The recovery of Mycobacterium avium subspecies paratuberculosis from the intestine of infected ruminants for proteomic evaluation.

J Microbiol Methods. 2008 May 14;

Authors: Egan S, Lanigan M, Shiell B, Beddome G, Stewart D, Vaughan J, Michalski WP

Johne's disease is a slowly developing intestinal disease, primarily of ruminants, caused by Mycobacterium avium subspecies paratuberculosis. The disease contributes to significant economic losses worldwide in agricultural industry. Analysis of bacterial proteomes isolated directly from infected animals can provide important information about the repertoire of proteins present during infection and disease progression. In this study, M. avium subspecies paratuberculosis has been extracted from Johne's disease-infected cattle and goat intestinal tissue sections in a manner compatible with direct 2-DE proteomic analysis for comparison with in vitro-cultured bacteria. M. avium subspecies paratuberculosis was harvested from the submucosa and mucosa of intestinal sections and enriched from macerated tissue by hypotonic lysis, sonication and centrifugation through a viscosity gradient. Subsequent comparison of the proteomes of the in vivo- and in vitro-derived bacteria identified a number of proteins that were differentially expressed. Among them, a number of hypothetical proteins of unknown function and a hypothetical fatty acyl dehydrogenase (FadE3_2) and 3-hydroxyacyl-CoA dehydrogenase, possibly important for in vivo metabolism, utilising the pathway for the beta-oxidation of fatty acids. PMID: 18547663 [PubMed - as supplied by publisher]

Polymerase chain reaction is superior to serology for the diagnosis of acute Mycoplasma pneumoniae infection and reveals a high rate of persistent infection.

BMC Microbiol. 2008 Jun 11;8(1):93

Authors: Nilsson AC, Bjorkman P, Persson K

ABSTRACT: BACKGROUND: Diagnosis of Mycoplasma pneumoniae (MP) infection is traditionally based on serology, which may require more than two weeks for diagnostic antibodies to develop. PCR-based methods offer earlier diagnosis. During a community outbreak of MP infection, we compared semi-nested and real-time PCR of oropharyngeal swabs with serology for diagnosis of MP infection at different time points after disease onset. PCR-positive individuals were followed longitudinally to assess the persistence of MP DNA in throat secretions. We also studied carriage of MP among household contacts and school children. RESULTS: MP infection was diagnosed in 48 of 164 patients with respiratory tract infection. Forty-five (29%) had detectable MP DNA in oropharynx. A significant increase in MP IgG IgG titre or MP IgM antibodies was detected in 44/154 (27%) subjects. Two MP PCR-positive patients lacked antibody responses. Sera were missing from another two patients. The agreement between serology and PCR was good, kappa = 0.90. During the first three weeks after disease onset the performance of PCR was excellent and all patients but one were detected. In contrast, only 21% of the patients with confirmed MP infection were positive by serum 1 during the first symptomatic week (56% during the second and 100% during the third week). Only 1/237 (0.4%) school children was positive by PCR. This child had respiratory symptoms. Eighteen of 22 (75%) symptomatic household contacts were MP PCR positive. Persistence of MP DNA in the throat was common. Median time for carriage of MP DNA was 7 weeks after disease onset (range 2 days - 7 months). Adequate antibiotic treatment did not shorten the period of persistence. Bacterial load, measured by quantitative real-time PCR declined gradually, and all followed patients eventually became PCR-negative. CONCLUSIONS: PCR is superior to serology for diagnosis of MP infection during the early phases of infection. Persistent, sometimes long-term, carriage of MP DNA in the throat is common following acute infection, and is not affected by antibiotic therapy. Asymptomatic carriage of MP even during an outbreak is uncommon. PMID: 18547431 [PubMed - as supplied by publisher]

Rapid diagnosis of bacterial meningitis in children with fluorescence quantitative polymerase chain reaction amplification in the bacterial 16S rRNA gene.

Eur J Pediatr. 2008 Jun 12;

Authors: Duan QJ, Shang SQ, Wu YD

Polymerase chain reaction (PCR) techniques have been increasingly used to detect microbial DNA in cerebrospinal fluid (CSF) for the diagnosis of bacterial meningitis. In order to determine the rapidity, sensitivity and specificity of 16S rRNA-based fluorescence quantitative polymerase chain reaction (FQ-PCR), 16S rRNA-based FQ-PCR, CSF bacterial culture and CSF routine analysis were compared in the diagnosis of bacterial meningitis in children. Twenty children who were clinically suspected of bacterial meningitis were included in this study. A total of 2.0 ml of CSF was collected from every child and was subjected to 16S rRNA-based FQ-PCR, CSF culture and CSF routine analysis. Bacterial DNA copies and the cycle threshold (CT) value of the 16S rRNA-based FQ-PCR was recorded, and the results were compared with CSF culture and CSF routine analysis. Seven children were found to be positive with a rate of 35% (7/20) when detected with 16S rRNA-based FQ-PCR and four children displayed a positive rate of 20% (4/20) with the CSF culture method. These two groups displayed a significant difference, with a p-value of 0.002. The method of 16S rRNA-based FQ-PCR demonstrated a high specificity when compared to the standard microbes. A negative correlation was noted between the CT value and the bacteria DNA copies, and the CT value was indicative of the seriousness of bacterial meningitis. 16S rRNA-based FQ-PCR was proved to be a more rapid, sensitive and specific method compared with CSF culture and it should have promising usage in the diagnosis of bacterial meningitis. PMID: 18548276 [PubMed - as supplied by publisher]

From no expression to high-level soluble expression in Escherichia coli by screening a library of the target proteins with randomized N-termini.

Methods Mol Biol. 2008;426:187-95

Authors: Kim KH, Yang JK, Waldo GS, Terwilliger TC, Suh SW

For structural studies by x-ray crystallography and nuclear magnetic resonance it is important for the target protein to be available in large quantity and high purity. Escherichia coli expression systems remain the most versatile and convenient means to produce a large quantity of recombinant proteins. Unfortunately, some proteins fail to be expressed in E. coli or are expressed in an insoluble form. To overcome the difficulty of no expression or expression at a very low level, a simple and efficient approach of screening a library of variants of a target protein with randomized N-termini was devised. In this method, a few N-terminal residues are randomized by designing a mixture of oligonucleotides for the forward PCR primer and we fuse the library in front of green fluorescent protein, which serves as a reporter for the target protein expression level and folding yield. In favorable cases this approach can result in high-level soluble expression of recombinant proteins in E. coli. This chapter describes the results of a test of this approach with a bacterial protein (the HI0952 gene product) that is not well expressed in E. coli. PMID: 18542864 [PubMed - in process]