Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system

In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied. Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH. We observed 100% sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100% specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system. Comparing the results obtained in LJ we observed 100% sensitivity for RMP (80-100), followed by INH-95% (81.8-99.1), EMB-94.7% (71.9-99.7), and 100% specificity for all drugs tested except for PZA-98.3 (89.5-99.9) at 95% confidence interval. The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories.     

Evaluation of negative results of BacT/Alert 3D automated blood culture system

Although automated continuous-monitoring blood culture systems are both rapid and sensitive, false-positive and false-negative results still occur. The objective of this study, then, was to evaluate negative results occurring with BacT/Alert 3D blood culture systems. A total of 1032 samples were cultured with the BacT/Alert 3D automated blood culture system, using both aerobic (FA) and anaerobic (FN) [corrected] media, and 128 of these samples yielded positive results. A total of 904 negative blood samples were then subcultured in 5% sheep blood agar, eosin methylene blue, chocolate agar, and sabouraud-dextrose agar. Organisms growing on these subcultures were subsequently identified using both Vitek32 (bioMerieux, Durham, NC) and conventional methods. Twenty four (2.6%) of the 904 subcultures grew on the subculture media. The majority (83.3%) of these were determined to be gram-positive microorganisms. Fourteen (58.3%) were coagulase-negative staphylococci, two (8.3%) were Bacillus spp., one (4.2%) was Staphylococcus aureus, and one (4.2%) was identified as Enterococcus faecium. Streptococcus pneumoniae and Neisseria spp. were isolated together in two (8.3%) vials. Gram-negative microorganisms comprised 12.5% of the subcultures, of which two (8.3%) were found to be Pseudomonas aeruginosa, and one (4.2%) was Pseudomonas fluorescens. The other isolate (4.2%) was identified as Candida albicans. We conclude that the subculture of negative results is valuable in the BacT/Alert 3D system, especially in situations in which only one set of blood cultures is taken.     

BacT／Aler3D全自动血培养仪的临床应用及评价

目的评价全自动血培养仪BacT／Aler3D的临床应用情况。方法对BacT／Aler3D全自动血培养仪检测的1853份标本,其检出的阳性率、病原菌种类及阳性检出时间进行了评估。结果1853份血培养分离出病原菌189株,阳性率为10．2％,分离出病原菌23种,最快检出时间为2h,24h以内阳性率为68．5％,48h内阳性率为87．3％,72h以内阳性率为92．3％。结论BacT／Aler3D全自动血培养仪提高血培养的阳性率,检出的细菌种类多,污染机会少,缩短阳性检出时间,而且操作简便,结果快速、准确。     

Restriction-modification system in bacteriophage MB78

Restriction-modification system is present in bacteria to protect the cells against phage infection. Interestingly, the bacteriophage MB78, a virulent phage of Salmonella typhimurium possesses restriction-modification system. Permissive host transformed with plasmid having the genomic fragment of MB78 carrying the putative restriction-modification genes severely restrict the growth of the phage 9NA. Growth of phage MB78 is also restricted to some extent. However, the temperate phage P22 is not restricted at all. Cloning of the the putative restriction-modification genes has been done in both orientations in different vectors. The clones carrying the genes in the same orientation as that of the lacZ in pUC19 are mostly unstable. However, those are stable when cloned in opposite orientation. Viability of the transformants is strain-, orientation-, and medium-dependent. The two genes have also been cloned individually/separately. Hosts carrying only the modification gene do not restrict growth of phages while the hosts carrying only the restriction gene do. The former produces stable transformants while the latter produces very unstable transformants which were viable only upto 36 h or so. The colonies carrying modification gene were normal looking while those carrying the restriction gene were tiny, flat, and looked distressed resembling very much the clones carrying bacterial restriction-modification system. Amplification of the genes and subsequent cloning in expression vector will be carried out for characterization of the enzymes.     

Influence of test duration on the sensitivity of the two-bottle choice test

The long-term two-bottle choice test is commonly used as a simple screen to examine the acceptance of taste solutions by rodents. As part of an investigation of factors influencing the sensitivity of the two-bottle choice test, we determined the extent to which test duration influenced test sensitivity. C57BL6/J and 129X1/SvJ mice received four series of eight two-bottle tests, with each test lasting 1, 2, 4 or 6 days. Each series involved sequential tests with water, 2 mM saccharin, 5 and 50 mM citric acid, 30 and 300 micro M quinine hydrochloride, 75 mM NaCl and 10% ethanol. There were significant differences between the strains in intake of saccharin, 5 and 50 mM citric acid, NaCl and ethanol in 4 and 6 day tests, but only saccharin and ethanol in 2 day tests, and 5 mM citric acid and ethanol in 1 day tests. To compare the sensitivity of the tests, we developed an analytical approach based on the comparison of deviations of individual 129X1/SvJ mice from the C57BL6/J strain mean. Our results suggest that to discriminate between strains or treatments when using 'standard' laboratory conditions and methods, 1 day tests are generally inadequate and 2 day tests are useful only if large effects are anticipated. Tests lasting 4 or 6 days are more sensitive, but conducting 6 day tests provides little additional benefit and sometimes is detrimental relative to conducting 4 day tests.     

Effects of preincubation temperature on the detection of fastidious organisms in delayed-entry samples in the BacT/ALERT 3D blood culture system

This study evaluates the effect of preincubation on delayed-entry samples for fastidious organisms including the HACEK group, Streptococcus species, Neisseria meningitidis, Haemophilus species and Corynebacterium species for the BacT/ALERT 3D System (bioMérieux) using the FA (aerobic) medium.Bottles were inoculated with two different concentrations (0.5 McFarland and a 1:100,000 dilution) of each organism and either loaded into the system immediately or stored at 4 °C, room temperature (RT) or 37 °C for 24 hours (h) prior to loading.The detection rate (DR) was 92.5% for bottles loaded immediately for both concentrations with a mean time to detection (TTD) of 26.7 h (standard deviation (SD): 14.7 h) for the low concentration and 9.21 h (SD: 5.3 h) for the high concentration. Preincubation at 4 °C did not affect the DR for either of the two concentrations in comparison to no preincubation. The DR at RT was 90.0% for the low concentration and 83.6% for the high concentration. At 37 °C the DR was 76.3% and 66.3% for the low and the high concentrations respectively. The average TTD was inversely correlated with the preincubation temperature. An incubation of four days was sufficient, with the exception of Eikenella corrodens and Gemella sanguinis. The serotype of Streptococcus pneumoniae or Neisseria meningitidis did not influence the TTD. Kingella kingae remained undetected.For the retrieval of the above mentioned bacteria we recommend storage of bottles at room temperature. In case of erroneous storage at 37 °C subcultivation is advisable.All cases with a negative result on day four should be reevaluated and eventually new material for alternative diagnostic procedures should be retrieved.     

Rapid test for assay of ozone sensitivity in Escherichia coli

Summary A rapid test devised for assay of ozone sensitivity in Escherichia coli is described. The detection of new mutants, either more resistant or more sensitive than wild type strain to ozone, and the genetic analysis of ozone recombinants are now possible. Results confirm that ozone resistance is probably involved with DNA repair mechanisms; and show that ozone and ultraviolet light inhibit the cell division capacity of lon mutants in a similar way.

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Résumé Une méthode permettant de déterminer rapidement la susceptibilité de Escherichia coli à l'ozone est décrite. Grâce à ce test rapide, il est maintenant possible de détecter de nouveaux mutants, plus résistants ou plus sensibles à l'ozone que la souche de type sauvage, et de procéder à l'analyse de recombinants pour ce caractère. Les résultats obtenus confirment le rôle probable des enzymes de réparation de l'ADN dans la résistance à l'ozone; et montrent que l'ozone inhibe la capacité de division cellulaire de mutants lon de façon comparable à la lumière ultraviolette.

     

Direct mechano-stress sensitivity of TRPM7 channel.

It has recently been shown that shear stress augments the heterologously expressed TRPM7 channel activity by exocytosis-mediated incorporation of TRPM7 into the plasma membrane. On the other hand, our recent study has shown that the TRPM7-like channel endogenously expressed in HeLa cells is activated by membrane expansion induced by membrane stretch or osmotic cell swelling. Thus, the present study was aimed at exploring the possibility that the heterogously expressed TRPM7 channel is activated directly by membrane expansion in a manner independent of exocytosis. Here, whole-cell currents of the TRPM7 channel heterologously expressed in HEK293T cells were found to be augmented not only by perfusion of bath solution but also by osmotic swelling even under the conditions where exocytotic events can hardly take place in the cytosol dialyzed with ATP-free, Ca(2+)-free and EGTA-containing pipette solution. In addition, shear stress-induced augmentation was not affected by a blocker of vesicular protein traffic, brefeldin A. Furthermore, in cell-free patches, membrane stretch directly augmented single-channel activity of TRPM7 by increasing Po value at < or = 20 mV. We thus conclude that the TRPM7 channel can be directly activated by mechano-stress in a manner independent of exocytosis-mediated incorporation of this channel protein into the plasma membrane.     

Prior bleeding enhances the sensitivity of the in vivo micronucleus test

It has been reported that the sensitivity of the in vivo mouse bone marrow micronucleus test can be increased by inducing erythropoiesis with exogenous erythropoietin prior to treatment (Suzuki et al., 1989). In these studies we demonstrate that removing approximately 0.5 ml of blood from an adult male BDF1 mouse, another method for increasing the rate of erythropoiesis, synergistically increased the frequency of bone marrow micronucleated polychromatic erythrocytes induced by mitomycin C, with maximal enhancement occurring when the mutagen was given 24 h after bleeding. This enhancement response was also demonstrated for benzo[a]pyrene and dimethylnitrosamine but not for 2-acetylaminofluorene. These results indicate that bleeding mice prior to chemical treatment is a simple method for increasing the sensitivity of the micronucleus assay.     

The Performance of the Four Anaerobic Blood Culture Bottles BacT/ALERT-FN, -FN Plus,BACTEC-Plus and -Lytic in Detection of Anaerobic Bacteria and Identification by Direct MALDI-TOF MS

Detection and identification of anaerobic bacteria in blood cultures (BC) is a well-recognized challenge in clinical microbiology. We studied 100 clinical anaerobic BC isolates to evaluate the performance of BacT/ALERT-FN, -FN Plus (BioMérieux), BACTEC-Plus and -Lytic (Becton Dickinson BioSciences) BC bottles in detection and time to detection (TTD) of anaerobic bacteria. BACTEC Lytic had higher detection rate (94/100, 94%) than BacT/ALERT FN Plus (80/100, 80%) (p<0.01) in the studied material. There was no significant difference in detection of anaerobic bacteria among the remaining bottle types. The 67 anaerobic bacteria that signalled positive in all four bottle types were analyzed to compare the time to detection (TTD) and isolates were directly identified by MALDI-TOF MS. There was a significant difference in TTD among the four bottle types (p<0.0001). The shortest median TTD was 18 h in BACTEC Lytic followed by BacT/ALERT FN (23.5 h), BACTEC Plus (27 h) and finally BacT/ALERT FN Plus (38 h) bottles. In contrast, MALDI-TOF MS performed similarly in all bottle types with accurate identification in 51/67 (76%) BacT/ALERT FN, 51/67 (76%) BacT/ALERT FN Plus, 53/67 (79%) BACTEC Plus and 50/67 (75%) BACTEC Lytic bottles. In conclusion, BACTEC Lytic bottles have significantly better detection rates and shorter TTD compared to the three other bottle types. The anaerobic BC bottles are equally suitable for direct MALDI-TOF MS for rapid and reliable identification of common anaerobic bacteria. Further clinical studies are warranted to investigate the performance of anaerobic BC bottles in detection of anaerobic bacteria and identification by direct MALDI-TOF MS.     

Rapid insulin sensitivity test (RIST).

A rapid insulin sensitivity test (RIST) was recently introduced to assess insulin action in vivo (H. Xie, L. Zhu, Y.L. Zhang, D.J. Legare, and W.W. Lautt. J. Pharmacol. Toxicol. Methods, 35: 77-82. 1996). This technical report describes the current recommended standard operating procedure for the use of the RIST in rats based upon additional experience with approximately 100 tests. We describe the manufacture and use of an arterial-venous shunt that allows rapid multiple arterial samples and intravenous administration of drugs. The RIST procedure involves determination of a stable arterial glucose baseline to define the ideal euglycemic level to be maintained following a 5-min infusion of insulin, with the RIST index being the amount of glucose required to be infused to maintain euglycemia over the test period. Insulin administration by a 5-min infusion is preferable to a 30-s bolus administration. No significant difference was determined between the use of Toronto pork-beef or human insulin. Four consecutive RISTs were carried out in the same animal over 4-5 h with no tendency for change with time. The RIST index is sufficiently sensitive and reproducible to permit establishment of insulin dose-response curves and interference of insulin action by elimination of hepatic parasympathetic nerves, using atropine. This technical report provides the current recommended standard operating procedure for the RIST.     

Disgust sensitivity is negatively associated with immune system activity in early pregnancy: Direct support for the Compensatory Prophylaxis Hypothesis

According to the Compensatory Prophylaxis Hypothesis (CPH), disgust may be considered a part of the behavioral immune system, adjusting as a function of immunocompetence. Early pregnancy involves modulation of a complex network of various immune-related factors, but only a few studies so far have focused on disgust sensitivity in pregnant women in the context of the CPH. This study aimed to examine associations between disgust sensitivity and immune activity indices, cytokine levels, and white blood cell (WBC) count in pregnant women. The sample included 78 women in the 1st trimester of pregnancy. Higher disgust sensitivity (Disgust Scale-Revised; DS-R) was significantly associated with decreased levels of IL-1β, IL-2 IL-4, IL-7, IL-17, Eotaxin, MCP-1 (MCAF), and RANTES in blood serum. This model explained 17.5% of the total DS-R score variability. Using the DS-R subscales, the Contamination disgust was significantly associated with levels of FGF basic, IFN-γ, IL-1β, IL-2, IL-4, IL-7, IL-17A, G-CSF, MCP-1 (MCAF), MIP-1α, PDGF-BB, and RANTES, and the Core disgust was significantly associated with levels of IL-1β, IL-2, IL-4, IL-7, IL-17A, Eotaxin, G-CSF, IP-10, MCP-1 (MCAF), PDGF-BB, and TNF-α. Disgust sensitivity was not associated with WBC count. Disgust may reflect and compensate for insufficient immune adaptation in early pregnancy, suggesting the potential clinical significance of this common prenatal symptom.