Analysis of genes encoding D-alanine:D-alanine ligase-related enzymes in Leuconostoc mesenteroides and Lactobacillus spp.

Degenerate oligodeoxyribonucleotides complementary to sequences encoding conserved amino acid (aa) motifs in D-alanine:D-alanine ligases (Ddl) were used to amplify approx. 600-bp fragments from glycopeptide-resistant strains of Leuconostoc mesenteroides (Lm), Lactobacillus plantarum, La. salivarius and La. confusus, and from a susceptible strain of La. leichmannii. Comparison of the deduced aa sequences of the PCR products revealed that the Ddl-related enzymes of resistant Lm and Lactobacillus spp. are more akin to each other (47-63% aa identity) than to that of susceptible La. leichmannii (33-37% aa identity), indicating that the Ddl-related enzymes in these intrinsically resistant species of Gram+ bacteria exhibit structural differences with those in susceptible species. The Ddl-related enzymes, VanA and VanB, implicated in acquired resistance to glycopeptides in enterococci, were not closely related to their counterparts in Lm and Lactobacillus spp., as they displayed only 26-32% aa identity.

Identification and characterization of an aadB gene cassette at a secondary site in a plasmid from Acinetobacter.

Transformation studies showed that an aminoglycoside resistance gene, aadB, is carried on a 6.0-kb plasmid (pRAY) in a clinical isolate of Acinetobacter (strain SUN). The gene was cloned and sequenced. An analysis of the DNA sequencing data showed that although the aadB gene is part of cassette, it is not associated with an integron. Rather, the aadB cassette has recombined at a secondary site downstream of putative promoters on pRAY.

Genetic heterogeneity of Campylobacter concisus determined by pulsed field gel electrophoresis-based macrorestriction profiling.

In order to investigate the genetic diversity of Campylobacter concisus to assist molecular typing studies, the use of macrorestriction profiling was examined. A suitable protocol was developed that included the use of formaldehyde pretreatment to prevent DNA degradation, and restriction enzyme NotI for pulsed field gel electrophoresis-based genotyping. Subsequently, 53 strains of C. concisus, principally from cases of diarrhoea in children, were examined. Fifty-one distinct patterns were obtained, indicating the high discriminatory potential of the method. Patterns comprised between one and 14 restriction fragments, with type and reference strains of two well-defined genomospecies of oral and faecal origin containing six and 12 fragments respectively. Our results show that C. concisus is genetically diverse and suggest the species as currently defined to be a taxonomic continuum comprised of several genomospecies. The pulsed field gel electrophoresis typing method described here has considerable potential for molecular epidemiological studies of C. concisus and may be a useful adjunctive method for helping to resolve key taxonomic issues for this species.

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus.

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.

Aminoglycoside-O-phosphotransferase (APH (3'')) activity in a clinical isolate of Acinetobacter calcoaceticus.

An isolate of Acinetobacter calcoaceticus var. anitratus containing an aminoglycoside-aminocyclitol antibiotic-modifying enzyme, which phosphorylates streptomycin at the 3" position, is described. The enzyme, APH(3"), was identified on the basis of its substrate specificity; in particular, its ability to phosphorylate streptomycin but not streptidine.

High level kanamycin resistance associated with the hyperproduction of AAC(3)II and a generalised reduction in the accumulation of aminoglycosides in Acinetobacter spp.

The clinical isolate of Acinetobacter baumannii strain SAK contains an actively transcribed aacC2 gene and a latent aadB gene that encode the aminoglycoside-modifying enzymes, AAC(3)II and AAD(2"), respectively. In an attempt to activate the aadB gene, the strain was cultured in the presence of kanamycin which is a substrate for AAD(2"). Although it was possible to isolate kanamycin resistant derivatives these were not associated with detectable AAD(2") activity. Instead, there was a marked increase in the level of AAC(3)II activity which was associated with amplification of the aacC2 gene.

Characterization of the Acinetobacter plasmid, pRAY, and the identification of regulatory sequences upstream of an aadB gene cassette on this plasmid.

Primer extension analyses carried out to identify the transcription start site of an aadB gene, which is part of a gene cassette recombined at a secondary site on an Acinetobacter plasmid, pRAY, suggest that transcription control signals in Acinetobacter are similar but not identical to their counterparts in Escherichia coli. pRAY was sequenced. An AT-rich region, containing eight copies of the consensus sequence, AAAAAATAT, previously shown to be present in the origins of replication of other Acinetobacter plasmids, was predicted to be the origin of pRAY. The translation product of one of the 10 open reading frames identified on pRAY shows homology to the mobilization protein, MbeA.

Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli.

DNA sequencing data showed that five clinical isolates of Escherichia coli with reduced susceptibility to ceftazidime, ceftriaxone, and cefotaxime contain an ampC gene that is preceded by a strong promoter. Transcription from the strong promoter was 8- to 18-fold higher than that from the promoter from a susceptible isolate. RNA studies showed that mRNA stability does not play a role in the control of AmpC synthesis.

Identification of an Acinetobacter baumannii gene region with sequence and organizational similarity to Tn2670.

A chloramphenicol resistance gene was cloned from chromosomal DNA prepared from a clinical Acinetobacter baumannii isolate. Sequence analysis of this gene (cat) and the flanking DNA regions shows that this gene is linked to Tn21 and to IS1 in a manner similar to that found in Tn2670.

Molecular identification of Campylobacter concisus.

A 1.6-kb DNA fragment isolated from a Campylobacter concisus genomic library gave C. concisus-specific restriction fragment length patterns when it was used as a probe in hybridization studies. All of the strains tested, including type strains and clinical isolates, contained a 0.5-kb HindIII fragment that hybridized to the probe. DNA sequencing of the 1.6-kb fragment identified three open reading frames (ORFs). One of the ORFs encodes the carboxy terminus of GyrB, and the translational products of ORF2 and ORF3 showed similarity to hypothetical proteins, previously identified in Campylobacter jejuni. DNA-DNA hybridization studies with a fragment internal to ORF3 showed that this sequence was responsible for the signal observed with the 0.5-kb HindIII fragment. A rapid PCR assay was developed and evaluated. Primers that annealed to the extremities of the 1.6-kb fragment were used to obtain an amplicon of the correct size from both reference and clinical strains of C. concisus.

Use of 16S rRNA sequencing for identification of Actinobacillus ureae isolated from a cerebrospinal fluid sample.

Actinobacillus ureae, previously Pasteurella ureae, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to A. ureae. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene.

Genetic basis of quinolone resistance and epidemiology of resistant and susceptible isolates of porcine Campylobacter coli strains.

AIMS: The aims of this study were to investigate the epidemiology of quinolone-resistant and -susceptible porcine isolates of Campylobacter coli and to characterize the genetic basis of quinolone resistance. METHODS AND RESULTS: Penner serotyping and flagellin gene sequence polymorphisms were used to investigate the epidemiology of the C. coli isolates. A total of 55 isolates were included, of which 30 were paired resistant and susceptible isolates from 15 pigs. Amplification of gyrA, gyrB and parC, followed by direct sequencing of amplicons was used to identify mutations in the targets of quinolones. Overall, 31 of the isolates were resistant to ciprofloxacin (minimum inhibitory concentrations (MIC), 2- >or = 32 microg x ml(-1)). Thirteen DdeI-flaA profiles were observed and resistant and susceptible strains were identified for nine profiles. The majority of resistant strains exhibited either profile 1 or 6. While profile 1 comprised susceptible and resistant strains, all of the strains with profile 6 were resistant to ciprofloxacin. The serogroup (O:24) of the profile 6 strains was identical. The only other serogroup to be uniformly associated with quinolone resistance was O:5. Strains with this phenotype comprised a number of genotypes, including profile 1. Only four of the paired isolates from individual pigs had the same profile. The genetic basis of quinolone resistance was investigated in two strains with ciprofloxacin MICs of 2 and > or = 32 miccrog x ml(-1), respectively. The amino acid substitution of isoleucine for threonine at position 86 was identified in the GyrA proteins from both strains. No mutations were identified in the GyrB proteins. CONCLUSIONS: There was an association between two of the genotypes, serotypes 5 and 24, and quinolone resistance. The association between genotype, serotype and resistance in C. coli isolates has not been reported previously. Only the mutation in GyrA associated with quinolone resistance was identified. No mutations in GyrB were identified. Amplification products of parC were not obtained and it may be that this gene is not present in some Campylobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on the distribution of ciprofloxacin resistance between subtypes of C. coli.