Evaluation of high-resolution melting curve analysis of ligation-mediated real-time PCR, a rapid method for epidemiological typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) pathogens.

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks.

High abundance of virulence gene homologues in marine bacteria.

Marine bacteria can cause harm to single-celled and multicellular eukaryotes. However, relatively little is known about the underlying genetic basis for marine bacterial interactions with higher organisms. We examined whole-genome sequences from a large number of marine bacteria for the prevalence of homologues to virulence genes and pathogenicity islands known from bacteria that are pathogenic to terrestrial animals and plants. As many as 60 out of 119 genomes of marine bacteria, with no known association to infectious disease, harboured genes of virulence-associated types III, IV, V and VI protein secretion systems. Type III secretion was relatively uncommon, while type IV was widespread among alphaproteobacteria (particularly among roseobacters) and type VI was primarily found among gammaproteobacteria. Other examples included homologues of the Yersinia murine toxin and a phage-related 'antifeeding' island. Analysis of the Global Ocean Sampling metagenomic data indicated that virulence genes were present in up to 8% of the planktonic bacteria, with highest values in productive waters. From a marine ecology perspective, expression of these widely distributed genes would indicate that some bacteria infect or even consume live cells, that is, generate a previously unrecognized flow of organic matter and nutrients directly from eukaryotes to bacteria.

Uropathogenic Escherichia coli and tolerance to nitric oxide: the role of flavohemoglobin.

PURPOSE: NO has an important role as part of the innate host response against bacterial infections. Flavohemoglobin, which is encoded by the hmp gene, protects Escherichia coli against nitrosative stress. We compared the NO tolerance of UPEC and nonpathogenic strains, and examined the involvement of flavohemoglobin. MATERIALS AND METHODS: The E. coli K12 derivates HB101 and DH5alpha represent nonpathogenic strains, while J96 and IA2 represent UPEC strains. HB101 was used as the host for a pBR322 plasmid carrying the hmp gene. Bacterial tolerance to NO was evaluated by determining cfu. Flavohemoglobin expression was examined using Northern and Western blot analysis. RESULTS: In the stationary growth phase, J96 was significantly more tolerant to DETA/NO (Alexis Biochemical, Lausen, Switzerland) (1 mM) compared to HB101 (47% +/- 11% vs 6.4% +/- 3.1% cfu). In the exponential growth phase DETA/NO exposure resulted in 98% +/- 4.6% cfu for J96 and 74% +/- 7.6% cfu for IA2 compared to 15% +/- 5.9% for HB101 and 21% +/- 12% for DH5alpha. HB101 over expressing hmp showed increased tolerance to DETA/NO (0.5 mM) compared to WT HB101 (106% +/- 5.6% vs 67 +/- 6.2%, p <0.01). Northern and Western blot analysis demonstrated increased flavohemoglobin expression after DETA/NO exposure and the strongest expression in HB101 carrying hmp on a multicopy plasmid. CONCLUSIONS: UPEC strains were significantly more tolerant to DETA/NO than nonpathogenic strains, which suggests a correlation between virulence and NO tolerance. Flavohemoglobin expression increased after DETA/NO exposure in UPEC and in nonpathogenic strains.

Mycobacterium avium with the bird type IS1245 RFLP profile is commonly found in wild and domestic animals, but rarely in humans.

Cervical lymphadenitis is the main manifestation of Mycobacterium avium infection in immunocompetent children. Exposure to birds has been discussed as a source of infection. To clarify from where children acquire the infection, M. avium isolates from different origins were analysed with restriction fragment length polymorphism (RFLP) on insertion sequence IS1245, and compared by computer cluster correlation analysis. This molecular epidemiological tool has previously revealed a distinction between multiband profiles found mainly in strains from humans, and a 3-band/bird type profile in strains isolated mainly from birds. 32 isolates from children were compared with 28 isolates from adults and 45 isolates from animals. We found that 67% of the animal isolates had the bird type profile, also found in 1 sputum isolate from an adult. Strains from children showed only multiband profiles that did not differ significantly from profiles of isolates from adults. All but 2 bird isolates showed the bird type profile. Neither of the remaining 2, which had multiband profiles, clustered with the isolates from children. Our results indicate that the true reservoir of M. avium is unknown. Thus the question of whether or not M. avium can be incriminated as a zoonotic disease remains unanswered.

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions.

Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the alpha-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), beta-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.

Binding interactions of Escherichia coli with globotetraosylceramide (globoside) using a surface plasmon resonance biosensor.

Surface plasmon resonance with an alkane L1 chip was used to investigate the binding of uropathogenic Escherichia coli, carrying adhesion receptors, to globotetraosylceramide (globoside; GbO4). The immobilization of globoside was reproducible and resulted in a stable globoside layer on the L1 chip. The data indicated that the globoside-immobilized L1 chip could be used for studying interactions with live or chemically fixed E. coli. The results indicated that the dissociation time was significantly reduced in glutaraldehyde-fixed E. coli as compared to living cells. Overall, the report demonstrates the significance of the L1 chip in terms of sensitivity, specificity, handling, and speed when studying globoside/E. coli interactions. This model may assist in screening for compounds that can inhibit the binding of uropathogenic E. coli to glycolipid ligands on target cells.