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

Tapping the nucleotide pool of the host: novel nucleotide carrier proteins of Protochlamydia amoebophila

Protochlamydia amoebophila UWE25 is related to the Chlamydiaceae comprising major pathogens of humans, but thrives as obligate intracellular symbiont in the protozoan host Acanthamoeba sp. The genome of P. amoebophila encodes five paralogous carrier proteins belonging to the nucleotide transporter (NTT) family. Here we report on three P. amoebophila NTT isoforms, PamNTT2, PamNTT3 and PamNTT5, which possess several conserved amino acid residues known to be critical for nucleotide transport. We demonstrated that these carrier proteins are able to transport nucleotides, although substrate specificities and mode of transport differ in an unexpected manner and are unique among known NTTs. PamNTT2 is a counter exchange transporter exhibiting submillimolar apparent affinities for all four RNA nucleotides, PamNTT3 catalyses an unidirectional proton-coupled transport confined to UTP, whereas PamNTT5 mediates a proton-energized GTP and ATP import. All NTT genes of P. amoebophila are transcribed during intracellular multiplication in acanthamoebae. The biochemical characterization of all five NTT proteins from P. amoebophila in this and previous studies uncovered that these metabolically impaired bacteria are intimately connected with their host cell’s metabolism in a surprisingly complex manner

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

An enzyme immuno assay (EIA) for the detection of IgM antibodies to Coxiella burnetii

Naar aanleiding van het voorkomen van Q-koorts pneumonie bij Nederlandse militairen in Libanon werd besloten een sero- epidemiologisch onderzoek op te zetten om de incidentie van Q-koorts bij Nederlandse militairen in Libanon te bepalen. Na proefstudies met complementbindingsreactie en immunofluorescentie bleek de laatste de meest betrouwbare serologische techniek. Daar deze zeer bewerkelijk is werd besloten een enzyme immuno assay (EIA) te ontwikkelen. Beschreven worden het bereiden van een geschikt antigeen, diverse antisera en conjugaten en een EIA voor bepaling van IgM tegen phase II antigeen van Coxiella burnetii. Tevens wordt aangegeven in welke richting een EIA voor IgG gezocht kan worden.Abstract not availableDEF/IMG

Molecular Evidence for Association of Chlamydiales Bacteria with Epitheliocystis in Leafy Seadragon (Phycodurus eques), Silver Perch (Bidyanus bidyanus), and Barramundi (Lates calcarifer)

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent. Epitheliocystis is an infection of the gills and skin of many fish species. Sometimes it can be grossly visible as cyst-like lesions, and sometimes the cysts can be seen microscopically in gill squashes, but often the only way to detect it is through histology. Epitheliocystis has been reported worldwide, both from freshwater and marine species (12). This condition is usually benign; however, sometimes it can be associated with a high mortality, particularly in cultured fish (3, 4, 26). Due to swelling of the cells of the gills and the increase in mucus around heavily infected gills, fish can become lethargic and show respiratory distress. The causative agent of epitheliocystis replicates intracellularly in the cysts and, since 1969 epitheliocystis has been associated with chlamydia-like bacteria based on the ultrastructural characteristics of the content of the cysts (4). Attempts to identify the causative agent by using monoclonal antibodies have not been successful, and their results are often inconsistent. In 1999 we discovered many new chlamydia-like sequences by using a universal Chlamydiales 16S rRNA gene PCR (30). Because of the unconfirmed ultrastructural association of epitheliocystis with chlamydia-like organisms we started investigation of archived epitheliocystis material of leafy seadragon (Phycodurus eques) and silver perch (Bidyanus bidyanus) and a new case of epitheliocystis in barramundi (Lates calcarifer) using this Chlamydiales-specific PCR. Ultrastructural analysis of epitheliocystis in these fish species was described previously, in leafy seadragon by Langdon et al. (19), in silver perch by Frances et al. (10), and in barramundi by Anderson and Prior (2). After we communicated our first preliminary positive findings during the Tenth International Symposium on Human Chlamydial Infections in Antalya, Turkey, in 2002 (25), Draghi et al. (6) undoubtedly identified a chlamydia-like bacterium as the cause of epitheliocystis in farmed Atlantic salmon (Salmo salar) using DNA sequence analysis and in situ hybridization (ISH). They proposed the name “Candidatus Piscichlamydia salmonis” for this bacterium. We describe here the characterization of the epitheliocystis agents of leafy seadragon, silver perch, and barramundi by molecular and immunocytochemical methods

Screening rectal swabs for carbapenemase genes

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Detection of microorganisms in vessel waal specimens of the abdominal aorta: development of a PCR-assay in the absence of a gold standard.

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