East and West African milk products are reservoirs for human and livestock-associated Staphylococcus aureus.

Staphylococcus aureus frequently isolated from milk products in sub-Saharan Africa (SSA) is a major pathogen responsible for food intoxication, human and animal diseases. SSA hospital-derived strains are well studied but data on the population structure of foodborne S. aureus required to identify possible staphylococcal food poisoning sources is lacking. Therefore, the aim was to assess the population genetic structure, virulence and antibiotic resistance genes associated with milk-derived S. aureus isolates from Côte d'Ivoire, Kenya and Somalia through spa-typing, MLST, and DNA microarray analysis. Seventy milk S. aureus isolates from the three countries were assigned to 27 spa (7 new) and 23 (12 new) MLST sequence types. Milk-associated S. aureus of the three countries is genetically diverse comprising human and livestock-associated clonal complexes (CCs) predominated by the CC5 (n = 10) and CC30 (n = 9) isolates. Panton-Valentine leukocidin, toxic shock syndrome toxin and enterotoxin encoding genes were predominantly observed among human-associated CCs. Penicillin, fosfomycin and tetracycline, but not methicillin resistance genes were frequently detected. Our findings indicate that milk-associated S. aureus in SSA originates from human and animal sources alike highlighting the need for an overarching One Health approach to reduce S. aureus disease burdens through improving production processes, animal care and hygienic measures.

How to introduce medical ethics at the bedside - Factors influencing the implementation of an ethical decision-making model.

BACKGROUND: As the implementation of new approaches and procedures of medical ethics is as complex and resource-consuming as in other fields, strategies and activities must be carefully planned to use the available means and funds responsibly. Which facilitators and barriers influence the implementation of a medical ethics decision-making model in daily routine? Up to now, there has been little examination of these factors in this field. METHODS: A medical ethics decision-making model called METAP was introduced on three intensive care units and two geriatric wards. An evaluation study was performed from 7 months after deployment of the project until two and a half years. Quantitative and qualitative methods including a questionnaire, semi-structured face-to-face and group-interviews were used. RESULTS: Sixty-three participants from different professional groups took part in 33 face-to-face and 9 group interviews, and 122 questionnaires could be analysed. The facilitating factors most frequently mentioned were: acceptance and presence of the model, support given by the medical and nursing management, an existing or developing (explicit) ethics culture, perception of a need for a medical ethics decision-making model, and engaged staff members. Lack of presence and acceptance, insufficient time resources and staff, poor inter-professional collaboration, absence of ethical competence, and not recognizing ethical problems were identified as inhibiting the implementation of the METAP model. However, the results of the questionnaire as well as of explicit inquiry showed that the respondents stated to have had enough time and staff available to use METAP if necessary. CONCLUSIONS: Facilitators and barriers of the implementation of a medical ethics decision-making model are quite similar to that of medical guidelines. The planning for implementing an ethics model or guideline can, therefore, benefit from the extensive literature and experience concerning the implementation of medical guidelines. Lack of time and staff can be overcome when people are convinced that the benefits justify the effort.

Bifidobacterium thermophilum RBL67 impacts on growth and virulence gene expression of Salmonella enterica subsp. enterica serovar Typhimurium.

BACKGROUND: Bifidobacterium thermophilum RBL67 (RBL67), a human fecal isolate and health promoting candidate shows antagonistic and protective effects against Salmonella and Listeria spec. in vitro. However, the underlying mechanisms fostering these effects remain unknown. In this study, the interactions of RBL67 and Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15) were explored by global transcriptional analysis. RESULTS: Growth experiments were performed in a complex nutritive medium with controlled pH of 6.0 and suitable for balanced growth of both RBL67 and N-15. RBL67 growth was slightly enhanced in presence of N-15. Conversely, N-15 showed reduced growth in the presence of RBL67. Transcriptional analyses revealed higher expression of stress genes and amino acid related function in RBL67 in co-culture with N-15 when compared to mono-culture. Repression of the PhoP regulator was observed in N-15 in presence of RBL67. Further, RBL67 activated virulence genes located on the Salmonella pathogenicity islands 1 and 2. Flagellar genes, however, were repressed by RBL67. Sequential expression of flagellar, SPI 1 and fimbrial genes is essential for Salmonella infection. Our data revealed that RBL67 triggers expression of SPI 1 and fimbrial determinants prematurely, potentially leading to redundant energy expenditure. In the competitive environment of the gut such energy expenditure could lead to enhanced clearing of Salmonella. CONCLUSION: Our study provides first insights into probiotic-pathogen interactions on global transcriptional level and suggests that deregulation of virulence gene expression might be an additional protective mechanism of probiotica against infections of the host.

Effect of Bifidobacterium thermophilum RBL67 and fructo-oligosaccharides on the gut microbiota in Göttingen minipigs.

Modulating the gut microbiota via dietary interventions is a common strategy to enhance the natural defence mechanisms of the host. Several in vitro studies have highlighted the probiotic potential of Bifidobacterium thermophilum RBL67 (RBL67) selected for its anti-Salmonella effects. The present study aimed to investigate the impact of RBL67 alone and combined with fructo-oligosaccharides (FOS) on the gut microbiota of Göttingen minipigs. Minipigs were fed a basal diet supplemented with 8 g/d probiotic powder (1×109 CFU/g in skim milk matrix) (probiotic diet (PRO)), 8 g/d probiotic powder plus 8 g/d FOS (synbiotic diet (SYN)) or 8 g/d skim milk powder (control), following a cross-sectional study design. Faecal and caecal microbiota compositions were analysed with pyrosequencing of 16S rRNA genes and quantitative PCR. Metabolic activity in the caecum and colon was measured by HPLC. 16S rRNA gene amplicon sequencing revealed that minipig faeces show close similarity to pig microbiota. During the treatments and at the time of killing of animals, RBL67 was consistently detected in faeces, caecum and colon at numbers of 105-106 16S rRNA copies/g content after feeding PRO and SYN diets. At the time of killing of animals, significantly higher Bifidobacterium numbers in the caecum and colon of SYN-fed minipigs were measured compared with PRO. Our data indicate that the Göttingen minipig may be a suitable model for gut microbiota research in pigs. Data from this first in vivo study of RBL67 colonisation suggest that the combination with FOS may represent a valuable symbiotic strategy to increase probiotic bacteria levels and survival in gastrointestinal tracts for feed and food applications.

Synergistic effects of Bifidobacterium thermophilum RBL67 and selected prebiotics on inhibition of Salmonella colonization in the swine proximal colon PolyFermS model.

BACKGROUND: Probiotics and prebiotics are promising strategies to counteract Salmonella prevalence in swine. In the present study, we investigated the effects of prebiotics (fructo- (FOS), galacto- (GOS) and mannan- (MOS) oligosaccharides) and the bacteriocinogenic Bifidobacterium thermophilum RBL67 (RBL67) on Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15) colonization using the PolyFermS in vitro continuous fermentation model simulating the swine proximal colon. MATERIAL AND METHODS: The PolyFermS model was designed with a first-stage reactor containing immobilized fecal pig microbiota. This reactor continuously inoculated five parallel second-stage reactors, a control and four treatment reactors, all operated with proximal colon conditions. FOS and GOS (5.2 g/day), and MOS (half dosage) and RBL67 (10(8) copy numbers/mL applied daily) were tested on the ability of N-15 to colonize reactors, inoculated with the same microbiota. Reactor effluents were collected daily and analyzed for microbial composition (quantitative PCR and 454 pyrosequencing of 16S rRNA gene pool) and main metabolites (HPLC). RESULTS: RBL67 and N-15 were shown to stably colonize the system. Colonization of N-15 was strongly inhibited by FOS and GOS, whereas addition of RBL67 alone or combined with MOS showed intermediate results. However, the effect of FOS and GOS was enhanced when prebiotics were combined with a daily addition of RBL67. FOS and GOS increased the total short chain fatty acid production, especially acetate and propionate. RBL67 combined with FOS additionally stimulated butyrate production. CONCLUSIONS: Our study demonstrates the suitability of the porcine PolyFermS in vitro model to study nutritional effects of pro- and prebiotics on gut microbiota composition and activity. It can further be used to monitor Salmonella colonization. The inhibition effects of FOS and GOS on N-15 colonization are partly due to an increased acetate production, while further antimicrobial mechanisms may contribute to an enhanced inhibition with prebiotic-RBL67 combinations. A future direction of this work could be to understand the anti-Salmonella effects of Bifidobacterium thermophilum RBL67 in the presence of prebiotics to unravel the mechanism of this probiotic:pathogen interaction.

In vitro continuous fermentation model (PolyFermS) of the swine proximal colon for simultaneous testing on the same gut microbiota.

In vitro gut modeling provides a useful platform for a fast and reproducible assessment of treatment-related changes. Currently, pig intestinal fermentation models are mainly batch models with important inherent limitations. In this study we developed a novel in vitro continuous fermentation model, mimicking the porcine proximal colon, which we validated during 54 days of fermentation. This model, based on our recent PolyFermS design, allows comparing different treatment effects on the same microbiota. It is composed of a first-stage inoculum reactor seeded with immobilized fecal swine microbiota and used to constantly inoculate (10% v/v) five second-stage reactors, with all reactors fed with fresh nutritive chyme medium and set to mimic the swine proximal colon. Reactor effluents were analyzed for metabolite concentrations and bacterial composition by HPLC and quantitative PCR, and microbial diversity was assessed by 454 pyrosequencing. The novel PolyFermS featured stable microbial composition, diversity and metabolite production, consistent with bacterial activity reported for swine proximal colon in vivo. The constant inoculation provided by the inoculum reactor generated reproducible microbial ecosystems in all second-stage reactors, allowing the simultaneous investigation and direct comparison of different treatments on the same porcine gut microbiota. Our data demonstrate the unique features of this novel PolyFermS design for the swine proximal colon. The model provides a tool for efficient, reproducible and cost-effective screening of environmental factors, such as dietary additives, on pig colonic fermentation.

Monitoring horizontal antibiotic resistance gene transfer in a colonic fermentation model.

The human microbiota is suggested to be a reservoir of antibiotic resistance (ABR) genes, which are exchangeable between transient colonizers and residing bacteria. In this study, the transfer of ABR genes from Enterococcus faecalis to Listeria monocytogenes and to commensal bacteria of the human gut microbiota was demonstrated in a colonic fermentation model. In the first fermentation, an E. faecalis donor harboring the marked 50-kb conjugative plasmid pRE25(*) and a chromosomal marker was co-immobilized with L. monocytogenes and infant feces. In this complex environment, the transfer of pRE25(*) to L. monocytogenes was observed. In a second fermentation, only the E. faecalis donor and feces were co-immobilized. Enumeration of pRE25(*) and the donor strain by quantitative PCR revealed an increasing ratio of pRE25(*) to the donor throughout the 16-day fermentation, indicating the transfer of pRE25(*) . An Enterococcus avium transconjugant was isolated, demonstrating that ABR gene transfer to gut commensals occurred. Moreover, pRE25(*) was still functional in both the E. avium and the L. monocytogenes transconjugant and transmittable to other genera in filter mating experiments. Our study reveals that the transfer of a multiresistance plasmid to commensal bacteria in the presence of competing fecal microbiota occurs in a colonic model, suggesting that commensal bacteria contribute to the increasing prevalence of antibiotic-resistant bacteria.

Construction and characterization of Enterococcus faecalis CG110/gfp/pRE25*, a tool for monitoring horizontal gene transfer in complex microbial ecosystems.

Enterococci are among the most notorious bacteria involved in the spread of antibiotic resistance (ABR) determinants via horizontal gene transfer, a process that leads to increased prevalence of antibiotic-resistant bacteria. In complex microbial communities with a high background of ABR genes, detection of gene transfer is possible only when the ABR determinant is marked. Therefore, the conjugative multiresistance plasmid pRE25, originating from a sausage-associated Enterococcus faecalis, was tagged with a 34-bp random sequence marker spliced by tet(M). The plasmid constructed, designated pRE25(*) , was introduced into E. faecalis CG110/gfp, a strain containing a gfp gene as chromosomal marker. The plasmid pRE25(*) is fully functional compared with its parental pRE25, occurs at one to two copies per chromosome, and can be transferred to Listeria monocytogenes and Listeria innocua at frequencies of 6 × 10(-6) to 8 × 10(-8) transconjugants per donor. The markers on the chromosome and the plasmid enable independent quantification of donor and plasmid, even if ABR genes occur at high numbers in the background ecosystem. Both markers were stable for at least 200 generations, permitting application of the strain in long-running experiments. Enterococcus faecalis CG110/gfp/pRE25(*) is a potent tool for the investigation of horizontal ABR gene transfer in complex environments such as food matrices, biofilms or colonic models.

Validation of the PhysioQuant blood pressure measuring monitor in adults.

A new automatic blood pressure (BP) measuring device PhysioQuant (also marketed under the brand name Ergoscan) was evaluated according to the International Protocol for Validation of Blood Pressure Measuring Devices in adults issued by the European Society of Hypertension. BP values measured by the PhysioQuant were compared with BP readings from two independent observers. Thirty-three patients (13 male, 20 female) provided systolic and diastolic BP readings in the normotensive, borderline hypertensive, and hypertensive range. Their age varied between 29 and 93 years, their arm circumference between 23.5 and 37 cm. The device showed a mean (+/-SD) deviation from observer measurements of -0.1 (+/-3.4) mmHg for systolic and +1.1 (+/-3.9) mmHg for diastolic BP. The accuracy of the device did not vary according to BP values or other patient characteristics. The device passed all phases of the protocol and can be recommended following the regulation rules of the European Society of Hypertension.