing concentrated EPS-producing cultures are proposed.