Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-screen latex agglutination test

The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant S. aureus (MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSA strains displayed positive results only after induction of the mecA gene by exposure to methicillin. Both mecA PCR and MRSA-Screen displayed negative results among the methicillin-susceptible S. aureus strains (n = 106), as well as for Micrococcus spp. (n = 10), members of the family Enterobacteriaceae (n = 10), Streptococcus pneumoniae (n = 10), and Enterococcus spp. (n = 10) (specificity = 100%). Producing the same PBP2a antigen, all 10 methicillin-resistant Staphylococcus epidermidis strains score positived in both the latex test and the mecA PCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain to the species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity and sensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in the microbiology laboratory

Patient-to-patient spread of a single strain of Corynebacterium striatum causing infections in a surgical intensive care unit

Over a 12-month period, Corynebacterium striatum strains were isolated from clinical specimens from 14 patients admitted to a surgical intensive care unit. These isolates were identical by morphology and biotype and displayed the same antibiogram. Ten isolates were found to be the sole possible pathogen. These 10 isolates were from six patients, three of whom had signs of infection at the time of positive culture. Further typing was performed by random amplification of polymorphic DNA analysis, by which all strains were identical and were found to differ to various degrees from reference strains and from isolates found in clinical samples from other wards. In a case-control study the only independent risk factor for acquiring the strain was intubation for longer than 24 h (odds ratio, 20.09; 95

Characterization of multidrug-resistant, qnrB2-positive and extended-spectrum-b-lactamase-producing Salmonella Concord and Salmonella Senftenberg isolates

Objectives: To characterize plasmids and resistance genes of multidrug-resistant (MDR) Salmonella Senftenberg and Salmonella Concord isolated from patients in the Netherlands. Methods: The resistance genes of four MDR Salmonella isolates (three Salmonella Concord and one Salmonella Senftenberg) were identified by miniaturized microarray, PCR and sequencing. Plasmids were characterized by S1 nuclease-PFGE and PCR-based replicon typing (PBRT). Linkage between plasmids and genes was determined by conjugation experiments and microarray analysis. The genetic relationship between the three Salmonella Concord isolates was determined by XbaI-PFGE. Results: A large variety of resistance genes was detected, including qnrB2 and the b-lactamase genes bla TEM-1 and bla SHV-12 in all isolates; moreover all Salmonella Concord isolates also harboured bla CTX-M-15 . Salmonella Senftenberg harboured a large IncHI2 plasmid. The three Salmonella Concord isolates harboured two large plasmids typed as IncHI2 and IncA/C. Conclusions: We detected the first plasmid-mediated MDR Salmonella isolates in the Netherlands harbouring both qnr and extended-spectrum b-lactamase (ESBL) genes. In Salmonella Senftenberg one large plasmid (IncHI2) and in Salmonella Concord two large plasmids (IncHI2 and IncA/C) were responsible for the multidrug resistance

Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended

Comparison of DNA methylation patterns of parentally imprinted genes in placenta derived from IVF conceptions in two different culture media

Study question: Is there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) versus G5 culture medium? Summary answer: We found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium. What is known already: Animal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta. Study design, size, duration: To identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008-2013 in the Netherlands as reference material. Participants/materials, setting, methods: To identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects. Main results and the role of chance: We found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture medium groups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon. Limitations, reasons for caution: Due to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues. Wider implications of the findings: It has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed. Study funding/competing interest(s): This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest. Trial registration number: Placental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298

Attributable sources of community-acquired carriage of Escherichia coli containing β-lactam antibiotic resistance genes: a population-based modelling study

Background: Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), plasmid-mediated AmpC-producing E coli (pAmpC-EC), and other bacteria are resistant to important β-lactam antibiotics. ESBL-EC and pAmpC-EC are increasingly reported in animals, food, the environment, and community-acquired and health-care-associated human infections. These infections are usually preceded by asymptomatic carriage, for which attributions to animal, food, environmental, and human sources remain unquantified. Methods: In this population-based modelling study, we collected ESBL and pAmpC gene data on the Netherlands population for 2005–17 from published datasets of gene occurrences in E coli isolates from different sources, and from partners of the ESBL Attribution Consortium and the Dutch National Antimicrobial Surveillance System. Using these data, we applied an established source attribution model based on ESBL-EC and pAmpC-EC prevalence and gene data for humans, including high-risk populations (ie, returning travellers, clinical patients, farmers), farm and companion animals, food, surface freshwater, and wild birds, and human exposure data, to quantify the overall and gene-specific attributable sources of community-acquired ESBL-EC and pAmpC-EC intestinal carriage. We also used a simple transmission model to determine the basic reproduction number (R0) in the open community. Findings: We identified 1220 occurrences of ESBL-EC and pAmpC-EC genes in humans, of which 478 were in clinical patients, 454 were from asymptomatic carriers in the open community, 103 were in poultry and pig farmers, and 185 were in people who had travelled out of the region. We also identified 6275 occurrences in non-human sources, including 479 in companion animals, 4026 in farm animals, 66 in wild birds, 1430 from food products, and 274 from surface freshwater. Most community-acquired ESBL-EC and pAmpC-EC carriage was attributed to human-to-human transmission within or between households in the open community (60·1%, 95% credible interval 40·0–73·5), and to secondary transmission from high-risk groups (6·9%, 4·1–9·2). Food accounted for 18·9% (7·0–38·3) of carriage, companion animals for 7·9% (1·4–19·9), farm animals (non-occupational contact) for 3·6% (0·6–9·9), and swimming in freshwater and wild birds (ie, environmental contact) for 2·6% (0·2–8·7). We derived an R0 of 0·63 (95% CI 0·42–0·77) for intracommunity transmission. Interpretation: Although humans are the main source of community-acquired ESBL-EC and pAmpC-EC carriage, the attributable non-human sources underpin the need for longitudinal studies and continuous monitoring, because intracommunity ESBL-EC and pAmpC-EC spread alone is unlikely to be self-maintaining without transmission to and from non-human sources. Funding: 1Health4Food, Dutch Ministry of Economic Affairs, and the EU's Horizon-2020 through One-Health European Joint Programme.</p

Samenvatting ESBL-Attributieanalyse (ESBLAT) : Op zoek naar de bronnen van antibioticaresistentie bij de mens

Samenvatting over een project rondom ESBL's. Doelstellingen waren: vaststellen wat de bijdrage is van alle ESBL-reservoirs aan het dragerschap en infecties bij mensen en vaststellen wat de transmissieroutes zijn vanuit deze reservoirs naar de (geïnfecteerde) mens

Rapid Detection of Methicillin Resistance in Staphylococcus aureus Isolates by the MRSA-Screen Latex Agglutination Test

The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR (“gold standard”) for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant S. aureus (MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSA strains displayed positive results only after induction of the mecA gene by exposure to methicillin. Both mecA PCR and MRSA-Screen displayed negative results among the methicillin-susceptible S. aureus strains (n = 106), as well as for Micrococcus spp. (n = 10), members of the family Enterobacteriaceae (n = 10), Streptococcus pneumoniae (n = 10), and Enterococcus spp. (n = 10) (specificity = 100%). Producing the same PBP2a antigen, all 10 methicillin-resistant Staphylococcus epidermidis strains score positived in both the latex test and the mecA PCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain to the species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity and sensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in the microbiology laboratory