Rapid molecular characterization of Acinetobacter baumannii clones with rep-PCR and evaluation of carbapenemase genes by new multiplex PCR in Hospital District of Helsinki and Uusimaa.

Multidrug-resistant Acinetobacter baumannii (MDRAB) is an increasing problem worldwide. Prevalence of carbapenem resistance in Acinetobacter spp. due to acquired carbapenemase genes is not known in Finland. The purpose of this study was to examine prevalence and clonal spread of multiresistant A. baumannii group species, and their carbapenemase genes. A total of 55 Acinetobacter isolates were evaluated with repetitive PCR (DiversiLab) to analyse clonality of isolates, in conjunction with antimicrobial susceptibility profile for ampicillin/sulbactam, colistin, imipenem, meropenem, rifampicin and tigecycline. In addition, a new real-time PCR assay, detecting most clinically important carbapenemase genes just in two multiplex reactions, was developed. The assay detects genes for KPC, VIM, IMP, GES-1/-10, OXA-48, NDM, GIM-1, SPM-1, IMI/NMC-A, SME, CMY-10, SFC-1, SIM-1, OXA-23-like, OXA-24/40-like, OXA-58 and ISAbaI-OXA-51-like junction, and allows confident detection of isolates harbouring acquired carbapenemase genes. There was a time-dependent, clonal spread of multiresistant A. baumannii strongly correlating with carbapenamase gene profile, at least in this geographically restricted study material. The new carbapenemase screening assay was able to detect all the genes correctly suggesting it might be suitable for epidemiologic screening purposes in clinical laboratories.

An outbreak of CTX-M-15 -producing Escherichia coli, Enterobacter cloacae, and Klebsiella in a children's hospital in Finland.

Four different extended-spectrum ß -lactamase (ESBL)-producing bacteria from a pediatric surgery ward were studied. The presence of TEM-, SHV-, and CTX-M-type ß -lactamases was analyzed and the relatedness of the isolates studied with a repetitive PCR system (DiversiLab) and pulsed-fi eld gel electrophoresis (PFGE). Molecular analysis showed that a clonal dissemination of CTX-M-15-producing Escherichia coli and Enterobacter cloacae had occurred.

Staphylococcal Ecb protein and host complement regulator factor H enhance functions of each other in bacterial immune evasion.

Staphylococcus aureus is a major human pathogen causing more than a tenth of all septicemia cases and often superficial and deep infections in various tissues. One of the immune evasion strategies of S. aureus is to secrete proteins that bind to the central complement opsonin C3b. One of these, extracellular complement binding protein (Ecb), is known to interfere directly with functions of C3b. Because C3b is also the target of the physiological plasma complement regulator, factor H (FH), we studied the effect of Ecb on the complement regulatory functions of FH. We show that Ecb enhances acquisition of FH from serum onto staphylococcal surfaces. Ecb and FH enhance mutual binding to C3b and also the function of each other in downregulating complement activation. Both Ecb and the C-terminal domains 19-20 of FH bind to the C3d part of C3b. We show that the mutual enhancing effect of Ecb and FH on binding to C3b depends on binding of the FH domain 19 to the C3d part of C3b next to the binding site of Ecb on C3d. Our results show that Ecb, FH, and C3b form a tripartite complex. Upon exposure of serum-sensitive Haemophilus influenzae to human serum, Ecb protected the bacteria, and this effect was enhanced by the addition of the C-terminal domains 19-20 of FH. This finding indicates that the tripartite complex formation could give additional protection to bacteria and that S. aureus is thereby able to use host FH and bacterial Ecb in a concerted action to eliminate C3b at the site of infection.

Applicability of DiversiLab repetitive sequence-based PCR method in epidemiological typing of enterohemorrhagic Escherichia coli (EHEC).

Enterohemorrhagic Escherichia coli (EHEC) causes diarrhea, often with severe complications. Rapid and discriminatory typing of EHEC using advanced molecular methods is needed for determination of the genetic relatedness of clones responsible for foodborne outbreaks and for finding out the transmission sources of the outbreaks. This study evaluated the potential of DiversiLab, a semiautomated repetitive sequence-based polymerase chain reaction method for the genotyping of EHEC strains. A set of 52 EHEC strains belonging to 15 O:H serotypes was clustered into 10 DiversiLab groups. All of the O157 strains and one O55 strain were classified into the same group based on a 90% similarity threshold. The other serotypes were classified to their own DiversiLab group, with the exception of one R:H(-) strain that was grouped with O5:H(-) strains. In addition, O26 and O111 strains were grouped together but ultimately subdivided according to their O-serotypes based on a 95% similarity threshold. The O104 strain, which was associated with a major outbreak of hemolytic uremic syndrome in Germany in May 2011, was also classified independently. The DiversiLab performed well in identifying isolates, but the discriminatory power of the repetitive sequence-based polymerase chain reaction method was lower than that of pulsed-field gel electrophoresis. Analysis of 15 enteropathogenic E. coli (EPEC) strains revealed that some EPEC strains clustered together with EHEC strains. Therefore, the DiversiLab system cannot be used to discriminate between these pathogroups. In conclusion, DiversiLab is a rapid and easy system for the primary exclusion of unrelated EHEC strains based on their serotypes, but more discriminatory methods, such as pulsed-field gel electrophoresis, are needed for accurate typing of the EHEC strains.

Clinical isolates of Pseudomonas aeruginosa from superficial skin infections have different physiological patterns.

Pseudomonas aeruginosa are known to have a wide physiological potential allowing them to constantly populate diverse environments leading to severe infections of humans such as septicemia, leg ulcers, and burn wounds. We set out to probe physiological characteristics of P. aeruginosa isolates from diabetic leg ulcers collected from Helsinki metropolitan area. A total of 61 clinical isolates were obtained. Detailed phenotypic (physiological) characteristics [outer membrane (OM) permeability, membrane voltage, and activity of multidrug resistance pumps] were determined in several growth phases leading to the division of the analyzed set of P. aeruginosa strains into five distinct clusters including cells with similar physiological properties. In addition, their antibiotic resistance patterns and genetic heterogeneity were determined. Multiple isolates from the same patient were genetically very closely related and belonged to the same phenotypic cluster. However, genetically close isolates from different patients expressed very different phenotypic properties. The characteristics of infected patients seem to determine the growth environments for microorganisms that adapt by changing their physiological and/or genetic properties.

A selective broth enrichment combined with real-time nuc-mecA-PCR in the exclusion of MRSA.

We analyzed the performance of a selective enrichment broth combined with Taqman-based real-time duplex nuc-mecA-PCR to expedite the screening of methicillin-resistant Staphylococcus aureus (MRSA). We found the broth to be able to select MRSA strains (oxacillin MIC range 4-256 microg/ml) from MSSA strains. A total of 31 MRSA strains were found from 1250 clinical samples screened. The nuc-mecA-PCR was positive from all enrichment broths containing MRSA. From the remaining 1219 samples negative for MRSA on culture/subculture, 138 samples were nuc+/mecA+ in PCR. The sensitivity of the test was 93.5%, specificity 88.6%, positive predictive value 17.3%, and negative predictive value 99.8% as compared to culture. Thus, with this method, the negative MRSA results can be reliably reported within 24-48 h from sampling. The method is a practical additional alternative to those already described for the same purpose.

Detection of virulence genes of Clostridium difficile by multiplex PCR.

Clostridium difficile strains belonging to the PCR ribotype 027, pulse-field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027.

Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

Evaluation of phenotypic and molecular methods for screening and detection of methicillin-resistant Staphylococcus aureus.

An in-house PCR was compared with the GenoType MRSA and the MRSA EVIGENE tests, both of which corresponded 100% with the results of in-house PCR. The cefoxitin disk diffusion method was superior to both the oxacillin disk diffusion and minimum inhibitory concentration tests for predicting the mecA status.