A real-time PCR assay with improved specificity for detection and discrimination of all clinically relevant Bordetella species by the presence and distribution of three Insertion Sequence elements

<p>Abstract</p> <p>Background</p> <p>In Dutch laboratories molecular detection of <it>B. pertussis </it>and <it>B. parapertussis </it>is commonly based on insertion sequences <it>IS</it>481 and <it>IS</it>1001, respectively. Both IS elements are more widely spread among <it>Bordetella </it>species. Both <it>Bordetella </it><it>holmesii</it>, and <it>B. bronchiseptica </it>can harbour <it>IS</it>481. Also, <it>IS</it>1001 is found among <it>B. bronchiseptica</it>. <it>IS</it>481, and <it>IS</it>1001 based PCR thus lacks specificity when used for detection of specific <it>Bordetella spp</it>.</p> <p>Findings</p> <p>We designed a PCR based on <it>IS</it>1002, another IS element that is present among <it>Bordetella </it>species, and exploited it as a template in combination with PCR for <it>IS</it>481, and <it>IS</it>1001. In combining the PCRs for <it>IS</it>481, <it>IS</it>1001, and <it>IS</it>1002, and including an inhibition control, we were able to detect and discriminate all clinically relevant <it>Bordetella </it>species.</p> <p>Conclusions</p> <p>We developed an improved PCR method for specific detection of <it>B. pertussis</it>, <it>B. parapertussis, B. holmesii, and B. bronchiseptica.</it></p

Review of a major epidemic of methicillin-resistant Staphylococcus aureus: The costs of screening and consequences of outbreak management

Background: A major outbreak of methicillin-resistant Staphylococcus aureus (MRSA) occurred in locations C and Z of our hospital and lasted for several years. It affected 1,230 patients and 153 personnel. Methods: Outbreak management was installed according to the Dutch "search and destroy" policy. A rapid, high-throughput method for molecular screening of potential MRSA carriers was implemented. Outbreak isolates were retrospectively genotyped by pulsed field gel electrophoresis. Costs of molecular screening were compared with screening by culture. Results: Genotyping results revealed 4 distinct epidemic MRSA clones. Three were present in hospital C. Because of a merger of hospitals, these clones spread to hospital Z. Another clone of MRSA affected other health care-related institutions in the region. Because of the implementation of strict containment measures of the "search and destroy" policy, the annual number of tests decreased from 100,000 to 18,000. The disposables and reagents used in polymerase chain reaction technology are more expensive than those of conventional methods. However, the clinical and economic benefits of fast results in regard to expenses of the hospital clearly outweigh the higher costs of screening. Conclusion: The implementation of a rapid, high-throughput molecular screening system greatly contributed to the effectiveness of strict containment measures of the "search and destroy" policy. The major epidemic clones of MRSA in the outbreak were eradicated by this strategy

Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC

Background: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC.Methods: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates.Results: Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons.Conclusions: In this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures

Spread of carbapenem resistance by transposition and conjugation among Pseudomonas aeruginosa

The emergence of carbapenem-resistant Pseudomonas aeruginosa represents a worldwide problem. To understand the carbapenem-resistance mechanisms and their spreading among P. aeruginosa strains, whole genome sequences were determined of two extensively drug-resistant strains that are endemic in Dutch hospitals. Strain Carb01 63 is of O-antigen serotype O12 and of sequence type ST111, whilst S04 90 is a serotype O11 strain of ST446. Both strains carry a gene for metallo-β-lactamase VIM-2 flanked by two aacA29 genes encoding aminoglycoside acetyltransferases on a class 1 integron. The integron is located on the chromosome in strain Carb01 63 and on a plasmid in strain S04 90. The backbone of the 159-kb plasmid, designated pS04 90, is similar to a previously described plasmid, pND6-2, from Pseudomonas putida. Analysis of the context of the integron showed that it is present in both strains on a ~30-kb mosaic DNA segment composed of four different transposons that can presumably act together as a novel, active, composite transposon. Apart from the presence of a 1237-bp insertion sequence element in the composite transposon on pS04 90, these transposons show > 99% sequence identity indicating that transposition between plasmid and chromosome could have occurred only very recently. The pS04 90 plasmid could be transferred by conjugation to a susceptible P. aeruginosa strain. A second class 1 integron containing a gene for a CARB-2 β-lactamase flanked by an aacA4'-8 and an aadA2 gene, encoding an aminoglycoside acetyltransferase and adenylyltransferase, respectively, was present only in strain Carb01 63. This integron is located also on a composite transposon that is inserted in an integrative and conjugative element on the chromosome. Additionally, this strain contains a frameshift mutation in the oprD gene encoding a porin involved in the transport of carbapenems across the outer membrane. Together, the results demonstrate that integron-encoded carbapenem and carbapenicillin resistance can easily be disseminated by transposition and conjugation among Pseudomonas aeruginosa strains

Within-host and population transmission of blaOXA-48 in K. pneumoniae and E. coli

During a large hospital outbreak of OXA-48 producing bacteria, most K. pneumoniaeOXA-48 isolates were phenotypically resistant to meropenem or imipenem, whereas most E. coliOXA-48 isolates were phenotypically susceptible to these antibiotics. In the absence of molecular gene-detection E. coliOXA-48 could remain undetected, facilitating cross-transmission and horizontal gene transfer of blaOXA-48. Based on 868 longitudinal molecular microbiological screening results from patients carrying K. pneumoniaeOXA-48 (n = 24), E. coliOXA-48 (n = 17), or both (n = 40) and mathematical modelling we determined mean durations of colonisation (278 and 225 days for K. pneumoniaeOXA-48 and E. coliOXA-48, respectively), and horizontal gene transfer rates (0.0091/day from K. pneumoniae to E. coli and 0.0015/day vice versa). Based on these findings the maximum effect of horizontal gene transfer of blaOXA-48 originating from E. coliOXA-48 on the basic reproduction number (R0) is 1.9%, and it is, therefore, unlikely that phenotypically susceptible E. coliOXA-48 will contribute significantly to the spread of blaOXA-48. Copyright

Improved CARMA Locality Estimation Model for Peer List Reordering and Its Experimental Validation

The improvement of CARMA network model by addition of locality flavor as well as IPv6 support are concerned. The direct experimental evidence of the improvement of the efficiency of the peer-to-peer networks in terms of the network throughput using previously proposed CARMA locality awareness methods is established. The possible influences of the dynamics of the number of seeding and peering nodes in a swarm (including those directly connected to the test rig) are analyzed and taken into account. Main results indicate average 2,5 % improvement in transfer speed in comparison with clean unbiased transfer modes.Розглянуто вдосконалену реалізацію моделі CARMA, до якої додано новий клас локальності та обґрунтовано включення підтримки новітнього протоколу IPv6; також отримано пряме експериментальне підтвердження підвищення ефективності однорангових мереж із використанням попередньої версії оцінки локальності за моделлю CARMA. Проаналізовано та враховано вплив динаміки кількості завершених і незавершених вузлів у рої (включаючи ті, що прямо зв’язані з експериментальним стендом). Результати експериментів свідчать про підвищення середньої швидкості передачі на 2,5 % порівняно з немодифікованим режимом передачі.Рассмотрена усовершенствованная реализация модели CARMA, в которую добавлен новый класс локальности и обосновано включение поддержки новейшего протокола IPv6; также получено прямое экспериментальное подтверждение повышения эффективности одноранговых сетей с использованием предварительной версии оценки локальности по модели CARMA. Проанализировано и учтено влияние динамики количества завершенных и незавершенных узлов в рое (включая те, которые прямо связаны с экспериментальным стендом). Результаты экспериментов свидетельствуют о повышении средней скорости передачи на 2,5 % в сравнении с немодифицированным режимом передачи

Detection of novel chromosome-SCCmec variants in Methicillin Resistant Staphylococcus aureus and their inclusion in PCR based screening

Findings. To facilitate automation, a novel DNA extraction method for MRSA was adopted. The MRSA specific chromosome-SCCmec PCR was adapted, additional primers were added, and the performance was validated. From various laboratories in The Netherlands we received a total of 86 MRSA clinical isolates, that were negative in commercially available tests. We identified 14 MRSA strains with new variant chromosome-SCCmec junctions by sequence analysis. These MRSA strains appeared to carry SCCmec sequences with a high degree of homology to SCC regions of S. epidermidis and S. haemolyticus. All were included for detection in chromosome-SCCmec based PCR. Background: Efficient management of Methicillin Resistant Staphylococcus aureus (MRSA) in the hospital is needed to prevent dissemination. It is important that MRSA can be rapidly identified, and effective infection control measures can be initiated. Equally important is a rapid MRSA negative report, especially for patients in isolation. For negative screening we implemented fully automated high through-put molecular screening for MRSA. Conclusions: Fourteen variant chromosome-SCCmec junctions in MRSA, that are not detected in commercially available MRSA detection kits were added to our PCR to detect all currently known variant SCC-mec types of MRSA

Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences.

A total of 188 Bordetella strains were characterized by the electrophoretic mobilities of 15 metabolic enzymes and the distribution and variation in positions and copy numbers of three insertion sequences (IS). The presence or absence of IS elements within certain lineages was congruent with estimates of overall genetic relationships as revealed by multilocus enzyme electrophoresis. Bordetella pertussis and ovine B. parapertussis each formed separate clusters, while human B. parapertussis was most closely related to IS1001-containing B. bronchiseptica isolates. The results of the analysis provide support for the hypothesis that the population structure of Bordetella is predominantly clonal, with relatively little effective horizontal gene flow. Only a few examples of putative recombinational exchange of an IS element were detected. Based on the results of this study, we tried to reconstruct the evolutionary history of different host-adapted lineages

平成25年度KALCSプログラム事業の概要

BACKGROUND:The objective of our study was the development of a semi-quantitative real-time PCR to detect uropathogens. Two multiplex PCR reactions were designed to detect Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus mirabilis, Enterococcus faecalis, and Pseudomonas aeruginosa. 16S based PCR was performed in parallel to detect Gram-positive and Gram-negative bacteria. Firstly to identify non-targeted agents of infection in the same urine specimen, and secondly to quantify background flora. The method was evaluated in comparison with standard bacterial culture, and a commercial PCR kit for detection of uropathogens. FINDINGS:Analysis with a known panel of 116 clinical isolates yielded a PCR specificity of 100%. Analysis of urine specimens from 211 patients revealed a high correlation of PCR Cq values with both culture positivity and quantity. Concordance between PCR and culture was 98% when both methods yielded results. PCR was found to be more sensitive than culture. With a cut-off Cq value of 33, the negative predictive value of PCR was 94%. The 16S PCR confirmed most results. One specimen was positive by 16S PCR suggesting another cause of infection not detected by the specific PCR assays. CONCLUSION:We conclude that it is feasible to detect and identify uropathogens by multiplex real-time PCR assay