The phylogenetic relationships of the type strains of 9 Klebsiella species and 20 species from 11 genera of the family Enterobacteriaceae were investigated by performing a comparative analysis of the sequences of the 16S rRNA and rpoB genes. The sequence data were phylogenetically analysed by the neighbourjoining and parsimony methods. The phylogenetic inference of the sequence comparison confirmed that the genus Klebsiella is heterogeneous and composed of species which form three clusters that also included members of other genera, including Enterobacter aerogenes, Erwinia clusters I and II and Tatumella. Cluster I contained the type strains of Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. rhinoscleromatis and Klebsiella pneumoniae subsp. ozaenae. Cluster II contained Klebsiella ornithinolytica, Klebsiella planticola, Klebsiella trevisanii and Klebsiella terrigena, organisms characterized by growth at 10 degrees C and utilization of L-sorbose as carbon source. Cluster III contained Klebsiella oxytoca. The data from the sequence analyses along with previously reported biochemical and DNA-DNA hybridization data support the division of the genus Klebsiella into two genera and one genogroup. The name Raoultella is proposed as a genus name for species of cluster II and emended definitions of Klebsiella species are proposed.

The infra-specific phylogenetic diversity and genetic structure of both Klebsiella pneumoniae and Klebsiella oxytoca was investigated using a combination of randomly amplified polymorphic DNA (RAPD) analysis, sequencing of gyrA and parC genes, and automated ribotyping. After RAPD analysis with four independent primers of 120 clinical isolates collected from 22 European hospitals in 13 countries, K. pneumoniae isolates fell into three clusters and K. oxytoca isolates fell into two clusters, while Klebsiella planticola isolates formed a sixth cluster. Each cluster was geographically widespread. K. pneumoniae cluster I (KpI) accounted for 80% of the isolates of this species and included reference strains of the three subspecies K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis. Clusters KpII and KpIII were equally represented, as were the two K. oxytoca clusters. Individualization of each cluster was fully confirmed by phylogenetic analysis of gyrA and parC gene sequences. In addition, sequence data supported the evolutionary separation of K. pneumoniae from a phylogenetic group including K. oxytoca, Klebsiella terrigena, K. planticola and Klebsiella ornithinolytica. Automated ribotyping using Mlu I appeared suitable for identification of each Klebsiella cluster. The adonitol fermentation test was found to be useful for cluster identification in K. pneumoniae, since it was negative in all strains of clusters KpIII and in some KpII strains, but always positive in cluster KpI. The usefulness of gyrA and parC sequence data for population genetics and cluster identification in bacteria was demonstrated, even for the phylogenetic positioning of quinolone-resistant isolates.

Phylogenetic relations within the family Enterobacteriaceae were analyzed using partial dnaJ sequences of 165 strains belonging to 93 species from 27 enterobacterial genera. The dnaJ phylogeny was in relative agreement with that constructed by 16S rDNA sequences, but more monophyletic groups were obtained from the dnaJ tree than from the 16S rDNA tree. The degree of divergence of the dnaJ gene was approximately 6 times greater than that of 16S rDNA. Also, the dnaJ gene showed the most discriminatory power in comparison with tuf and atpD genes, facilitating clear differentiation of any 2 enterobacterial species by dnaJ sequence analysis. The application of dnaJ sequences to the identification was confirmed by assigning 72 clinical isolates to the correct enterobacterial species. Our data indicate that analysis of the dnaJ gene sequences can be used as a powerful marker for phylogenetic study and identification at the species level of the family Enterobacteriaceae.

Real-time polymerase chain reaction and sequencing were used to characterize a 506-bp-long DNA fragment internal to the gyrB gene (gyrBint). The sequences obtained from 32 Enterobacteriaceae-type strains and those available in the Genbank nucleotide sequence database (n = 24) were used as a database to identify 240 clinical enterobacteria isolates. Sequence analysis of the gyrBint fragment of 240 strains showed that gyrBint constitutes a discriminative target sequence to differentiate between Enterobacteriaceae species. Comparison of these identifications with those obtained by phenotypic methods (Vitek 1 system and/or Rapid ID 32E; bioM?rieux, Marcy l'Etoile, France) revealed discrepancies essentially with genera Citrobacter and Enterobacter. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter hormaechei strains by gyrBint sequencing. The direct sequencing of gyrBint would be useful as a complementary tool in the identification of clinical Enterobacteriaceae isolates.

The objectives of this study were to determine the distribution of Klebsiella species and phylogenetic groups in animal clinical samples and to determine the levels of antimicrobial resistance of animal Klebsiella clinical isolates. One hundred Klebsiella veterinary clinical isolates were identified using gyrA PCR-RFLP and rpoB gene sequencing as a confirmatory method. Klebsiella pneumoniae phylogenetic group KpI was dominant (78 isolates), but KpII, KpIII (K. variicola), K. oxytoca, K. planticola and K. terrigena were also represented. The relative frequencies in animal infections of Klebsiella species and phylogenetic groups were similar to those observed in human nosocomial infections, suggesting that similar ecological and molecular factors cause Klebsiella infections in both situations. Resistance was common against ampicillin (99%) and cephalexin (43%) but not against ceftazidime, ceftiofur, tetracycline, enrofloxacin, gentamicin and trimethoprim-sulfamethoxazole. Thirteen isolates resistant to three or more antimicrobials or combinations thereof were found, but acquired antimicrobial resistance remains lower among animal isolates than among human nosocomial isolates.

A new Klebsiella species, K. variicola, is proposed on the basis of total DNA-DNA hybridization, on the monophyly observed in the phylogenetic analysis derived from the sequences of rpoB, gyrA, mdh, infB, phoE and nifH genes and on distinct phenotypic traits. The bacteria from this new species seem to be genetically isolated from K. pneumoniae strains, do not ferment adonitol and were obtained from plants (such as banana, rice, sugar cane and maize) and hospitals. The type strain is F2R9T (= ATCC BAA-830T = CFNE 2004T).

Raoultella terrigena ATCC 33257, recently reclassified from the genus Klebsiella, is a drinking water isolate and belongs to a large group of non-typeable Klebsiella and Raoultella strains. Using an O-antiserum against a capsule-deficient mutant of this strain, we could show a high prevalence (10.5%) of the R. terrigena O-serotype among non-typeable, clinical Klebsiella and Raoultella isolates. We observed a strong serological cross-reaction with the K. pneumoniae O12 reference strain, indicating that a large percentage of these non-typeable strains may belong to the O12 serotype, although these are currently not detectable by the K. pneumoniae O12 reference antiserum in use. Therefore, we analyzed the O-polysaccharide (O-PS) structure and genetic organization of the wb gene cluster of R. terrigena ATCC 33257, and both confirmed a close relation of R. terrigena and K. pneumoniae O12. The two strains possess an identical O-PS, lipopolysaccharide core structure, and genetic organization of the wb gene cluster. Heterologous expression of the R. terrigena wb gene cluster in Escherichia coli K-12 resulted in the WecA-dependent synthesis of an O-PS reactive with the K. pneumoniae O12 antiserum. The serological data presented here suggest a higher prevalence of the O12-serotype among Klebsiella and Raoultella isolates than generally assumed.

Nitrogen-fixing bacteria isolated from root nodules of Medicago plants growing in the 10 km zone around the Chernobyl nuclear power plant were screened for the production of new water-soluble acidic exopolysaccharides (EPSs). The different strains belonged to the Enteriobacteriaceae family (Enterobacter ludwigii, Raoultella terrigena, Klebsiella oxytoca), except for one which belonged to the Rhizobiaceae family (Sinorhizobium meliloti). All of the bacteria produced highly viscous EPS with an average molecular weight comprised between 1 x 10(6) and 3 x 10(6) Da. Five different compositions of EPS were characterized by physico-chemical analyses and (1)H NMR spectroscopy: galactose/mannose (2/1), galactose/glucose (1/1), galactose/glucose/mannose (1/2/1), fucose/galactose/glucose (2/1/1) and fucose/galactose/glucose/mannose (2/2/1/1 or 1/1/2/4). Glucuronic acid, a charged monosaccharide, was also recovered in most of the different EPSs.

Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in gram-negative bacteria by simultaneously genotyping bla(TEM), bla(SHV), and bla(CTX-M). The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla(CTX-M) (76%), bla(SHV) (22%), or both (2%), whereas no ESBL variant of bla(TEM) was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).

Export of the Escherichia coli serotype O9a O-antigenic polysaccharides (O-PS) involves an ATP-binding cassette (ABC) transporter. The process requires a non-reducing terminal residue, which is recognized by a carbohydrate-binding module (CBM) appended to the C terminus of the nucleotide-binding domain of the transporter. Here, we investigate the process in Klebsiella pneumoniae serotype O12 (and Raoultella terrigena ATCC 33257). The O12 polysaccharide is terminated at the non-reducing end by a �]-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue. The O12 ABC transporter also binds its cognate O-PS via a CBM, and export is dependent on the presence of the terminal �]-Kdo residue. The overall structural architecture of the O12 CBM resembles the O9a prototype, but they share only weak sequence similarity, and the putative binding pocket for the O12 glycan is different. Removal of the CBM abrogated O-PS transport, but export was restored when the CBM was expressed in trans with the mutant CBM-deficient ABC transporter. These results demonstrate that the CBM-mediated substrate-recognition mechanism is evolutionarily conserved and can operate with glycans of widely differing structures.

Klebsiella pneumoniae, a Gram-negative bacillus that causes life-threatening infections in both hospitalized patients and ambulatory persons, can be classified into nine lipopolysaccharide (LPS) O-antigen serotypes. The O-antigen type has important clinical and epidemiological significance. However, K. pneumoniae O serotyping is cumbersome, and the reagents are not commercially available. To overcome the limitations of conventional serotyping methods, we aimed to create a rapid and accurate PCR method for K. pneumoniae O genotyping. We sequenced the genetic determinants of LPS O antigen from serotypes O1, O2a, O2ac, O3, O4, O5, O8, O9, and O12. We established a two-step genotyping scheme, based on the two genomic regions associated with O-antigen biosynthesis. The first set of PCR primers, which detects alleles at the wzm-wzt loci of the wb gene cluster, distinguishes between O1/O2, O3, O4, O5, O8, O9, and O12. The second set of PCR primers, which detects alleles at the wbbY region, further differentiates between O1, O2a, and O2ac. We verified the specificity of O genotyping against the O-serotype reference strains. We then tested the sensitivity and specificity of O genotyping in K. pneumoniae, using the 56 K-serotype reference strains with known O serotypes determined by an inhibition enzyme-linked immunosorbent assay (iELISA). There is a very good correlation between the O genotypes and classical O serotypes. Three discrepancies were observed and resolved by nucleotide sequencing--all in favor of O genotyping. The PCR-based O genotyping, which can be easily performed in clinical and research microbiology laboratories, is a rapid and accurate method for determining the LPS O-antigen types of K. pneumoniae isolates.

Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. �\-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of �]-Kdo residues links "group 2" capsular polysaccharides to (lyso)phosphatidylglycerol. �]-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo-dependent �\-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the �]-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype �]-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis in Raoultella terrigena The �]-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal �\/�] domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities.

Capsule is an important virulence factor in bacteria. A total of 78 capsular types have been identified in Klebsiella pneumoniae. However, there are limitations in current typing methods. We report here the development of a new genotyping method based on amplification of the variable regions of the wzc gene. Fragments corresponding to the variable region of wzc were amplified and sequenced from 76 documented capsular types of reference or clinical strains. The remaining two capsular types (reference strains K15 and K50) lacked amplifiable wzc genes and were proven to be acapsular. Strains with the same capsular type exhibited ��94% DNA sequence identity across the variable region (CD1-VR2-CD2) of wzc. Strains with distinct K types exhibited <80% DNA sequence identity across this region, with the exception of three pairs of strains: K22/K37, K9/K45, and K52/K79. Strains K22 and K37 shared identical capsular polysaccharide synthesis (cps) genes except for one gene with a difference at a single base which resulted in frameshift mutation. The wzc sequences of K9 and K45 exhibited high DNA sequence similarity but possessed different genes in their cps clusters. K52 and K79 exhibited 89% wzc DNA sequence identity but were readily distinguished from each other at the DNA level; in contrast, strains with the same capsular type as K52 exhibited 100% wzc sequence identity. A total of 29 strains from patients with bacteremia were typed by the wzc system. wzc DNA sequences confirmed the documented capsular type for twenty-eight of these clinical isolates; the remaining strain likely represents a new capsular type. Thus, the wzc genotyping system is a simple and useful method for capsular typing of K. pneumoniae.

The increase demand for fresh vegetables is causing an expansion of the market for minimally processed vegetables along with new recognized food safety problems. To gain further insight on this topic we analyzed the microbiological quality of Portuguese ready-to-eat salads (RTS) and their role in the spread of bacteria carrying acquired antibiotic resistance genes, food products scarcely considered in surveillance studies. A total of 50 RTS (7 brands; split or mixed leaves, carrot, corn) were collected in 5 national supermarket chains in Porto region (2010). They were tested for aerobic mesophilic counts, coliforms and Escherichia coli counts as well as for the presence of Salmonella and Listeria monocytogenes. Samples were also plated in different selective media with/without antibiotics before and after enrichment. The E. coli, other coliforms and Enterococcus recovered were characterized for antibiotic resistance profiles and clonality with phenotypic and genetic approaches. A high number of RTS presented poor microbiological quality (86%--aerobic mesophilic counts, 74%--coliforms, 4%--E. coli), despite the absence of screened pathogens. In addition, a high diversity of bacteria (species and clones) and antibiotic resistance backgrounds (phenotypes and genotypes) were observed, mostly with enrichment and antibiotic selective media. E. coli was detected in 13 samples (n=78; all types and 4 brands; phylogenetic groups A, B1 and D; none STEC) with resistance to tetracycline [72%; tet(A) and/or tet(B)], streptomycin (58%; aadA and/or strA-strB), sulfamethoxazole (50%; sul1 and/or sul2), trimethoprim (50%; dfrA1 or dfrA12), ampicillin (49%; blaTEM), nalidixic acid (36%), ciprofloxacin (5%) or chloramphenicol (3%; catA). E. coli clones, including the widespread group D/ST69, were detected in different samples from the same brand or different brands pointing out to a potential cross-contamination. Other clinically relevant resistance genes were detected in 2 Raoultella terrigena carrying a bla(SHV-2) and 1 Citrobacter freundii isolate with a qnrB9 gene. Among Enterococcus (n=108; 35 samples; Enterococcus casseliflavus--40, Enterococcus faecalis--20, Enterococcus faecium--18, Enterococcus hirae--9, Enterococcus gallinarum--5, and Enterococcus spp.--16) resistance was detected for tetracyclines [6%; tet(M) and/or tet(L)], erythromycin [3%; erm(B)], nitrofurantoin (1%) or ciprofloxacin (1%). The present study places ready-to-eat salads within the spectrum of ecological niches that may be vehicles for antibiotic resistance bacteria/genes with clinical interest (e.g. E. coli-D-ST69; bla(SHV-2)) and these findings are worthy of attention as their spread to humans by ingestion cannot be dismissed.

A cytoplasmatic phytase was purified about 410-fold to apparent homogeneity with a recovery of 28%. The enzyme is induceable under carbon limitation in the presence of phytate. It behaves as a monomeric protein of a molecular mass of about 40 kDa. The phytase is rather specific for phytate and exhibits optimal conditions for phytate degradation at pH 5.0 and 58 degrees C. Kinetic parameters for the hydrolysis of Na phytate are KM 300 microM and kcat 180 s-1 at 35 degrees C and pH 5.0. Phytate is hydrolyzed in a stepwise manner; the penta- and tetrakisphosphate were identified as I(1,2,4,5,6)P5 and I(1,2,5,6)P4. Consequently, this enzyme is a 3-phytase (EC 3.1.3.8).

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

The gnd gene, encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44), was sequenced in 87 strains of 15 species assigned to five nominal genera of the Enterobacteriaceae, including 36 isolates of Salmonella enterica and 32 strains of Escherichia coli. In S. enterica, the effective (realized) rate of recombination of horizontally transferred gnd sequences is only moderately higher than the rates for other chromosomal housekeeping genes. In contrast, recombination at gnd has occurred with such high frequency in Escherichia coli that the indicated evolutionary relationships among strains are not congruent with those estimated by sequence analysis of other genes and by multilocus enzyme electrophoresis. E. coli and S. enterica apparently have not exchanged gnd sequences, but those of several strains of E. coli have been imported from species of Citrobacter and Klebsiella. The relatively frequent exchange of gnd within and among taxonomic groups of the Enterobacteriaceae, compared with other housekeeping genes, apparently results from its close linkage with genes that are subject to diversifying selection, including those of the rfb region determining the structure of the O antigen polysaccharide.