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For case-control study 1, case patients were defined as individuals who had at least one MRSA-positive surveillance culture while staying in the ICU. For case-control study 2, case patients' definition included the presence of at least one MSSA positive surveillance culture during ICU stay. For both studies, the control group was formed by all individuals from the study period in which S.

Patient data were recovered from medical charts and laboratory files. Underlying conditions were defined following the guidelines in International Classification of Diseases [11]. Hospital admissions in the previous year and transfer from other hospitals were also recorded. All other data were analyzed from the day of admission to our hospital up to the isolation of S. Data included performance of surgery or other invasive procedure; use of steroids or other immune-suppressing drugs and use of antimicrobials. Time at risk was assessed using two different variables: "time in the hospital" defined as time from admission to the hospital up to the isolation of S.

Each variable was submitted to univariate analysis. For multivariable analysis, we used a stepwise forward selection process [13]. Variables were gradually inserted in the model according to their effect. A p value of 0. It was also set as the final limit for statistical significance. A total number of patients met the inclusion criteria for our study. Results from case-control 2 are presented in Table 2.

Determining which one of these factors is predominant in the epidemiology of a specific pathogen is primary for designing preventive strategies [14]. This finding has been reported by others, and points out to the occurrence of cross-transmission within the unit [15,16]. On the other hand, our results suggest that ecological pressure of antimicrobial use may also play a role in MRSA epidemiology in our hospital. A recent study by Weber et al. Interestingly, evidence from experimental studies suggests that sub-inibitory concentrations of quinolones enhance S.

Our findings slightly differ from those reported by Weber et al.


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While the use of ciprofloxacin increased the risk of MRSA colonization, levofloxacin had a protective effect. We hypothesize that the much greater activity of levofloxacin against Gram-positive pathogens may have been sufficient to inhibit MRSA colonization. Unfortunately, specimens from surveillance cultures were not routinely tested for susceptibility to quinolones in our hospital.

Perhaps this information could help us elucidate this issue. Additional risk factors for MRSA acquisition in ICUs have been identified by other authors: severity-of-illness and use of invasive particularly intravascular devices [19].

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Even though these variables were included in our analysis, they were not significant predictors for MRSA colonization. Case-case-control studies allow investigators to simultaneously analyze risk factors for the acquisition of resistant and susceptible bacterial strains [10]. Results from these studies can be grouped in three categories: a variables that are risk factors only for resistant strains; b variables that are risk factors only for susceptible strains; and c variables that are risk factors for both susceptible and resistant strains.

Results in category c should be interpreted as risk factors for the target organism as a whole. Age was negatively associated with MSSA carriage. We may speculate that elder patients were exposed to factors that prevented MSSA colonization and that were not assessed in our study. However, this effect was not identified.

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This was probably due to the small number of MSSA cases, which was a major limitation in our study. We have sought to identify risk factors that are amenable to interventions for containing its spread among critical patients in our hospital. They behave so because they are afraid the be killed or victim of a coup. They want a powerful nation to back them in case of aggression or trouble.

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African leaders would work in the interest of their people if they were not constantly stalked and bullied by colonial countries. I still have to find out the complete details about the amounts, the evaluation of the colonial benefits and the terms of payment imposed on the african countries, but we are working on that help us with info.

The final say is that of the French Treasury which has invested the foreign reserves of the African countries in its own name on the Paris Bourse. The two CFA banks are African in name, but have no monetary policies of their own. The countries themselves do not know, nor are they told, how much of the pool of foreign reserves held by the French Treasury belongs to them as a group or individually.

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The earnings of the investment of these funds in the French Treasury pool are supposed to be added to the pool but no accounting is given to either the banks or the countries of the details of any such changes. Gary K. To make things more tragic, France impose a cap on the amount of money the countries could borrow from the reserve. France has the first right to buy any natural resources found in the land of its ex-colonies. As consequence, in many of the french ex-colonies, all the majors economical assets of the countries are in the hand of french expatriates.

Interestingly, there was almost no overlap between the two groups; 78 of the 84 regulated transcripts in the myd88 mutants could not be found regulated in the WT larvae. Thus, nearly the entire transcriptional response signature of the host to the microbiome seems dependent on Myd88 signaling Supplementary Fig.

In contrast to the transcriptome response to colonization in a WT background, there was a very clearly discernable ontology signature in the transcriptomic response of the myd88 deficient larvae. These data seem to indicate an important role for Myd88 in maintaining metabolic homeostasis, which is sensitive to colonization status.

Considering our qPCR and in situ hybridization results, indicating that the gut and liver are important organs of myd88 transcriptional upregulation under germ-free conditions Fig. The germ-free and conventionalized larvae represent extremes on a scale of complexity of interactions between the host and the intestinal microbial communities, from an absence of microbial colonization and host-microbe interactions in germ-free larvae to the much greater complexity of uncharacterized communities in conventionalized larvae.


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  • To assess the effects of this complexity, we examined the impact of colonization by specific species on the transcriptomic response of the host. To this end, we compared the transcriptomic responses of germ-free larvae with two mono-associated groups of embryos, each colonized with a single commensal bacterial species. The species were Exiguobacterium ZWU phylum Firmicutes and Chryseobacterium ZOR phylum Bacteroidetes , both of which represent phylae of major research interest in humans and mice 33 , 34 , though not numerically dominant species in zebrafish intestinal communities 8 , 23 , Thus, the mono-associated colonization groups are likely to represent very different microbial communities to those of the conventionalized group, and any transcriptional regulation that is shared between such diverse colonization conditions is likely to be very robust to differences in the nature of colonizing microbial communities.

    Successful enteric colonization in mono-associated embryos was verified microscopically using the amine-reactive Dye-light fluorescent labeling system Fig. The transcriptomics results yielded and significantly regulated transcripts in response to mono-association with Chryseobacterium and Exiguobacterium , respectively, and show a very clear overlap with the host transcriptome response to conventionalization Fig.

    The magnitude of the responses in different conditions were more variable. Overlaying the sets of genes which met the regulation criteria of fold change and P -value yielded 65 genes shared among the three data-sets. All were regulated in the same direction, 60 down and 5 up Fig. This shows that many of the transcriptional effects regulated by colonization by a complex microbial community can be mimicked by mono-association, an exposure that must be considered drastically different in nature from conventionalization.

    The identified set of 65 shared genes can be used as a common marker set of shared transcriptional response which seems independent of the nature or treatment dose of the stimulating microbiota. Mono-associated treatment groups help refine the colonization sensitive genes. By plating homogenized embryos, the colonizing CFU burden was estimated to be approximately 70—75 for each, colonization patterns represent observations made in three independent colonization experiments.

    These 65 genes represent strong candidates for markers of the shared transcriptional response. Myd88 , along with several transcription factor encoding genes known to function downstream of Myd88, are among them. The sensitivity of myd88 transcription to colonization points to the presence of a sensing mechanism whereby the host responds to the presence of microbes. While analyzing the shared transcriptional response marker set Fig.


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    • Out of the 65 shared response transcripts, 11 were shared with the Tlr2 dependent transcription response set. In contrast, there was no overlap with a Tlr5 dependent transcription response set of the same study To investigate whether Tlr2 could be involved in the microbial suppression of myd88 transcription, we performed qPCR analysis to assess transcription of myd88 and two other shared transcriptional response markers in conventionalized and germ-free larvae of Tlr2 loss of function mutant fish line tlr2 sa , see materials and methods compared to wildtype siblings Fig.

      The results show that the down regulation of myd88 transcription in response to colonization only occurs in the WT siblings and not in Tlr2 mutants. In the Tlr2 mutant, the regulation of the two other selected markers of the shared transcriptional response, cebpb and fosl1a , was no longer significant. We performed extensive confocal laser scanning microscopy of isolated intestines of L-plastin stained Tlr2 mutant and control fish to verify that Tlr2 was involved in mediating the immune regulation in the intestine.

      These data show that the microbiome induced intestinal immune regulation, affecting intestinal leukocyte populations, was no longer apparent in Tlr2 mutants Fig. Taken together these data suggest the presence of a microbiome sensitive immune regulatory mechanism, functioning through Tlr2 and Myd88 to regulate intestinal leukocyte status in response to colonization. Microbiome transcriptional control of myd88 is dependent on Tlr2.

      Our results show that germ-free conditions are characterized by a lower number of neutrophils in the intestines compared to conventionalized conditions, in line with previous observations from similar zebrafish studies 9. We show that the neutrophil responses of the intestine are dependent on Myd Additionally, we show a significant, Myd88 dependent, elevation of macrophage presence in the intestine under germ-free conditions. The fact that the elevated intestinal macrophage infiltration in germ-free conditions is dependent on Myd88 is intriguing, as the canonical function of Myd88 is to initiate inflammatory responses upon TLR stimulation, which is supposed to be absent under germ-free conditions.

      To resolve this conundrum, we analyzed the system wide transcriptomic responses mounted by the host in response to microbial colonization. Unexpectedly, we found that myd88 , along with several other genes known to be regulated in a Myd88 dependent manner during bacterial infection, is among the down regulated genes in conventionalized larvae. Having defined a gene marker set of the transcriptional response signature of the host to the microbiome and considering that suppressed myd88 expression is a key element of this response, we investigated the function of Myd88 in the host response to microbial colonization.

      Results from myd88 deficient mutants revealed that Myd88 signaling is indispensable for this response. This dependence on Myd88 could reflect the simplicity of the larval colonization system we used, that is defined by never-fed conditions and thereby eliminating a number of confounding factors. While this experimental set-up allows us to draw strong conclusions regarding Myd88 dependence and innate immune cell responses, extrapolation of the results to more complex, conditions of microbiome interactions and colonization in the presence of an adaptive immune system and feeding should be done with caution.

      Nevertheless, the model is a good complement to present murine models as this model allows different factors to be added in sequentially. This can aid in dissecting the influences deriving from different factors such as specific microbial species, host genotypes or diet. Using a gnotobiotic system we investigated whether the identified gene marker set for the transcriptional response signature of the host to the microbiome is dependent on microbiome diversity. The transcriptional response signature is remarkably similar, and, in most cases, the same genes are regulated in the same direction when comparing two mono-associations with conventionalized conditions.

      This is interesting given that the conventionalized communities, though not characterized, are assumed to be dominated by gamma proteobacteria which have been found to be numerically dominant in the intestines of young zebrafish larvae 36 , while the Bacteroidetes and Firmicutes are much less prominent.