Production by Clostridium difficile Isolates
M. Scholing, L. Thiel, G.B. Linde
GGD Amsterdam, Streeklaboratorium, Amsterdam
Introduction: Clostridium difficile associated disease
(CDAD) is an urgent threat to public health. Reliable rapid toxin detection on stool samples and C. difficile isolates is necessary to control outbreaks. Toxogenic culture performed on unformed stool specimen is the gold standard for diagnosing CDAD. The method of confirming toxin production by C. difficile isolates is not specified in guidelines and many laboratories use either an enzyme immunoassay, the cytotoxicity neutralization assay (CNA) or
PCR detecting genes encoding for toxin A and B. To speed up toxicity confirmation we compared the C. DIFF QUIK CHEK
COMPLETE (Quik Chek) directly on C. difficile isolates with our current practice, VIDAS Clostridium difficile A & B (Vidas) on overnight liquid culture.
Methods: C. difficile culture was routinely performed on
Cycloserine Cefoxitin Fructose Agar (bioMrieux, France). Isolates identified as C. difficile by colony morphology, Gramstaining and proline aminopeptidase were subse- quently incubated overnight in Brain Heart Infusion (BHI) broth. To confirm toxin production 200l BHI broth was used for the Vidas assay (bioMrieux, France), an automated ELFA detecting C. difficile toxin A and B. This method was compared by subjecting five C. difficile colonies directly to the Quik Chek (Techlab, USA), a rapid enzyme immunoassay for the detection of glutamate dehydrogenase and C. difficile toxin A and B. In addition, direct testing of stool samples by Quik Check according manufacturers instructions was performed.
Results: We cultured C. difficile isolates from 39 stool
samples. 26 Isolates were confirmed to produce toxin by the Vidas, 6 isolates revealed an equivocal result and 7 tested negative. The Quik Chek was positive in 32/32 strains that tested positive or equivocal in the Vidas (sensitivity 100%) and was negative in 7/7 strains that tested negative in the Vidas (specificity 100%). The sensitivity of the Quik Chek directly on stool samples was 25/32 (78%) if compared with toxogenic culture confirmed by the Vidas.
Conclusion: To our knowledge this is the first report of
confirmation of toxogenic culture. In this context the Quik Chek is a rapid and reliable alternative to the Vidas.
P022
Effects of N deposition on diazotrophic activity and distri- bution of microorganisms associated with Sphagnum magellanicum
M.A.R. Kox, C. Lüke, E. van Elzen, C. Fritz, M.S.M. Jetten, L.P.M. Lamers, K.F. Ettwig
Institute for Water and Wetland Research, Radboudumc, Dept. of Ecological Microbiology, Nijmegen
Introduction: Sphagnum-dominated peatlands are
important carbon (C) sinks and greenhouse gas filters.1
These ecosystems are generally N-limited, but increasing levels of atmospheric N-deposition due to anthropogenic activities have the potential to shift them from C-sinks to C-sources.2 N-deposition effects on plant communities
have been well studied, but little is known about the effects on the N-fixing microbial community associated with Sphagnum spp. in peatlands.3
Objectives: We aim to quantify the short-term and
memory’ effects of high N-deposition on diazotrophic activity and community associated with Sphagnum magel- lanicum in ombrotrophic bogs. Ultimately we want to understand the influence of N-deposition on ecosystem functioning
Materials and methods: N2 fixation rates associated with S. magellanicum from pristine and high-N-deposition sites were measured by acetylene reduction assays, calibrated with 15N
2 (with and without C2H2 ). DNA was extracted
from the same samples and diazotrophic community diversity was analysed by clone library construction of nifH genes and by amplicon sequencing of nifH genes (Ion Torrent PGMTM).
Results & Conclusion: N-fertilisation had a negative effect
on N2 fixation rates in mosses from both pristine sites and areas with high N-deposition. Labelling showed that acetylene decreased 15N
2 uptake, questioning the usefulness
of this commonly used method. Community diversity analyses showed a diverse diazotrophic community, of which the majority of nifH sequences belonged to a cluster without cultured representatives and unclear affiliation.
References
1. Gorham E. Ecol. appl. 1991;1:182-95. 2. Bragazza l, et al. PNAS. 2006;103,19386-9. 3. Andersen R, et al. Soil Biol Biochem. 2012;57,979-94.
P023
The pathology of antibiotic-induced intestinal outgrowth by multi-drug resistant Enterococcus faecium
A.P.A. Hendrickx, J. Top, M.J.M. Bonten, R.J.L. Willems
University Medical Center Utrecht, Dept. of Medical Microbiology, Utrecht
Introduction: Enterococcus faecium was considered a
commensal of the microbiota within the large intestine of the human gastrointestinal tract, but is now recognized as a multi-drug resistant (MDR) pathogen. In hospitals, patients are typically administered antibiotics that perturb and reduce the intestinal commensal microbiota, leading to outgrowth of MDR-E. faecium, subsequent spread, infections, and hospital outbreaks. However, intestinal blooming’ byMDR-E. faecium has not been thoroughly studied. Therefore, the objective was to analyze the pathology of antibiotic-induced outgrowth of MDR-E.
faecium within the large intestine.
Methods:. Four BALB/c mice were treated for 2 days with
ceftriaxon prior inoculation with 1x1010 CFU’s of MDR-E.
faecium E1162 (Group A), and left on cefoxitin antibiotics
during the experiment. Group B is similar as Group A, but animals did not receive antibiotics. Three mice were left untreated (Group C). Colonization of E1162was monitored by enumeration of CFU’s from faeces. After 10 days intestinal parts were: (1) formalin fixed, paraffine embedded (FFPE), thin sectioned and subjected to Gram-, Hematoxylin and Eosin- (H&E), and Periodic acid-Schiff (PAS)-staining followed by light microscopy (LM); and (2) fixed in 2% glutaraldehyde, serially dehydrated, and analyzed by scanning electron microscopy (SEM).
Results: Enumerating CFU’s demonstrated that Group A
mice were highly colonized with E1162 (10x1010 CFU/gram
faeces), while Group B mice were low-level colonized (1x105
CFU/gram faeces), and Group C revealed no enterococci. SEM on cecum and colon tissue confirmed the presence of only cocci on the epithelial cells (Group A) in high levels, and Group B +C revealed dense microbiota consisting of various rod-shaped bacteria, cocci and spirochetes. In addition, LM on H&E-, Gram- and PAS-stained FFPE cecum and colon sections of Group A mice showed absence of microbiota and enrichment of enterococci aligned onto the thin mucus layer and surrounding the undigested faecal material. In the colon, enterococci were predomi- nantly present in areas of mucus secreting Goblet cells and at the apical side of columnar epithelium. Group B + C mice had a very diverse Gram-negative and -positive microbiota separated by a thick mucus layer from the epithelium.
Conclusion: Antibiotics reduce the intestinal microbiota
and mucus layer and allow MDR-E. faecium to bloom in the gut. The finding that enterococci predominate at the apical
side of the epithelium and surround faeces likely enables the bacteria to interact with host receptors and to spread after defecation.
P024
Capability of nitrification and denitrification by
Methylacidiphilum fumariolicum SolV to handle nitrosative
stress
S. Mohammadi, A. Pol, M. Jetten, H. op den Camp
IWWR Radboudumc, Dept. of Microbiology, Nijmegen
Methane is a powerful greenhouse gas, which is released to the atmosphere both from natural and anthropogenic sources. Understanding sources and sinks of methane is important for future models of climate change on our planet. Methane oxidizing microbes are one of the most important biological sinks of methane. Methanotrophic bacteria are distinctive in their ability to exploit methane as the only carbon and energy source. Recently, the new acidiphilic methanotrophic bacterium Methylacidiphylum
fumariolicum SolV belonging to the Planctomycetes/
Verrucomicrobia/Chlamydiae superphylum was discovered, and it has been shown that its growth is dependent on the presence of lanthanides.
Methane and ammonia are structurally comparable molecules. Thus, methanotrophs and nitrifiers have many features in common. They use many similar important enzymes, most particularly the ammonia monooxygenase/ particulate methane monooxygenase enzyme family. The two groups are suggested to have a common evolu- tionary history. Nitrifiers are able to oxidize methane, and methanotrophs are capable of nitrification. In the draft genome of strain SolV, genes for nitrite reduction (nirK) and nitric oxide reduction (norB, norC), were identified but the gene to encode nitrous oxide reductase was absent. A
haoAB gene cluster encoding hydroxylamine oxidase was
also identified, suggesting the ability of nitrification and nitrosative stress handling.
In order to study the effects of nitrosative stress on strain SolV two chemostat bioreactors were used: (a) In the first bioreactor, cells were grown on methane, and nitrate was used as the nitrogen source. (b) In the second reactor, cells were grown on hydrogen, and ammonium was used as the nitrogen source. In both conditions, oxygen was limited. Using 15N stable isotopes in combination with a nitric oxide
analyser and GC-MS, we showed that the cells grown on methane (nitrate limited condition; pH 6.2) are able to perform denitrification by converting nitrite to nitric oxide and further to nitrous oxide in the absence of oxygen. Moreover, washed cells from the same chemostat reactor were used in batch tests at pH 6.2. Once ammonium was added to these cells with methane concentration ranging from 0 to 5% (v/v), nitrite production was measured.
Interestingly, we showed that nitrite production rate increases while methane concentration decreases, suggesting the capability of nitrification by strain SolV. Furthermore, we demonstrated that the cells grown on hydrogen (N-source: ammonium) convert ammonium to significant amounts of nitrite at pH 5 - 5.5 in the oxygen limited condition. Moreover, a rapid production of nitric oxide and decrease of nitrite concentration were observed when oxygen was absent, suggesting the action of a denitrification pathway converting nitrite to nitric oxide. Additionally, a decrease in nitric oxide levels resulted in an increase in nitrous oxide concentrations, suggesting conversion of nitric oxide to nitrous oxide by nitric oxide reduction enzymes under anoxic condition.
This study showed that strain SolV, performs nitrification, when methane is limited or completely absent, and carries out denitrification by converting nitrite to nitric oxide and further to nitrous oxide under anoxic conditions. These observations also indicate that strain SolV is capable of handling nitrosative stress.
P025
Comparison of the Copan Eswab™ with the Medical Wire S-Transwab™ for microbial nucleic acid testing
N.M. van Maarseveen, M. Zegers, R. Neuteboom
Rijnstate, Dept. of Medical Microbiology and Immunology, Velp
Introduction: With the purchase of a new sample
processing unit (Copan WASP) we had to replace our current sample collection swab (Medical Wire S-Transwab) with the Copan Eswab. The Medical Wire S-Transwab is a foam-tipped swab, whereas the Copan Eswab is a nylon flocked swab. Both swab systems contain modified Liquid Amies medium as transport medium.
In this study we compared the Copan Eswab with the Medical Wire S-Transwab for microbial nucleic acid testing by analyzing different storage times and temperatures.
Methods: Suspensions were made of several viral
isolates (Influenza A, Influenza B, Respiratory Syncytial Virus, Herpes Simplex Virus, Varicella Zoster Virus) and bacterial strains (Neisseria gonorrhoeae, Mycoplasma
genitalium and Legionella pneumophila). Each suspension
was aliquoted (100l/ well) in a 96-well plate and subse- quently absorbed by either the Eswab or the S-Transwab. After absorption, swabs were inserted in their respective transport medium and incubated at two different storage temperatures (room temperature and 4C) and different storage times (0 hours, 24 hours, 48 hours and 72 hours). After incubation, 200 l of transport medium was used for RNA/DNA extraction using the bioMrieux easyMAG extraction system followed by an in-house multiplex real-time PCR assay on an ABI 7500 system. For Neisseria
gonorrhoeae, DNA extraction was performed with the
Abbott mSample Preparation SystemDNA on the Abbott
m2000sp system, followed by the Abbott RealTime CT/
NG assay.
Results: For Influenza A, Influenza B, Herpes Simplex
Virus, Varicella Zoster Virus, Neisseria gonorrhoeae and
Mycoplasma genitalium the Eswab and the S-Transwab
yielded similar results for up to 72 hours of storage, irrespective of the storage temperature.
For the Respiratory Syncytial Virus, Ct-values slightly increased over time with approximately 2-3 Ct-values in both the Eswab and the S-Transwab. This was observed for both storage temperatures.
Only for Legionella pneumophila a difference was observed between the two swabs. For the Eswab mean Ct-values were consistently lower than the S-Transwab, with a mean Ct-value of 32.1 for the Eswab and 34.5 for the S-Transwab. This difference in Ct-values was seen for all different storage times and temperatures.
Conclusion: For all targets, with the exception of
Respiratory Syncytial Virus and Legionella pneumophila, the Copan Eswab and the Medical Wire S-Transwab performed equally well and yielded stable results for at least 72 hours, irrespective of the storage temperature. Thereby, both swabs seem suitable for use in microbial nucleic acid testing.
P026
Terminal anaerobic processes in hypersaline soda lakes
D.Y. Sorokin
TU Delft, Dept. of Biotechnology, Delft
Soda lakes represent a unique double extreme habitat characterized by high salt and alkalinity of its soda brines. Despite severe conditions, two decades of intense micro- biological studies demonstrated a fully functional and very diverse prokaryotic system in soda lakes even close to soda saturation. Until recently, however, the two key terminal anaerobic processes of microbial sulfidogenesis and methanogenesis at extremely halo-alkaline conditions have been poorly understood.
Our 8 years study of anoxic sediments of hypersaline soda lakes is south-western Siberia in general demon- strated sulfidogenesis is a dominant terminal electron sink. The rates of sulfide production from sulfate, thiosulfate and sulfur were comparable with the rates in marine sediments and only significantly lowered at salt saturation. Sulfate reduction was most vulnerable to salt inhibition as compared to thiosulfate and sulfur reduction. Maximum activity was detected with formate as e-donor and elemental sulfur as e-donor, while sulfate reduction rates were the lowest. The key players were represented by obligately natronophilic SRB of the orders
Desulfovibrionales and Desulfobacterales. The lithotrophic
growth by thiosulfate and sulfite disproportionation was a common trait among the soda lake SRB. Acetate was directly utilized as electron donor only at sulfur-reducing conditions, while at sulfate-reducing conditions acetate was oxidized syntrophically. Bacterial sulfur reduction at moderate salinity was a function of a very specialized group of fatty acid-utilizing sulfur-reducing natronophilic bacteria from the class Chrysiogenetes, while at soda- saturated conditions sulfur-respiring natronoarchaea have been discovered for the first time.
Methanogenesis in soda lakes, similar to other saline sulfate-rich habitats was mostly active with methylated substrates. However, in case of limitation of SRB activity, also lithotrophic and even acetoclastic methanogenesis was manifesting. In the absence of SRB competition, the potential rates of methanogenesis from formate were close to the rates in marine sediments. Totally unexpected, however, was the fact that lithotrophic methanogenesis was possible in nearly saturated soda brines. The main players responsible for the methylotrophic process in soda lakes were identified as obligately natronophilic Methanolobus (moderate salinity) and Methanosalsum (extreme salinity), while obligate natronophilic representatives of the genus
Methanocalculus were universally dominating the litho-
trophic process from low to extreme salinity.
Overall results so far demonstrated that, despite generally very low energy efficiency, the secondary anaerobes still manage to function at doubly extreme conditions of soda brines. In that respect, the soda lakes seem to be funda- mentally different from hypersaline habitats with neutral pH.
P027
Towards understanding the molecular basis of the foreign body response and biomaterial-associated infections
M. Riool, V. Jaspers, L. de Boer, O.W. Stockhammer, S.A.J. Zaat
Academic Medical Center, Dept. of Medical Microbiology, Amsterdam
Introduction: Infection of inserted or implanted medical
devices (‘biomaterials’) can have disastrous consequences, including removal of the device. Implantation of a biomaterial provokes an inflammatory response known as the ‘foreign body response’. Staphylococcus epidermidis is the major cause of biomaterial associated infection (BAI), which in absence of a foreign body hardly ever cause infection. Formation of biofilms on the biomaterial surface is generally considered the main reason for these persistent infections, but Staphylococcus epidermidis has been shown to survive inside macrophages around biomaterials implanted in mice (Boelens et al. 2000), and was retrieved
from peri-catheter tissue in humans (Broekhuizen et
al. 2008), showing that the tissue around implants is a
second niche for infection. In order to understand the high infection-susceptibility of tissue around biomaterials, we aimed to unravel the molecular basis of the foreign body reaction and of biomaterial-associated infection. We assessed the gene expression underlying the foreign body response to titanium over time and the influence of S.
epidermidis on this response.
Methods: Four experimental groups were compared in the
biomaterial-associated infection mouse model: a) sham surgery (no implantation of a biomaterial), b) implantation of a titanium biomaterial, c) sham surgery with an S.
epidermidis infection, and d) implantation of a titanium
biomaterial combined with an S. epidermidis infection. At 1 and 6 hours and 2, 4, 9, 14 and 21 days, bacterial coloni- zation, histology and gene expression were analyzed. To characterize multiple cellular immune responses in single microscopic slides of mouse tissue with both implant and bacterial infection, an immunohistological staining protocol using multiple spectral imaging was developed. Gene expression was recorded using Affimetrix Mouse Gene-ST microarrays.
Results: The histology and gene expression patterns
showed distinct differences between sham (i.e. only surgery) and biomaterial groups possibly related to the foreign body response, and between biomaterial without and with infection. The analysis of the expressed gene sets is presently ongoing.
Conclusion: These results are a powerful start towards
understanding the molecular basis of the foreign body response and biomaterial-associated infection.
P028
Detection of oxacillinases and other beta-lactamases in Acinetobacter baumannii using LC-MS/MS
H. Trip1, J.A. Majchrzykiewicz-Koehorst1, A.G. Hulst1, K.
Mende2, C.K. Murray2, H.J. Jansen3, A. Paauw1
1TNO, CBRN Protection, Rijswijk, 2Brooke Army Medical
Center, Fort Sam Houston, San Antonio, USA, 3Ministry of
Defence, CEAG
Introduction: For effective treatment of bacterial
infections, rapid identification of antibiotic resistance against first line antibiotics has become of increasing importance, especially due to the emergence and fast spread of antibiotic resistance. The nosocomial pathogen
Acinetobacter baumannii, intrinsically already resistant to
first and second generation cephalosporins, has a high capacity of acquiring antibiotic resistance. Therefore the spread of resistance against carbapenems, a class of last resort beta-lactam antibiotics, has raised great concerns. Carbapenem resistance in A. baumannii is mainly caused
by expression of oxacillinases, which can be subdivided in different subclasses such as OXA-23-like and OXA-54- like. In this study, LC-MS/MS was employed to detect and identify oxacillinases and other beta-lactamases in clinical isolates of A. baumannii, with the goal to evaluate its potential for detection of antibiotic resistance.
Methods: Thirty isolates, acquired from a military
hospital (Brooke Army Medical Center,US), were tested for resistance against three carbapenems and ceftazidime, a third generation cephalosporin, by classical growth inhibition tests. Each isolate was PCR tested for 15 different beta-lactamase genes. LC-MS/MS analysis was performed on whole cell lysates, following a fast sample preparation