Application of Siderotyping for Characterization of Pseudomonas tolaasii and “Pseudomonas reactans” Isolates Associated with Brown Blotch Disease of Cultivated Mushrooms

Pyoverdine isoelectric focusing analysis and pyoverdine-mediated iron uptake were used as siderotyping methods to analyze a collection of 57 northern and central European isolates of P. tolaasii and “P. reactans.” The bacteria, isolated from cultivated Agaricus bisporus or Pleurotus ostreatus mushroom sporophores presenting brown blotch disease symptoms, were identified according to the white line test (W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 47:401–407, 1979) and their pathogenicity towards A. bisporus and were grouped into siderovars according to the type of pyoverdine they produced. Seventeen P. tolaasii isolates were recognized, which divided into two siderovars, with the first one containing reference strains and isolates of various geographical origins while the second one contained Finnish isolates exclusively. The 40 “P. reactans” isolates divided into eight siderovars. Pyoverdine isoelectric focusing profiles and cross-uptake studies demonstrated an identity for some “P. reactans” isolates, with reference strains belonging to the P. fluorescens biovars II, III, or V. Thus, the easy and rapid methods of siderotyping proved to be reliable by supporting and strengthening previous taxonomical data. Moreover, two potentially novel pyoverdines characterizing one P. tolaasii siderovar and one “P. reactans” siderovar were found.     

Evidence for genotypic differences between the two siderovars of Pseudomonas tolaasii,cause of brown blotch disease of the cultivated mushroom Agaricus bisporus

Sixteen representative isolates of Pseudomonas tolaasii, the causal agent of brown blotch of the cultivated mushroom Agaricus bisporus, were previously assigned to two siderovars (sv1 and sv2) on the basis of pyoverdines synthesized. Each isolate was pathogenic and produced a typical white line precipitate when cultured adjacent to Pseudomonas "reactans" strain LMG 5329. These 16 isolates of P. tolaasii, representing sv1 and sv2, were further characterized using genotypic methods to examine the relationships between the isolates. Rep-PCR studies revealed two distinct patterns from these isolates, which were consistent with the siderovar grouping. Ribotyping differentiated P. tolaasii LMG 2342T (sv1) and PS 3a (sv2) into two distinct ribotypes. A pair of primers, targeted to a 2.1-kb fragment of tl1 (encoding a tolaasin peptide synthetase), yielded the same PCR product from P. tolaasii LMG 2342T (sv1) and PS 22.2 (sv1), but not from PS 3a (sv2). Southern blot analysis indicated that homologues of tl1 are present in PS 3a, but the pattern of hybridization differed from PS 22.2 and LMG 2342T. Sequence determination and analysis of the internally transcribed spacer region ITSI for P. tolaasii LMG 2342T, LMG 6641, and PS 3a strains further supported the presence of the two siderovars. It is concluded that considerable genotypic differences exist among Finnish isolates of P. tolaasii causing brown blotch disease on the cultivated mushroom, which is in agreement with the phenotypic diversity highlighted through previous siderotyping studies.     

Siderotyping of Antarctic fluorescent Pseudomonas strains.

Five fluorescent Pseudomonas strains isolated from Antarctica have been previously recognized as producing three structurally different pyoverdines. In the present work, siderotyping procedures have been used to classify these strains, together with 1282 isolates of different origins, into siderovars. The strain biodiversity encountered within each siderovar, as well as the potential taxonomic value of the siderovars, are described and discussed. It is concluded that a majority of antarctic strains are commonly distributed worldwide. One strain, however, presenting a particular pyoverdine structure found in a unique other isolate, was apparently much more specific to cold environment.     

The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans"

A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans". This interaction has been considered as highly specific. However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T. Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P. tolaasii(T), produced a typical white precipitate when streaked towards P. "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P. tolaasii. We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P. tolaasii.     

Effects of Caenorhabditis elegans (Nematoda: Rhabditidae) on the spread of the bacterium Pseudomonas tolaasii in mushrooms (Agaricus bisporus)

The effects of mass-produced saprobic rhabditid nematodes, Caenorhabditis elegans on the spread of the bacterial blotch pathogen, Pseudomonas tolaasii , were studied in mushroom growth chambers. C. elegans significantly reduced the intensity of blotch on sporophores. Repeated isolations of the bacterial flora from the gut of C. elegans recovered from mushroom sporophores during cropping, revealed the presence of Pseudomonas fluorescens biovar reactans . All the isolates of P. fluorescens biovar reactans isolated from nematodes were antagonists of P. tolaasii .
C. elegans produced much larger populations in monoxenic cultures with P. fluorescens biovar reactans than with P. tolaasii . It is suggested that as C. elegans selects P. fluorescens biovar reactans rather than P. tolaasii as a food substrate it probably spreads the antagonist in the mushroom crop and may contribute to the control of bacterial blotch.     

Genetic diversity in pseudomonads associated with cereal cultures infected with basal bacteriosis

The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S-23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae ("Syringae" cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici ("Fluorescens" cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the "Fluorescens" cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.     

Siderophore typing,a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads

A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, "Pseudomonas mosselii," "Pseudomonas palleronii," Pseudomonas rhodesiae, "Pseudomonas salomonii," Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.     

Identification of exopolysaccharides produced by fluorescent pseudomonads associated with commercial mushroom (Agaricus bisporus) production.

The acidic exopolysaccharides (EPSs) from 63 strains of mushroom production-associated fluorescent pseudomonads which were mucoid on Pseudomonas agar F medium (PAF) were isolated, partially purified, and characterized. The strains were originally isolated from discolored lesion which developed postharvest on mushroom (Agaricus bisporus) caps or from commercial lots of mushroom casing medium. An acidic galactoglucan, previously named marginalan, was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens (biovars III and V) isolated from casing medium. One biovar II strain (J1) of P. fluorescens produced alginate, a copolymer of mannuronic and guluronic acids, and one strain (H13) produced an apparently unique EPS containing neutral and amino sugars. Of 10 strains of the pathogen "P. gingeri," the causal agent of mushroom ginger blotch, 8 gave mucoid growth on PAF. The "P. gingeri" EPS also was unique in containing both neutral sugar and glucuronic acid. Mucoid, weakly virulent strains of "P. reactans" produced either alginate or marginalan. All 10 strains of the pathogen P. tolaasii, the causal agent of brown blotch of mushrooms were nonnmucoid on PAF. Production of EPS by these 10 strains plus the 2 nonmucoid strains of "P. gingeri" also was negative on several additional solid media as well as in two broth media tested. The results support our previous studies indicating that fluorescent pseudomonads are a rich source of novel EPSs.     

Characterization by 16S rRNA sequence analysis of pseudomonads causing blotch disease of cultivated Agaricus bisporus

Bacterial blotch of Agaricus bisporus has typically been identified as being caused by either Pseudomonas tolaasii (brown blotch) or Pseudomonas gingeri (ginger blotch). To address the relatedness of pseudomonads able to induce blotch, a pilot study was initiated in which pseudomonads were selectively isolated from mushroom farms throughout New Zealand. Thirty-three pseudomonad isolates were identified as being capable of causing different degrees of discoloration (separable into nine categories) of A. bisporus tissue in a bioassay. These isolates were also identified as unique using repetitive extragenic palindromic PCR and biochemical analysis. Relationships between these 33 blotch-causing organisms (BCO) and a further 22 selected pseudomonad species were inferred by phylogenetic analyses of near-full-length 16S rRNA gene nucleotide sequences. The 33 BCO isolates were observed to be distributed throughout the Pseudomonas fluorescens intrageneric cluster. These results show that in addition to known BCO (P. tolaasii, P. gingeri, and Pseudomonas reactans), a number of diverse pseudomonad species also have the ability to cause blotch diseases with various discolorations. Furthermore, observation of ginger blotch discoloration of A. bisporus being independently caused by many different pseudomonad species impacts on the homogeneity and classification of the previously described P. gingeri.     

Distribution and evolution of ferripyoverdine receptors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium, which is also able to cause severe opportunistic infections in humans. The colonization of the host is importantly affected by the production of the high-affinity iron (III) scavenging peptidic siderophore pyoverdine. The species P. aeruginosa can be divided into three subgroups ('siderovars'), each characterized by the production of a specific pyoverdine and receptor (FpvA). We used a multiplex PCR to determine the FpvA siderovar on 345 P. aeruginosa strains from environmental or clinical origin. We found about the same proportion of each type in clinical strains, while FpvA type I was slightly over-represented (49%) in environmental strains. Our multiplex PCR also detected the presence or absence of an additional receptor for type I pyoverdine (FpvB). The fpvB gene was in fact present in the vast majority of P. aeruginosa strains (93%), regardless of their siderovar or their origin. Finally, molecular analyses of fpvA and fpvB genes highlighted a complex evolutionary history, probably linked to the central role of iron acquisition in the ecology and virulence of P. aeruginosa .     

Preliminary description of biocidal (syringomycin) activity in fluorescent plant pathogenic Pseudomonas species

Strains representing the fluorescent plant pathogenic Pseudomonas spp., Ps. agarici , Ps. asplenii , Ps. avellanae , Ps. beteli , Ps. caricapapayae , Ps. cichorii , Ps. corrugata , Ps. ficuserectae , Ps. flectens , Ps. fuscovaginae , Ps. marginalis , Ps. meliae , Ps. savastanoi , Ps. syringae , Ps. tolaasii and Ps. viridiflava were tested for biocidal activity using Aspergillus niger as assay organism. Inhibitory behaviour was found in strains of Ps. asplenii , Ps. blatchfordae , Ps. cichorii , Ps. corrugata , Ps. fuscovaginae , Ps. marginalis , Ps. marginalis pv. pastinacea , Ps. syringae pv. syringae , Ps. syringae pv. aptata , Ps. syringae pv. atrofaciens , Ps. syringae pv. lapsa , Ps. tolaasii , and strains of a Pseudomonas sp. pathogenic to Actinidia , in the Ps. savastanoi genomic sp. Antifungal activity could be identified with the production of members of the syringomycin family of toxins by strains in Ps. syringae , Ps. asplenii and Ps. fuscovaginae . These toxin reactions support suggestions made elsewhere of the synonymy of the latter two species. In a preliminary characterization using tests for stability to heat, protease, acid and alkaline treatments, unknown toxins consistent with syringomycin-like toxins the strains from Actinidia speciesColour RGB 0,0,128. The toxins from Ps. cichorii and from Ps. corrugata differed in their reactions from all other agents. Pseudomonas tolaasii produces the antifungal compound tolaasin. The white line reaction with ' Ps. reactans ', a test for tolaasin production by strains of Ps. tolaasii , was confirmed as specific for this compound. Some of these low molecular weight toxins may be produced by some of these plant pathogenic strains.     

Biological characterization of white line-inducing principle (WLIP) produced by Pseudomonas reactans NCPPB1311

The biological activities of the lipodepsipeptides (LDP) white line-inducing principle (WLIP), produced by Pseudomonas reactans NCPPB1311, and tolaasin I, produced by R tolaasii NCPPB2192, were compared. Antimicrobial assays showed that both LDP inhibited the growth of fungi-including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.--chromista, and gram-positive bacteria. Assays of the two LDP on blocks of Agaricus bisporus showed their capacity to alter the mushrooms' pseudo-tissues though WLIP was less active than that of tolaasin I. Contrary to previous studies, tolaasin I was found to inhibit the growth of gram-negative bacteria belonging to the genera Escherichia, Erwinia, Agrobacterium, Pseudomonas, and Xanthomonas. The only gram-negative bacterium affected by WLIP was Erwinia carotovora subsp. carotovora. Both WLIP and tolaasin I caused red blood cell lysis through a colloid-osmotic shock mediated by transmembrane pores; however, the haemolytic activity of WLIP was greater than that of tolaasin I. Transmembrane pores, at a concentration corresponding to 1.5 x C50, showed a radius between 1.5 and 1.7 +/- 0.1 nm for WLIP and 2.1 +/- 0.1 nm for tolaasin I. The antifungal activity of WLIP together with the finding that avirulent morphological variants of P. reactans lack WLIP production suggests that WLIP may play an important role in the interaction of the producing bacterium P. reactans and cultivated mushrooms.     

Genetic and functional heterogeneities among fluorescent Pseudomonas isolated from environmental samples

Fluorescent Pseudomonas from diverse environmental samples including wastes were identified and screened for the solubilization of tricalcium phosphate, indole-3-acetic acid (IAA), production and inhibition of extracellular N-acylhomoserine lactone (AHLs) and characterized for their siderophores. Genotypic analysis by amplified rDNA restriction analysis (ARDRA) and BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) typing resulted respectively in 14 ARDRA types and 24 different BOX-types with diverse incidence among the analyzed strains. Based on 16S rRNA sequence analysis the isolates were assigned to P. aeruginosa, P. otitidis, P. plecoglossicida, P. mosselii, P. monteilii, P. koreensis, P. taiwanenesis, P. frederiksbergensis and P. graminis. Of the 66 isolates, 56 (84.85%) isolates solubilized tri-calcium phosphate (TCP), 53 (80.30%) isolates produced plant growth hormone IAA, 62 (94%) produced bacteriocin and 34 (52%) isolates produced extracellular N-acylhomoserine lactone while 30 (45%) isolates were able to interfere with N-acylhomoserine lactone. Isolates were clustered into 17 siderotypes and (59)Fe cross-incorporation experiments permitted assignment of all siderotypes but two into well-defined siderovars.     

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 microM). This effect was not detected for tolaasin I at the concentrations tested (< 28 microM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.     

Genetic Diversity in Pseudomonads Associated with Cereal Cultures Infected with Basal Bacteriosis

The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S–23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae (“Syringae” cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici (“Fluorescens” cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the “Fluorescens” cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 537–544.Original Russian Text Copyright © 2005 by Bobrova, Milyutina, Troitskii.     

Pyocine Typing of Clinical Strains of Pseudomonas aeruginosa

A total of 954 clinical isolates of Pseudomonas aeruginosa were typed by their ability to produce pyocines. The strains of Pseudomonas were isolated from urines, bloods, sputa, stools, and miscellaneous infectious exudates or tissue of patients of the Mayo Clinic and four associated hospitals. About 80% of the typable strains could be grouped into three major pyocine types: A (30.9%), B (34.8%), and D (14.1%). These large groups could be divided into subtypes by using additional indicator strains. There was no significant difference in the distribution of types by either institutional or specimen source, except that urine specimens yielded the highest percentage of one type. By this procedure, 93% of all isolates could be typed. Repeated typing of serially transferred strains indicated that the procedure has a high degree of reliability. Several strains exhibited extreme fluctuation in inhibition pattern. The procedure is a simple and reliable method to monitor the patterns of nosocomial infections due to P. aeruginosa.     

Production and comparison of peptide siderophores from strains of distantly related pathovars of Pseudomonas syringae and Pseudomonas viridiflava LMG 2352

The production of peptide siderophores and the variation in siderophore production among strains of Pseudomonas syringae and Pseudomonas viridiflava were investigated. An antibiose test was used to select a free amino acid-containing agar medium favorable for production of fluorescent siderophores by two P. syringae strains. A culture technique in which both liquid and solid asparagine-containing culture media were used proved to be reproducible and highly effective for inducing production of siderophores in a liquid medium by the fluorescent Pseudomonas strains investigated. Using asparagine as a carbon source appeared to favor siderophore production, and relatively high levels of siderophores were produced when certain amino acids were used as the sole carbon and energy sources. Purified chelated siderophores of strains of P. syringae pv. syringae, P. syringae pv. aptata, P. syringae pv. morsprunorum, P. syringae pv. tomato, and P. viridiflava had the same amino acid composition and spectral characteristics and were indiscriminately used by these strains. In addition, nonfluorescent strains of P. syringae pv. aptata and P. syringae pv. morsprunorum were able to use the siderophores in biological tests. Our results confirmed the proximity of P. syringae and P. viridiflava; siderotyping between pathovars of P. syringae was not possible. We found that the spectral characteristics of the chelated peptide siderophores were different from the spectral characteristics of typical pyoverdins. Our results are discussed in relation to the ecology of the organisms and the conditions encountered on plant surfaces.     

Expression of Pectinase Activity Among Aspergillus Flavus Isolates from Southwestern and Southeastern United States

Aspergillus flavus is a widely distributed filamentous fungus that contaminates crops with the potent carcinogen aflatoxin. This species can be divided into S and L strains on the basis of sclerotial morphology. During crop infection, A. flavus can secrete a large array of hydrolytic enzymes. These include pectinase, which aids fungal spread through plant tissues. A survey of pectinase expression by soil isolates derived from different regions of the United States revealed geographic polymorphisms. Strain L isolates from Arizona produced moderate to high levels of a specific pectinase P2c, while S strain isolates produced variable amounts of P2c. In contrast, L strain isolates from southeastern U.S. yielded variable P2c production, while S strain isolates consistently expressed high P2c levels. These results were corroborated by pectinase surveys of additional collections of A. flavus from soil and cottonseed. Expression patterns for P2c and pectinmethylesterase were evaluated for a select number of isolates using an isoelectric focusing technique. Clear zone reactions from the pectinase plate assay corresponded to the presence of P2c, while red ring reactions corresponded to the lack of P2c. Commercial cottonseed infected by S strain isolates frequently contained aflatoxin, even when infected by S strain isolates that did not produce pectinase P2c. Thus, although P2c-lacking isolates have reduced invasiveness, these isolates still have sufficient pathogenicity to cause aflatoxin contamination.     

The genotypic diversity of Pseudomonas brassicacearum populations isolated from roots of Arabidopsis thaliana: influence of plant genotype

Pseudomonas brassicacearum is a newly described bacterial species isolated from the rhizosphere of Arabidopsis thaliana. The P. brassicacearum populations were isolated from the rhizosphere of two ecotypes of A. thaliana (Wassilewskija (WS) and Columbia (COL)), a mutant of Columbia impaired in starch metabolism (pgm mutant), and a genetically distant plant (wheat), grown in a French eutric cambisol (Méréville). The strains were isolated on semi-selective media. Their diversity was assessed using repetitive extragenic palindromic (REP)-PCR profiling and their affiliation to the P. brassicacearum species using ARDRA and siderotyping. A total of 379 strains isolated in two experiments were clustered into 68 REP-genotypes. Statistical analysis showed that the genetic structure of the P. brassicacearum populations was homogeneous for strains isolated from different plants of the same genotype within the same experiment, but significantly differed across the four tested plant genotypes. Comparison of the REP-genotype distributions showed that some bacterial genotypes were poorly represented, whereas others were strongly stimulated by plant roots.     

A 5-year epidemiological study of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a medium- and long-stay neurological unit

Thirty-eight different strains of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL Kp), isolated from urine and pus samples of 38 patients hospitalized in a medium- and long-stay neurology department between 1 January 1992 and 31 December 1996, were analysed by antibiotic resistance phenotyping, DNA macrorestriction by pulsed-field electrophoresis and isoelectric focusing of beta-lactamases. An epidemiological survey was conducted to identify risk factors for infection by ESBL Kp in this setting. The 38 isolates were distributed into 13 antibiotypes, three of which predominated (13, six and six isolates). The DNA macrorestriction pattern identified 15 genotypes, four of which predominated (11, six, four and four isolates). A combination of the two typing methods revealed several epidemic clones that emerged consecutively. Two main types of ESBL (SHV-2 and CTX-1) were identified by isoelectric focusing, the former predominating. The case-control study showed that the length of hospital stay, degree of malnutrition and dependency, and urinary sphincter status were the main factors significantly associated with ESBL Kp isolation.