- Volume 56, Issue 2, 2007

Acinetobacter baumannii is a major nosocomial pathogen and frequent cause of hospital-acquired pneumonia, surgical wound infections and sepsis. As very little is known of the endotoxic potential of A. baumannii lipopolysaccharide (LPS) with respect to human cells or of its ability to stimulate inflammatory signalling via human Toll-like receptors (TLRs), the biological activity of these endotoxins was investigated in human monocytic THP-1 cells and in TLR-deficient HEK-293 cells transfected with human TLR2 and TLR4 constructs. Endotoxins derived from five clinical isolates of A. baumannii and one of Acinetobacter ‘genomospecies 9’ showed high potency, which was comparable to that of Escherichia coli strain R1 NCTC 13114 LPS, in the induction of the Limulus amoebocyte reaction and interleukin 8 and tumour necrosis factor alpha release from THP-1 cells. Whole UV-killed cells of A. baumannii and Acinetobacter ‘genomospecies 9’ stimulated both TLR2- and TLR4-dependent signalling, whereas pure endotoxins of all investigated strains induced signalling via TLR4, but not TLR2.

Escherichia coli O123 strains express a broad spectrum of phenotypes, H serotypes and virulence markers and are able to colonize and to cause disease in different hosts including humans. In this study, two subtypes of E. coli O123 antigen (group I and group II) have been identified based on their cross-reactions with other E. coli O antigens. Investigation of the relationship between O123 group I and group II strains by O serotyping and Fourier transform infrared spectroscopy of whole bacteria revealed surface structural differences between these two groups of E. coli O123 strains. Nucleotide sequence analysis of the O-antigen gene clusters of two E. coli O123 strains representing O123 group I and group II revealed no change at the amino acid level. These findings indicate that the differences in the surface structures of group I and group II strains are not related to genetic heterogeneity in their O-antigen gene clusters. A PCR assay based on O123 antigen-specific wzx and wzy genes was developed and found to be suitable for reliable detection of all subtypes of E. coli O123 strains, which bears an advantage over traditional serological detection.

Frequent outbreaks of Pichia anomala fungaemia in paediatric patients have warranted the development of a rapid identification system for this organism. This study describes a specific PCR-based method targeting the rRNA gene intergenic spacer region 1 (IGS1) for rapid identification of Pichia anomala isolates and characterization at the strain level. These methods of species identification and strain typing were used on 106 isolates of Pichia anomala (77 from a previously described outbreak and 29 isolated post-outbreak from the same hospital). Using conventional morphological and biochemical methods, 11 strains isolated during the outbreak were misidentified as P. anomala. blast analysis of sequences of internal transcribed spacer (ITS) regions of rRNA genes confirmed that they were Pichia guilliermondii (eight isolates) and Debaryomyces hansenii (three isolates). Strain typing of Pichia anomala isolates confirmed the previous finding of a point-source outbreak. The results suggest that IGS sequences and their polymorphisms could be exploited for similar typing methods in other organisms.

The occurrence and genetic variability of Candida parapsilosis isolates in two Hungarian hospitals, located in Debrecen and Pécs, were examined. Among the 209 Candida isolates examined, 20 were found to belong to C. parapsilosis sensu lato, based on morphological, physiological and molecular data. The frequency of occurrence of C. parapsilosis isolates (9.6 %) was lower than that observed in Europe but higher than that observed previously in Hungary. The genetic variability of C. parapsilosis sensu lato isolates was also examined using random amplified polymorphic DNA (RAPD) analysis and sequence analysis of the intergenic transcribed spacer (ITS) region of the rRNA gene cluster. The genetic variability of the isolates was relatively high, as revealed by RAPD analysis. Two isolates were found to belong to the recently described Candida metapsilosis species (C. parapsilosis group III), based on ITS sequence data, RAPD analysis and phenotypic data. These two isolates could also be distinguished from C. parapsilosis sensu stricto isolates using a primer pair developed for the detection of C. parapsilosis group I isolates. To the best of the authors' knowledge, this is the first report on the identification of C. metapsilosis from bloodstream infection.

The closely related Clostridium novyi and Clostridium botulinum types C and D are of current interest because of their association with serious infections in injecting drug users (C. novyi type A) and equine and feline dysautonomias (C. botulinum types C/D). The species are defined by the major toxins they produce: the α toxin of C. novyi, and the type C and D neurotoxins of C. botulinum (BoNT/C and BoNT/D). The other major toxin produced by this group, and previously thought to be restricted to the botulinum types, is the chromosomally encoded C2 – a binary toxin consisting of two components, I and II. In the current study 44 of these clostridia from the authors' culture collection were investigated – most of which had been identified previously by conventional biochemical tests as ‘C. novyi type A’. The aim was to check the distribution of toxin genes by PCR to see if the identities were consistent with the genes carried, and to ascertain if the C2 gene was only found in authentic C. botulinum strains. Several combinations of the species-defining genes and the two components of the C2 genes were detected. Only the authentic BoNT/C- and BoNT/D-positive C. botulinum strains and one of two non-neurotoxic variants of type C carried genes for both components of the C2 toxin. Of the remaining 40 C. novyi type A-like strains, the gene for the α toxin was found in 22, with 19 of these also possessing the gene for component I (16) or component II (3) but not both. In the α toxin-negative strains (22), both of the C2 genes were detected in 5 strains (3 C. botulinum), with component I in 11 strains and neither gene in 6 strains.

Bloodstream infections are life-threatening conditions which require timely initiation of appropriate antimicrobial therapy. The accuracy of direct disk diffusion susceptibility testing of positive blood cultures was investigated, including for the first time β-lactam/β-lactam-inhibitor combination antibiotics. Results of direct testing, following the guidelines of the Clinical and Laboratory Standards Institute, were compared to standard microtitre broth dilution susceptibility testing of the subcultured isolate on the Merlin MICRONAUT system. Altogether, 758 isolates and 4930 organism/antibiotic combinations from 590 patients were evaluated. With regard to Gram-positive cocci (n=532), agreement between both methods was found in 93.9 % of cases, with 1.6 % very major, 1.1 % major and 2.6 % minor errors. For Gram-negative rods (n=226), agreement was found in 91.9 % of cases, with 1.2 % very major, 0.7 % major and 6.3 % minor errors. When applying the breakpoints of the Deutsches Institut für Normung for interpretation of MICRONAUT tests, agreement of direct disk diffusion with standard testing decreased to 82.4 % in Gram-negative rods, with 3.6 % very major, 0.5 % major and 13.4 % minor errors. A high rate of disagreement was observed with oxacillin and gentamicin in Gram-positive cocci, and with cefuroxime, amoxycillin/clavulanate and piperacillin/tazobactam in Gram-negative rods. In conclusion, the limitations of direct disk diffusion testing of positive blood cultures must be kept in mind by the clinical microbiologist and should, where necessary, be communicated to the clinician to ensure adequate treatment of severely ill patients.

The egc locus of Staphylococus aureus harbours two enterotoxin genes (seg and sei) and three enterotoxin-like genes (selm, seln and selo). Between the sei and seln genes are located two pseudogenes, ψent1 and ψent2, or the selu or selu v gene. While these two alternative sei–seln intergenic regions can be distinguished by PCR, to date, DNA sequencing has been the only confirmatory option because of the very high degree of sequence similarity between egc loci bearing the pseudogenes and the selu or selu v gene. In silico restriction enzyme digestion of genomic regions encompassing the egc locus from the 3′ end of the sei gene through the 5′ first quarter of the seln gene allowed pseudogene- and selu- or selu v-bearing egc loci to be distinguished by PCR-RFLP. Experimental application of these findings demonstrated that endonuclease HindIII cleaved PCR amplimers bearing pseudogenes but not those with a selu or selu v gene, while selu- or selu v-bearing amplimers were susceptible to cleavage by endonuclease HphI, but not by endonuclease HindIII. The restriction enzyme BccI cleaved selu- or selu v-harbouring amplimers at a unique restriction site created by their signature 15 bp insertion compared with pseudogene-bearing amplimers, thereby allowing distinction of these egc loci. PCR-RFLP analysis using these restriction enzymes provides a rapid, easy to interpret alternative to DNA sequencing for verification of PCR findings on the nature of an egc locus type, and can also be used for the primary identification of the intergenic sei–seln egc locus type.

A total of 99 isolates out of 370 colonization factor (CF)-positive, well-characterized enterotoxigenic Escherichia coli (ETEC) strains belonging to 13 different CF types isolated from diarrhoeal patients admitted to the hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, were tested. The isolates were selected at random based on expression of the major CFs prevailing in Dhaka, Bangladesh, from 1996 to 1998. These isolates were characterized by O-antigenic serotyping, randomly amplified polymorphic DNA (RAPD) analysis and biochemical fingerprinting using the PhenePlate (PhP) system. The 99 ETEC isolates belonged to 10 O serogroups, the predominant ones being O6 (n=28), O115 (n=20) and O128 (n=20). Most isolates of serogroup O6 (CS1+CS3, 11/14; CS2+CS3, 5/8) belonged to the same PhP/RAPD type (H/f), whereas other isolates of serogroup O6 (n=12) belonged to different PhP/RAPD types (Si/f and F/c). Eleven serogroup O128 (CFA/I) isolates belonged to the same PhP/RAPD type (E/b), whereas the other O128 isolates formed different PhP/RAPD types. Fifteen (75 %) serogroup O115 isolates (together with fourteen isolates from serogroups O25, O114, O142 and O159) demonstrated two closely related common groups by PhP typing (A and A1) and belonged to the same PhP/RAPD type (A/a). Three major clonal groups were identified among the ETEC strains in this study, largely based on O-antigenic type, CF expression pattern and toxin profile.

Group B streptococcus (GBS) is an important aetiological agent of serious neonatal infections. A rapid and sensitive method for the detection of GBS colonization in pregnant women at delivery could make intrapartum screening for GBS possible. A real-time PCR method targeting the sip gene of GBS in pregnant women at delivery has been evaluated. The performance of the real-time PCR was compared with optimized GBS culture. Separate vaginal and rectal swabs were collected from women hospitalized at the delivery department at St Olavs Hospital, Trondheim, Norway, from January 15 through May 2005. The specimens were cultured on selective blood agar plates and in selective broth and examined by real-time PCR. Of samples from 251 women, 87 (34.7 %) were GBS positive by culture and 86 (34.3 %) were positive by PCR. Using GBS culture as the ‘gold standard’, the sensitivity of real-time PCR was 0.97 (95 % confidence interval 0.90–0.99) and specificity was 0.99 (95 % confidence interval 0.97–1.00). In two women the PCR was positive and the culture negative. Additional analysis using cylE PCR substantiates that these two women were true GBS carriers with negative GBS culture. The rate of GBS colonization was lower in vaginal specimens than in rectal specimens both by culture and PCR. The real-time PCR assay is fast, highly sensitive and specific for detecting GBS colonization in pregnant women at delivery, and has the potential for intrapartum detection of GBS colonization. Both vaginal and rectal samples are required to achieve highest possible detection rate.

The mechanisms of resistance to macrolides in 51 erythromycin-resistant clinical isolates of Streptococcus pyogenes collected from 1997 through 2003 in Seoul, Korea were evaluated. They were characterized by their antimicrobial susceptibility, phenotype (using triple-disc and induction tests), resistance genotype, emm genotyping (M typing) and phylogenetic analysis. Erythromycin resistance was observed in 23 % of isolates. Inducible phenotype was the most common (iMLS, 51 %, 26 strains), followed by the constitutive phenotype (cMLS, 31 %, 16 strains) and the M phenotype (18 %, 9 strains). Eight of twenty-six iMLS isolates exhibited the iMLS-C phenotype. The remaining 18 isolates gave small inhibition zones (<12 mm) around all three discs, and mild blunting of the spiramycin and clindamycin zones of inhibition proximal to the erythromycin disc. They showed remarkable inducibility in erythromycin and clindamycin resistance. The MIC90 of erythromycin and clindamycin rose from 8 to >128 μg ml−1 and from 0.5 to >128 μg ml−1, respectively. Their resistance characteristics did not fit into any known iMLS subtype reported so far in the literature. So, it was named as an iMLS-D, new subtype. All of these iMLS-D strains harboured the erm(B) gene, demonstrated the emm12 genotype, except one, and formed a tight cluster in a phylogenetic tree, with 89.2 to 100 % sequence homology, suggesting that they are closely related. Nine of sixteen cMLS strains had the emm28 genotype, which had been reported to be associated with multiple drug resistance.

Between January and September 2003, 39 isolates of the family Enterobacteriaceae with phenotypically positive Vitek 1 extended-spectrum beta-lactamase (ESBL) test results were collected, originating from patients of two hospitals in Saxony, Germany. Plasmid DNA was isolated and screened by PCR for the presence of genes encoding beta-lactamases of SHV, TEM and CTX-M types. To differentiate ESBL and non-ESBL among SHV and TEM genes, detailed analysis of PCR products was performed. Twenty-four strains carried SHV-2, SHV-5 or SHV-12 genes. In a further 11 strains a CTX-M gene was detected. The CTX-M genes could be affiliated to the CTX-M-1 and CTX-M-9 cluster by RFLP analysis. In the case of four Klebsiella oxytoca isolates, hyperproduction of the chromosomal beta-lactamase K1 was inferred, because genes of the above-mentioned types were not detected. The strains contained plasmid DNA between 45 and 160 kb in size. Common plasmid restriction patterns among SHV-5 producers provided evidence of horizontal spread. Twenty strains had a MIC for cefotaxime of ⩽4 mg l−1, 18 strains had the same MIC for ceftazidime, and nine strains had this MIC of >4 mg l−1 for both antibiotics. The ESBL phenotypes often coincided with ciprofloxacin or gentamicin resistance.

Cells of Mycobacterium avium strain A5 adhered to plasticized polyvinyl chloride catheter tubing and grew at low nutrient concentration, consistent with reports of catheter-associated M. avium infection. Starting with initial cell densities of 1–2×106 c.f.u. ml−1, biofilms of approximately 350 c.f.u. cm−2 formed within 24 h at room temperature. Growth rates of cells in biofilms were exponential and equal to 2.45 days doubling time. Rates were exponential for 1–2 weeks incubation and reached cell densities of 6.5×104 c.f.u. cm−2 by 4 weeks. Cells grown in catheter biofilms were significantly more resistant to clarithromycin and rifamycin than cells grown in suspension.