Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp® DNA Mini Kit, Reischl et al.’s method and FTA® Elute. FTA® Elute significantly showed the highest median DNA extraction efficiency of 76.9% for E. coli and 108.9% for S. aureus. The Reischl et al. method and QIAamp® DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA® Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall, the FTA® Elute method was found to be the most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity, and transport and storage conditions

Properties, production, and applications of camelid single-domain antibody fragments

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications

Design of a randomised controlled trial on immune effects of acidic and neutral oligosaccharides in the nutrition of preterm infants: carrot study

<p>Abstract</p> <p>Background</p> <p>Prevention of serious infections in preterm infants is a challenge, since prematurity and low birth weight often requires many interventions and high utility of devices. Furthermore, the possibility to administer enteral nutrition is limited due to immaturity of the gastrointestinal tract in the presence of a developing immune system. In combination with delayed intestinal bacterial colonisation compared with term infants, this may increase the risk for serious infections. Acidic and neutral oligosaccharides play an important role in the development of the immune system, intestinal bacterial colonisation and functional integrity of the gut. This trial aims to determine the effect of enteral supplementation of acidic and neutral oligosaccharides on infectious morbidity (primary outcome), immune response to immunizations, feeding tolerance and short-term and long-term outcome in preterm infants. In addition, an attempt is made to elucidate the role of acidic and neutral oligosaccharides in postnatal modulation of the immune response and postnatal adaptation of the gut.</p> <p>Methods/Design</p> <p>In a double-blind placebo controlled randomised trial, 120 preterm infants (gestational age <32 weeks and/or birth weight <1500 gram) are randomly allocated to receive enteral acidic and neutral oligosaccharides supplementation (20%/80%) or placebo supplementation (maltodextrin) between day 3 and 30 of life. Primary outcome is infectious morbidity (defined as the incidence of serious infections). The role of acidic and neutral oligosaccharides in modulation of the immune response is investigated by determining the immune response to DTaP-IPV-Hib(-HBV)+PCV7 immunizations, plasma cytokine concentrations, faecal Calprotectin and IL-8. The effect of enteral acidic and neutral oligosaccharides supplementation on postnatal adaptation of the gut is investigated by measuring feeding tolerance, intestinal permeability, intestinal viscosity, and determining intestinal microflora. Furthermore, short-term and long-term outcome are evaluated.</p> <p>Discussion</p> <p>Especially preterm infants, who are at increased risk for serious infections, may benefit from supplementation of prebiotics. Most studies with prebiotics only focus on the colonisation of the intestinal microflora. However, the pathways how prebiotics may influence the immune system are not yet fully understood. Studying the immune modulatory effects is complex because of the multicausal risk of infections in preterm infants. The combination of neutral oligosaccharides with acidic oligosaccharides may have an increased beneficial effect on the immune system. Increased insight in the effects of prebiotics on the developing immune system may help to decrease the (infectious) morbidity and mortality in preterm infants.</p> <p>Trial registration</p> <p>Current Controlled Trials ISRCTN16211826.</p

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

Characterisation of Australian MRSA Strains ST75- and ST883-MRSA-IV and Analysis of Their Accessory Gene Regulator Locus

Background: Community-acquired methicillin-resistant Staphylococcus aureus have become a major problem in Australia. These strains have now been isolated throughout Australia including remote Indigenous communities that have had minimal exposure to healthcare facilities. Some of these strains, belonging to sequence types ST75 and ST883, have previously been reported to harbour highly divergent alleles of the housekeeping genes used in multilocus sequence typing. Methodology/Principal Findings: ST75-MRSA-IV and ST883-MRSA-IV isolates were characterised in detail. Morphological features as well as 16S sequences were identical to other S. aureus strains. Although a partial rnpB gene sequence was not identical to previously known S. aureus sequences, it was found to be more closely related to S. aureus than to other staphylococci. Isolates also were screened using diagnostic DNA microarrays. These isolates yielded hybridisation results atypical for S. aureus. Primer directed amplification assays failed to detect species markers (femA, katA, sbi, spa). However, arbitrarily primed amplification indicated the presence of unknown alleles of these genes. Isolates could not be assigned to capsule types 1, 5 or 8. The allelic group of the accessory gene regulator (agr) locus was not determinable. Sequencing of a region of agrB, agrC and agrD (approximately 2,100 bp) revealed a divergent sequence. However, this sequence is more related to S. aureus agr alleles I and IV than to agr sequences from other Staphylococcus species. The predicted autoinducing peptide (AIP) sequence of ST75 was identical to that of agr group I, while the predicted AIP sequence of ST883 was identical to agr group IV. Conclusions/Significance: The genetic properties of ST75/ST883-MRSA may be due to a series of evolutionary events in ancient insulated S. aureus strains including a convergent evolution leading to agr group I- or IV-like AIP sequences and a recent acquisition of SCCmec IV elements

Antibody responses in furunculosis patients vaccinated with autologous formalin-killed Staphylococcus aureus

Autologous vaccines (short: autovaccines) have been used since the beginning of the 20th century to treat chronic staphylococcal infections, but their mechanisms of action are still obscure. This prospective pilot study involved four patients with furunculosis who were vaccinated with autologous formalin-killed Staphylococcus aureus cells. Vaccines were individually prepared from the infecting S. aureus strain and repeatedly injected subcutaneously in increasing doses over several months. We characterized the virulence gene repertoire and spa genotype of the infecting and colonising S. aureus strains. Serum antibody responses to secreted and surface-bound bacterial antigens were determined by two-dimensional immunoblotting and flow-cytometry based assays (Luminex®). All patients reported clinical improvement. Molecular characterization showed that all strains isolated from one patient over time belonged to the same S. aureus clone. Already before treatment, there was robust antibody binding to a broad range of staphylococcal antigens. Autovaccination moderately boosted the IgG response to extracellular antigens in two patients, while the antibody response of the other two patients was not affected. Similarly, vaccination moderately enhanced the antibody response against some staphylococcal surface proteins, e.g. ClfA, ClfB, SdrD and SdrE. In summary, autovaccination only slightly boosted the pre-existing serum antibody response, predominantly to bacterial surface antigens

Molecular Typing and Phenotype Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Blood in Taiwan

BACKGROUND: Staphylococcus aureus causes a variety of severe infections such as bacteremia and sepsis. At present, 60-80% of S. aureus isolates from Taiwan are methicillin resistant (MRSA). It has been shown that certain MRSA clones circulate worldwide. The goals of this study were to identify MRSA clones in Taiwan and to correlate the molecular types of isolates with their phenotypes. METHODS: A total of 157 MRSA isolates from bacteremic patients were collected from nine medical centers. They were typed based on polymorphisms in agr, SCCmec, MLST, spa, and dru. Phenotypes characterized included Panton-Valentine leucocidin (pvl), inducible macrolide-lincosamide-streptogramin B resistance (MLSBi), vancomycin (VA) and daptomycin (DAP) minimal inhibitory concentrations (MIC), and superantigenic toxin gene profiles. Difference between two consecutive samples was determined by Mann-Whitney-U test, and difference between two categorical variables was determined by Fisher's exact test. RESULTS: Four major MRSA clone complexes CC1, CC5, CC8, and CC59 were found, including 4 CC1, 9 CC5, 111 CC8, and 28 CC59 isolates. These clones had the following molecular types: CC1: SCCmecIV and ST573; CC5: SCCmecII and ST5; CC8: SCCmecIII, ST239, and ST241, and CC59: SCCmecIV, SCCmecV(T), ST59, and ST338. The toxin gene profiles of these clones were CC1: sec-seg-(sei)-sell-selm-(seln)-selo; CC5: sec-seg-sei-sell-selm-(seln)-selp-tst1; CC8: sea-selk-selq, and CC59: seb-selk-selq. Most isolates with SCCmecV(T), ST59, spat437, and dru11 types were pvl(+) (13 isolates), while multidrug resistance (≥4 antimicrobials) were associated with SCCmecIII, ST239, spa t037, agrI, and dru14 (119 isolates) (p<0.001). One hundred and twenty four isolates with the following molecular types had higher VA MIC: SCCmecII and SCCmecIII; ST5, ST239, and ST241; spa t002, t037, and t421; dru4, dru10, dru12, dru13, and dru14 (p<0.05). No particular molecular types were found to be associated with MLSBi phenotype. CONCLUSIONS: Four major MRSA clone complexes were found in Taiwan. Further studies are needed to delineate the evolution of MRSA isolates

Differences in avoidable mortality between migrants and the native Dutch in the Netherlands

BACKGROUND: The quality of the healthcare system and its role in influencing mortality of migrant groups can be explored by examining ethnic variations in 'avoidable' mortality. This study investigates the association between the level of mortality from 'avoidable' causes and ethnic origin in the Netherlands and identifies social factors that contribute to this association. METHODS: Data were obtained from cause of death and population registries in the period 1995–2000. We compared mortality rates for selected 'avoidable' conditions for Turkish, Moroccan, Surinamese and Antillean/Aruban groups to native Dutch. RESULTS: We found slightly elevated risk in total 'avoidable' mortality for migrant populations (RR = 1.13). Higher risks of death among migrants were observed from almost all infectious diseases (most RR > 3.00) and several chronic conditions including asthma, diabetes and cerebro-vascular disorders (most RR > 1.70). Migrant women experienced a higher risk of death from maternity-related conditions (RR = 3.37). Surinamese and Antillean/Aruban population had a higher mortality risk (RR = 1.65 and 1.31 respectively), while Turkish and Moroccans experienced a lower risk of death (RR = 0.93 and 0.77 respectively) from all 'avoidable' conditions compared to native Dutch. Control for demographic and socioeconomic factors explained a substantial part of ethnic differences in 'avoidable' mortality. CONCLUSION: Compared to the native Dutch population, total 'avoidable' mortality was slightly elevated for all migrants combined. Mortality risks varied greatly by cause of death and ethnic origin. The substantial differences in mortality for a few 'avoidable' conditions suggest opportunities for quality improvement within specific areas of the healthcare system targeted to disadvantaged groups