Numerical taxonomy ofKlebsiella pneumoniae strains isolated from clinical and nonclinical sources

A total of 180 clinical and nonclinical isolates ofKlebsiella pneumoniae, for which 99 characteristics were recorded, were subjected to numerical taxonomy analysis. Of these strains, 172 clustered into five major groups, with an overall similarity of 64%. Intragroup similarities ranged from 77 to 82%, with the subgroups corresponding to the speciesK. pneumoniae sensu stricto, K. oxytoca, andKlebsiella spp. 1, 2, and 3. Biochemical tests useful in distinguishing the species included production of indole, degradation of pectate, growth at 10°C, fecal coliform response, production of urease, fermentation of inulin andd-tartrate, utilization ofl-arginine and gentisate andm-hydroxybenzoate, and pigment formation ond-gluconate ferric citrate agar.     

Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates

Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)₅- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)₅ and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)₅ and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)₅ and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.     

Incidence of Klebsiella Species in Surface Waters and Their Expression of Virulence Factors

To investigate the occurrence of different Klebsiella spp. in aquatic environments, a total of 208 samples of natural surface waters was examined. From half (53%) of these samples, 123 Klebsiella strains were isolated, the most common species being Klebsiella pneumoniae. A comparison of these isolates to a group of 207 clinical K. pneumoniae isolates demonstrated that water isolates of K. pneumoniae, unlike those of K. oxytoca and K. planticola, are as capable as clinical isolates of expressing putative virulence factors such as serum resistance and capsular polysaccharides, pili, and siderophores.     

The β-tubulin gene as a molecular phylogenetic marker for classification and discrimination of the <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">sensu stricto</Emphasis> complex

The Saccharomyces sensu stricto complex comprises seven very closely related species. In this study, we compared the use of two different phylogenetic markers, the 26S rDNA and β-tubulin genes, for discriminating phylogenetic relationships among Saccharomyces sensu stricto strains using sequencing as well as RFLP methods. The average sequence similarity for the β-tubulin gene (90.0%) among seven strains was significantly less than that for 26S rDNA (98.6%). This result demonstrates that β-tubulin gene sequences provided higher resolution than 26S rDNA sequences. Species-specific restriction profiles of the Saccharomyces strains were obtained by cutting them with the Tsp509I enzyme. Our data indicate that phylogenetic relationships between these strains are best resolved using sequencing or RFLP analysis of the β-tubulin gene. The β-tubulin gene sequence data reported in this paper appear in the GenBank nucleotide sequence database with the following accession numbers: FJ238316–FJ238341.     

Metabolism of tannin-protein complex by facultatively anaerobic bacteria isolated from koala feces

The metabolic pathways involved in degradation of tannin-protein complex (T-PC) were investigated in various facultatively anaerobic bacteria, with specific reference to fecal isolates from the koala including T-PC-degrading enterobacteria (T-PCDE),Streptococcus bovis, Klebsiella pneumoniae, andK. oxytoca. It was demonstrated that T-PCDE andS. bovis biotype I were capable of degrading protein complexed with gallotannin (a hydrolyzable tannin), but not that complexed with quebracho (a condensed tannin). Subsequent studies showed that these strains metabolized gallic acid to pyrogallol. Strains ofKlebsiella pneumoniae andK. oxytoca, which did not degrade T-PC, also metabolized gallic acid into pyrogallol. Pyrogallol was not degraded by any strains studied, but it was not detected in fresh feces of the koalas. The majority of strains isolated from feces could degrade phloroglucinol. Based on these findings, we propose that members of the gut microflora of the koala cooperate in the degradation of T-PC.     

Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains

Summary Glycerol-fermenting anaerobes were enriched with glycerol at low and high concentrations in order to obtain strains that produce 1,3-propanediol. Six isolates were selected for more detailed characterization; four of them were identified as Citrobacter freundii, one as Klebsiella oxytoca and one as K. pneumoniae. The Citrobacter strains formed 1.3-propanediol and acetate and almost no by-products, while the Klebsiella strains produced varying amounts of ethanol in addition and accordingly less 1,3-propanediol. Enterobacterial strains of the genera Enterobacter, Klebsiella, and Citrobacter from culture collections showed similar product patterns except for one group which formed limited amounts of ethanol, but no propanediol. Seven strains were grown in pH-controlled batch cultures to determine the parameters necessary to evaluate their capacity for 1,3-propanediol production. K. pneumoniae DSM 2026 exhibited the highest final concentration (61 g/l) and the best productivity (1.7 g/l h) whereas C. freundii Zu and K2 achieved only 35 g/l and 1.4 g/l h, respectively. The Citrobacter strains on the other hand gave somewhat better yields which were very close to the theoretical optimum of 65 mol %. Offprint requests to: H. Biebl     

Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces

There have been an increasing number of reports of yeast systemic infection involving Saccharomyces cerevisiae strains. The development of a rapid and reliable diagnostic tool is therefore warranted in order to explore the distribution of S. cerevisiae as an opportunistic pathogen in humans. In this study, we designed and validated five primer sets targeting the 26S rRNA gene of S. cerevisiae and the S. sensu stricto complex using 26 yeast strains. Among them, two sets of primers specifically amplified the 26S rRNA gene and the ITS region of S. cerevisiae strains, and three sets were specific for amplifying the same genes in the S. sensu stricto complex. After determining the optimal conditions of two primer pairs for quantitative real time PCR, human fecal samples were analyzed to examine the distribution of S. cerevisiae and the S. sensu stricto complex. It was possible to detect a single cell of S. cerevisiae in environmental sample. Qualitative PCR revealed that out of eleven fecal samples tested, one sample contained S. cerevisiae and four samples contained the S. sensu stricto complex. Quantitative real time PCR revealed that the target gene copy numbers of S. cerevisiae and the S. sensu stricto complex were 0.84 and 2.44 respectively, in 1 ng of DNA from the bulk fecal community.     

Phylogeography and phylogeny of the genus Acanthonyx (Decapoda,Epialtidae) in the north‐east Atlantic and Mediterranean

The genus Acanthonyx Latreille, 1828 (Majoidea, Epialtidae) contains 17 or 18 known species, depending on competing taxonomic views, that are widely distributed across the world. Morphologically, most species look superficially alike, and therefore, similar taxonomic concepts have been described under different names. Consequently, there is today a considerable list of synonyms, which further complicates the taxonomy of the genus Acanthonyx. In this study, we conducted a phylogeographical and phylogenetic analysis of populations of the genus Acanthonyx in the NE Atlantic, Mediterranean and Macaronesia using the mitochondrial cytochrome oxidase subunit I (COI) and the nuclear 28S rRNA loci. Our phylogenetic and phylogeographical results revealed that Acanthonyx lunulatus sensu lato is a complex of three distinct lineages: one corresponding to the previously described Acanthonyx brevifrons, another to A. lunulatus sensu stricto and a third to a yet undescribed group. Whereas our results confirms that A. brevifrons deserves the status of a species, as it can be easily distinguishable from A. lunulatus by a few morphological traits, we could not find any such traits suitable for the discrimination between A. lunulatus sensu stricto and the third lineage. Furthermore, the degree of COI divergence between this lineage and A. lunulatus is below average levels for Decapoda species. Yet, no shared haplotypes have been detected between them. The differences found in the nuclear gene (indels), together with the sympatric occurrence of the two forms, prompt for a more detailed analysis of this group. Overall, the results show that significant genetic differentiation between specimens with similar morphology occurs in the Epialtidae, thus reinforcing the importance of combining morphological and genetic tools to fully resolve the taxonomy of these decapods.     

Copper-resistant bacteria from industrial effluents and their role in remediation of heavy metals in wastewater

Six copper-resistant bacterial strains were isolated from wastewater of tanneries of Kasur and Rohi Nala. Two strains tolerated copper at 380 mg/L, four up to 400 mg/L. Three strains were identified as members of the genusSalmonella; one strain was identified asStreptococcus pyrogenes, one asVagococcus fluvialis and the last was identified asEscherichia coli. The pH and temperature optimum for two of them were 7.0 and 30 °C, respectively; four strains had corresponding optima at 7.5 and 37 °C, respectively. All bacterial isola-tes showed resistance against Ag⁺ (280–350 mg/L), Co²⁺ (200–420), Cr^VI (280–400), Cd²⁺ (250–350), Hg²⁺ (110–200), Mn²⁺ (300–380), Pb²⁺ (300–400), Sn²⁺ (480–520) and Zn²⁺ (300–450). Largesized plasmids (>20 kb), were detected in all of the strains. After the isolates were cured of plasmids with ethidium bromide, the efficiency of curing was estimated in the range of 60–90%. Reference strain ofE. coli was transformed with the plasmids of the bacterial isolates which grew in Luria-Bertani medium containing 100 mg/L Cu²⁺. The capability to adsorb and afterwards accumulate Cu²⁺ inside their cells was assayed by atomic absorption spectrophotometer; all bacterial cells had the ability to adsorb 50–80 % of the Cu²⁺ and accumulate 30–45 % Cu²⁺ inside them after 1 d of incubation.     

Proteomic Analysis of the Effect of Cyanide on Klebsiella oxytoca

Cyanide has been proved to be degraded by Klebsiella oxytoca. In order to examine the physiological responses of cyanide degradation by this bacterium, two-dimensional (2-DE) electrophoresis approach and MALDI–TOF–MS allow us to identify 106 proteins spots that were significantly altered in the presence of 1 mM cyanide in relative to that in 1 mM ammonia when K. oxytoca grown at the late-log phase. Among them, 27 proteins were successfully identified. These proteins were involved in carbohydrate metabolism, nucleotide metabolism, amino acid metabolism, nitrogen metabolism, stress responses, oxidation–reduction reactions, transporters, and miscellaneous function. Some proteins related with regulation of nitrogen assimilation pathways (glutamine synthetase), oxidative stress repairing (catalase), and protection (neutral trehalase and glycosyltransferase) could improve the effectiveness of cyanide biodegradation. Although the nitrogenase was suggested to participate in cyanide degradation in our previous study, this enzyme induction was not observed as expected. These findings could provide new insights into the inducible mechanisms underlying the capacity of K. oxytoca to tolerate cyanide stress.     

Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

Phage Associated Bacteriocins Reveal a Novel Mechanism for Bacteriocin Diversification in Klebsiella

Ninety-six isolates of Klebsiella pneumoniae and K. oxytoca were recovered from wild mammals in Australia. 14.6% of these bacteria produce killing phenotypes that suggest the production of bacteriocin toxins. Cloning and sequencing of the gene clusters encoding two of these killing phenotypes revealed two instances of a bacteriocin associated with a bacteriophage gene, the first such genetic organization described. The newly identified klebicin C gene cluster was discovered in both K. pneumoniae and K. oxytoca. The newly identified klebicin D gene cluster was detected in K. oxytoca. Protein sequence comparisons and phylogenetic inference suggest that klebicin C is most closely related to the rRNase group of colicins (such as colicin E4), while klebicin D is most closely related to the tRNase group of colicins (such as colicin D). The klebicin C and D gene clusters have similar genetic and regulatory organizations. In both cases, an operon structure is inferred consisting of a phage-associated open reading frame and klebicin activity and associated immunity genes. This novel bacteriophage/bacteriocin organization may provide a novel mechanism for the generation of bacteriocin diversity in Klebsiella.Reviewing Editor: Prof. David Guttman     

Azole-resistant Aspergillus fumigatus is highly prevalent in the environment of Vietnam,with marked variability by land use type

Azole-resistant environmental Aspergillus fumigatus presents a threat to public health but the extent of this threat in Southeast Asia is poorly described. We conducted environmental surveillance in the Mekong Delta region of Vietnam, collecting air and ground samples across key land-use types, and determined antifungal susceptibilities of Aspergillus section Fumigati (ASF) isolates and azole concentrations in soils. Of 119 ASF isolates, 55% were resistant (or non-wild type) to itraconazole, 65% to posaconazole and 50% to voriconazole. Azole resistance was more frequent in A. fumigatus sensu stricto isolates (95%) than other ASF species (32%). Resistant isolates and agricultural azole residues were overrepresented in samples from cultivated land. cyp51A gene sequence analysis showed 38/56 resistant A. fumigatus sensu stricto isolates carried known resistance mutations, with TR₃₄/L98H most frequent (34/38).     

Clonal Transmission of ESBL-Producing <Emphasis Type="Italic">Klebsiella</Emphasis> spp. at a University Hospital in Brazil

The present study was designed to determine the prevalence and extended-spectrum β-lactamase (ESBL) types in clinical isolates of Klebsiella spp. at a university hospital located in the Brazilian southern region (Ribeirão Preto, São Paulo) as well as their antibiotic susceptibility and genetic profiles. This study included 147 non-repeat Klebsiella spp. isolates collected from January to June 2000, of which 23 K. pneumoniae and 8 K. oxytoca were selected as ESBL producers by using the Oxoid combination disk method and Etest ESBL strip. β-lactamases were characterized by IEF, PCR and sequencing assays using primers for ESBL genes. Antibiotic susceptibility was evaluated by MicroScan system. Dissemination of two major clones of ESBL-producing Klebsiella spp. occurred in the hospital. According to the results obtained in this study there was a clonal spread of CTX-M-producing K. oxytoca in five clinics and dissemination of ESBL-producing K. pneumoniae in the nursery and pediatrics wards.     

Molecular genetic characterization of bacterial isolates causing brown blotch on cultivated mushrooms in Japan

The polymerase chain-reaction (PCR) was used to amplify 16S ribosomal DNA (16S rDNA) from bacteria, identified asPseudomonas tolaasii orP. fluorescens, causing brown blotch on cultivated mushrooms in Japan. PCR-amplified 16S rDNA was analyzed on the basis of nucleotide sequence and restriction fragment length polymorphisms (RFLP) to determine the specific identity of isolates. Banding patterns obtained through PCR using primers corresponding to repetitive extragenic palindromic sequences of enteric bacteria (REP-PCR) were used to determine the relatedness of conspecific isolates. AllP. tolaasii isolates and a mushroom pathogen identified asP. fluorescens had identical RFLP patterns and partial 16S sequences, and are considered conspecific. An isolate ofP. fluorescens from creamery wastes (IFO 3507) differed slightly from isolates ofP. tolaasii in both 16S sequence (0.8%) and RFLP patterns (d=0.08), and had almost entirely different REP-PCR bands (d=0.88–1.0). Phylogenetic analyses based on 16S sequences indicated thatP. tolaasii andP. fluorescens are close members ofPseudomonas sensu stricto. REP-PCR shows promise in characterizing isolates pathogenic on different mushroom crops. Two isolates ofP. tolaasii pathogenic onPleurotus ostreatus had identical banding patterns, but three isolates fromLentinula edodes showed the greatest diversity. Contribution No. 312 of the Tottori Mycological Institute, Totori, Japan.     

Isolation and characterization of novel denitrifying alkalithermophiles, AT-1 and AT-2

Two novel denitrifying alkalithermophilic bacteria, AT-1 and AT-2, were isolated from manure-amended soil. The isolates grew at 35–65°C with an optimum temperature at 50–60°C, and pH 6.5–10.0 with an optimum pH at 9.5. Both isolates were Gram-positive, facultative anaerobic, non-motile rod-shaped bacteria. A phylogenetic analysis based on 16S rRNA sequence data indicated that both AT-1 and AT-2 are members of the genus Anoxybacillus. DNA-DNA hybridization revealed moderate relatedness between AT-1 and AT-2 and one phylogenetically related strain, A. pushchinensis K1 (69.5 and 69.1%, respectively). Comparative analysis of morphology and biochemical characteristics of the two isolates also showed similarity to A. pushchinensis K1. Based on these results, we identified AT-1 and AT-2 as A. pushchinensis. To our knowledge, this is the first report of denitrifying bacterium isolated from alkalithermophilic Anoxybacillus spp.     

A new cryptic species of <Emphasis Type="Italic">Aponurus</Emphasis> Looss, 1907 (Digenea: Lecithasteridae) from Mediterranean goatfish (Teleostei: Mullidae)

The status of the trematode Aponurus laguncula Looss, 1907 in the western Mediterranean is re-assessed by means of a comparative morphological study and rDNA sequences based on newly collected material. A. laguncula (sensu stricto) is redescribed from Trachinus draco L. and a new cryptic species of the ‘A. laguncula complex’, Aponurus mulli n. sp., is described on the basis of abundant material from Mullus barbatus L. (type-host) and M. surmuletus L. off the Spanish Mediterranean coasts. The new species is differentiated from A. laguncula (sensu stricto) by its: significantly larger, elongate body, with maximum width at the level of the ventral sucker; shorter forebody; distinctly larger sinus-sac, seminal receptacle and seminal vesicle, with the latter also being more elongate; vesicular pars prostatica; more anteriorly located vitellarium, which consists of eight globular follicles; and distinctly smaller eggs, which are also smaller in relation to body size and have both their opercular and anopercular poles rounded. The variability and the allometric growth of the morphological characters in the new species were studied in detail, resulting in additional distinguishing features. Sequences of the large subunit rRNA (28S) gene (domains D1–D3) and ITS2 rRNA gene region for the new species have been submitted to GenBank in order to enhance future studies on species differentiation within the ‘A. laguncula complex’.     

Antibiotic Resistance and Its Transfer Among Clinical and Nonclinical Klebsiella Strains in Botanical Environments

A total of 183 isolates of Klebsiella from drinking water, market vegetables, wood, sawdust, industrial effluents, and human and animal origin were examined for susceptibility to 10 antibacterial agents. Incidence of resistance to two or more antibiotics tested was: 65% of the human clinical isolates, 59% among bovine mastitis, and 24% among the nonclinical isolates. The five different multiple resistance patterns among nonclinically derived Klebsiella were also found among the human and bovine mastitis isolates. Statistical analyses revealed that patterns of resistance among Klebsiella isolates from drinking water, market vegetables, and industrial effluents were highly correlated with each other and with resistance patterns of human clinical isolates. Antibiotic resistance was transferred between Klebsiella growing in two habitat-simulated environments (growing radish plants and aqueous sawdust suspensions). Transconjugants were detected in 5 of 21 and 6 of 21 mating pairs, respectively. Average transconjugants/donor ranged from 10⁻³ to 10⁻⁶ in Penassay broth, from 10⁻⁶ to 10⁻⁷ on radish plants, and from 10⁻⁵ to 10⁻⁸ in sawdust suspensions. Although antibiotic resistance transfer under simulated environmental conditions can occur, regrowth of clinical strains is probably the major cause for the widespread occurrence of antibiotic-resistant Klebsiella in the nonclinical environment.