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International audience; The treatment of osteomyelitis, a destructive inflammatory process caused by bacterial infections to bone tissue, is one of the most critical challenges of orthopedics and bone regenerative medicine. The standard treatment consists of intense antibiotic therapies combined with tissue surgical debridement and the application of a bone defect filler material. Unfortunately, commercially available candidates, such as gentamicin-impregnated polymethylmethacrylate cements, possess very poor pharmacokinetics (i.e., 24 hours burst release) and little to no regenerative potential. Fostered by the intrinsic limitations associated with conventional treatments, alternative osteostimulative biomaterials with local drug delivery have recently started to emerge. In this study, we propose the use of a polycaprolactone-silica sol–gel hybrid material as carrier for the delivery of rifampicin, an RNA-polymerase blocker often used to treat bone infections, and of osteostimulative silicate ions. The release of therapeutic agents from the material is dual, offering two separate and simultaneous effects, and decoupled, meaning that the kinetics of rifampicin and silicate releases are independent from each other. A series of hybrid formulations with increasing amounts of rifampicin was prepared. The antibiotic loading efficacy, as well as the release profiles of rifampicin and silicates were measured. The characterization of cell viability and differentiation of rat primary osteoblasts and antibacterial performance were also performed. Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa and Escherichia coli were selected due to their high occurrence in bone infections. Results confirmed that rifampicin can be successfully loaded within the hybrids without significant degradation and that it is possible to tailor the antibiotic release according to need. Once in a physiological environment, the rapid release of silicates was associated with optimal cell proliferation and the overexpression of osteoblastic differentiation. Simultaneously, rifampicin is delivered over the course of several weeks with significant inhibition of all tested strains. In particular, the materials caused a growth reduction of 7–10 orders of magnitude in Staphylococcus aureus, the major strain responsible for osteomyelitis worldwide. Our data strongly suggest that PCL/silica hybrids are a very promising candidate to develop bone fillers with superior biological performance compared to currently available options. Thanks to their unique synthesis route and their dual tailored release they can promote bone regeneration while reducing the risk of infection for several weeks upon implantation.

International audience; Interest has been rekindled in the old antibiotic fosfomycin, partly because of its ability to penetrate biofilm. Using a transcriptomic approach, we investigated the modifications induced by fosfomycin in sessile cells of a clinical Staphylococcus aureus isolated from a device-associated infection. Cells still able to form biofilm after 4 h of incubation in the presence of subinhibitory concentrations of fosfomycin and cells from 24-h-old biofilm later submitted to fosfomycin had 6.77% and 9.41%, respectively, of differentially expressed genes compared with their antibiotic-free control. Fosfomycin induced mostly downregulation of genes assigned to nucleotide, amino acid and carbohydrate transport, and metabolism. Adhesins and capsular biosynthesis proteins encoding genes were downregulated in fosfomycin-grown biofilm, whereas the murein hydrolase regulator lgrA and a D-lactate dehydrogenase-encoding gene were upregulated. In fosfomycin-treated biofilm, the expression of genes encoding adhesins, the cell wall biosynthesis protein ScdA, and to a lesser extent the fosfomycin target MurA was also decreased. Unattached cells surrounding fosfomycin-grown biofilm showed greater ability to form aggregates than their counterparts obtained without fosfomycin. Reducing their global metabolism and lowering cell wall turnover would allow some S. aureus cells to grow in biofilm despite fosfomycin stress while promoting hyperadherent phenotype in the vicinity of the fosfomycin-treated biofilm.

GYNOPHILUS (Lcr REGENERANS) is a live biotherapeutic product (LBP) aimed at restoring the vaginal microbiome and contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35. In this study, the LBP formulation and manufacturing process significantly enhanced the anti-Candida activity of L. rhamnosus Lcr35, with a complete loss of viability of the yeast after 48 h of coincubation. Sodium thiosulfate (STS), one excipient of the product, was used as a potentiator of the anti-Candida spp. activity of Lactobacilli. This contact-independent phenomenon induced fungal cell disturbances, as observed by electron microscopy observations. Nonverbal sensory experiments showed clear odor dissimilarities between cocultures of L. rhamnosus Lcr35 and C. albicans in the presence and absence of STS, suggesting an impact of odor-active metabolites. A volatolomic approach allowed the identification of six odor-active compounds, including one sulfur compound that was identified as S-methyl thioacetate (MTA). MTA was associated with the antifungal effect of Lcr35, and its functional link was established in vitro. We show for the first time that the LBP GYNOPHILUS, which is a highly active product in the reduction of vulvovaginal candidiasis, requires the presence of a sulfur compound to fully achieve its antifungal effect.

Copper is an essential trace element required for human life, and is involved in several physiological mechanisms. Today researchers have found and confirmed that Cu has biological properties which are particularly useful for biomaterials applications. Indeed, Cu exhibits antibacterial functions, provides angiogenic ability and favors osteogenesis; these represent major key points for ideal biomaterial integration and the healing process that follows. Copper-doped biomaterials thus present an interesting alternative to the massive use of prophylactic antibiotics to prevent infection complications following a surgical act for bone tissue reconstruction. This review describes the bio-functional advantages offered by Cu and focuses on the antibacterial, angiogenic and osteogenic properties of Cu-doped biomaterials used in orthopedic applications. The chemical role of doping - used in all families of biomaterials (ceramics, composites and metals) - is the main concern of this article. Other aspects, independent of doping, which may be important for optimizing biomaterial efficiency (in particular surface structuration), are also mentioned here, although they are not the focus of this review.

In most environments, microorganisms evolve in a sessile mode of growth, designated as biofilm, which is characterized by cells embedded in a self-produced extracellular matrix. Although a biofilm is commonly described as a "cozy house" where resident bacteria are protected from aggression, bacteria are able to break their biofilm bonds and escape to colonize new environments. This regulated process is observed in a wide variety of species; it is referred to as biofilm dispersal, and is triggered in response to various environmental and biological signals. The first part of this review reports the main regulatory mechanisms and effectors involved in biofilm dispersal. There is some evidence that dispersal is a necessary step between the persistence of bacteria inside biofilm and their dissemination. In the second part, an overview of the main methods used so far to study the dispersal process and to harvest dispersed bacteria was provided. Then focus was on the properties of the biofilm-dispersed bacteria and the fundamental role of the dispersal process in pathogen dissemination within a host organism. In light of the current body of knowledge, it was suggested that dispersal acts as a potent means of disseminating bacteria with enhanced colonization properties in the surrounding environment.

Background: Klebsiella pneumoniae is a major cause of community-acquired and nosocomial infections. This germ is responsible for acute and chronic infections, most of which are due to its ability to adhere to medical implants and form a biofilm. The objective of this work is to study the interaction between clinical isolates of K. pneumoniae and abiotic surfaces (medical devices) and some factors influencing biofilm formation. Methods: Over a period of 2 years, 115 strains of K. pneumoniae were isolated from medical devices CHU Tlemcen, most of which had a high level of resistance to cephalosporins 1st, 2nd, and 3rd generation. Their capacity to form biofilm was assessed using two 3 techniques: TCP, TP, and RCA. We determined in vitro the effects of three antimicrobial agents against planktonic and biofilm forms of K. pneumoniae. The presence of MrkD genes was detected by polymerase chain reaction (PCR). Results: According to the studied (TCP, TP, RCA) strains of K .pneumoniae isolated from urinary catheters have proved very good, forming the biofilm to those isolated from other medical devices. 24 of 115 isolated strains showed a clear difference in antibiotic susceptibility between planktonic populations and biofilm populations. They were 10-20 times higher. All strains presented a highly hydrophilic character and adhesion 2-10 times greater in PVC with respect to glass support. The MrkD gene (detected by PCR) responsible for biofilm formation was found in 22 strains of K. pneumoniae, which may explain their adhesion and therefore their pathogenicity. Conclusion: Our results show the great ability of K.pneumoniae strains to form a biofilm on medical devices, and the isolates were at least 10 times more resistant than their planktonic counterparts. In addition, we showed that the presence of type 3-encoding gene mrkD was associated with high adhesion indexes.

Interest has been rekindled in the old antibiotic fosfomycin, partly because of its ability to penetrate biofilm. Using a transcriptomic approach, we investigated the modifications induced by fosfomycin in sessile cells of a clinical Staphylococcus aureus isolated from a device-associated infection. Cells still able to form biofilm after 4 h of incubation in the presence of subinhibitory concentrations of fosfomycin and cells from 24-h-old biofilm later submitted to fosfomycin had 6.77% and 9.41%, respectively, of differentially expressed genes compared with their antibiotic-free control. Fosfomycin induced mostly downregulation of genes assigned to nucleotide, amino acid and carbohydrate transport, and metabolism. Adhesins and capsular biosynthesis proteins encoding genes were downregulated in fosfomycin-grown biofilm, whereas the murein hydrolase regulator lgrA and a D-lactate dehydrogenase-encoding gene were upregulated. In fosfomycin-treated biofilm, the expression of genes encoding adhesins, the cell wall biosynthesis protein ScdA, and to a lesser extent the fosfomycin target MurA was also decreased. Unattached cells surrounding fosfomycin-grown biofilm showed greater ability to form aggregates than their counterparts obtained without fosfomycin. Reducing their global metabolism and lowering cell wall turnover would allow some S. aureus cells to grow in biofilm despite fosfomycin stress while promoting hyperadherent phenotype in the vicinity of the fosfomycin-treated biofilm.

International audience; Azo dyes are widely used in industries and their release in the environment contributes to the pollution of effluents. The authors aim to develop a new eco-friendly water treatment method for the degradation of azo dyes based on in situ magnetic separation and immobilisation of bacterial cells. The immobilisation was achieved using superparamagnetic Fe3O4 nanoparticles and offers the possibility of reusing bacteria by magnetic separation for several degradation cycles. The iron–oxide nanoparticles were synthesised by reverse co-precipitation. The Gram-positive bacteria Bacillus subtilis were immobilised using iron–oxide nanoparticles by adsorption and then separated with an external magnetic field. Transmission electron microscopy observation showed that the particles' diameter was ∼20 nm with a narrow size distribution. Moreover, the iron–oxide nanoparticles were adsorbed onto the surface in order to coat the cells. B. subtilis has proved its ability to decolorise and degrade several azo dyes at different values of pH, with the highest decolorisation rate for Congo red. Furthermore, immobilised cells have a degradation activity similar to that of free cells. The system provided a degradation rate up to 80% and could be reused for seven batch cycles.

Biofilm-dispersal is a key determinant for further dissemination of biofilm-embedded bacteria. Recent evidence indicates that biofilm-dispersed bacteria have transcriptional features different from those of both biofilm and planktonic bacteria. In this study, the in vitro and in vivo phenotypic properties of Klebsiella pneumoniae cells spontaneously dispersed from biofilm were compared with those of planktonic and sessile cells. Biofilm-dispersed cells, whose growth rate was the same as that of exponential planktonic bacteria but significantly higher than those of sessile and stationary planktonic forms, colonized both abiotic and biotic surfaces more efficiently than their planktonic counterparts regardless of their initial adhesion capabilities. Microscopy studies suggested that dispersed bacteria initiate formation of microcolonies more rapidly than planktonic bacteria. In addition, dispersed cells have both a higher engulfment rate and better survival/multiplication inside macrophages than planktonic cells and sessile cells. In an in vivo murine pneumonia model, the bacterial load in mice lungs infected with biofilm-dispersed bacteria was similar at 6, 24 and 48 h after infection to that of mice lungs infected with planktonic or sessile bacteria. However, biofilm-dispersed and sessile bacteria trend to elicit innate immune response in lungs to a lesser extent than planktonic bacteria. Collectively, the findings from this study suggest that the greater ability of K. pneumoniae biofilm-dispersed cells to efficiently achieve surface colonization and to subvert the host immune response confers them substantial advantages in the first steps of the infection process over planktonic bacteria.

In most environments, microorganisms evolve in a sessile mode of growth, designated as biofilm, which is characterized by cells embedded in a self-produced extracellular matrix. Although a biofilm is commonly described as a "cozy house" where resident bacteria are protected from aggression, bacteria are able to break their biofilm bonds and escape to colonize new environments. This regulated process is observed in a wide variety of species; it is referred to as biofilm dispersal, and is triggered in response to various environmental and biological signals. The first part of this review reports the main regulatory mechanisms and effectors involved in biofilm dispersal. There is some evidence that dispersal is a necessary step between the persistence of bacteria inside biofilm and their dissemination. In the second part, an overview of the main methods used so far to study the dispersal process and to harvest dispersed bacteria was provided. Then focus was on the properties of the biofilm-dispersed bacteria and the fundamental role of the dispersal process in pathogen dissemination within a host organism. In light of the current body of knowledge, it was suggested that dispersal acts as a potent means of disseminating bacteria with enhanced colonization properties in the surrounding environment.

Isolates of Klebsiella pneumoniae are responsible for opportunistic infections in humans, particularly of the urinary respiratory tracts. These bacteria express type 3 fimbriae that have been implicated in binding to eukaryotic cells and matrix proteins. Twenty four (24) K. pneumoniae strains isolated from medical devices were studied. Their capacity to form biofilm was assessed using two types of materials, polyvinylchloride (PVC) and glass of microfermenter in static or kinetic conditions. Strains with adherence to PVC also cling strongly to glass slides. We determined the in vitro effects of three antimicrobial agents against planktonic and biofilm forms of K. pneumoniae and we demonstrated that isolates of the biofilm form were at least 10-25 times more resistant than the planktonic form. Most strains of K. pneumoniae harbored the mrkD gene and exhibited a strong ability to adhere to inert surfaces. Key words: Biofilm, Fimbriae, Klebsiella pneumoniae, polyvinylchloride (PVC), microfermenter.

International audience; Recognition of bacteria by metazoans is mediated by receptors that recognize different types of microorganisms and elicit specific cellular responses. The soil amoebae Dictyostelium discoideum feeds upon a variable mixture of environmental bacteria, and it is expected to recognize and adapt to various food sources. To date, however, no bacteria-sensing mechanisms have been described. In this study, we isolated a Dictyostelium mutant (fspA KO) unable to grow in the presence of non-capsulated Klebsiella pneumoniae bacteria, but growing as efficiently as wild-type cells in the presence of other bacteria, such as Bacillus subtilis. fspA KO cells were also unable to respond to K. pneumoniae and more specifically to bacterially secreted folate in a chemokinetic assay, while they responded readily to B. subtilis. Remarkably, both WT and fspA KO cells were able to grow in the presence of capsulated LM21 K. pneumoniae, and responded to purified capsule, indicating that capsule recognition may represent an alternative, FspA-independent mechanism for K. pneumoniae sensing. When LM21 capsule synthesis genes were deleted, growth and chemokinetic response were lost for fspA KO cells, but not for WT cells. Altogether, these results indicate that Dictyostelium amoebae use specific recognition mechanisms to respond to different K. pneumoniae elements.

International audience; Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47-48%) were rare sequences (

Probiotics are administered as complex manufactured products and yet most studies on probiotic bacterial strains have been performed with native culture strains. Little is known about the influence of industrial processes on the properties of the microorganisms. In this study, we comparatively assessed the characteristics of the probiotic bacterial strain Lactobacillus rhamnosus (Lcr35(®)) together with four of its commercial formulations, including three intestinal formulas (BACILOR with Lcr Restituo(®) packet and capsule and FLOREA Lcr Lenio(®)) and one vaginal formula (GYNOPHILUS Lcr Regenerans(®)). Lcr35(®) grown from the intestinal formulas displayed increased resistance to acidic pH and bile stress, especially FLOREA (Lcr Lenio(®)), which showed a 4.5log higher number of viable bacteria compared to the results obtained with the control native Lcr35(®) strain. Adhesion to intestinal cells was significantly higher with Lcr Restituo(®) packet and Lcr Restituo(®) capsule vs Lcr35(®). Bacteria from the vaginal formulation GYNOPHILUS had increased ability to metabolize glycogen thereby increasing lactic acid production. In vitro growth inhibition of the pathogen Candida albicans was significantly higher with bacteria from the vaginal formulation (4.5 log difference) and in the presence of vaginal epithelial cells than with the native strain. Our results show that the manufacturing process influences strain properties and should therefore be adapted according to the strain and the therapeutic indication.

All accessible mucous membranes of the human body are colonized by an abundant and diversified microbial flora called microbiota. Recent studies have shown that these microorganisms, long regarded as purely commensal, have essential beneficial effects on human health. Thus, numerous human ailments are linked to dysbiosis; that is, imbalances in the microflora composition. The administration of probiotic microorganisms could, in some situations, provide substantial relief from such disorders. These live microorganisms, which, according to the definition, confer a health benefit to the host when administered in adequate amounts, are often derived from human flora and belong mostly to lactic acid bacteria, in particular to the genus Lactobacillus . The constant improvement of knowledge of the role of human microbiota and the growing popularity of probiotics are now opening the door to new prophylactic and therapeutic strategies in human health.

In humans, Klebsiella pneumoniae is a saprophytic bacterium of the nasopharyngeal and intestinal mucosae that is also frequently responsible for severe nosocomial infections. Two major factors of virulence, capsular polysaccharide (CPS) and lipopolysaccharide (LPS) O antigen, are involved in mucosal colonization and the development of infections. These bacterial surface structures are likely to play major roles in interactions with the mucosal immune system, which are orchestrated by a network of surveillance based on dendritic cells (DCs). To determine the roles of K. pneumoniae CPS and LPS in the DC response, we investigated the response of immature human monocyte-derived DCs to bacterial challenge with a wild-type strain and its isogenic mutants deficient in CPS or LPS O-antigen production. As observed by flow cytometry and confocal laser microscopy, the rate of phagocytosis was inversely proportional to the amount of CPS on the bacterial cell surface, with LPS playing little or no role. The K. pneumoniae wild-type strain induced DC maturation with upregulation of CD83, CD86, and TLR4 and downregulation of CD14 and DC-SIGN. With CPS mutants, we observed a greater decrease in DC-SIGN, suggesting a superior maturation of DCs. In addition, incubation of DCs with CPS mutants, and to a lesser extent with LPS mutants, resulted in significantly higher Th1 cytokine production. Combined, our findings suggest that K. pneumoniae CPS, by hampering bacterial binding and internalization, induces a defective immunological host response, including maturation of DCs and pro-Th1 cytokine production, whereas the LPS O antigen seems to be involved essentially in DC activation.

Objectives. The ability of a probioticLactobacillus rhamnosusstrain (Lcr35) to adhere to cervical and vaginal cells and to affect the viability of two main vaginosis-associated pathogens,Prevotella bivia, Gardnerella vaginalis, as well asCandida albicanswas investigated.Methods. Adhesion ability was determined in vitro with immortalized epithelial cells from the endocervix, ectocervix, and vagina. Coculture experiments were performed to count viable pathogens cells in the presence of Lcr35.Results. Lcr35 was able to specifically and rapidly adhere to the three cell lines. In coculture assays, a decrease in pathogen cell division rate was observed as from 4 hours of incubation and bactericidal activity after a longer period of incubation, mostly withP. bivia.Conclusion. The ability of Lcr35 to adhere to cervicovaginal cells and its antagonist activities against vaginosis-associated pathogens suggest that this probiotic strain is a promising candidate for use in therapy.

Interaction with cells is believed to be a crucial step in the process of colonization by Klebsiella pneumoniae. In this study, we investigated the adhesion properties of 17 extended-spectrum beta-lactamases (ESBLs) producing clinical isolates using 2 intestinal cells (Int-407 and HT-29 cells) and components of the extracellular matrix (ECM) (fibrinogen, collagen, fibronectin). Since the capsule formed by K. pneumoniae potentially masks bacterial cell surface components, we created mutants defective in capsular synthesis. The levels of adhesion of the acapsulated mutants were higher than their corresponding wild-type strains for 7 and 4 isolates with Int-407 and HT-29 cells, respectively, whereas 2 mutants displayed lower adhesion indexes with HT-29 cells. Nine mutants adhered at higher levels than their corresponding wild-type strains to all 3 components of ECM tested and 13 only to collagen. Transfer by mating of ESBL-encoding plasmids did not correlate with the acquisition of adhesive properties by the transconjugants, which suggests that these plasmids were not involved in adhesion expression. The presence of ESBL-encoding plasmids alters the basal adhesion capacity of the recipient strain, and cured strains adhered more than the parental strains. The possessing of capsule or plasmid-encoded factors would have a bacterial cost, including a weaker capacity for adhesion.

The antimicrobial activity of essential oils from Vetiveria zizanioides and Vetiveria nigritana roots was tested against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa, and compared to the activity of clove oil. The results showed a low activity against Gram-negative bacilli; but a higher efficiency was observed against Gram-positive cocci, with MIC values amounting to that of clove oil. The antifungal activity of oils was also tested on Candida albicans; the MIC values were approximately identical for the two oils, and quite similar to those of clove oil.

We report here the genome sequence of Klebsiella pneumoniae CH1034, a sequence type 6 (ST6) strain isolated in 2012 from a central venous catheter of a hospitalized patient.

International audience; Treatment of orthopaedic infections remains challenging owing to the inability of antibiotics to eradicate biofilms and prevent their regrowth. The present study characterized the effects of 12 antibiotics on in vitro biofilm formed by a representative strain of meticillin-susceptible Staphylococcus aureus (MSSA) isolated from a bone infection. Determination of the minimum biofilm eradication concentrations indicated that in vitro eradication of 24 h-old biofilms required concentrations up to 51,200 times higher than MICs. The influence of the same panel of antibiotics was also investigated on biofilm formation at concentrations including the breakpoints, by numbering viable cells in the suspensions (individual cells) and the biofilm biomass. Except for fusidic acid, the presence of antibiotics during the initial steps of biofilm formation resulted in significant decreases in the number of sessile viable bacteria at the highest concentrations tested. Ceftarolin, daptomycin, fosfomycin, gentamicin, ofloxacin, rifampicin and vancomycin were the most effective drugs. Confocal microscopy analysis indicated that daptomycin was more efficient at bacteria lysis than gentamicin and vancomycin. However, viable individual cells were still detectable in the assays performed with ceftarolin, fosfomycin, ofloxacin, rifampicin and vancomycin at concentrations for which no sessile cells were detected. Although none of the molecules tested was effective at classical therapeutic concentrations against 24 h-old MSSA biofilms, all except fusidic acid were able to impair biofilm formation at concentrations near the breakpoints. However, presence of viable individual unattached cells could imply a significant risk of microbial dissemination and increased risk of infections.

The asymmetric synthesis of the trifluoromethyl analogue of (4S)-4,5-dihydroxy-2,3-pentanedione [(4S)-DPD], the metabolic precursor of the bacterial quorum sensing (QS) autoinducer AI-2, is described. The trifluoromethyl group was introduced by treating (trifluoromethyl)trimethylsilane with an α-methylene ester resulting from a Baylis–Hillman reaction between (tert-butyldimethylsilyloxy)acetaldehyde and a vinylamide derived from Oppolzer’s sultam reagent. The α-diketonic moiety is formed during the last step by reductive ozonolysis of the hemiketal form of an α-methylene trifluoromethyl ketone. Biological evaluation shows that this analogue is a weaker inducer of QS in Vibrio harveyi bacteria than DPD. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

The first step in nosocomial infections due to Klebsiella pneumoniae is colonization of the patient's gastrointestinal (GI) tract. In a previous work, signature-tagged mutagenesis was used in a murine model to identify 13 genes required for efficient colonization, two of which were involved in urea metabolism. The role of urease was further investigated by the construction and analysis of an isogenic urease-deficient mutant. The behavior of both the wild-type strain and the urease-deficient mutant was tested under hostile conditions, reproducing stresses encountered in the GI environment. The wild-type strain had an acid tolerance response (ATR) to inorganic acid, was resistant to organic acids (38.5% survival) and was able to survive concentrations of bile encountered in vivo. The absence of urease did not affect the resistance of K. pneumoniae to acid and bile stresses, but the enhanced adhesion response to Int-407 cells after exposure to bile observed with the wild-type strain was no longer detected with the urease mutant. When tested in the murine intestinal colonization model, both strains were mainly recovered in the large intestine parts, and the mutant was impaired in its colonization capacities, but only when tested in competition with the wild-type strain. These findings emphasize the prominent role played by metabolic function in the colonization process of such a complex ecosystem as the host GI tract.

Quorum sensing is a process by which bacteria communicate by using secreted chemical signaling molecules called autoinducers. Many bacterial species modulate the expression of a wide variety of physiological functions in response to changes in population density by this mechanism. In this study, the opportunistic pathogen Klebsiella pneumoniae was observed to secrete type 2 signaling molecules. A homologue of luxS , the gene required for AI-2 synthesis in Vibrio harveyi , was isolated from the K. pneumoniae genome. A V. harveyi bioassay showed the luxS functionality in K. pneumoniae and its ability to complement the luxS -negative phenotype of Escherichia coli DH5α. Autoinducer activity was detected in the supernatant, and maximum expression of specific messengers detected by quantitative reverse transcription-PCR analysis occurred during the late exponential phase. The highest levels of AI-2 were observed in minimal medium supplemented with glycerol. To determine the potential role of luxS in colonization processes, a K. pneumoniae luxS isogenic mutant was constructed and tested for its capacity to form biofilms in vitro on an abiotic surface and to colonize the intestinal tract in a murine model. No difference was observed in the level of intestinal colonization between the wild-type strain and the luxS mutant. Microscopic analysis of biofilm structures revealed that the luxS mutant was able to form a mature biofilm but with reduced capacities in the development of microcolonies, mostly in the early steps of biofilm formation. These data suggest that a LuxS-dependent signal plays a role in the early stages of biofilm formation by K. pneumoniae .

Klebsiella pneumoniae is an opportunistic pathogen responsible for nosocomial infections. Both resistance to multiple antibiotics and the expression of virulence factors are likely to be involved in the physiopathological process. In this study, 227 isolates of K. pneumoniae collected over a 1-year period in a teaching hospital in Clermont-Ferrand, France, were investigated for their antibiotic resistance pattern and the presence of several potential virulence traits. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) indicated that most of the isolates were phylogenetically unrelated. When tested in an in vitro adhesion assay with Int-407 intestinal cells, the median adhesion index was 5.5x10(4) bacteria/cm(2) (range, 2.0x10(2)-3.4x10(5)). Isolates resistant to cefoxitin, chloramphenicol, and quinolones showed significantly lower adhesion indexes. The frequency of mutagenesis conferring resistance to rifampicin was low for most of the isolates. The median mutagenesis frequency was 1.0x10(-8) (range, 2.5x10(-9)-3.2x10(-6)) at 24 h and 1.1x10(-8) (range, 1.8x10(-9)-1.2x10(-5)) at 7 days. In contrast, isolates resistant to cefoxitin, chloramphenicol, and tetracycline showed a significantly greater ability to mutate. These results suggest a link between adhesion capabilities and resistance to certain antibiotics. They furthermore indicate that strains with a high mutagenesis capacity are more likely to acquire antibiotic resistance genes. The high pathogenicity island of Yersinia was detected in 16.3% of the strains and was more often associated with isolates resistant to nalidixic acid and augmentin.

The colonization by the probiotic Lactobacillus casei subsp. rhamnosus Lcr35 of the gastrointestinal tracts of mice and humans was studied. The mice were orally given 10 9 CFU of Lcr35 either once or three times at 24-h intervals. A 16S ribosomal nucleic probe used in hybridization assays detected Lcr35 in the feces of mice for up to 3 days after the feeding, at a level of 10 8 to 10 9 CFU/g of feces. In the human assay, 12 healthy volunteers were enrolled in a randomized trial and ingested Lcr35 at a dosage of 10 8 or 10 10 or 10 12 CFU every day for 7 days. Then, after a 3-week posttreatment period, there was a second intake period similar to the first one. Analysis of fecal samples showed significant increases in the number of lactobacilli during the first intake period, whatever the dose given. The greatest increases were observed in subjects harboring the lowest indigenous population of Lcr35-like bacteria. During the 3-week posttreatment period, the number of CFU slightly decreased over time, and an increase, although not a statistically significant one, was observed during the second test period. These findings suggest that Lcr35 is able to survive within the gastrointestinal tract.

Klebsiella pneumoniae is an opportunistic pathogen responsible for nosocomial infections that initially colonize the intestinal tract of patients. Signature-tagged mutagenesis was used to identify genes required for this function. A library of 2,200 mutants was analyzed for the inability of the mutants to survive in a murine model of intestinal colonization and to adhere to human intestinal cells (Int-407) in vitro. Twenty-nine attenuated mutants were selected for further analyses after competition assays against the wild-type strain. Whatever the screening model, most of the transposon insertions occurred in genes involved in metabolic pathways, membrane transport, DNA metabolism, transcriptional regulation, and unknown functions. Only one mutant was attenuated in both the murine colonization and the in vitro adhesion models, and the sequence disrupted by the transposon had homology to adhesin-encoding genes of Haemophilus sp.

Twenty-nine Enterococcus faecalis isolates from patients with endocarditis or bacteremia or from stools of healthy volunteers were investigated for their ability to adhere to Int-407 and Girardi heart cell lines and for the presence of known enterococcal virulence factors. Eight strains (27.6%) adhered predominantly to Int-407 cells. The adherence of enterococci was enhanced by proteolytic digestion, suggesting that some cell binding components become surface-exposed after treatment with trypsin. The occurrence of known potential virulence factors of enterococci among these strains was determined and was as follows: enterococcal surface protein (72.4%), gelatinase (58.6%), aggregation substance (48.3%) and cytolysin (17.2%). Bacterial adherence was not significantly associated with any of these virulence factors.

The influence of the industrial process on the properties of probiotics, administered as complex manufactured products, has been poorly investigated. In the present study, we comparatively assessed the cell wall characteristics of the probiotic strain Lactobacillus rhamnosus Lcr35® together with three of its commercial formulations with intestinal applications. Putative secreted and transmembrane-protein-encoding genes were initially searched in silico in the genome of L. rhamnosus Lcr35®. A total of 369 candidate genes were identified which expressions were followed using a custom Lactobacillus DNA chip. Among them, 60 or 67 genes had their expression either upregulated or downregulated in the Lcr Restituo® packet or capsule formulations, compared to the native Lcr35® strain. Moreover, our data showed that the probiotic formulations (Lcr Lenio®, Lcr restituo® capsule and packet) showed a better capacity to adhere to intestinal epithelial Caco-2 cells than the native Lcr35® strain. Microbial (MATS) tests showed that the probiotic was an electron donor and that they were more hydrophilic than the native strain. The enhanced adhesion capacity of the active pharmaceutical ingredients (APIs) to epithelial Caco-2 cells and their antipathogen effect could be due to this greater surface hydrophilic character. These findings suggest that the manufacturing process influences the protein composition and the chemical properties of the cell wall. It is therefore likely that the antipathogen effect of the formulation is modulated by the industrial process. Screening of the manufactured products' properties would therefore represent an essential step in evaluating the effects of probiotic strains.

International audience; Competition and cooperation phenomena occur within highly interactive biofilm communities and several non-biocides molecules produced by microorganisms have been described as impairing biofilm formation. In this study, we investigated the anti-biofilm capacities of an ubiquitous and biofilm producing bacterium, Klebsiella pneumoniae. Cell-free supernatant from K. pneumoniae planktonic cultures showed anti-biofilm effects on most Gram positive bacteria tested but also encompassed some Gram negative bacilli. The anti-biofilm non-bactericidal activity was further investigated on Staphylococcus epidermidis, by determining the biofilm biomass, microscopic observations and agglutination measurement through a magnetic bead-mediated agglutination test. Cell-free extracts from K. pneumoniae biofilm (supernatant and acellular matrix) also showed an influence, although to a lesser extend. Chemical analyses indicated that the active molecule was a high molecular weight polysaccharide composed of five monosaccharides: galactose, glucose, rhamnose, glucuronic acid and glucosamine and the main following sugar linkage residues [→ 2)-α-L-Rhap-(1 →]; [→ 4)-α-L-Rhap-(1 →]; [α-D-Galp-(1 →]; [→ 2,3)-α-D-Galp-(1 →]; [→ 3)-β-D-Galp-(1 →] and, [→ 4)-β-D-GlcAp-(1 →]. Characterization of this molecule indicated that this component was more likely capsular polysaccharide (CPS) and precoating of abiotic surfaces with CPS extracts from different serotypes impaired the bacteria-surface interactions. Thus the CPS of Klebsiella would exhibit a pleiotropic activity during biofilm formation, both stimulating the initial adhesion and maturation steps as previously described, but also repelling potential competitors.

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids ( stx 2 -restriction fragment length polymorphism [RFLP], stx 2 gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx 2 -RFLP and stx 2 variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx 2 -RFLP, stx 2 variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.

The interest of probiotics as remedies for a broad number of gastrointestinal and other infectious diseases has gained wide interest over the last few years, but little is known about their underlying mechanism of action. In this study, the probiotic activities of a human isolate of Lactobacillus casei subsp. rhamnosus strain (Lcr35) were investigated. Using intestinal Caco-2 cell line in an in vitro model, we demonstrated that this strain exhibited adhesive properties. The inhibitory effects of Lcr35 organisms on the adherence of three pathogens, enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli (ETEC) and Klebsiella pneumoniae, were determined. A decrease in the number of adhering pathogens was observed, using either preincubation, postincubation or coincubation of the pathogens with Lcr35. Moreover, the antibacterial activities of cell-free Lcr35 supernatant was examined against nine human pathogenic bacteria, ETEC, EPEC, K. pneumoniae, Shigella flexneri, Salmonella typhimurium, Enterobacter cloacae, Pseudomonas aeruginosa, Enterococcus faecalis and Clostridium difficile. The growth of all strains was inhibited, as measured by determining the number of viable bacteria over time, but no bactericidal activity was detected in this in vitro assay. Together, these findings suggest that this probiotic strain could be used to prevent colonization of the gastrointestinal tract by a large variety of pathogens.

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.

From August 1996 to May 1997, six verotoxin-producing Escherichia coli (VTEC) strains were isolated from stool specimens of adults suffering from hemolytic-uremic syndrome (HUS). All the isolates were stx 2 positive and belonged to different serotypes: O6:H4, O91:H10, O91:H21, O rough:H16, OX3:H−, and O nontypeable:H−. The enterohemolysin (Ehly)-encoding genes were detected in two isolates, and none of the isolates harbors the intimin (Eae)-encoding gene. These findings suggest that stx 2 -positive non-O157:H7 VTEC is a major cause of HUS in adults and that several sources of pathogens are responsible for local endemic infections.

Attempts to transform wild-type strains of Klebsiella pneumoniae using the electroporation method resulted in very low efficiencies. Transformation assays performed with plasmid pBR328 resulted in 104 transformants/μg DNA using a fresh isolate of K. pneumoniae, and only with bacteria harvested at the beginning of the growth curve (≈108 CFU/ml). This low efficiency is presumably due to the presence of bacterial exopolysaccharides. We investigated the effect of substances reducing polysaccharide production on the electroporation efficiency. Addition of sodium salicylate (187 mM), bismuth subsalicylate (0.25 mM) or ethylene glycol bis (2-aminoethyl ether) tetraacetate (EGTA) (0.4 mM) to the culture medium had no or little effect upon transformation effeciencies. However, up to 108 transformants/μg DNA were obtained by adding 0.7 mM of ethylene diamine tetra acetate (EDTA) to the culture medium and harvesting bacteria in the early growth phase.

Aggregative adhesion of Klebsiella pneumoniae LM3 to Intestine-407 (Int-407) cells was studied. Adhesive capacities were affected by the bacterial growth phase (with a maximum of adherence obtained during the exponential phase), temperature, multiplicity of infection, and length of incubation with Int-407 cells. Adhesion occurred through a cytochalasin D-sensitive process and was greatly reduced after treatment of Int-407 with cycloheximide, indicating that aggregative adhesion requires active participation of Int-407 cells. Transmission electron microscopy revealed that adherent bacteria were surrounded by a capsule-like material, apparently involved in both bacterium-Int-407 cell and bacterium-bacterium adherence. Examination with a scanning electron microscope showed interactions of intestinal cell microvilli with bacteria and formation in 3 h of a fibrous network within and around the bacterial clusters. We speculate that aggregative adhesion of K. pneumoniae mediated by a capsule-like extracellular material might explain the persistence of these strains inside the host gastrointestinal tract.

International audience; River biofilms are assemblies of autotrophic and heterotrophic microorganisms that can be affected by pollutants such as those found in watersheds and wastewater treatment plants. In the laboratory, experimental biofilms were formed from river water, and their overall composition was investigated. Denaturing gradient gel electrophoresis (DGGE) and cytometry were used to assess the richness and diversity of these communities. The software Cytostack (available on request) was developed to treat and analyze the cytometric data. Measurements of chlorophyll-a and carotenoids were used to assess the global composition of the photoautotrophic community, whereas proteins, polysaccharides (PS) content, and esterase activities were used to assess overall changes in the mixed communities. We evaluated the effects that 3 weeks of treatment with the herbicides diuron and glyphosate (10μgL(-1)) had on these biofilms. Exposed to diuron, bacterial communities adapted, changing their composition. Glyphosate inhibited growth of one autotrophic community but caused no chlorophyll deficit. As a whole, the biofilm acted as a micro-ecosystem, able to regulate and maintain a constant level of photosynthetic pigment through the structural adaptation of the autotrophic community. These results are one more proof that microbial diversity of aquatic biofilms is influenced by chemical stresses, potentially leading to disturbances within the ecosystems.

Objectives A clone of CTX-M-15-producing Escherichia coli has recently been reported to be spreading through Europe and Africa. The aim of this work was to thoroughly characterize this clone. Materials and methods Representative isolates of this clone were subjected to multilocus sequence typing, O typing, virulence gene detection, adhesion assay on human cells, biofilm production assay and mouse lethality assay. Results The clone: (i) belongs to a unique B2 phylogenetic subgroup encompassing the pyelonephritogenic diffusely adhering EC7372 strain; (ii) exhibits a specific O25b molecular subtype; (iii) is identical to the E. coli clone O25:H4-ST131 producing CTX-M-15; (iv) produces biofilm; and (v) is highly virulent in mice despite lacking classical extraintestinal pathogenicity islands (except for high pathogenicity island) and the afa/dra gene. Conclusions The CTX-M-15-producing E. coli diffusing clone is associated with a high level of antibiotic resistance and with high virulence, showing that, under certain selective pressures, the previously observed trade-off between resistance and virulence may not apply.

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae ; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.

International audience; The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases of biofilm development. A 2200 K. pneumoniae mutant library previously obtained by signature-tagged mutagenesis was screened in static and dynamic culture models to detect clones impaired at early and/or mature stages of biofilm formation. A total of 28 mutants were affected during late phases of biofilm formation, whereas 16 mutants displayed early adhesion defect. These mutants corresponded to genes involved in potential cellular and DNA metabolism pathways and to membrane transport functions. Eight mutants were deficient in capsule or LPS production. Gene disruption and microscopic analyses showed that LPS is involved in initial adhesion on both glass and polyvinyl-chloride and the capsule required for the appropriate initial coverage of substratum and the construction of mature biofilm architecture. These results give new insight into the bacterial factors sequentially associated with the ability to colonize an abiotic surface and reveal the dual roles played by surface exopolysaccharides during K. pneumoniae biofilm formation.

Klebsiella pneumoniae is a common cause of urinary tract infections (UTIs) and pneumonia, especially in immunocompromised individuals. Epidemiological studies have revealed that K. pneumoniae infections are frequently preceded by gastrointestinal colonization and the gastrointestinal tract is believed to be the most important reservoir for transmission of the bacteria. To identify genes involved in the ability of K. pneumoniae to colonize the intestine and infect the urinary tract, a novel multi-screening signature-tagged mutagenesis (MS-STM) assay was implemented. In the MS-STM assay, PCR-amplified tags present in the inoculum as well as recovered pools from each infection model are simultaneously subjected to hybridization using each specific tag as a probe. Therefore, screenings of a mutant library in more than one infection model is significantly eased compared to the traditional signature-tagged mutagenesis methodology. From a total of 1,440 K. pneumoniae transposon mutants screened, 13 mutants were identified as attenuated in intestinal colonization as well as the UTI model. In addition, six mutants attenuated only in the UTI model were identified. Transposon insertion sites in attenuated mutants were, among others, in genes encoding well-known K. pneumoniae virulence factors such as lipopolysaccharide and capsule, as well as in genes of unknown function.

Some strains of Escherichia coli are involved in enteric infections in both adults and children. However the classical diagnostic methods can not differentiate pathogenic from nonpathogenic E. coli, because of the lack of phenotypic differences. In this study, we developed multiplex PCR in order to amplify fragments of specific virulence genes of the five main E. coli pathotypes. Fragments of the expected size were obtained using previously or newly designed primers and allowed identification of 10 virulence genes in only 5 reactions. This method was applied to the detection of pathogenic E. coli isolated from 90 patients’ stools specimens during an 18‐month survey. Patients were suffering from diarrhea or hemolytic uremic syndrome and in 13 cases (14.4%), an enterovirulent E. coli strain was detected. This diagnostic method could therefore represent an important technique in clinical laboratories which lack standard tests for these pathogens. J. Clin. Lab. Anal. 15:100–103, 2001. © 2001 Wiley‐Liss, Inc.

The effect of subinhibitory concentrations of roxithromycin on the adhesion of three strains of Klebsiella pneumoniae to Int-407 cells was studied. Adherence was markedly inhibited and the effect was increased when roxithromycin was added to the cell culture medium rather than to the bacterial growth medium. Several assays were performed in order to understand the mechanism by which roxithromycin exerted this inhibitory effect. The greatest effect was obtained when roxithromycin was concentrated in the extracellular compartment; when roxithromycin was concentrated in the intracellular compartment, the inhibitory effect was reduced. The analysis of adhesion factors of bacteria showed that exposure to roxithromycin did not alter their apparent structure or quantity. Roxithromycin appears to interfere in the interaction between bacteria and eukaryotic receptors.

An enterotoxigenic Escherichia coli strain (strain 2230), isolated from a patient with acute infantile diarrhea, was found to adhere only to the brush border of human intestinal epithelial cells. This strain does not hemagglutinate human, bovine, chicken, or guinea pig erythrocytes. The adhesion of E. coli 2230 appears to be mediated by a nonfimbrial bacterial surface protein of 16,000 daltons which can be extracted by heating the bacteria at 60 degrees C for 20 min. This surface protein is implicated as an adhesive factor because pretreatment of enterocytes with this protein extract completely inhibits the adhesion of E. coli 2230. This adhesive factor is serologically distinct from other adhesive factors found in enterotoxigenic E. coli strains. A plasmid DNA of 66 megadaltons is involved in the synthesis of this nonfimbrial adhesive factor.

The afimbrial adhesive factor from human enterotoxigenic Escherichia coli strain 2230 was isolated. The adhesin was removed from the cells by heat treatment. The purification procedure included ammonium sulfate precipitation and column chromatography on Sepharose CL-6B in the presence of guanidine hydrochloride. The purified adhesin was a 16-kDa protein with an isoelectric point of 4.4. The amino acid sequence of the N-terminal 29 amino acid residues showed significant homology with that of several fimbrial proteins of E. coli.

The adhesive properties of 69 enteropathogenic Escherichia coli (EPEC) strains were studied. The strains were isolated from diarrheal stools of infants in Burundi, Africa, and identified by serotyping with 12 classical EPEC O serogroup antisera. A test for adhesion to HEp-2 cells revealed that 52% of the strains showed localized adherence and 13% diffuse adherence. Localized adherence phenotype was found in previously described serogroups O86, O111, O125, O127, O128 and O142; strains belonging to another serogroup, O126, also exhibited localized adherence. Use of an EPEC adherence factor DNA probe in colony hybridization and in Southern blot techniques revealed that all strains exhibiting localized adherence and no strain exhibiting diffuse adherence produced a positive reaction; the genes were localized on high molecular weight plasmids (50-70 megadaltons). In vitro adhesion tests with human enterocytes performed concurrently with all 69 strains showed that only six of them adhered. These strains belonged to the O26, O117, O125, O128 and O142 serogroups. The adhesin CFA/I was detected only in the O128 Escherichia coli. The strain of serogroup O142 exhibited both adhesion to human enterocytes and localized adherence to HEp-2 cells, which suggests that the adhesive systems of enterotoxigenic and enteropathogenic Escherichia coli may coexist.

Escherichia coli strains were isolated from 228 children with diarrhea in Senegal from 1982 to 1984. Among these E. coli involved in cases of diarrhea, we found that 20.3% were enteropathogenic E. coli. Only 3.9% of the strains adhered to the brush borders of human intestinal enterocytes, and they belonged to different serotypes. All these adhesion-positive strains possessed genes encoding for the heat-stable enterotoxin, but their adhesive factors were different regarding serology with anti-colonization factor sera, hemagglutination patterns, electron microscopy structures, or major surface protein subunits.

Intestinal colonization by Klebsiella, Enterobacter, and Serratia (KES) strains is a crucial step in the development of nosocomial infections. We studied the adhesive properties, antibiotic resistance, and involvement in colonization or infection of 103 KES clinical isolates: 30 Klebsiella pneumoniae (29%), 16 Klebsiella oxytoca (15%), 30 Enterobacter aerogenes (29%), 14 Enterobacter cloacae (14%), and 13 Serratia sp. (13%) isolates. Half of them were resistant to several antimicrobial agents, including aminoglycosides and beta-lactam antibiotics. A total of 27 of 30 K. pneumoniae isolates (90%) adhered to the human cell line Intestine-407 (Int-407), while none of the K. oxytoca or E. aerogenes isolates and only 2 of the E. cloacae isolates adhered. Three adhesive patterns were observed for K. pneumoniae: an aggregative adhesion in 57% of the isolates, a diffuse adhesion in only one isolate, and a new pattern, localized adhesion, in 30% of the isolates. While most of the sensitive strains adhered with the aggregative phenotype, the localized pattern was associated with resistant K. pneumoniae isolates producing the CAZ-5 beta-lactamase. Furthermore, 45% of such localized-adhesion isolates were involved in severe infections. The distributions of type 1 and type 3 fimbriae, enteroaggregative E. coli, and cf29, pap, and afa/Dr adhesin-encoding genes were determined by using specific DNA probes. No relationship was found between the adhesive pattern and the production of specific fimbriae, suggesting that several unrecognized adhesive factors are involved. Our study indicates that special adhesive properties associated with resistance to antimicrobial agents could account for the pathogenicity of certain nosocomial strains.