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Background: Before 2012, established national surveillance systems in the Netherlands were not able to provide a timely, comprehensive epidemiological view on nosocomial outbreaks. The Healthcare-associated Infections and AntiMicrobial Resistance Monitoring Group (SO-ZI/AMR) was initiated in 2012 for timely national nosocomial outbreak monitoring and risk assessment. This paper aims to describe the achievements of the SO-ZI/AMR by presenting characteristics of outbreaks reported in 2012-2021., Methods: Hospitals and, since 2015, long-term care facilities (LTCF) were requested to report outbreaks when (1) continuity of care was threatened, or (2) transmission continued despite control measures. A multi-disciplinary expert panel (re-)assessed the public health risk of outbreaks during monthly meetings, using 5 severity phases and based on data collected via standardised questionnaires. We descriptively studied the panel's consensus-based severity classification, distribution of (highly resistant) microorganisms, and duration and size of outbreaks between April 2012 and December 2021., Results: In total, 353 hospital outbreaks and 110 LTCF outbreaks were reported. Most outbreaks (hospitals: n = 309 (88%), LTCF: n = 103 (94%)) did not progress beyond phase 1 (no public health implications, outbreak expected to be controlled within two months), one hospital outbreak reached phase 4 (insufficient/ineffective response: possible public health threat, support offered). Highly resistant microorganisms (HRMO) were involved in 269 (76%) hospital and 103 (94%) LTCF outbreaks. Most outbreaks were caused by methicillin-resistant Staphylococcus aureus (MRSA; n = 93 (26%) in hospitals, n = 80 (72%) in LTCF), vancomycin-resistant Enterococcus faecium (VRE; n = 116 (33%) in hospitals, n = 2 (2%) in LTCF) and highly resistant Enterobacterales (n = 41 (12%) in hospitals, n = 20 (18%) in LTCF). Carbapenemase-producing gram-negative bacteria were involved in 32 (9.1%) hospital and five (4.5%) LTCF outbreaks. In hospitals, VRE outbreaks had the longest duration (median 2.3; range 0.0-22.8 months) and widest range of affected patients (median 9; range 2-483)., Conclusions: The SO-ZI/AMR provided national insight into the characteristics of nosocomial outbreaks over the past decade. HRMO outbreaks - mostly caused by MRSA, VRE (in hospitals) and highly resistant Enterobacterales - occurred regularly, but most of them were controlled quickly and did not develop into a public health threat. The SO-ZI/AMR has become a solid monitoring body, essential to assess risks and raise awareness of potential HRMO threats., (© 2023. The Author(s).)

Background: Infection control link nurses (ICLN) disseminate knowledge on infection prevention topics to their peers. Little is known about how they succeed and thereby contribute to infection prevention in daily practise., Aim: To explore the experiences of infection control link nurses regarding their role in acute care hospitals and identify perceived facilitators and best practices., Methods: We conducted a qualitative study with semi-structured individual and focus group interviews with ICLN. The effect of COVID-19 on the ICLN role was added as a topic in focus group interviews during the pandemic., Results: Twenty-six ICLN working in acute care hospitals were interviewed. ICLN perceived their role as to identify, monitor, facilitate and inform their colleagues on infection prevention topics related to their ward. Their experiences vary from feeling challenged and wonder how to get started, to feeling confident and taking initiatives that lead to ward-based improvements. When inspired by each other and supported by infection control practitioners or managers, ICLN feel empowered to initiate more activities to improve practice. During the COVID-19 pandemic, ICLN felt their responsibilities were magnified. When transferred to another ward, the focus on the ICLN role seemed dispersed., Discussion: Empowered ICLN adjust and operationalize infection prevention policies to fit the conditions of their specific wards and provide practical instructions and feedback to their peers which enable better compliance to infection prevention policies. Support and inspiration from other ICLN, infection control practitioners and management contribute to this empowerment and consequently to taking impactful initiatives to improve practice., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Background: The Netherlands Donor Feces Bank provides standardized ready-to-use donor faecal suspensions for faecal microbiota transplantation treatment of patients with recurrent Clostridioides difficile infection., Objective: The purpose of this study was evaluation of safety, feasibility and outcome of faecal microbiota transplantation facilitated by a national stool bank., Methods: The methods used included: observational cohort study of donors and recipients of faecal suspensions; assessment of donor screening and patient selection performed by an expert panel of medical microbiologists, gastroenterologists and infectious disease specialists; and patient outcome evaluated at different timepoints after faecal microbiota transplantation., Results: Of 871 volunteers who registered as a potential faeces donor, 16 (2%) became active donors. Nine donors stopped or were excluded after a mean donation period of 5.7 months. In 2016-2019, 47 (27%) of 176 requests for faecal microbiota transplantations were deemed not indicated by the expert panel. In total, 129 patients with recurrent C. difficile infection were treated with 143 faecal suspensions in 40 different hospitals. The cure rate at two months after a single infusion was 89% (107/120). Of 84 patients, long-term follow-up (median 42 weeks) was available and sustained cure was achieved in 61 (73%). Early C. difficile infection relapses (within two months after faecal microbiota transplantation) and late recurrences (after more than two months) occurred more frequently in patients who received non- C. difficile antibiotics within three weeks after faecal microbiota transplantation and in moderately to severely immunocompromised patients. Of 21 patients with C. difficile infection after faecal microbiota transplantation, 14 were cured with anti- C. difficile antibiotics and seven with a second transplantation. No faecal microbiota transplantation-related serious adverse events were observed, but gastro-intestinal complaints (nausea, abdominal pain or diarrhoea) persisted in 32% of the treated patients at long-term follow-up., Conclusion: Faecal suspensions provided by a centralized stool bank, supported by a multidisciplinary expert team, resulted in effective, appropriate and safe application of faecal microbiota transplantation for recurrent C. difficile infection., Level of Evidence: Level II, prospective cohort study.

Molecular typing has become indispensable in the detection of nosocomial transmission of bacterial pathogens and the identification of sources and routes of transmission in outbreak settings, but current methods are labor-intensive, are difficult to standardize, or have limited resolution. Whole-genome multilocus sequence typing (wgMLST) has emerged as a whole-genome sequencing (WGS)-based gene-by-gene typing method that may overcome these limitations and has been applied successfully for several species in outbreak settings. In this study, genus-, genetic-complex-, and species-specific wgMLST schemes were developed for Citrobacter spp., the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, and Klebsiella pneumoniae and used to type a national collection of 1,798 extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E) isolates obtained from patients in Dutch hospitals. Genus-, genetic-complex-, and species-specific thresholds for genetic distance that accurately distinguish between epidemiologically related and unrelated isolates were defined for Citrobacter spp., the E. cloacae complex, E. coli, and K. pneumoniae wgMLST was shown to have higher discriminatory power and typeability than in silico MLST. In conclusion, the wgMLST schemes developed in this study facilitate high-resolution WGS-based typing of the most prevalent ESBL-producing species in clinical practice and may contribute to further elucidation of the complex epidemiology of antimicrobial-resistant Enterobacteriaceae wgMLST opens up possibilities for the creation of a Web-accessible database for the global surveillance of ESBL-producing bacterial clones., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Klebsiella pneumoniae is emerging as an important nosocomial pathogen due to its rapidly increasing multidrug resistance, which has led to a renewed interest in polymyxin antibiotics, such as colistin, as antibiotics of last resort. However, heteroresistance (i.e., the presence of a subpopulation of resistant bacteria in an otherwise susceptible culture) may hamper the effectiveness of colistin treatment in patients. In a previous study, we showed that colistin resistance among extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates emerged after the introduction of selective digestive tract decontamination (SDD) in an intensive care unit (ICU). In this study, we investigated heteroresistance to colistin among ESBL-producing K. pneumoniae isolates by using population analysis profiles (PAPs). We used whole-genome sequencing (WGS) to identify the mutations that were associated with the emergence of colistin resistance in these K. pneumoniae isolates. We found five heteroresistant subpopulations, with colistin MICs ranging from 8 to 64 mg/liter, which were derived from five clonally related, colistin-susceptible clinical isolates. WGS revealed the presence of mutations in the lpxM, mgrB, phoQ, and yciM genes in colistin-resistant K. pneumoniae isolates. In two strains, mgrB was inactivated by an IS3-like or ISKpn14 insertion sequence element. Complementation in trans with the wild-type mgrB gene resulted in these strains reverting to colistin susceptibility. The MICs for colistin-susceptible strains increased 2- to 4-fold in the presence of the mutated phoQ, lpxM, and yciM alleles. In conclusion, the present study indicates that heteroresistant K. pneumoniae subpopulations may be selected for upon exposure to colistin. Mutations in mgrB and phoQ have previously been associated with colistin resistance, but we provide experimental evidence for roles of mutations in the yciM and lpxM genes in the emergence of colistin resistance in K. pneumoniae., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: Little is known about the validity and reliability of expert assessments of the quality of antimicrobial prescribing, despite their importance in antimicrobial stewardship. We investigated how infectious disease doctors' assessments compared with a reference standard (modal expert opinion) and with the assessments of their colleagues., Methods: Twenty-four doctors specialized in infectious diseases or clinical microbiology (16 specialists and 8 residents) from five hospitals were asked to assess the appropriateness of antimicrobial agents prescribed for a broad spectrum of indications in 56 paper cases. They were instructed how to handle guideline applicability and deviations. We created a reference standard of antimicrobial appropriateness using the modal assessment of 16 specialists. We calculated criterion validity and interrater and intrarater overall and specific agreement with an index expert (senior infectious disease physician) and analysed the influence of doctor characteristics on validity., Results: Specialists agreed with the reference standard in 80% of cases (range 75%-86%), with a sensitivity and specificity of 75% and 84%, respectively. This did not differ by clinical specialty, hospital or years of experience, and residents had similar results. Specialists agreed with the index expert in 76% of cases and the index expert agreed with his previous assessments in 71% of cases., Conclusions: Doctors specialized in infectious diseases and clinical microbiology assess the appropriateness of antimicrobials prescribed for a broad spectrum of indications with acceptable agreement and validity, regardless of their experience or hospital of employment. However, there is room for improvement, which merits attention in multidisciplinary discussions and education., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavourable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction., Methods: Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy., Results: After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin-deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked reduction of phagocytes., Conclusions: This new meningitis model permits detailed analysis and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.

Background: An outbreak of Clostridium difficile ribotype 027 infection (CDI) occurred at an university hospital, involving 19 departments. To determine what hospital-associated factors drove the outbreak of this particular strain we performed a case-control study., Methods: Cases (n = 79), diagnosed with CDI due to C. difficile ribotype 027 were matched for age and treating medical specialty to four control patients (n = 316). Patients diagnosed with CDI due to other ribotypes were included as a second control group. A random selection of C. difficile ribotype 027 strains (n = 10) was genotyped by Whole Genome Sequencing (WGS)., Findings: WGS showed the outbreak was likely caused by a single strain of C. difficile (two or less single-nucleotide variants between isolates). Ninety-five percent of cases had used antibiotics, compared to 56% of controls. Previous admission to the intensive care unit (ICU) (OR: 2.4, 95% CI 1.0-5.6), longer length of stay (LOS), and recent hospital admission were associated with CDI ribotype 027. Cases were less likely to have been admitted to a ward with a known isolated CDI patient (OR: 0.2, 95% CI 0.1-0.6). Analysis of patients who stayed at the ICU (35 cases; 51 controls), indicated that the use of selective decontamination of the digestive tract (SDD) and a longer LOS in the ICU were associated with CDI risk., Interpretation: In this large outbreak, any antibiotic use, including SDD use, appeared as a prerequisite for acquisition of the outbreak strain. The role of use of SDD and prolonged stay on the ICU could not be disentangled, but both factors can play a biologically plausible role in C. difficile acquisition and infection.

Travel to (sub)tropical countries is a well-known risk factor for acquiring resistant bacterial strains, which is especially of significance for travellers from countries with low resistance rates. In this study we investigated the rate of and risk factors for travel-related acquisition of extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E), ciprofloxacin-resistant Enterobacteriaceae (CIPR-E) and carbapenem-resistant Enterobacteriaceae. Data before and after travel were collected from 445 participants. Swabs were cultured with an enrichment broth and sub-cultured on selective agar plates for ESBL detection, and on plates with a ciprofloxacin disc. ESBL production was confirmed with the double-disc synergy test. Species identification and susceptibility testing were performed with the Vitek-2 system. All isolates were subjected to ertapenem Etest. ESBL and carbapenemase genes were characterized by PCR and sequencing. Twenty-seven out of 445 travellers (6.1%) already had ESBL-producing strains and 45 of 445 (10.1%) travellers had strains resistant to ciprofloxacin before travel. Ninety-eight out of 418 (23.4%) travellers acquired ESBL-E and 130 of 400 (32.5%) travellers acquired a ciprofloxacin-resistant strain. Of the 98 ESBL-E, predominantly Escherichia coli and predominantly blaCTX-M-15, 56% (55/98) were resistant to gentamicin, ciprofloxacin and co-trimoxazole. Multivariate analysis showed that Asia was a high-risk area for ESBL-E as well as CIPR-E acquisition. Travellers with diarrhoea combined with antimicrobial use were significantly at higher risk for acquisition of resistant strains. Only one carbapenemase-producing isolate was acquired, isolated from a participant after visiting Egypt. In conclusion, travelling to Asia and diarrhoea combined with antimicrobial use are important risk factors for acquiring ESBL-E and CIPR-E., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

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.)

Objectives: The objectives of this study were to determine the prevalence of carriage of ESBL-producing Enterobacteriaceae (ESBL-E) in a representative sample of the general adult Dutch community, to identify risk factors and to gain understanding of the epidemiology of these resistant strains., Methods: Adults enrolled in five general practices in Amsterdam were approached by postal mail and asked to fill in a questionnaire and to collect a faecal sample. Samples were analysed for the presence of ESBL-E. ESBL genes were characterized by PCR and sequencing. Strains were typed using MLST and amplified fragment length polymorphism (AFLP) and plasmids were identified by PCR-based replicon typing. Risk factors for carriage were investigated by multivariate analysis., Results: ESBL-E were found in 145/1695 (8.6%) samples; 91% were Escherichia coli. Most ESBL genes were of the CTX-M group (blaCTX-M-1 and blaCTX-M-15). MLST ST131 was predominant and mainly associated with CTX-M-15-producing E. coli. One isolate with reduced susceptibility to ertapenem produced OXA-48. In multivariate analyses, use of antimicrobial agents, use of antacids and travel to Africa, Asia and Northern America were associated with carriage of ESBL-E, in particular strains with blaCTX-M-14/15., Conclusions: This study showed a high prevalence of ESBL-E carriage in the general Dutch community. Also, outside hospitals, the use of antibiotics was a risk factor; interestingly, use of antacids increased the risk of carriage. A major risk factor in the general population was travel to countries outside Europe, in particular to Asia, Africa and Northern America., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objective: To identify patients who benefit most from Staphylococcus aureus screening and decolonization treatment upon admission., Background: S. aureus carriers are at increased risk of developing surgical-site infections with S. aureus. Previously, we demonstrated in a randomized, placebo-controlled trial (RCT) that these infections can largely be prevented by detection of carriage and decolonization treatment upon admission. In this study, we analyzed 1- and 3-year mortality rates in both treatment arms of the RCT to identify patient groups that should be targeted when implementing the screen-and-treat strategy., Methods: Three years after enrolment in the RCT, mortality dates of all surgical patients were checked. One- and 3-year mortality rates were calculated for all patients and for various subgroups., Results: After 3 years, 44 of 431 (10.2%) and 43 of 362 (11.9%) patients had died in the mupirocin/chlorhexidine and placebo groups, respectively. No significant differences in mortality rates were observed between the treatment groups or the subgroups according to type of surgery. In the subgroup of patients with clean procedures (382 cardiothoracic, 167 orthopedic, 61 vascular, and 56 other), mupirocin/chlorhexidine reduced 1-year mortality: 11 of 365 (3.0%) died in the mupirocin/chlorhexidine versus 21 of 301 (7.0%) in the placebo group [hazard ratio = 0.38 (95% CI: 0.18-0.81)]., Conclusions: Detection and decolonization of S. aureus carriage not only prevents S. aureus surgical-site infections but also reduces 1-year mortality in surgical patients undergoing clean procedures. Such patients with a high risk of developing S. aureus infections should therefore be the primary target when implementing the screen-and-treat strategy in clinical practice.

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.)

Background: Invasive aspergillosis is the most common life-threatening opportunistic invasive mycosis in immunocompromised patients. A test for invasive aspergillosis should neither be too invasive nor too great a burden for the already weakened patient. The serum galactomannan enzyme-linked immunosorbent assay (ELISA) seems to have the potential to meet both requirements., Objectives: To obtain summary estimates of the diagnostic accuracy of galactomannan detection in serum for the diagnosis of invasive aspergillosis., Search Methods: We searched MEDLINE, EMBASE and Web of Science with both MeSH terms and text words for both aspergillosis and the sandwich ELISA. We checked the reference lists of included studies and review articles for additional studies. We conducted the searches in February 2014., Selection Criteria: We included cross-sectional studies, case-control designs and consecutive series of patients assessing the diagnostic accuracy of galactomannan detection for the diagnosis of invasive aspergillosis in patients with neutropenia or patients whose neutrophils are functionally compromised. The reference standard was composed of the criteria given by the European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group (MSG)., Data Collection and Analysis: Two review authors independently assessed quality and extracted data. We carried out meta-analysis using the bivariate method. We investigated sources of heterogeneity by adding potential sources of heterogeneity to the model as covariates., Main Results: We included 54 studies in the review (50 in the meta-analyses), containing 5660 patients, of whom 586 had proven or probable invasive aspergillosis. When using an optical density index (ODI) of 0.5 as a cut-off value, the sensitivity of the test was 82% (73% to 90%) and the specificity was 81% (72% to 90%). At a cut-off value of 1.0 ODI, the sensitivity was 72% (65% to 80%) and the specificity was 88% (84% to 92%). At a cut-off value of 1.5 ODI, the sensitivity was 61% (47% to 75%) and the specificity was 93% (89% to 97%). None of the potential sources of heterogeneity had a statistically significant effect on either sensitivity or specificity., Authors' Conclusions: If we used the test at a cut-off value of 0.5 ODI in a population of 100 patients with a disease prevalence of 9% (overall median prevalence), two patients who have invasive aspergillosis would be missed (sensitivity 82%, 18% false negatives), and 17 patients would be treated unnecessarily or referred unnecessarily for further testing (specificity 81%, 19% false negatives). If we used the test at a cut-off value of 1.5 in the same population, that would mean that four invasive aspergillosis patients would be missed (sensitivity 61%, 39% false negatives), and six patients would be treated or referred for further testing unnecessarily (specificity 93%, 7% false negatives). These numbers should, however, be interpreted with caution because the results were very heterogeneous.

Current international guidelines lack definite conclusions regarding repeat stool sampling for the detection of toxigenic Clostridium difficile. We assessed the value of repeat sampling and compared the diagnostic yield in an epidemic to a non-epidemic setting. Consecutive fecal samples obtained during two time frames were analyzed using direct stool immunoassay toxin testing (enzyme immunoassay [EIA]), direct stool real-time PCR toxin gene testing, and toxigenic culture. Samples collected within 7 days of the initial sample were considered repeat tests. In the epidemic setting 989 patients were analyzed, and in the non-epidemic setting 1,015. In the epidemic setting 204 patients had two or more specimens included for analysis and in the non-epidemic setting 287 patients. In the epidemic setting 136 samples yielded a positive results, either by EIA or toxigenic culture; of these, 108 were positive according to EIA and 123 according to toxigenic culture. In the first test round 98 (90.7%, 95% CI 85.3 to 96.2), 114 (92.7%, 88.1 to 97.3), and 126 (92.6%, 88.3 to 97.0) positives were detected. Subsequent test rounds yielded 10 (9.3%, 3.8 to 14.7), 9 (7.3%, 2.7 to 11.9), and 10 (7.4%, 3.0 to 11.7) extra positives. In the non-epidemic setting EIA, toxigenic culture and PCR detected 33, 66, and 83 positives. The three tests combined 93 detected positives. In the first test round 30 (90.9%, 81.1 to 100.7), 63 (95.5%, 90.4 to 110.5), 76 (91.6%, 85.6 to 97.5), and 87 (93.5%, 88.6 to 98.5) positives were detected. Subsequent test rounds yielded 3 (9.1%, -0.7 to 18.9), 3 (4.5%, -0.5 to 9.6), 7 (8.4%, 2.5 to 14.4), and 6 (6.5%, 1.5 to 11.4) extra positives. In conclusion, repeat testing resulted in 4.5% to 9.3% extra positives. No significant difference between the settings studied could be demonstrated. Repeat sampling and multimodality testing may be chosen in an outbreak situation to detect all cases, effectively controlling nosocomial spread.

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.

Objectives: The aim of the present study was to determine the prevalence and to characterize extended-spectrum β-lactamases- and/or carbapenemases-producing Enterobacteriaceae among Enterobacteriaceae isolated from retail chicken meat in Zagazig, Egypt., Methods: One hundred and six Enterobacteriaceae isolates were collected from retail chicken meat samples purchased in Zagazig, Egypt in 2013. Species identification was done by MALDI-TOF MS. Screening for ESBL-E was performed by inoculation of isolates recovered from meat samples onto the EbSA (Cepheid Benelux, Apeldoorn, the Netherlands) selective screening agar. ESBL production was confirmed by combination disc diffusion test with clavulanic acid (Rosco, Taastrup, Denmark). Carbapenemases production was confirmed with double disk synergy tests. Resistance genes were characterized by PCR with specific primers for TEM, SHV, and CTX-M and carbapenemases (KPC, NDM, OXA-48, IMP and VIM). PCR products of CTX-M genes were purified and sequenced. Phylogenetic grouping of E. coli was performed by a PCR-based method., Results: Of these 106 isolates 69 (65.09%) were ESBL producers. Twelve (11.32%) of these isolates were also phenotypically class B carbapenemases producer. TEM genes were detected in 61 (57.55%) isolates. 49 (46.23%) isolates harbored CTX-M genes, and 25 (23.58%) carried genes of the SHV family. All CPE belonged to the NDM group. The predominant CTX-M sequence type was CTX-M-15 (89.80%). The majority (80%) of the ESBL-EC belonged to low virulence phylogroups A and B1., Conclusions: This is the first study from Egypt reporting high rates of ESBLs and carbapenemases (65.09% and 11.32%, respectively) in Enterobacteriaceae isolated from retail chicken meat. These results raise serious concerns about public health and food safety as retail meat could serve as a reservoir for these resistant bacteria which could be transferred to humans through the food chain.

Objectives: The aim of the study was to investigate the prevalence of extended-spectrum β-lactamase and carbapenemase production among Enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection., Methods: Ninety-four Enterobacteriaceae blood culture isolates from Egyptian patients with suspected blood stream infection were collected, one isolate per patient. Identification of bacterial isolates was performed with MALDI-TOF (MS-based Vitek MS system, bioMerieux). Screening for ESBLs and carbapenemases production was done with the Vitek 2 system (bioMérieux). ESBL production was confirmed using the combined disk diffusion method for cefotaxime, ceftazidime, and cefepime, all with and without clavulanic acid (Rosco). Real-time PCR and sequencing were used to characterize the resistance genes. The phylogenetic groups of E. coli were identified by a PCR-based method., Results: Of the 94 Enterobacteriaceae isolates 46 (48.93%) showed an ESBL phenotype. One Enterobacter spp isolate was ESBL-producer and meropenem-resistant. The genetic analysis showed that CTX-M was present in 89.13% (41/46) of the ESBL-producing Enterobacteriaceae, whereas TEM and SHV were detected in 56.52% (26/46) and 21.74% (10/46) respectively (47.83%) of the ESBL-producing isolates were multidrug resistant (MDR). Eleven out of 30 ESBL-producing E-coli isolates were assigned to phylogroup B2, followed by groups B1 (8 isolates), A (6 isolates) and D (5 isolates)., Conclusions: The high ESBL-E rates (48.93%) found in this study together with the identification of one carbapenem-resistant Enterobacter spp isolate is worrisome. Our results indicate that systems for monitoring and detection of ESBL-producing bacteria in Egyptian hospitals have to be established. Also strict hospital infection control policies with the restriction of the consumption of extended-spectrum cephalosporins are necessary.

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.)

Objectives: A rapid test to diagnose Clostridium difficile infection (CDI) on hospital wards could minimize common but critical diagnostic delay. Field asymmetric ion mobility spectrometry (FAIMS) is a portable mass spectrometry instrument that quickly analyses the chemical composition of gaseous mixtures (e.g., above a stool sample). Can FAIMS accurately distinguish C. difficile-positive from -negative stool samples?, Methods: We analyzed 213 stool samples with FAIMS, of which 71 were C. difficile positive by microbiological analysis. The samples were divided into training, test, and validation samples. We used the training and test samples (n=135) to identify which sample characteristics discriminate between positive and negative samples, and to build machine learning algorithms interpreting these characteristics. The best performing algorithm was then prospectively validated on new, blinded validation samples (n=78). The predicted probability of CDI (as calculated by the algorithm) was compared with the microbiological test results (direct toxin test and culture)., Results: Using a Random Forest classification algorithm, FAIMS had a high discriminatory ability on the training and test samples (C-statistic 0.91 (95% confidence interval (CI): 0.86-0.97)). When applied to the blinded validation samples, the C-statistic was 0.86 (0.75-0.97). For samples analyzed ≤7 days of collection (n=76), diagnostic accuracy was even higher (C-statistic: 0.93 (0.85-1.00)). A cutoff value of 0.32 for predicted probability corresponded with a sensitivity of 92.3% (95% CI: 77.4-98.6%) and specificity of 86.0% (78.3-89.3%). For even fresher samples, discriminatory ability further increased., Conclusions: FAIMS analysis of unprocessed stool samples can differentiate between C. difficile-positive and -negative samples with high diagnostic accuracy.

Objectives: The objective of this study was to determine the prevalence of pAmpC beta-lactamases in community-acquired Gram negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains., Methods: Fecal samples were obtained from community-dwelling volunteers. Participants also returned a questionnaire for analysis of risk factors. Screening for pAmpC was performed with selective enrichment broth and a selective screening agar. Confirmation of AmpC-production was performed with two double disc combination tests: cefotaxime and ceftazidime with either boronic acid or cloxacillin as inhibitor. Multiplex PCR was used as gold standard for detection of pAmpC. 16S rRNA PCR and AFLP were performed as required, plasmids were identified by PCR-based replicon typing. Questionnaire results were analyzed with SPSS, version 20.0., Results: Fecal samples were obtained from 550 volunteers; mean age 51 years (range: 18-91), 61% were females. pAmpC was present in seven E. coli isolates (7/550, 1.3%, 0.6-2.7 95% CI): six CMY-2-like pAmpC and one DHA. ESBL-encoding genes were found in 52/550 (9.5%, 7.3-12.2 95% CI) isolates; these were predominantly blaCTX-M genes. Two isolates had both ESBL and pAmpC. Admission to a hospital in the previous year was the only risk factor we identified., Conclusions: Our data indicate that the prevalence of pAmpC in the community seems still low. However, since pAmpC-producing isolates were not identified as ESBL producers by routine algorithms, there is consistent risk that further increase of their prevalence might go undetected.

Background: Availability of a patient data management system (PDMS) has created the opportunity to develop trigger-based electronic surveillance systems (ESSs). The aim was to evaluate a semi-automated trigger-based ESS for the detection of ventilator-associated pneumonia (VAP) and central line-associated blood stream infections (CLABSIs) in the intensive care., Methods: Prospective comparison of surveillance was based on a semi-automated ESS with and without trigger. Components of the VAP/CLABSI definition served as triggers. These included the use of VAP/CLABSI-related antibiotics, the presence of mechanical ventilation or an intravenous central line, and the presence of specific clinical symptoms. Triggers were automatically fired by the PDMS. Chest X-rays and microbiology culture results were checked only on patient days with a positive trigger signal from the ESS. In traditional screening, no triggers were used; therefore, chest X-rays and culture results had to be screened for all patient days of all included patients. Patients with pneumonia at admission were excluded., Results: A total of 553 patients were screened for VAP and CLABSI. The incidence of VAP was 3.3/1,000 ventilation days (13 VAP/3,927 mechanical ventilation days), and the incidence of CLABSI was 1.7/1,000 central line days (24 CLABSI/13.887 central line days). For VAP, the trigger-based screening had a sensitivity of 92.3%, a specificity of 100%, and a negative predictive value of 99.8% compared to traditional screening of all patients. For CLABSI, sensitivity was 91.3%, specificity 100%, and negative predictive value 99.6%., Conclusions: Pre-selection of patients to be checked for signs and symptoms of VAP and CLABSI by a computer-generated automated trigger system was time saving but slightly less accurate than conventional surveillance. However, this after-the-fact surveillance was mainly designed as a quality indicator over time rather than for precise determination of infection rates. Therefore, surveillance of VAP and CLABSI with a trigger-based ESS is feasible and effective.

Objectives: Early and rapid identification of Clostridium difficile infections (CDI) is important to prevent transmission. In this study we assessed the diagnostic accuracy of a trained detection dog for detecting CDI cases on hospital wards in an outbreak setting., Methods: During a CDI outbreak in a large Dutch university hospital, we screened affected hospital wards repeatedly with a trained detection dog. The dog's response was compared to the clinical diagnosis, supported by laboratory results., Results: During a total of 9 hospital visits, the dog performed 651 screenings involving 371 participants. The dog correctly identified 12 out of 14 CDI cases [sensitivity 86% (95% confidence interval (CI): 56-97%)] and 346 out of 357 CDI negative participants [specificity of 97% (95% CI: 94-98%)]. Interestingly, of the 11 CDI negative participants that were 'falsely' indicated by the dog as positive, 2 (18%) did actually developed CDI during the 3 months of follow-up after the detection period; compared to only 12 of the 346 participants (3.5%) that the dog identified as C. difficile negative (p = 0.06)., Conclusion: A trained detection dog can accurately detect CDI in hospitalized patients during an outbreak. A (repeated) positive dog response is a strong indication of a CDI episode coming, be it the next day or possibly up to a month., (Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

To determine whether extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) are present in retail raw vegetables in Amsterdam, the Netherlands, we collected 119 samples of 15 different types of vegetables from various sources. After culture, strain identification and susceptibility testing, ESBL-encoding genes were characterised by a microarray. Four of the 15 vegetable types were contaminated with ESBL-E. Seven samples (6 %) yielded ESBL-E. Three bla CTX-M-15, one bla CTX-M-1, two genes of the CTX-M-9 group and one SHV ESBL-encoding gene were found. The ESBL genes were similar to what is found in enterobacterial strains from human origin. Therefore, raw vegetables might be a source of resistance genes for the enterobacterial strains found in humans.

The composition of the gut microbiota is associated with various disease states, most notably inflammatory bowel disease, obesity and malnutrition. This underlines that analysis of intestinal microbiota is potentially an interesting target for clinical diagnostics. Currently, the most commonly used sample types are feces and mucosal biopsy specimens. Because sampling method, storage and processing of samples impact microbiota analysis, each sample type has its own limitations. An ideal sample type for use in routine diagnostics should be easy to obtain in a standardized fashion without perturbation of the microbiota. Rectal swabs may satisfy these criteria, but little is known about microbiota analysis on these sample types. In this study we investigated the characteristics and applicability of rectal swabs for gut microbiota profiling in a clinical routine setting in patients presenting with various gastro-intestinal disorders. We found that rectal swabs appeared to be a convenient means of sampling the human gut microbiota. Swabs can be performed on demand, whenever a patient presents; swab-derived microbiota profiles are reproducible, whether they are gathered at home by patients or by medical professionals in an outpatient setting and may be ideally suited for clinical diagnostics and large-scale studies.

Introduction: Infectious complications and especially anastomotic leakage (AL) severely impede the recuperation of patients following colorectal cancer (CRC) surgery. When the normal gut barrier fails, as in AL, pathogenic microorganisms can enter the circulation and may cause severe sepsis which is associated with substantial mortality. Moreover, AL has a negative impact on the CRC prognosis. Selective decontamination of the digestive tract (SDD) employs oral nonabsorbable antibiotics to eradicate pathogenic microorganisms before elective tumour resection., Methods: In this multicentre randomised clinical trial, perioperative SDD in addition to standard antibiotic prophylaxis is compared with standard antibiotic prophylaxis alone in patients with CRC who undergo elective surgical resection with a curative intent. The SDD regimen consists of colistin, tobramycin and amphotericin B. The primary objectives of this randomised clinical trial are to evaluate if perioperative SDD reduces the incidence of clinical AL and its septic consequences as well as other infectious complications. A main secondary objective is improvement of the cancer-free survival. A total of 762 patients will be included in total for sufficient power., Conclusion: It is hypothesised that SDD will reduce clinical AL thereby reducing the morbidity and the mortality in CRC patients., Funding: The trial is investigator-initiated, investigator-driven and supported by the Dutch Digestive Foundation (WO 11-06) and the private Posthumus Meyes Fund., Trial Registration: The trial is registered at ClinicalTrials.gov: NCT01740947.

Objectives: The aim of this study was to compare the current screening methods and to evaluate confirmation tests for phenotypic plasmidal AmpC (pAmpC) detection., Methods: For this evaluation we used 503 Enterobacteriaceae from 18 Dutch hospitals and 21 isolates previously confirmed to be pAmpC positive. All isolates were divided into three groups: isolates with 1) reduced susceptibility to ceftazidime and/or cefotaxime; 2) reduced susceptibility to cefoxitin; 3) reduced susceptibility to ceftazidime and/or cefotaxime combined with reduced susceptibility to cefoxitin. Two disk-based tests, with cloxacillin or boronic acid as inhibitor, and Etest with cefotetan-cefotetan/cloxacillin were used for phenotypic AmpC confirmation. Finally, presence of pAmpC genes was tested by multiplex and singleplex PCR., Results: We identified 13 pAmpC producing Enterobacteriaceae isolates among the 503 isolates (2.6%): 9 CMY-2, 3 DHA-1 and 1 ACC-1 type in E. coli isolates. The sensitivity and specificity of reduced susceptibility to ceftazidime and/or cefotaxime in combination with cefoxitin was 97% (33/34) and 90% (289/322) respectively. The disk-based test with cloxacillin showed the best performance as phenotypic confirmation method for AmpC production., Conclusions: For routine phenotypic detection of pAmpC the screening for reduced susceptibility to third generation cephalosporins combined with reduced susceptibility to cefoxitin is recommended. Confirmation via a combination disk diffusion test using cloxacillin is the best phenotypic option. The prevalence found is worrisome, since, due to their plasmidal location, pAmpC genes may spread further and increase in prevalence.

Methicillin-resistant Staphylococcus aureus (MRSA) has rapidly emerged worldwide, affecting both healthcare and community settings, and intensive livestock industry. The efficient control of MRSA strongly depends on its adequate laboratory detection. This guideline provides recommendations on the appropriate use of currently available diagnostic laboratory methods for the timely and accurate detection of MRSA in patients and healthcare workers. Herewith, it aims to standardise and improve the diagnostic laboratory procedures that are used for the detection of MRSA in Dutch medical microbiology laboratories.

Introduction: Gastrointestinal surgery is associated with a high incidence of infectious complications. Selective decontamination of the digestive tract is an antimicrobial prophylaxis regimen that aims to eradicate gastrointestinal carriage of potentially pathogenic microorganisms and represents an adjunct to regular prophylaxis in surgery., Material and Methods: Relevant studies were identified using bibliographic searches of MEDLINE, EMBASE, and the Cochrane database (period from 1970 to November 1, 2012). Only studies investigating selective decontamination of the digestive tract in gastrointestinal surgery were included., Results: Two randomized clinical trials and one retrospective case-control trial showed significant benefit in terms of infectious complications and anastomotic leakage in colorectal surgery. Two randomized controlled trials in esophageal surgery and two randomized clinical trials in gastric surgery reported lower levels of infectious complications., Conclusion: Selective decontamination of the digestive tract reduces infections following esophageal, gastric, and colorectal surgeries and also appears to have beneficial effects on anastomotic leakage in colorectal surgery. We believe these results provide the basis for a large multicenter prospective study to investigate the role of selective decontamination of the digestive tract in colorectal surgery.

Background: The significance of isolation of herpes simplex virus (HSV) type 1 from the lower respiratory tract in critically ill patients on mechanical ventilation is still unclear. In the current study, we used polymerase chain reaction techniques to quantify HSV-1 to further evaluate its role., Objectives: The hypothesis was that high loads reflect invasive pulmonary disease related to prolonged mechanical ventilation and increased mortality, as opposed to shedding from the upper respiratory tract, which leads to lower viral loads., Study Design: We prospectively studied 77 consecutive patients admitted to the intensive care unit and analyzed 136 tracheal aspirates or bronchoalveolar lavage fluids, taken when clinically indicated in the diagnostic workup of fever, radiologic pulmonary infiltrates, progressive respiratory insufficiency or combinations. Samples were cultured for bacteria and yeasts according to routine microbiological methods and HSV-1 loads were determined by real time quantitative PCR. Viral loads were expressed per number of cells recovered., Results: HSV-1 load was directly related to the simplified acute physiology score II (rs=0.47, P=0.04) when the first specimen taken proved positive for HSV-1. HSV-1 positivity concurred with Candida spp. colonization. Patients with and without a HSV-1 load did not differ with respect to pulmonary and systemic courses and vital outcomes., Conclusions: The data suggest that HSV-1 in the lower respiratory tract originates from shedding in the upper respiratory tract in about 30% of critically ill patients, following immune suppression and reactivation, without invasively infecting the lung. No attributable mortality was observed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Selective decontamination of the digestive tract (SDD) selectively eradicates aerobic Gram-negative bacteria (AGNB) by the enteral administration of oral nonabsorbable antimicrobial agents, i.e., colistin and tobramycin. We retrospectively investigated the impact of SDD, applied for 5 years as part of an infection control program for the control of an outbreak with extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in an intensive care unit (ICU), on resistance among AGNB. Colistin MICs were determined on stored ESBL-producing K. pneumoniae isolates using the Etest. The occurrence of both tobramycin resistance among pathogens intrinsically resistant to colistin (CIR) and bacteremia caused by ESBL-producing K. pneumoniae and CIR were investigated. Of the 134 retested ESBL-producing K. pneumoniae isolates, 28 were isolated before SDD was started, and all had MICs of <1.5 mg/liter. For the remaining 106 isolated after starting SDD, MICs ranged between 0.5 and 24 mg/liter. Tobramycin-resistant CIR isolates were found sporadically before the introduction of SDD, but their prevalence increased immediately afterward. Segmented regression analysis showed a highly significant relationship between SDD and resistance to tobramycin. Five patients were identified with bacteremia caused by ESBL-producing K. pneumoniae before SDD and 9 patients thereafter. No bacteremia caused by CIR was found before SDD, but its occurrence increased to 26 after the introduction of SDD. In conclusion, colistin resistance among ESBL-producing K. pneumoniae isolates emerged rapidly after SDD. In addition, both the occurrence and the proportion of tobramycin resistance among CIR increased under the use of SDD. SDD should not be applied in outbreak settings when resistant bacteria are prevalent.

The aim of this study was to determine the rate of carriage of ESBL-producing Enterobacteriaceae (ESBL-E) in the community in the Netherlands and to gain understanding of the epidemiology of these resistant strains. Faecal samples from 720 consecutive patients presenting to their general practitioner, obtained in May 2010, and between December 2010 and January 2011, were analysed for presence of ESBL-E. Species identification and antibiotic susceptibility testing were performed according to the Dutch national guidelines. PCR, sequencing and microarray were used to characterize the genes encoding for ESBL. Strain typing was performed with amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Seventy-three of 720 (10.1%) samples yielded ESBL-producing organisms, predominantly E. coli. No carbapenemases were detected. The most frequent ESBL was CTX-M-15 (34/73, 47%). Co-resistance to gentamicin, ciprofloxacin and cotrimoxazole was found in (9/73) 12% of the ESBL-E strains. AFLP did not show any clusters, and MLST revealed that CTX-M-15-producing E. coli belonged to various clonal complexes. Clonal complex ST10 was predominant. This study showed a high prevalence of ESBL-producing Enterobacteriaceae in Dutch primary care patients with presumed gastrointestinal discomfort. Hence, also in the Netherlands, a country with a low rate of consumption of antibiotics in humans, resistance due to the expansion of CTX-M ESBLs, in particular CTX-M-15, is emerging. The majority of ESBL-producing strains do not appear to be related to the international clonal complex ST131., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

Background: The worldwide prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is increasing rapidly both in hospitals and in the community. A connection between ESBL-producing bacteria in food animals, retail meat, and humans has been suggested. We previously reported on the genetic composition of a collection of ESBL-producing Escherichia coli (ESBL-EC) from chicken meat and humans from a restricted geographic area. Now, we have extended the analysis with plasmid replicons, virulence factors, and highly discriminatory genomic profiling methods., Methods: One hundred forty-five ESBL-EC isolates from retail chicken meat, human rectal carriers, and blood cultures were analyzed using multilocus sequence typing, phylotyping, ESBL genes, plasmid replicons, virulence genes, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE)., Results: Three source groups overlapped substantially when their genetic composition was compared. A combined analysis using all variables yielded the highest resolution (Wilks lambda [Λ]: 0.08). Still, a prediction model based on the combined data classified 40% of the human isolates as chicken meat isolates. AFLP and PFGE showed that the isolates from humans and chicken meat could not be segregated and identified 1 perfect match between humans and chicken meat., Conclusions: We found significant genetic similarities among ESBL-EC isolates from chicken meat and humans according to mobile resistance elements, virulence genes, and genomic backbone. Therefore, chicken meat is a likely contributor to the recent emergence of ESBL-EC in human infections in the study region. This raises serious food safety questions regarding the abundant presence of ESBL-EC in chicken meat.

Objective: To investigate whether a dog's superior olfactory sensitivity can be used to detect Clostridium difficile in stool samples and hospital patients., Design: Proof of principle study, using a case-control design., Setting: Two large Dutch teaching hospitals., Participants: A 2 year old beagle trained to identify the smell of C difficile and tested on 300 patients (30 with C difficile infection and 270 controls)., Intervention: The dog was guided along the wards by its trainer, who was blinded to the participants' infection status. Each detection round concerned 10 patients (one case and nine controls). The dog was trained to sit or lie down when C difficile was detected., Main Outcome Measures: Sensitivity and specificity for detection of C difficile in stool samples and in patients., Results: The dog's sensitivity and specificity for identifying C difficile in stool samples were both 100% (95% confidence interval 91% to 100%). During the detection rounds, the dog correctly identified 25 of the 30 cases (sensitivity 83%, 65% to 94%) and 265 of the 270 controls (specificity 98%, 95% to 99%)., Conclusion: A trained dog was able to detect C difficile with high estimated sensitivity and specificity, both in stool samples and in hospital patients infected with C difficile.

Cyanovirin-N (CV-N) is a mannose-binding lectin that inhibits HIV-1 infection by blocking mannose-dependent target cell entry via C-type lectins. Like HIV-1, Mycobacterium tuberculosis expresses mannosylated surface structures and exploits C-type lectins to gain cell access. In this study, we investigated whether CV-N, like HIV-1, can inhibit M. tuberculosis infection. We found that CV-N specifically interacted with mycobacteria by binding to the mannose-capped lipoglycan lipoarabinomannan. Furthermore, CV-N competed with the C-type lectins DC-SIGN and mannose receptor for ligand binding and inhibited the binding of M. tuberculosis to dendritic cells but, unexpectedly, not to macrophages. Subsequent in vivo infection experiments in a mouse model demonstrated that, despite its activity, CV-N did not inhibit or delay M. tuberculosis infection. This outcome argues against a critical role for mannose-dependent C-type lectin interactions during the initial stages of murine M. tuberculosis infection and suggests that, depending on the circumstances, M. tuberculosis can productively infect cells using different modes of entry.

Mycobacterial pathogens use specialized type VII secretion (T7S) systems to transport crucial virulence factors across their unusual cell envelope into infected host cells. These virulence factors lack classical secretion signals and the mechanism of substrate recognition is not well understood. Here we demonstrate that the model T7S substrates PE25/PPE41, which form a heterodimer, are targeted to the T7S pathway ESX-5 by a signal located in the C terminus of PE25. Site-directed mutagenesis of residues within this C terminus resulted in the identification of a highly conserved motif, i.e., YxxxD/E, which is required for secretion. This motif was also essential for the secretion of LipY, another ESX-5 substrate. Pathogenic mycobacteria have several different T7S systems and we identified a PE protein that is secreted by the ESX-1 system, which allowed us to compare substrate recognition of these two T7S systems. Surprisingly, this ESX-1 substrate contained a C-terminal signal functionally equivalent to that of PE25. Exchange of these C-terminal secretion signals between the PE proteins restored secretion, but each PE protein remained secreted via its own ESX secretion system, indicating that an additional signal(s) provides system specificity. Remarkably, the YxxxD/E motif was also present in and required for efficient secretion of the ESX-1 substrates CFP-10 and EspB. Therefore, our data show that the YxxxD/E motif is a general secretion signal that is present in all known mycobacterial T7S substrates or substrate complexes.

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.