Beta‐lactam antibiotics: from antibiosis to resistance and bacteriologyKONG, KOK‐FAI; SCHNEPER, LISA; MATHEE, KALAI
doi: 10.1111/j.1600-0463.2009.02563.xpmid: 20041868
Kong K‐F, Schneper L, Mathee K. Beta‐lactam antibiotics: from antibiosis to resistance and bacteriology. APMIS 2010; 118: 1–36. This review focuses on the era of antibiosis that led to a better understanding of bacterial morphology, in particular the cell wall component peptidoglycan. This is an effort to take readers on a tour de force from the concept of antibiosis, to the serendipity of antibiotics, evolution of beta‐lactam development, and the molecular biology of antibiotic resistance. These areas of research have culminated in a deeper understanding of microbiology, particularly in the area of bacterial cell wall synthesis and recycling. In spite of this knowledge, which has enabled design of new even more effective therapeutics to combat bacterial infection and has provided new research tools, antibiotic resistance remains a worldwide health care problem.
Herpes simplex virus type‐2 in Egyptian patients with bladder cancer or cystitisBADAWI, HALA; AHMED, HANEM; ABOUL FADL, LAILA; HELMI, AMIRA; FAM, NEVINE; DIAB, MANAL; ISMAIL, AHMED; BADAWI, AFKAR; SABER, MOHAMED
doi: 10.1111/j.1600-0463.2009.02556.xpmid: 20041869
Badawi H, Ahmed H, Aboul Fadl L, Helmi A, Fam N, Diab M, Ismail A, Badawi A, Saber M. Herpes simplex virus type‐2 in Egyptian patients with bladder cancer or cystitis. APMIS 2010; 118: 37–44. The present study was designed to investigate the prevalence of herpes simplex virus type‐2 (HSV‐2) in Egyptian patients with bladder cancer or cystitis and to evaluate the performance of different diagnostic HSV‐2 assays. The study included 50 patients: 27 with bladder cancer (group I), 23 with cystitis (group II) and 20 subjects as controls (group III). HSV‐2 DNA was detected using polymerase chain reaction (PCR) on bladder tissue and buffy coat cells (BCC). Electron microscopic studies (EMS) on BCC and ELISAs for IgM, IgG and specific glycoprotein G‐2 (gG‐2) IgG were performed. HSV‐2 DNA was detected by PCR on bladder tissue biopsies in 29.6% and 21.7% of group I and II respectively and it was also detected by PCR on BCC in 22.2% and 21.7% of group I and II respectively. EMS revealed HSV like particles in 16.6% of cases. IgG, specific gG‐2 IgG and IgM were detected in 30%, 16% and 6% of cases respectively. The different assays were evaluated in relation to PCR on bladder tissue biopsies. The gG‐2‐based ELISA and EMS on BCC were found to be highly specific (97.3% and 100% respectively), with similar low sensitivity of ≈54%. PCR on BCC was the most sensitive assay. The association of HSV‐2 with bladder cancer is suggested especially in schistosomal patients.
Quantification of Chlamydia pneumoniae in cultured human macrophages and HL cells: comparison of real‐time PCR, immunofluorescence and ELISA methodsPOIKONEN, KARI; LAJUNEN, TAINA; SILVENNOINEN‐KASSINEN, SYLVI; LEINONEN, MAIJA; SAIKKU, PEKKA
doi: 10.1111/j.1600-0463.2009.02557.xpmid: 20041870
Poikonen K, Lajunen T, Silvennoinen‐Kassinen S, Leinonen M, Saikku P. Quantification of Chlamydia pneumoniae in cultured human macrophages and HL cells: comparison of real‐time PCR, immunofluorescence and ELISA methods. APMIS 2010; 118: 45–8. Chlamydia pneumoniae is an intracellular gram‐negative bacterium, which replicates only in eukaryotic cells. Quantification of C. pneumoniae in cell culture is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally, this has been performed by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary, and multiple inclusions are also seen. Therefore, methods are needed to quantify exact amounts of C. pneumoniae in cells. Here, we describe a new method based on the real‐time PCR quantification of chlamydial genomes adjusted to the number of human genomes in cultures. In human epithelial (HL) cell cultures, the C. pneumoniae inclusion numbers and the ratio of C. pneumonia genomes/human genome (Cpn/Hum) correlated significantly (r = 0.978, p < 0.001); thus with HL cells, both methods are usable. However, in macrophage cultures, the correlation was weaker (r = 0.133, p = 0.036) and we recommend PCR quantification for exact measurements.
Phosphatase inhibitors with anti‐angiogenic effect in vitroSYLVEST, LENE; BENDIKSEN, CHRISTINE DAM; HOUEN, GUNNAR
doi: 10.1111/j.1600-0463.2009.02561.xpmid: 20041871
Sylvest L, Bendiksen CD, Houen G. Phosphatase inhibitors with anti‐angiogenic effect in vitro. APMIS 2010; 118: 49–59. Levamisole has previously been identified as an inhibitor of angiogenesis in vitro and in vivo, but the mechanism behind the anti‐angiogenic behavior has not yet been established. However, one known effect of levamisole is the inhibition of alkaline phosphatase, and this fact encouraged us to test other phosphatase inhibitors for their anti‐angiogenic effects by using the same method as used to identify levamisole: an ELISA‐based co‐culture angiogenesis assay giving quantitative and qualitative results. Historically, intracellular phosphatases have been associated with the downregulation of signaling pathways, and kinases with their upregulation, but lately, the phospatases have also been coupled to positive signaling, which is why inhibition of phosphatases has become associated with anti‐tumorigenic and anti‐angiogenic effects. The results obtained in this work reveal several agents with anti‐angiogenic potential and give a strong indication that phosphatase inhibition is linked to anti‐angiogenic activity. An apparent disruption of endothelial tube formation was seen for seven of eight phosphatase inhibitors tested in the angiogenesis assay. By looking at the morphological results, it was seen that most of the inhibitors impaired proliferation and elongation of the endothelial cells, which still had a differentiated appearance. One inhibitor, PTP inhibitor IV, seemed to impair endothelial cell differentiation and induced the same morphology as when cells were treated with levamisole, although at a 200 times lower concentration than that of levamisole. Hence, our work points out compounds with a potential that may be of use in the search for new medical products for the treatment of malignant tumors, or other conditions where angiogenesis plays a central role.
Comparison of methods of DNA extraction for real‐time PCR in a model of pleural tuberculosisSANTOS, ANA; CREMADES, ROSA; RODRÍGUEZ, JUAN CARLOS; GARCÍA‐PACHÓN, EDUARDO; RUIZ, MONTSERRAT; ROYO, GLORIA
doi: 10.1111/j.1600-0463.2009.02558.xpmid: 20041872
Santos A, Cremades R, Rodríguez JC, García‐Pachón E, Ruiz M, Royo G. Comparison of methods of DNA extraction for real‐time PCR in a model of pleural tuberculosis. APMIS 2010; 118: 60–5. Molecular methods have been reported to have different sensitivities in the diagnosis of pleural tuberculosis and this may in part be caused by the use of different methods of DNA extraction. Our study compares nine DNA extraction systems in an experimental model of pleural tuberculosis. An inoculum of Mycobacterium tuberculosis was added to 23 pleural liquid samples with different characteristics. DNA was subsequently extracted using nine different methods (seven manual and two automatic) for analysis with real‐time PCR. Only two methods were able to detect the presence of M. tuberculosis DNA in all the samples: extraction using columns (Qiagen) and automated extraction with the TNAI system (Roche). The automatic method is more expensive, but requires less time. Almost all the false negatives were because of the difficulty involved in extracting M. tuberculosis DNA, as in general, all the methods studied are capable of eliminating inhibitory substances that block the amplification reaction. The method of M. tuberculosis DNA extraction used affects the results of the diagnosis of pleural tuberculosis by molecular methods. DNA extraction systems that have been shown to be effective in pleural liquid should be used.
Vancomycin‐resistant Enterococcus spp.: validation of susceptibility testing and in vitro activity of vancomycin, linezolid, tigecycline and daptomycinRATHE, MATHIAS; KRISTENSEN, LISE; ELLERMANN‐ERIKSEN, SVEND; THOMSEN, MARIANNE KRAGH; SCHUMACHER, HELGA
doi: 10.1111/j.1600-0463.2009.02559.xpmid: 20041873
Rathe M, Kristensen L, Ellermann‐Eriksen S, Thomsen MK, Schumacher H. Vancomycin‐resistant Enterococcus spp.: validation of susceptibility testing and in vitro activity of vancomycin, linezolid, tigecycline and daptomycin. APMIS 2010; 118: 66–73. Vancomycin‐resistant enterococci (VRE) have emerged to become a significant nosocomial pathogen. However, detection may be challenging and treatment possibilities are limited. Reports of resistance to linezolide, daptomycin and tigecycline underline the need for reliable susceptibility testing with respect to these compounds. We evaluated the in vitro activity of vancomycin, linezolid, daptomycin and tigecycline against a panel of VRE and vancomycin‐susceptible enterococci by broth microdilution (BMD). Etest for determination of minimum inhibitory concentration of these four antibiotics and two disc diffusion assays for detecting VRE and for susceptibility testing against tigecycline and linezolid were evaluated. Before susceptibility testing, all isolates were classified by polymerase chain reaction as vanA or vanB gene positive or vanA/B gene negative. Linezolid, daptomycin and tigecycline had excellent in vitro activity towards all isolates. For daptomycin and tigecycline, the overall agreement between BMD and Etest was suboptimal. For both disc diffusion assays, use of current break points was inadequate to detect vancomycin resistance for isolates carrying the vanB gene. Inspection of the inhibition zone for a diffuse edge, as recommended, accurately predicted presence of the vanB gene.
A selective broth enrichment combined with real‐time nuc‐mecA ‐PCR in the exclusion of MRSAPASANEN, TANJA; KORKEILA, MAIJA; MERO, SOINTU; TARKKA, EVELIINA; PIIPARINEN, HELI; VUOPIO‐VARKILA, JAANA; VAARA, MARTTI; TISSARI, PÄIVI
doi: 10.1111/j.1600-0463.2009.02562.xpmid: 20041874
Pasanen T, Korkeila M, Mero S, Tarkka E, Piiparinen H, Vuopio‐Varkila J, Vaara M, Tissari P. A selective broth enrichment combined with real‐time nuc‐mecA‐PCR in the exclusion of MRSA. APMIS 2010; 118: 74–80. We analyzed the performance of a selective enrichment broth combined with Taqman‐based real‐time duplex nuc‐mecA‐PCR to expedite the screening of methicillin‐resistant Staphylococcus aureus (MRSA). We found the broth to be able to select MRSA strains (oxacillin MIC range 4–256 μg/ml) from MSSA strains. A total of 31 MRSA strains were found from 1250 clinical samples screened. The nuc‐mecA‐PCR was positive from all enrichment broths containing MRSA. From the remaining 1219 samples negative for MRSA on culture/subculture, 138 samples were nuc+/mecA+ in PCR. The sensitivity of the test was 93.5%, specificity 88.6%, positive predictive value 17.3%, and negative predictive value 99.8% as compared to culture. Thus, with this method, the negative MRSA results can be reliably reported within 24–48 h from sampling. The method is a practical additional alternative to those already described for the same purpose.