Fosfomycin Etest for Enterobacteriaceae: Interobserver and interlaboratory agreement.

OBJECTIVES: The increasing use of fosfomycin requires reliable susceptibility testing in clinical practice. The reference standard, agar dilution (AD), is rarely used in routine settings. The fosfomycin Etest (BioMérieux) is frequently used, although reading MICs can be hampered by the interpretation of the growth of macrocolonies in the inhibition zone. We investigated the interobserver (IO), interlaboratory (IL), and interobserver-interlaboratory (IOIL) agreement of the fosfomycin Etest and evaluated the agreement with AD. METHODS: Etests were performed for 57 extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae of four bacterial species (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Enterobacter cloacae) in two laboratories. Photographs of fosfomycin Etests were interpreted by four observers following manufacturer's instructions. RESULTS: Essential agreement (EA) and categorical agreement (CA) between Etest and AD were 57% and 89% (κ-value 0.68), respectively, with an underestimation of Etest interpretations compared with AD of 0.26 (95% confidence interval [CI] 0.03-0.48) 2-fold dilutions. Between Etest observations, IO-EA and -CA were reached in 82% and 94% of comparisons; IL-EA and -CA in 38% and 85% of comparisons; and IOIL-EA and -CA in 40% and 85% of comparisons, respectively. Agreement of the Etest with AD and between Etests was better for E. coli than for other species. Ignoring all macrocolonies and haze during Etest interpretation improved the agreement with AD (CA κ-value 0.80) and between Etests (CA κ-value from 0.68 to 0.81). CONCLUSIONS: In this study on 57 ESBL-producing Enterobacteriaceae, IOIL agreement was low with an EA of 40% and a CA of 85%, affected most by IL agreement and to a lesser extent by IO agreement.

Comparison of MALDI-TOF MS and AFLP for strain typing of ESBL-producing Escherichia coli.

Typing of bacterial isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) potentially provides an efficient on-site method to monitor the spread of antibiotic-resistant bacteria and rapidly detect outbreaks. We compared MALDI-MS typing results to those of amplified fragment length polymorphism (AFLP) in a collection of 52 ESBL-producing Escherichia coli, isolated in a Dutch nursing home with an on-going outbreak of ST131 E. coli. Specific MALDI types were defined based on spectral data from four replicate colony samples of isolates grown on Columbia agar using multivariate statistical procedures. Type-specific superspectra were computed for four E .coli MALDI-types and tested for the potential of rapid and automated typing. The effect of different incubation conditions on typing performance was tested by analysing five isolates incubated for 24 h and 48 h on five different media. Types defined based on MALDI spectra were largely in agreement with the AFLP results, although some MALDI types comprised of more than one AFLP type. In particular, isolates belonging to ST131 showed distinct mass patterns. The proportion of isolates correctly assigned was substantially lower for isolates incubated on Sabouraud-dextrose and Drigalski agars for 24 h, and for those incubated for 48 h (all media). Our results show that the identification of type-specific peaks potentially allows direct typing of isolates belonging to specific clonal lineages. Both incubation time and media affected type assignment, suggesting that there is a need for a careful standardization of incubation time and culturing conditions when developing MALDI-typing schemes for E. coli.

Rectal Carriage of Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae in Hospitalized Patients: Selective Preenrichment Increases Yield of Screening.

This study evaluated the added value of selective preenrichment for the detection of rectal carriage of extended-spectrum-beta-lactamase-producing Enterobacteriaceae (ESBL-E). ESBL-E rectal carriage was identified in 4.8% of hospitalized patients, and 25.9% of ESBL-E rectal carriers were identified with selective preenrichment only.

Extended-spectrum ß-lactamase producing Klebsiella spp. in chicken meat and humans: a comparison of typing methods.

Recently, chicken meat was identified as a plausible source of extended-spectrum ß-lactamase (ESBL) -producing Escherichia coli in humans. We investigated the relatedness of ESBL-producing Klebsiella spp. in chicken meat and humans. Furthermore, we tested the performance of SpectraCell RA(®) (River Diagnostics), a new typing method based on Raman spectroscopy, in comparison with multilocus sequence typing (MLST) for Klebsiella pneumoniae. Twenty-seven phenotypically and genotypically confirmed ESBL-producing Klebsiella spp. isolates were typed with MLST and SpectraCell RA. The isolates derived from chicken meat, human rectal swabs and clinical blood cultures. In the 22 ESBL-producing K. pneumoniae isolates, CTX-M15 was the predominant genotype, found in five isolates of human origin and in one chicken meat isolate. With MLST, 16 different STs were found, including five new STs. Comparing the results of SpectraCell RA with MLST, we found a sensitivity of 70.0% and a specificity of 81.8% for the new SpectraCell RA typing method. Therefore, we conclude that SpectraCell RA is not a suitable typing method when evaluating relationships of ESBL-producing Klebsiella spp. at the population level. Although no clustering was found with isolates of chicken meat and human origin containing the same ESBL genes, MLST showed no clustering into distinctive clones of isolates from chicken meat and human origin. More studies are needed to elucidate the role of chicken meat in the rise of ESBL-producing Klebsiella spp. in humans.

Evaluation of brilliance MRSA 2 agar for detection of methicillin-resistant Staphylococcus aureus in clinical samples.

We compared 2 chromogenic media (Oxoid Brilliance MRSA 2 agar [Thermo Fisher Scientific] and MRSA-ID [bioMérieux]) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in 1,368 hospital samples. For both media, broth enrichment was essential to obtain satisfactory diagnostic performance. Although with direct cultures only, the diagnostic performance (particularly sensitivity) of Brilliance MRSA 2 agar appears better than that of MRSA-ID, no difference in sensitivity or specificity between the media was detected after inclusion of an enrichment step.

Comparison of SpectraCell RA typing and multilocus sequence typing for extended-spectrum-ß-lactamase-producing Escherichia coli.

Multilocus sequence typing (MLST) is one of the most reliable methods for typing of Escherichia coli, including extended-spectrum-ß-lactamase-producing E. coli (ESBL-EC). We investigated the performance of a new typing method, SpectraCell RA (River Diagnostics, Madison, WI), in comparison on MLST on a well-defined collection of ESBL-EC isolates obtained from chicken meat and humans. Ninety-two ESBL-EC isolates obtained from meat and 59 ESBL-EC isolates obtained from human rectal swabs and clinical blood cultures were typed using MLST and SpectraCell RA. The sensitivity and specificity of SpectraCell RA were calculated, using MLST as a reference method. Subsequently, the results of SpectraCell RA were used to determine the relatedness of ESBL-EC isolates from chicken and humans. Using MLST as the gold standard, the performance of SpectraCell RA was evaluated for 3 different cutoff values: 0.99975, 0.99955, and 0.99935. Depending on the cutoff value, the sensitivity was mediocre to unacceptably low, with values of 9.4%, 43.9%, and 66.7%, respectively. When sensitivity increased, the specificity decreased rapidly, from 95.6% to 69.8% and 34.4%, respectively. Also, the number of clusters containing both human and meat samples varied from 0 (0.0%) to 14 (38.9%). Our study shows that SpectraCell RA is not a suitable typing method for ESBL-EC when evaluating relationships of ESBL-EC at the population level.

Effect of manganese in test media on in vitro susceptibility of Enterobacteriaceae and Acinetobacter baumannii to tigecycline.

We assessed the effect of increasing manganese concentrations in test media (0.001 to 1,024 mg/liter) on MICs of tigecycline. For both broth microdilution (BMD) and Etests, this effect was negligible for physiological concentrations, but MICs increased when concentrations exceeded 8 mg/liter. Susceptibility testing should be performed on media with standardized low manganese content.

Evaluation of the DiversiLab typing method in a multicenter study assessing horizontal spread of highly resistant gram-negative rods.

The worldwide prevalence of highly resistant Gram-negative rods (HR-GNR) is increasing rapidly. Reliable typing methods are needed to detect and control outbreaks and to monitor the effectiveness of infection control programs in endemic situations. In this study, we investigated the performance of the DiversiLab typing method in comparison with the amplified fragment length polymorphism (AFLP) typing method. Six hundred fifty-three HR-GNR isolates, which were obtained during a 6-month prospective survey in 18 Dutch hospitals, were typed by AFLP and DiversiLab. Subsequently, the sensitivity and specificity of DiversiLab were calculated, using AFLP as the reference method. In addition, results were compared by means of epidemiological linkage, and Cohen's kappa for agreement was calculated. DiversiLab considered significantly more isolates (275) to belong to a cluster than AFLP (198) (P < 0.001). In direct comparison, the sensitivity was 83.8%, and the specificity was 78.6%. When epidemiological linkage was included in the analysis, DiversiLab considered eight isolates as secondary cases, which were considered unique in AFLP. Only two secondary cases, according to AFLP, were missed by DiversiLab. This results in a kappa for agreement of 0.985. In daily practice, a typing method has to be used in combination with epidemiological information. When this was done, DiversiLab was shown to be a reliable method for the typing of HR-GNR. This, in combination with the ease of use and the speed, makes DiversiLab an appropriate method for screening in routine clinical practice. When a cluster is suspected and the consequences of these findings are substantial, a confirmatory analysis should be performed.

Highly resistant gram-negative microorganisms: incidence density and occurrence of nosocomial transmission (TRIANGLe Study).

OBJECTIVES: The objectives of this study were to determine the incidence density and the occurrence of horizontal spread of highly resistant gram-negative rods (HR-GNRs) in Dutch hospitals. The factors that influence these outcome measures were also investigated. METHODS: All patients with HR-GNRs, as determined by sample testing, who were hospitalized in 1 of 18 hospitals during a 6-month period (April through October 2007) were included in this study. For all available isolates, the species was identified, susceptibility was determined (including the presence of extended-spectrum ß-lactamases [ESBLs]), and molecular typing was performed. On the basis of a combination of species identification, molecular typing, and epidemiological data, the occurrence of nosocomial transmission was determined. RESULTS: The mean incidence density of patients with HR-GNRs was 55 per 100,000 patient-days (cumulative incidence, 39 per 10,000 patients admitted). A facility being a university hospital was a statistically significant (P = .03) independent determinant of a higher incidence of patients with HR-GNRs. The majority of HR-GNR isolates were ESBL producers. The adjusted transmission index-the ratio between secondary and primary cases-in the participating hospitals ranged from 0.0 to 0.2. The overall adjusted transmission index of HR-GNRs was 0.07. No determinants for a higher transmission index were identified. DISCUSSION: The nosocomial transmission rate of HR-GNRs was relatively low in all hospitals where well-established transmission-based precautions were used. The incidence density of patients with HR-GNRs was higher in university hospitals, probably due to the patient population and the complexity of the care provided.

Laboratory detection of extended-spectrum-beta-lactamase-producing Enterobacteriaceae: evaluation of two screening agar plates and two confirmation techniques.

The worldwide prevalence of extended-spectrum-beta-lactamase-producing ESBL-producing Enterobacteriaceae (ESBL-E) is increasing, making the need for optimized detection techniques more urgent. In this study we investigated the performance of two ESBL-E screening and two ESBL-E confirmation techniques. In accordance with the Dutch national guidelines (www.wip.nl), a collection of 642 highly resistant Enterobacteriaceae strains, as identified by Vitek2, was used to test the performances of two screening techniques (EbSA ESBL agar plate and ChromID ESBL agar plate) and of two confirmation techniques (MIC-strip ESBL and Vitek2 ESBL test panel). The individual test results were compared by using Etest, followed by a combination disk test if Etest results were inconclusive. Among group 1 isolates (Escherichia coli, Klebsiella spp., Proteus spp., Salmonella spp., and Shigella spp.) 291 (57.6%) were ESBL-E, versus 65 (47.4%) in group 2 (Enterobacter spp., Citrobacter spp., Morganella morganii, Serratia spp., and Providencia spp.). The sensitivities of all four tests for group 1 were comparable (EbSA, 96.6%; ChromID, 97.3%; MIC-strip, 99.6%; and Vitek2, 95.1%). The specificities of the EbSA and ChromID were the same (93.9%). However, the confirmation techniques produced many inconclusive test results, which reduces the applicability in routine laboratories. Only the two screening agar plates were validated for ESBL testing of group 2 microorganisms. They showed comparable sensitivities; however, the EbSA screening agar plate had a significantly higher specificity (78.6% versus 44.3%). In conclusion the screening agar plates performed better than the two confirmation techniques. The EbSA agar plate had the best overall performance.

Clinical evaluation of Bio-Rad MRSASelect™ medium for the detection of livestock-associated methicillin-resistant Staphylococcus aureus.

Bio-Rad MRSASelect™ medium was evaluated for its ability to recover methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples of pig farmers and their household members. In total, 257 samples were inoculated on Bio-Rad MRSASelect™ medium with and without broth enrichment and on bioMérieux MRSA ID with broth enrichment. A sample was considered to be positive if at least one of the media grew MRSA. The sensitivity of Bio-Rad MRSASelect™ medium without broth enrichment was 63.9%. With broth enrichment, the sensitivity increased to 98.4%. The specificity was 95.4% both with and without broth enrichment. In conclusion, Bio-Rad MRSASelect™ medium as well as MRSA ID medium are reliable methods to detect MRSA carriage when used in combination with broth enrichment. The directly inoculated MRSASelect™ medium was statistically significantly less sensitive than the two media after broth enrichment.

Evaluation of a fourth-generation latex agglutination test for the identification of Staphylococcus aureus.

In this study, we evaluated a fourth-generation agglutination assay (Staph Plus; DiaMondiaL[DML]) for the rapid identification of Staphylococcus aureus. First, comparison with three third-generation assays (Slidex Staph Plus, bioMérieux; Staphaurex Plus, Murex Diagnostics; Pastorex Staph-Plus, Sanofi Diagnostics Pasteur) was performed on a predefined strain collection: 265 coagulase-negative staphylococci (CNS), 266 methicillin-resistant S. aureus (MRSA) and 262 methicillin-susceptible S. aureus (MSSA) strains ("strain study"). Second, patient material-derived strains (883 CNS, 847 MSSA and 135 MRSA) were tested concurrently with both the DML and Slidex assays ("daily practice study"). In the strain study, the overall sensitivity and specificity of the DML, Slidex, Staphaurex and Pastorex assays were 99.2% and 100%, 98.1% and 100%, 95.2% and 100%, and 98.2% and 98.8%, respectively. Using the respective tests, the result was indeterminate in 0.0%, 0.6%, 0.4% and 1.5% of the strains. Overall, the sensitivity of the DML and Slidex assays were comparable in both sub-studies. However, in MRSA strains, the sensitivity of the DML assay was significantly lower than the Slidex assay. The specificity of the Slidex assay was significantly higher than the DML assay. However, the percentage of indeterminate results was much higher for the Slidex than the DML assay. In conclusion, the presumptive identification of S. aureus by the DML assay proved to be equal to third-generation latex agglutination assays.

In vitro activity of tigecycline against methicillin-resistant Staphylococcus aureus, including livestock-associated strains.

The in vitro activity of tigecycline was determined using a well-defined collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 202), including 33 livestock-associated strains. Susceptibility testing was performed using the Etest system. Among the 202 MRSA strains, three (1.5%) had a minimum inhibitory concentration (MIC) value for tigecycline greater than 0.5 mg/l, which are considered to be resistant. When these strains were tested using Iso-Sensitest medium, the MICs were substantially lower and no resistance was found. This discrepancy warrants further investigations into the preferred test conditions for tigecycline. In conclusion, tigecycline showed good activity against MRSA strains in vitro.

Performance of Oxoid Brilliance MRSA medium for detection of methicillin-resistant Staphylococcus aureus: an in vitro study.

Oxoid Brilliance MRSA was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus. A well-defined collection of staphylococci was used (n = 788). After 20 h incubation, the sensitivity was 99.6% and the specificity was 97.3%. This new medium is a highly sensitive method of screening for MRSA.

Coexpression of GSK-3beta corrects phenotypic aberrations of dorsal root ganglion cells, cultured from adult transgenic mice overexpressing human protein tau.

Coexpression of constitutively active GSK-3beta[S9A] rescued the axonal pathology induced by overexpression of human tau in transgenic mice (Spittaels et al., (2000) J. Biol. Chem. 275, 41340-41349). We isolated dorsal root ganglion (DRG) neuronal cultures from adult tau4R- and tau4R x GSK-3beta-transgenic mice to define the mechanisms at the cellular and subcellular level. DRG from tau4R-transgenics showed a reduced sprouting capacity while density and stability of microtubules in the axonal processes were significantly increased. Video-enhanced contrast microscopy demonstrated a dramatic inhibition of fast axonal transport. Coexpression of GSK-3beta increased tau phosphorylation and reversed the effects on microtubule stability and saltatory motion. In DRG from GSK-3beta single transgenics, increased tau phosphorylation was evident without any major effects on microtubule stability or axonal transport. These observations support the hypothesis that excess tau competed with motor-proteins for binding to microtubules and/or that a rigid microtubular system inhibits axonal transport.

In vitro correlation between two colorimetric assays and the pyruvic acid consumption by fibroblasts cultured to determine the sodium laurylsulfate cytotoxicity.

The target of this research was to determine the cytotoxicity of sodium laurylsulfate on single-layer cultures of human fibroblasts, using two colorimetric methods (neutral red and MTT tests) and the evaluation of the pyruvic acid consumption by the cells. For the determination of the cytotoxicity by colorimetric tests, we have determined the absorbance at 540 nm using a spectrophotometer. Pyruvic acid, present in the culture medium, is the mitochondria's C3 energetic metabolite. So, a measure of the cell's consumption of pyruvic acid was developed. The reaction is as follows: Pyruvic acid + NADH --> Lactic acid + NAD+ and the enzyme employed is the LDH (lactate dehydrogenase). This method can be used to measure cytotoxicity, proliferation, and the cell's activation. The method is rapid, precise, and lacks any toxic byproduct. The absorbance was measured using a spectrophotometer at 340 nm. The consumption of pyruvic acid follows upon the fibroblast's growth. Sodium laurylsulfate cytotoxicity test after 24 h shows that the NR colorimetric test and the pyruvic acid consumption are correctly correlated (r = 0.91, alpha = 0.05). This dosage can be used to study the barrier properties of the corneocyte layer without destroying the artificial skin.

[Streptococcus bovis septicemia and colonic cancer]. / Septicémie à Streptococcus bovis et cancer colique.

The authors report the case of a patient who developed a Streptococcus Bovis septicemia in the post-operative course of a surgical cure of an incarcerated umbilical hernia. There were no other complaints. A barium enema showed a malignant tumor of the descending colon. There were hepatic metastases. No complementary treatment was undertaken. The patient died shortly after. In the literature colonic proliferative lesions in association with this germ is reported in a total of 36% of proliferative lesions (15% of cancers and 21% of adenomas). It is concluded that the discovery of a malignant or premalignant proliferative lesion in one third of the cases justifies the exploration of the colon by barium enema and/or colonoscopy in the case of Streptococcus Bovis septicemia. The discovery of other digestive lesions (malignant or not) seems to be coincidental.