InfectoGnostics Research Campus

Jena, Germany

InfectoGnostics Research Campus

Jena, Germany

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Klinger-Strobel M.,Jena University Hospital | Stein C.,Jena University Hospital | Stein C.,InfectoGnostics Research Campus | Forstner C.,Jena University Hospital | And 5 more authors.
International Journal of Antimicrobial Agents | Year: 2017

Biofilms are the preferred environment of micro-organisms on various surfaces such as catheters and heart valves, are associated with numerous difficult-to-treat and recurrent infections, and confer an extreme increase in antibiotic tolerance to most compounds. The aim of this study was to evaluate how colistin affects both the extracellular biofilm matrix and the embedded bacteria in biofilms of methicillin-resistant Staphylococcus aureus (MRSA), a species with intrinsic resistance to colistin, and colistin-susceptible Escherichia coli. Biofilms of MRSA and E. coli were treated with different concentrations of colistin. The minimum biofilm eradication concentration (MBEC) and the effectiveness of colistin at reducing the planktonic fraction were defined as the remaining viable bacteria measured as CFU/mL. In addition, biofilm-embedded cells were LIVE/DEAD-stained and were analysed by confocal laser scanning microscopy (CLSM). Quantification of the biofilm CLSM images was conducted using an open-access in-house algorithm (qBA). In contrast to MRSA, E. coli biofilms and planktonic cells were significantly reduced by colistin in a concentration-dependent manner. Nevertheless, colistin has been shown to exert a matrix-reducing effect following treatment both in laboratory strains and clinical isolates of MRSA and E. coli. Because exposure to colistin rapidly triggered the emergence of highly resistant clones, monotherapy with colistin should be applied with caution. These results suggest that colistin destabilises the biofilm matrix structure even in species with intrinsic colistin resistance, such as S. aureus, leading to the release of planktonic cells that are more susceptible to antibiotics. © 2017 Elsevier B.V. and International Society of Chemotherapy

Brandt C.,Jena University Hospital | Brandt C.,Infectognostics Research Campus | Zander E.,University of Cologne | Pfeifer Y.,Robert Koch Institute | And 8 more authors.
Future Microbiology | Year: 2016

Aim: To countermeasure the global spread of β-lactamases, we developed a rapid molecular test for the highly variable OXA-β-lactamases that allows minimizing the time to effective treatment. Methods: OXA-mRNA was specifically enriched from total RNA using group-specific biotinylated DNA probes and streptavidin-coated magnetic beads. Phylogenetic OXA groups were distinguished by PCR product size. Results: This mRNA fishing method is highly sensitive, yielding specific results from as little as 1 ng total RNA. It enables discrimination of OXA-extended substrate spectrum β-lactamases and carbapenemases and the semi-quantitative detection of highly expressed ISAba1-controlled variants. Conclusion: Targeting mRNA with specific probes on magnetic beads will allow for adaptation to automated systems, such as point-of-care diagnostics. © 2016 Future Medicine Ltd.

PubMed | Infectognostics Research Campus, University of Cologne, Jena University Hospital and Robert Koch Institute
Type: | Journal: Future microbiology | Year: 2016

To countermeasure the global spread of -lactamases, we developed a rapid molecular test for the highly variable OXA--lactamases that allows minimizing the time to effective treatment.OXA-mRNA was specifically enriched from total RNA using group-specific biotinylated DNA probes and streptavidin-coated magnetic beads. Phylogenetic OXA groups were distinguished by PCR product size.This mRNA fishing method is highly sensitive, yielding specific results from as little as 1 ng total RNA. It enables discrimination of OXA-extended substrate spectrum -lactamases and carbapenemases and the semi-quantitative detection of highly expressed ISAba1-controlled variants.Targeting mRNA with specific probes on magnetic beads will allow for adaptation to automated systems, such as point-of-care diagnostics.

Raji M.A.,Alfaisal University | Garaween G.,Alfaisal University | Ehricht R.,Alere Technologies GmbH | Ehricht R.,InfectoGnostics Research Campus | And 7 more authors.
Frontiers in Microbiology | Year: 2016

Limited data exist from the Gulf Cooperation Council states on the prevalence and population dynamics of Staphylococcus aureus colonizing livestock or contaminating retail meat. This study was designed to determine the presence and genetic characteristics of Staphylococcus aureus isolated from raw retail meat sold in Riyadh, Saudi Arabia. Over a period of 9 months, different raw retail meat types were aseptically processed using the double broth enrichment technique, characteristic colonies from chromogenic and mannitol salt agar were further identified using conventional methods. Susceptibility to 9 antibiotics was determined using the disc diffusion technique. Interpretation of inhibition zone was done according to Clinical and Laboratory Standards Institute guidelines. Molecular characterization was carried out using the StaphyType DNA microarray technology. Twenty-five meat samples yielded Staphylococcus aureus isolates. Camel meat had the highest contamination rate with Methicillin resistant Staphylococcus aureus (MRSA) (20%) and Methicillin susceptible Staphylococcus aureus (28%), while poultry meat had the least contamination rate with MRSA (4%). The MRSA isolates were grouped into 4 clonal complexes (CCs) namely CC1-MRSA-IV/SCCfus (n = 2), CC15-MRSA-V/SCCfus (n = 4), CC80-MRSA-IV/PVL+ (n = 5), and CC88-MRSA-IV/PVL+ (n = 2). All CC15-MRSA-V/SCCfus isolates were obtained from camel meat. This is the first study to demonstrate the novel CC15-MRSA-V/SCCfus in retail camel meat. We recommend that surveillance studies should be incorporated in public health and food hygiene programs. © 2016 Raji, Garaween, Ehricht, Monecke, Shibl and Senok.

Senok A.,Alfaisal University | Ehricht R.,Alere Technologies GmbH | Ehricht R.,InfectoGnostics Research Campus | Monecke S.,Alere Technologies GmbH | And 4 more authors.
New Microbes and New Infections | Year: 2016

Changes in the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) continue to be reported. This study was carried out to characterize MRSA isolates in Saudi Arabia. MRSA isolates causing nosocomial infections (n = 117) obtained from 2009–2015 at a tertiary-care facility in Riyadh, Saudi Arabia, were studied. Molecular characterization of isolates was carried out using the StaphyType DNA microarray (Alere Technologies, Jena, Germany). Fourteen clonal complexes (CC) were identified, with the most common being CC80 (n = 35), CC6 (n = 15), CC5 (n = 13) and CC22 (n = 12). With the exception of nine ST239 MRSA-III isolates, all others were of community-associated MRSA lineages. The following strains are identified for the first time in Saudi Arabia: ST8-MRSA-IV [PVL+/ACME+], USA300 (n = 1); ST72-MRSA-IV USA700 (n = 1); CC5-MRSA-IV, [PVL+/edinA+], WA MRSA-121 (n = 1); CC5-MRSA-V+SCCfus, WA MRSA-14/109 (n = 2), CC97-MRSA-IV, WA MRSA-54/63; CC2250/2277-MRSA-IV and WA MRSA-114. CC15-MRSA (n = 3) was identified for the first time in clinical infection in Saudi Arabia. None of the isolates harboured vancomycin resistance genes, while genes for resistance to mupirocin and quaternary ammonium compounds were found in one and nine isolates respectively. Fifty-seven isolates (48.7%) were positive for Panton-Valentine leukocidin genes. While the staphylokinase (sak) and staphylococcal complement inhibitor (scn) genes were present in over 95% of the isolates, only 37.6% had the chemotaxis-inhibiting protein (chp) gene. Increasing occurrence of community-acquired MRSA lineages plus emergence of pandemic and rare MRSA strains is occurring in our setting. Strict infection control practices are important to limit the dissemination of these MRSA strains. © 2016 The Authors

Braun S.D.,Alere Technologies GmbH | Braun S.D.,InfectoGnostics Research Campus | Ahmed M.F.E.,Mansoura University | El-Adawy H.,Institute of Bacterial Infections and Zoonoses | And 11 more authors.
Frontiers in Microbiology | Year: 2016

Introduction: Industrial livestock farming is a possible source of multi-resistant Gram-negative bacteria, including producers of extended spectrum beta-lactamases (ESBLs) conferring resistance to 3rd generation cephalosporins. Limited information is currently available on the situation of ESBL producers in livestock farming outside of Western Europe. A surveillance study was conducted from January to May in 2014 in four dairy cattle farms in different areas of the Nile delta, Egypt. Materials and Methods: In total, 266 samples were collected from 4 dairy farms including rectal swabs from clinically healthy cattle (n = 210), and environmental samples from the stalls (n = 56). After 24 h pre-enrichment in buffered peptone water, all samples were screened for 3rd generation cephalosporin-resistant Escherichia coli using Brilliance™ ESBL agar. Suspected colonies of putatively ESBL-producing E. coli were sub-cultured and subsequently genotypically and phenotypically characterized. Susceptibility testing using the VITEK-2 system was performed. All suspect isolates were genotypically analyzed using two DNA-microarray based assays: CarbDetect AS-1 and E. coli PanType AS-2 kit (ALERE). These tests allow detection of a multitude of genes and their alleles associated with resistance toward carbapenems, cephalosporins, and other frequently used antibiotics. Serotypes were determined using the E. coli SeroGenotyping AS-1 kit (ALERE). Results: Out of 266 samples tested, 114 (42.8%) ESBL-producing E. coli were geno- and phenotypically identified. 113 of 114 phenotypically 3rd generation cephalosporin-resistant isolates harbored at least one of the ESBL resistance genes covered by the applied assays [blaCTX-M15 (n = 105), blaCTX-M9 (n = 1), blaTEM (n = 90), blaSHV (n = 1)]. Alarmingly, the carbapenemase genes blaOXA-48 (n = 5) and blaOXA-181 (n = 1) were found in isolates that also were phenotypically resistant to imipenem and meropenem. Using the array-based serogenotyping method, 66 of the 118 isolates (55%) could be genotypically assigned to O-types. Conclusion: This study is considered to be a first report of the high prevalence of ESBL-producing E. coli in dairy farms in Egypt. ESBL-producing E. coli isolates with different underlying resistance mechanisms are common in investigated dairy cattle farms in Egypt. The global rise of ESBL- and carbapenemase-producing Gram-negative bacteria is a big concern, and demands intensified surveillance. © 2016 Braun, Ahmed, El-Adawy, Hotzel, Engelmann, Weiβ, Monecke and Ehricht.

Braun S.D.,Alere Technologies GmbH | Braun S.D.,InfectoGnostics Research Campus | Dorneanu O.S.,Grigore T. Popa University of Medicine and Pharmacy | VremerA T.,Grigore T. Popa University of Medicine and Pharmacy | And 6 more authors.
Future Microbiology | Year: 2016

Aim: Limited information is currently available about the prevalence of carbapenemase-producing Enterobacteriaceae (CPE) in Romania. Materials & methods: Routine tests of 1,993 clinical isolates at a hospital in Iaşi yielded 46 isolates that were resistant to carbapenems. All 46 isolates were phenotypically and genotypically analyzed using VITEK-2 and DNA microarray-based assays. Results: Isolates were assigned to Klebsiella pneumoniae and Enterobacter cloacae. For 39 isolates, carbapenem resistance was confirmed and 37 harbored at least one carbapenem resistance gene. Two isolates were probably resistant due to AmpC β-lactamases in combination with a porin loss. The overall concordance between detected phenotype and genotype was 95%. Conclusion: Our data show that carbapenemase-producing isolates with different underlying resistance mechanisms are still rare in Iaşi, but the global rise of CPE warrants intensified surveillance. © 2016 Future Medicine Ltd.

Monecke S.,TU Dresden | Monecke S.,Alere Technologies GmbH | Monecke S.,InfectoGnostics Research Campus | Jatzwauk L.,Dresden University Hospital | And 11 more authors.
PLoS ONE | Year: 2016

SCCmec elements are very important mobile genetic elements in Staphylococci that carry beta-lactam resistance genes mecA/mecC, recombinase genes and a variety of accessory genes. Twelve main types and a couple of variants have yet been described. In addition, there are also other SCC elements harbouring other markers. In order to subtype strains of methicillin-resistant S. aureus (MRSA) based on variations within their SCCmec elements, 86 markers were selected from published SCC sequences for an assay based on multiplexed primer extension reactions followed by hybridisation to the specific probes. These included mecA/mecC, fusC, regulatory genes, recombinase genes, genes from ACME and heavy metal resistance loci as well as several genes of unknown function. Hybridisation patterns for published genome or SCC sequences were theoretically predicted. For validation of the microarray based assay and for stringent hybridisation protocol optimization, real hybridization experiments with fully sequenced reference strains were performed modifying protocols until yielded the results were in concordance to the theoretical predictions. Subsequently, 226 clinical isolates from two hospitals in the city of Dresden, Germany, were characterised in detail. Beside previously described types and subtypes, a wide variety of additional SCC types or subtypes and pseudoSCC elements were observed as well as numerous composite elements. Within the study collection, 61 different such elements have been identified. Since hybridisation cannot recognise the localisation of target genes, gene duplications or inversions, this is a rather conservative estimate. Interestingly, some widespread epidemic strains engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III, CC5-, CC22-, CC30-, and CC45-MRSA-IV or CC398-MRSA-V. Conversely, identical SCC elements were observed in different strains with SCCmec IVa being spread among the highest number of Clonal Complexes. The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as high-throughput screening tool for the detection of putative new SCC types or variants that warrant further investigation and sequencing. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing. It also raises the question on scale and speed of the evolution of SCC elements. © 2016 Monecke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Smith S.,Nigerian Institute of Medical Research | Braun S.,Alere Technologies GmbH | Braun S.,InfectoGnostics Research Campus | Akintimehin F.,University of Lagos | And 8 more authors.
Molecular and Cellular Probes | Year: 2016

Microarray-based serogenotyping, antimicrobial susceptibility tests and the detection of relevant resistance genes were performed on isolates of Salmonella spp. from retail meat samples obtained in Lagos, Nigeria.Out of 151 meat samples, 33 Salmonella isolates were obtained. Nine different Salmonella serovars (S. Amoutive, S. Bargny, S. Drac, S. Ealing, S. Urbana, S. Hadar, S. Nyborg, S. Anatum and S. Havana) were identified by microarray-based serogenotyping and confirmed afterwards using classical serotyping. Antibiotic susceptibility tests with 17 antibiotics showed that almost all isolates were fully susceptible to this panel.The results of this study indicated a high prevalence of Salmonella in retail meat, the presence of some previously rather rarely described Serovars in retail meat samples from Lagos, and a need to monitor for Salmonella and their antibiotic resistance determinants. The microarray-based system used herein proved to be perfectly suited as epidemiological tool to replace classical serotyping. © 2016 Elsevier Ltd.

Kohler C.,Friedrich Loeffler Institute for Medical Microbiology | Dunachie S.J.,Mahidol University | Dunachie S.J.,University of Oxford | Muller E.,Alere Technologies GmbH | And 9 more authors.
PLoS Neglected Tropical Diseases | Year: 2016

Background: The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking. Methods and Principal Findings: In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response. Conclusions: Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis. © 2016 Kohler et al.

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