Emergence of the quinolone resistance-mediating gene aac(6')-Ib-cr in extended-spectrum-beta-lactamase-producing Klebsiella isolates collected in Slovenia between 2000 and 2005.

Seventy-four nonrepetitive uropathogenic fluoroquinolone-resistant or -intermediate extended-spectrum-beta-lactamase-producing Klebsiella isolates from Slovenia were screened for the presence of plasmid-mediated quinolone resistance genes. None of the known qnr genes were detected. The aac(6')-Ib-cr allele was detected on plasmids from 25 transconjugants for which the ciprofloxacin MIC was higher than for the recipient Escherichia coli strain.

Haloarchaeal communities in the crystallizers of two adriatic solar salterns.

Solar salterns operate only for short dry periods of the year in the north shore of the Adriatic Sea because of its relatively humid and cold Mediterranean climate. In a previous paper, we showed that the NaCl precipitation ponds (crystallizers) of Northern Adriatic Secovlje salterns have different haloarchaeal populations from those typically found in dry and hot climates such as Southern Spain. To check whether there is a common pattern of haloarchaeal diversity in these less extreme conditions, diversity in crystallizers of other Adriatic solar salterns in Ston, Croatia was ascertained by molecular and culture methods. In addition, the cultivation approach was used to further describe haloarchaeal diversity in both salterns. Over the period of two solar salt collection seasons, isolates related to species of the genera Haloferax, Haloarcula, and Haloterrigena were recovered from both salterns. Within the same sampling effort, relatives of the genus Halorubrum and a Natrinema-like isolate were cultivated from Slovenian Secovlje salterns while Halobacterium related isolates were obtained from the Croatian Ston salterns. Concurrent with our previous findings, a library of Croatian saltern crystallizer PCR-amplified 16S rRNA genes was dominated by sequences related to the genus Halorubrum. The microbial community structure was similar in both salterns but diversity indices showed greater values in Slovenian salterns when compared with Croatian salterns.

Sequence analysis of the plasmid pColG from the Escherichia coli strain CA46.

The complete 4715 nucleotide sequence of the plasmid pColG from the Escherichia coli strain CA46, which was originally assumed to code for colicin G activity, has been determined. Based on the nucleotide sequence homology of the 1828bp replication region, with an average G+C content of 48%, pColG was classified as a ColE1-like plasmid. Computer assisted analysis of the remaining 2887bp nucleotide sequence with an average G+C content of 34% revealed three putative OFRs. To find out whether one or all of the three ORFs code for a possible bacteriocin, a DNA fragment encompassing these ORFs has been cloned and the recombinant colonies tested for bacteriocin production. None of the colonies had an inhibitory activity against E. coli strains DH5, HB101 and MC4100. The assumption that the plasmid pColG from the E. coli strain CA46 codes for a bacteriocin thus could not be confirmed.

Diversity of halophilic archaea in the crystallizers of an Adriatic solar saltern.

Haloarchaeal diversity in the crystallizers of Adriatic Secovlje salterns was investigated using gene fragments encoding 16S rRNA and bacteriorhodopsin as molecular markers. Screening of 180 clones from five gene libraries constructed for each gene targeted revealed 15 different 16S rRNA and 10 different bacteriorhodopsin phylotypes, indicating higher haloarchaeal diversity than previously reported in such hypersaline environments. Furthermore, results of rarefaction analysis indicated that analysis of an increasing number of clones would have revealed additional diversity. Finally, most sequences from the crystallizers grouped within the Halorubrum branch, whereas square-shaped 'Haloquadratum' relatives, repeatedly reported to dominate crystallizer communities, were rare. Presence of such special and diverse haloarchaeal community could be attributed to the Secovlje salterns rare continuous short-cycling salt production mechanism.

Distribution of Clostridium difficile variant toxinotypes and strains with binary toxin genes among clinical isolates in an American hospital.

Genetic variants of Clostridium difficile have been reported with increasing frequency, but their true incidence is unknown. C. difficile strains have been classified into variant toxinotypes according to variations in the pathogenicity locus encoding the major virulence factors, toxins A and B. Some strains produce an additional toxin, binary toxin CDT. This survey of clinical isolates (153) from patients in a single hospital set out to ascertain the distribution of variant toxinotypes and strains possessing binary toxin genes. A PCR-RFLP-based method of toxinotyping identified 123 (80.4 %) isolates as toxinotype 0, 13 (8.5 %) strains as non-toxigenic and 17 (11.1 %) as belonging to variant toxinotypes. Binary toxin genes were amplified by PCR in nine strains (5.8 %), all of which were variant toxinotypes. Toxin variants of C. difficile are pathogenic and commonly isolated and need to be considered when evaluating new diagnostic testing strategies for C. difficile disease.

Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins.

Pathogenic strains of Clostridium difficile commonly produce two large clostridial toxins (LCTs), A and B, virulence factors responsible for C. difficile disease. Some strains have been reported to produce an additional toxin, a binary toxin designated CDT. Binary toxin has cytotoxic effects on cells in culture, but its role in human disease is not yet defined. In this study we examined the frequency of binary toxin genes (cdtB and cdtA) among C. difficile isolates that do not produce LCTs (A(-) B(-)) from a large United States-based collection organized by restriction endonuclease analysis (REA) typing. Of 58 strains tested, 9 (15.5%) were cdtB and cdtA positive, including 4 of 46 (8.7%) non-LCT-producing REA groups, with an estimated prevalence of at least 2% of all non-LCT-producing isolates within the collection. Five of the binary toxin-positive strains belonged to toxinotype XI, which does not produce LCTs but has minor parts of the LCT coding region or pathogenicity locus (PaLoc). We describe two new binary toxin-positive variants, one without any remnant of the LCT genes. This previously unknown variation was found in three isolates that were unrelated by REA typing. LCT-negative, binary toxin-positive strains were isolated from symptomatic and asymptomatic patients and from the hospital environment.

New types of toxin A-negative, toxin B-positive strains among Clostridium difficile isolates from Asia.

A total of 56 C. difficile strains were selected from 310 isolates obtained from different hospitals in Japan and Korea and from healthy infants from Indonesia. Strains that had been previously typed by pulsed-field gel electrophoresis and PCR ribotyping, were characterized by toxinotyping and binary toxin gene detection. When toxinotyped, 35 strains were determined to be toxinotype 0, whereas 21 strains showed variations in toxin genes and could be grouped into 11 variant toxinotypes. Six of the toxinotypes had been described before (I, III, IV, VIII, IX, and XII). In addition, five new toxinotypes were defined (XVI to XX). Three of the new toxinotypes (XVIII, XIX, and XX) vary only in repetitive regions of tcdA and produce both toxins. In two strains from toxinotypes XVI and XVII, the production of TcdA could not be detected with commercial immunological kits. Strain J9965 (toxinotype XVII) was in PaLoc similar but not identical to another known A(-)B(+) strain, C. difficile 8864. Strain SUC 36 (toxinotype XVI), on the other hand, was similar to well-defined group consisting of toxinotypes V, VI, and VII, which thus far includes only A(+)B(+) strains. Toxinotypes XVI and XVII represent two new groups of A(-)B(+) strains. Strains of the well-known A(-)B(+) group from toxinotype VIII have a nonsense mutation at the beginning of tcdA gene, and the introduction of a stop codon at amino acid position 47 results in nonproduction of TcdA. The 5'-end sequence of tcdA in two newly described A(-)B(+) strains does not contain an identical mutation. The prevalence of variant C. difficile strains varied greatly among nine hospitals. Only five strains from four different hospitals were positive in PCR for amplification of the binary toxin gene.

Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes.

Toxinotyping and PCR ribotyping are two methods that have been used to type Clostridium difficile isolates. Toxinotyping is based on PCR-RFLP analysis of a 19 kb region encompassing the C. difficile pathogenicity locus. PCR ribotyping is based on comparison of patterns of PCR products of the 16S-23S rRNA intergenic spacer region. Representative strains (101) from a C. difficile PCR ribotype library and 22 strains from previously described toxinotypes were analysed to compare ribotyping with toxinotyping. Within this panel of strains all 11 toxinotypes (0-X) described previously and an additional 5 novel toxinotypes (XI-XV) were observed. PCR ribotyping and toxinotyping correlated well and usually all strains within a given ribotype had similar changes in toxin genes. The new toxinotype XI comprises strains that did not express toxins TcdA or TcdB at detectable levels, but contained part of the tcdA gene. Strains of toxinotype XII exhibit changes only in the 5' end of the tcdB gene. Toxinotype XIV is composed of strains that have a large insertion at the beginning of the tcdA gene. A total of 25 of the 89 tested PCR ribotypes of C. difficile contained variant strains. It was estimated that they represent 7.7% of the total number of strains in the Anaerobe Reference Unit collection.