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Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We describe a new phenotypic test to detect beta-lactamases. This assay is based on diffusion of beta-lactam/beta-lactamase through a bacterial filter. Beta-lactam hydrolysis on (the other side of) the filter leads to a change in antibiotic susceptibility, which can be measured by disc diffusion tests. We illustrate its ease of use to detect beta-lactamases of different classes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To establish a possible role for the natural environment in the transmission of clinically relevant AMR bacteria to humans, a literature review was conducted to systematically collect and categorize evidence for human exposure to extended-spectrum β-lactamase-producing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus spp. in the environment. In total, 239 datasets adhered to inclusion criteria. AMR bacteria were detected at exposure-relevant sites (35/38), including recreational areas, drinking water, ambient air, and shellfish, and in fresh produce (8/16). More datasets were available for environmental compartments (139/157), including wildlife, water, soil, and air/dust. Quantitative data from exposure-relevant sites (6/35) and environmental compartments (11/139) were scarce. AMR bacteria were detected in the contamination sources (66/66) wastewater and manure, and molecular data supporting their transmission from wastewater to the environment (1/66) were found. The abundance of AMR bacteria at exposure-relevant sites suggests risk for human exposure. Of publications pertaining to both environmental and human isolates, however, only one compared isolates from samples that had a clear spatial and temporal relationship, and no direct evidence was found for transmission to humans through the environment. To what extent the environment, compared to the clinical and veterinary domains, contributes to human exposure needs to be quantified. AMR bacteria in the environment, including sites relevant for human exposure, originate from contamination sources. Intervention strategies targeted at these sources could therefore limit emission of AMR bacteria to the environment.

This national population-based study was conducted as part of the development of a national automated surveillance system for hospital-acquired bacteraemia and ascertains the utilization of blood cultures (BCs). A primary objective was to understand how local differences may affect interpretation of nationwide surveillance for bacteraemia. From the Danish Microbiology Database, we retrieved all BCs taken between 2010 and 2013 and linked these to admission data from the National Patient Registry. In total, 4 587 295 admissions were registered, and in 11%, at least one BC was taken. Almost 50% of BCs were taken at admission. The chance of having a BC taken declined over the next days but increased after 4 days of admission. Data linkage identified 876 290 days on which at least one BC was taken; 6.4% yielded positive results. Ten species, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Pseudomonas aeruginosa, Candida albicans, Enterobacter cloacae and Klebsiella oxytoca, accounted for 74.7% of agents for this purpose classified as pathogenic. An increase in BCs and positive BCs was observed over time, particularly among older patients. BCs showed a seasonal pattern overall and for S. pneumoniae particularly. A predominance of male patients was seen for bacteraemias due to S. aureus, E. faecium and K. pneumoniae. Minor differences in BCs and positive BCs between departments of clinical microbiology underpin the rationale of a future automated surveillance for bacteraemia. The study also provides important knowledge for interpretation of surveillance of invasive infections more generally., (Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Honey has potent activity against both antibiotic-sensitive and -resistant bacteria, and is an interesting agent for topical antimicrobial application to wounds. As honey is diluted by wound exudate, rapid bactericidal activity up to high dilution is a prerequisite for its successful application. We investigated the kinetics of the killing of antibiotic-resistant bacteria by RS honey, the source for the production of Revamil® medical-grade honey, and we aimed to enhance the rapid bactericidal activity of RS honey by enrichment with its endogenous compounds or the addition of antimicrobial peptides (AMPs). RS honey killed antibiotic-resistant isolates of Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus faecium, and Burkholderia cepacia within 2 h, but lacked such rapid activity against methicillin-resistant S. aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. It was not feasible to enhance the rapid activity of RS honey by enrichment with endogenous compounds, but RS honey enriched with 75 μM of the synthetic peptide Bactericidal Peptide 2 (BP2) showed rapid bactericidal activity against all species tested, including MRSA and ESBL E. coli, at up to 10-20-fold dilution. RS honey enriched with BP2 rapidly killed all bacteria tested and had a broader spectrum of bactericidal activity than either BP2 or honey alone.

The human intestinal microbiota is known to play an important role in human health and disease, and with the advent of novel molecular techniques, disease-specific variations in its composition have been found. However, analysis of the intestinal microbiota has not yet been applicable in large-scale clinical research or routine diagnostics because of the complex and expensive nature of the techniques needed. Here, we describe a new PCR-based profiling technique for high-throughput analysis of the human intestinal microbiota, which we have termed IS-pro. This technique combines bacterial species differentiation by the length of the 16S-23S rDNA interspace region with instant taxonomic classification by phylum-specific fluorescent labeling of PCR primers. We validated IS-pro in silico, in vitro, and in vivo, on human colonic biopsies and feces, and introduced a standardized protocol for data analysis. IS-pro is easy to implement in general clinical microbiological laboratories with access to capillary gel electrophoresis, and the high-throughput nature of the test makes analysis of large numbers of samples feasible. This combination renders IS-pro ideally suited for use in clinical research and routine diagnostics.

We evaluated the use of a trypticase soy broth (TSB) for improving detection of extended-spectrum-beta-lactamase-producing (ESBL(+)) bacteria. Preenrichment of throat and rectal swabs in TSB prior to inoculation on solid medium doubled the number of ESBL(+) bacteria detected in samples obtained from patients in our intensive care unit.

Background: Antibiotic resistance among microbes urgently necessitates the development of novel antimicrobial agents. Since ancient times, honey has been used successfully for treatment of infected wounds, because of its antibacterial activity. However, large variations in the in vitro antibacterial activity of various honeys have been reported and hamper its acceptance in modern medicine., Methods: We assessed the in vitro bactericidal activity of Revamil (Bfactory), a medical-grade honey produced under controlled conditions, and assessed its efficacy for reduction of forearm skin colonization in healthy volunteers in a within-subject-controlled trial., Results: With Bacillus subtilis as a test strain, we demonstrated that the variation in bactericidal activity of 11 batches of medical-grade honey was <2-fold. Antibiotic-susceptible and -resistant isolates of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Klebsiella oxytoca were killed within 24 h by 10%-40% (vol/vol) honey. After 2 days of application of honey, the extent of forearm skin colonization in healthy volunteers was reduced 100-fold (P < .001), and the numbers of positive skin cultures were reduced by 76% (P < .001)., Conclusions: Revamil is a promising topical antimicrobial agent for prevention or treatment of infections, including those caused by multidrug-resistant bacteria.

* Sexual reproduction does not occur in bacteria. The point of departure in bacterial reproduction is always that one individual divides itself into two identical descendants. * In the bacterial world, however, there is certainly exchange of hereditary characteristics (DNA). This type of exchange is called horizontal gene transfer. * There are 3 basic ways for the exchange of DNA between bacteria: conjugation, transduction and natural transformation. Each of these has its specific impact on the species. * During conjugation, a piece of DNA is copied in one bacterium and transferred to another via a temporary connection, a conjugative pilus. In this way, for example, a particular gene that codes for resistance against antibiotics can be transmitted. * In transduction, the transfer of DNA takes place with the aid of bacteriophages. The gene that codes for the toxin produced by Vibrio cholerae is spread by transduction. * In transformation, DNA that is located outside the cell is fragmented and imported into the cell, after which, via recombination, the DNA replaces a piece of original DNA in the chromosome of the host. Transformation is responsible for, among other things, antigen variation in the pneumococcal capsule. Antigen variation helps the pneumococci to resist the immune response leading to the forming of antibodies and adequate opsonisation.

We evaluated a modified fluorescence in situ hybridization (FISH) assay for rapid (<1 h) identification of microorganisms in growth-positive blood cultures. The results were compared to those of the standard FISH technique and conventional culturing. The rapid identification of microorganisms with modified FISH can have important effects on clinical management of patients with bloodstream infections.

Rapid identification of microorganisms in blood cultures is required to optimize empirical treatment at an early stage. Fluorescence in situ hybridization (FISH) can reduce the time to identification of microorganisms in growth-positive blood cultures. In this study, we evaluated the performance, time to identification, and potential clinical benefits of FISH compared to those of conventional culture methods in routine practice. After Gram staining, blood culture fluids were simultaneously further identified with FISH and with conventional culture methods. Results and points in time of FISH and culture identification (provisional and final identifications) were collected and compared. For 91% of microorganisms, the genus or family name was identified, and for 79%, the species name could be attributed. The sensitivity and specificity of the individual probes exceeded 95%, except for the Enterobacteriaceae probe (sensitivity, 89%). Cross-hybridization was obtained with the Klebsiella pneumoniae probe for Klebsiella oxytoca. The time gains of FISH and final culture identification were more than 18 h for bacteria and 42 h for yeasts. With FISH, Staphylococcus aureus was differentiated from coagulase-negative staphylococci 1.4 h faster than by provisional identification (P < 0.001). In conclusion, FISH allows rapid and reliable identification of the majority of microorganisms in growth-positive blood cultures. The substantial time gain of identification with FISH may allow same-day adjustment of antimicrobial therapy, and FISH is especially useful if no provisional identification is obtained. With further extension of the number of probes and a reduction in turnaround time, FISH will become a very useful diagnostic tool in the diagnosis of bloodstream infections.

Background and Objectives: A universal quantitative real-time polymerase chain reaction (PCR), based on bacterial 16S rDNA, to detect bacterial contamination of platelet concentrates (PCs), was developed previously and compared with automated culturing. In the present study, this real-time PCR method was evaluated to determine the optimal sampling time for screening of bacterial contamination in PCs., Materials and Methods: Routinely prepared PCs were spiked with suspensions of Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Propionibacterium acnes to 1, 10 and 100 colony-forming units (CFU)/ml and stored at room temperature for 7 days. The presence of bacteria in these PCs was monitored by quantitative real-time PCR. As a reference method (additional control), BacT/Alert automated culturing was used. For PCR, 1-ml aliquots were drawn from all (spiked) PCs on days 0, 1, 2, 3, 6 and 7 of storage. As a control, triplicate samples (10 ml) were inoculated into aerobic and anaerobic BacT/Alert culture bottles immediately after spiking (day 0) and after storage for 1, 2, 3, 6 or 7 days., Results: With quantitative real-time PCR, all bacterial species tested were reproducibly detected on day 1 after spiking at original concentrations of 10 and 100 CFU/ml. Bacteria were also detected on day 1 from PCs spiked with an initial concentration of 1 CFU/ml, except for E. coli, which was detected in only one of the three samples and P. aeruginosa, for which analysis was not performed on day 1. With the reference method, bacteria were detected in culture bottles (inoculated on day 0) within a mean time of 20.1 h, with the exception of P. acnes which was detected at a mean time of 102.3 and 49.3 h (for original spiking concentrations of 10 and 100 CFU/ml respectively)., Conclusions: PCR enables the rapid detection of low initial numbers of bacteria in PCs. For reliable detection, our results support that sampling of PCs for real-time PCR screening should not be carried out earlier than 1 day after preparation (48 h after blood collection). Importantly, the real-time PCR approach has the potential to be used before the release of PCs from the blood centre or shortly before they are transfused in the hospital.

Background: Based on real-time polymerase chain reaction (PCR) technology, a broad-range 16S rDNA assay was validated and its performance was compared to that of an automated culture system to determine its usefulness for rapid routine screening of platelet concentrates (PCs)., Study Design and Methods: The presence of bacteria in pooled PCs was routinely assessed in an automated culturing system (BacT/ALERT, bioMerieux). The PCR assay was performed with DNA extracted from the same samples as used for culturing. DNA extraction was performed with a automated extraction system (MagNA Pure, Roche Diagnostics). PCR amplification was performed with a set of universal primers and probe targeting eubacterial 16S rDNA., Results: A total of 2146 PCs were tested. Eighteen (0.83%) samples were found to be contaminated. These samples were positive for the presence of bacteria by both methods. All contaminants were identified as bacteria belonging to the common human skin flora. These included Propionibacterium spp. (n = 7), Staphylococcus spp. (n = 6), Bacillus spp. (n = 2), Micrococcus spp. (n = 2), and Peptostreptococcus spp. (n = 1). Estimation of the bacterial load in PCs by real-time PCR showed that the initial levels of contamination varied between 13.6 and 9 x 10(4) colony-forming unit equivalents per PCR procedure., Conclusions: Compared to culture in the BacT/ALERT system, the PCR assay had a sensitivity of 100 percent and a specificity of 100 percent. This real-time PCR assay has a much shorter turnaround time of 4 hours, which offers the possibility to test and obtain results on PCs before release or the day they are transfused. This would permit the withdrawal of contaminated PCs before transfusion.

New techniques have emerged for the detection of bacteria in blood, because the blood culture as gold standard is slow and insufficiently sensitive when the patient has previously received antibiotics or in the presence of fastidious organisms. DNA-based techniques, hybridisation probes, and PCR-based detection or protein-based detection by mass spectroscopy are aimed at rapid identification of bacteria and provide results within 2 h after the first signal of growth in conventional blood cultures. Also, detection of microorganisms directly in blood by pathogen-specific or broad-range PCR assays (eubacterial or panfungal) shows promising results. Interpretation is complex, however, because of detection of DNA rather than living pathogens, the risk of interfering contamination, the presence of background DNA in blood, and the lack of a gold standard. As these techniques are emerging, clinical value and cost-effectiveness have to be assessed. Nevertheless, molecular assays are expected eventually to replace the current conventional microbiological techniques for detection of bloodstream infections.

The widespread use of antibiotics for treatment of bacterial infections has lead to the emergence of resistant human pathogens. Great differences have been documented between European countries in the use of systemic antibiotics. In parallel, significant differences in levels of resistant pathogens have been documented. In order to investigate whether differences in antibiotic use influence the level of antimicrobial resistance of the subgingival microflora, microorganisms from the subgingival plaque of untreated patients with adult periodontitis in The Netherlands (n = 30) and Spain (n = 31) were compared. Blood agar plates containing breakpoint concentrations of penicillin, amoxicillin, amoxicillin and clavulanate, metronidazole, erythromycin, azithromycin, clindamycin and tetracycline were used to determine the proportion of bacteria from the subgingival plaque that was resistant to these antibiotics. In the Spanish patients, statistically significant higher mean levels of resistance were found for penicillin, amoxicillin, metronidazole, clindamycin and tetracycline. The mean number of different bacterial species growing on the selective plates was higher in the Spanish patients, as was the percentage of resistant strains of most periodontal pathogens. A striking difference was observed in the frequency of occurrence of tetracycline-resistant periodontal pathogens. In Spain, 5 patients had > 3 tetracycline resistant periodontal pathogens, whereas this was not observed in any of the Dutch patients. It is concluded that the widespread use of antibiotics in Spain is reflected in the level of resistance of the subgingival microflora of adult patients with periodontitis.