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Enterobacteria species are common causes of hospital-acquired infections, which are associated with high morbidity and mortality rates. Immunocompromised patients such as solid organ transplant (SOT) recipients are especially at risk because they are frequently exposed to antibiotics in the course of their treatments. In this work, we used a collection of 106 Escherichia coli, 78 Klebsiella pneumoniae, 25 Enterobacter spp., and 24 Citrobacter spp. multidrug resistant strains isolated from transplant patients (hepatic, renal or renal/pancreatic) in order to examine their ability to adhere in vitro to HT-29 human colon cells, and to determine if some adhesive characteristics are associated with prevalence and persistence of these strains. A total of 33 E. coli (31%), 21 K. pneumoniae (27%), 7 Enterobacter spp. (28%), and 5 Citrobacter spp. (21%), adhered to the colon epithelial cells. Two main adherence patterns were observed in the four species analyzed, diffuse adherence, and aggregative adherence. Under transmission electronic microscopy (TEM), most bacteria lacked visible fimbria on their surface, despite their strong adherence to epithelial cells. None of the strains studied was able to induce any cytotoxic effect on HT-29 cells although some of them strongly colonizing both cells and glass coverslips at high density. Some of the strains failed to adhere to the epithelial cells but adhered strongly to the cover-slide, which shows that microscopy studies are mandatory to elucidate the adherence of bacteria to epithelial cells in vitro, and that quantitative assays using colony forming unit (CFUs) counting need to be supplemented with pictures to determine definitively if a bacterial strain adheres or not to animal cells in vitro. We report here, for the first time, the aggregative adherence pattern of two multidrug resistant (MDR) Citrobacter freundii strains isolated from human patients; importantly, biofilm formation in Citrobacter is totally dependent on the temperature; strong biofilms were formed at room temperature (RT) but not at 37°C, which can play an important role in the colonization of hospital surfaces. In conclusion, our results show that there is a great variety of adhesion phenotypes in multidrug-resistant strains that colonize transplanted patients.

Introduction:Acinetobacter baumannii is an opportunistic nosocomial pathogen associated with multiple infections. This pathogen usually colonizes (first stage of microbial infection) host tissues that are in contact with the external environment. As one of the sites of entry in human hosts is the gastrointestinal tract, the pathogen must be capable of tolerating bile salts. However, studies analyzing the molecular characteristics involved in the response to bile salts in clinical strains of A. baumannii are scarce.Material and Methods: Microbiological and transcriptional studies (arrays and RT-PCR) in the response to bile salts were carried out in isogenic (A. baumanni ΔadeB ATCC 17978 and A. baumannii ΔadeL ATCC 17978) and clinical strains from clone ST79/PFGE-HUI-1 which is characterized by lacking the AdeABC efflux pump and by overexpression the AdeFGH efflux pump.Results and Discussion: In presence of bile salts, in addition to the glutamate/aspartate transporter were found overexpressed in A. baumannii ΔadeB ATCC 17978, the virulence factors (surface motility, biofilm, and Type VI Secretion System) which are associated with activation of the Quorum Sensing system. Overexpression of these factors was confirmed in clinical strains of clone ST79/PFGE-HUI-1.Conclusions: This the first study about the adaptive response to bile salts investigating the molecular and microbiological characteristics in response to bile salts of an isogenic model of A. baumannii ATCC 17978 and clinical isolates of A. baumannii (clinical strains of ST79/PFGE-HUI-1) lacking the main RND efflux pump (AdeABC). Clinical isolates of A. baumannii lacking the AdeABC efflux pump (clone ST79/PFGE-HUI-1) displayed a new clinical profile (increased invasiveness) possibly associated with the response to stress conditions (such as the presence of bile salts).