A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides.

BACKGROUND: The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target. RESULTS: Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM. CONCLUSIONS: This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.

Group Assessments to Help Build Online Learning Communities in Biomedical Science Distance Learning Programmes.

Introduction: Biomedical Science distance learning courses offer flexibility in study while in employment. Asynchronous and self-learning approaches are common within such courses and often student-student interaction is limited. The aims of this study were to establish learning communities, develop confidence in participating in online teamwork and foster an appreciation of transferable skills including digital capabilities through remote group activities. Materials and Methods: Two cohorts of students (n = 20/n = 21) were enrolled in a microbiology module of an IBMS accredited MSc distance learning course. Groups of 4-5 students produced a digital output relating to current global infection-related issues, namely, assignment 1, production of a slide deck, which peers could use as learning resources and assignment 2, a voiceover PowerPoint debate, and infographic, voting assessment and peer/self-marking. Students also prepared reflections using written format and a FlipGrid video-recording. A qualitative content analysis was conducted on reflections from all students. Students completed a pre- and post-assignment survey focused on the development of transferable skills for the biomedical sector. Results: Students' skills and confidence increased following completion of the group assignment, as evident from the pre- and post-questionnaire responses, namely, possession of digital skills and digital creation abilities (29% v 83%), applying for jobs which require digital skills (54% v 89%), talking about examples of using digital media during job interviews (21% v 78%) and demonstration of creativity during assignment tasks (33% v 90%). Critical thinking was more commonly demonstrated during the debate in comparison to the slide deck activity (p = 0.001). The importance of developing digital skills, was higher following completion of the group activities (p = 0.03). Students reflected on the value of the group activities in relation to knowledge acquisition (85%, 86%), collegiality (70%, 71%), digital skills development (80%, 90%), the fact that the activities were enjoyable (70%, 67%) and the development of peer interaction and support (50%, 67%) in relation to assignment 1 and 2, respectively. Discussion: Increasingly digital technologies are being used in the healthcare sector resulting in updated HCPC Standards of Proficiency. This study highlights that virtual group activities promote the establishment of supportive learning communities and the development of transferable skills including digital capabilities for application within the biomedical science workplace.

UVc-irradiation sublethal stress does not alter antibiotic susceptibility of staphylococci (MRSA, MSSA, and coagulase-negative staphylococci) to ß-lactam, macrolide, and fluoroquinolone antibiotic agents.

Skin tanning, either by exposure to natural sunlight or through use of UV sunbeds, has become a popular practice in the US, where it is estimated that approximately 1 million times per day someone in the US uses UV radiation for skin tanning, equating to 30 million Americans (circa 10% of the US population) who use a tanning bed. As well as exposing the host to periods of UV radiation, such practices also expose commensal skin bacteria, including Staphylococcus aureus, to such UV radiation. Previous work has indicated that environmental stresses on bacteria may lead to an upregulation of stress responses, in an attempt for the organism to combat the applied stress and remain viable. UV light may act as an environmental stress on bacteria, and so it was the aim of this study to examine the effect of UVc light on the antibiotic susceptibility of commensal skin bacteria, to determine if UV radiation would increase the antibiotic resistance of such skin flora and thus lead to a potential skin flora with increased antibiotic resistance. Previously, it has been shown that UVc light has a greater mutational effect on bacteria compared to lower-energy UV forms, including UVa and UVb light. Therefore, we decided to employ UVc light in our study to amplify the potential for mutational events occurring in skin staphylococci organisms (n=8) including methicillin-sensitive Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (n=4), and coagulase-negative staphylococci (Staphylococcus haemolyticus) (n=2) were exposed to varying degrees of sublethal radiation via UVc light, and their minimum inhibitory concentration (MIC) susceptibility was determined by broth dilution assay against three classes of commonly used antibiotics, namely ß-lactams (penicillin), macrolides (erythromycin), and fluoroquinolones (ciprofloxacin). There was no significant difference between antibiotic susceptibility before UVc exposure and until maximum sublethal stress, prior to cell death due to fatal UVc exposure with the cells. These results indicate that UV environmental stress/exposure does not upregulate antibiotic resistance, and therefore these data indicate that UVc radiation does not lead to a more antibiotic-resistant population in the staphylococci organisms post-exposure.

Age-stratified Bayesian analysis to estimate sensitivity and specificity of four diagnostic tests for detection of Cryptosporidium oocysts in neonatal calves.

There is no gold standard diagnostic test for the detection of bovine cryptosporidiosis. Infection is usually highest in 2-week-old calves, and these calves also excrete high numbers of oocysts. These factors may give rise to variations in the sensitivity and specificity of the various diagnostic tests used to detect infection in calves of various ages. An age-stratified Bayesian analysis was carried out to determine the optimum diagnostic test to identify asymptomatic and clinical Cryptosporidium sp. infection in neonatal calves. Fecal samples collected from 82 calves at 1 week, 2 weeks, 3 weeks, and 4 weeks of age were subjected to the following tests: microscopic examination of smears stained with either phenol-auramine O or fluorescein isothiocyanate (FITC)-conjugated anti-Cryptosporidium monoclonal antibody, nested-PCR, and quantitative real-time PCR. The results confirmed a high prevalence of Cryptosporidium sp. infection, as well as a high level of oocyst excretion, in 2-week-old calves. The sensitivities of all the tests varied with the age of the calves. Quantitative real-time PCR proved to be the most sensitive and specific test for detecting infection irrespective of the age of the calf. The microscopic techniques were the least sensitive and exhibited only moderate efficiency with 2-week-old calves excreting large numbers of oocysts, the majority of which were diarrheic. It was concluded that, when interpreting the results of routine tests for bovine cryptosporidiosis, cognizance should be taken of the sensitivity of the tests in relation to the age of the calves and stage of infection.

Molecular characterization and phylogenetic analysis of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE gene loci in viridans group streptococci isolated from adult patients with cystic fibrosis.

OBJECTIVES: Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs). METHODS: The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis. RESULTS: Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ≥8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%). CONCLUSIONS: Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.

Zoonoses associated with petting farms and open zoos.

The popularity of open farms and petting zoos has increased markedly over the last 5 years, with most children in developed countries now having the opportunity to visit such a facility at some stage in their childhood, either through school or family visits. The open access policy of these establishments allows visitors to be in direct contact with animals such as sheep (lambs), goats, cats (kittens), dogs (puppies), and birds and to have the opportunity to feed such animals. This contact may lead to the transmission of microbial pathogens from animals to humans, e.g., Escherichia coli O157:H7, resulting in human disease. This review outlines the causal organisms associated with such zoonoses, a description of previous outbreaks at farms and zoos, as well as infection control measures to help prevent such zoonotic infections.

Antibacterial activity of honey against community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA).

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has now been described globally, as a clinically significant pathogen, particularly associated with skin and soft tissue infections, including abscesses, cellulitis and furunculosis. The recent emergence of CA-MRSA combined with its predominant presentation associated with skin and soft tissue infection, the previous literature indicating honey as an effective treatment of healthcare-associated HA-MRSA-related wound infection, as well as honey's ease of topical application, make the current study timely and of interest to healthcare practitioners involved with wound management. Although previous studies have examined the antimicrobial activity of honey against HA-MRSA, such data are limited regarding the activity of honey against this emerging type of MRSA. CA-MRSA (n=6 isolates), was examined for its susceptibility to natural honey (n=3 honey produced from bees in Northern Ireland and one commercial French honey). Results demonstrated that all honey was able to reduce the cultural count of all CA-MRSA from approximately 10(6) colony-forming units (cfus) (mean = 6.46 log10 cfu/g) to none detectable within 24h of co-culture of separate CA-MRSA organisms individually with all four-honey types examined. Subsequent non-selective enrichment of honey demonstrated that inoculated honey remained positive for CA-MRSA until 72h postinoculation, after which point no culturable organisms could be detected. This study demonstrated that, in vitro, these natural products had an antimicrobial activity against the CA-MRSA organisms tested. Further studies are now required to demonstrate if this antimicrobial activity has any clinical application.

Bactericidal activity of denture-cleaning formulations against planktonic health care-associated and community-associated methicillin-resistant Staphylococcus aureus.

The aim of this study was to examine the survival dynamics of several epidemic health care-associated (HA) and community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) in planktonic state in widely employed denture-cleaning solutions. The bacteriocidal activity of five widely employed denture-cleaning formulations were examined against five phage-types of HA-MRSA (EMRSA 15, EMRSA 16, Irish 1, Irish 2, unique type), as well as a CA-MRSA strain, in this study. Viable MRSA cells (circa 10(5) cfu/mL) were coincubated with optimum recommended working concentrations of denture-cleaning solutions for up to 17 hours (overnight). Recovery experiments were unable to isolate any of the inoculated MRSA organisms 10 minutes post inoculation. The significance and impact of this short study indicates that HA-MRSA and CA-MRSA are not able to remain culturable for 10 minutes in planktonic form, in commonly used denture-cleaning formulations widely available on the UK High Street, suggesting that these formulations may be useful in lowering the numbers of MRSA. Further work is however required to examine the more complex survival dynamics of MRSA in naturally derived denture biofilm, associated with dental plaque and the use of such cleaning formulations.

The epidemiology of antibiotic resistance in Campylobacter.

Antibiotic resistance, particularly with the fluoroquinolones and macrolide antibiotics, has now emerged globally with thermophilic campylobacters, including Campylobacter jejuni and C. coli, giving rise to concerns about how these organisms have acquired such resistance characteristics, as well as consequences for human and animal treatment. This review examines (i) the clinical epidemiology of antibiotic resistance in human and animal thermophilic campylobacters, (ii) an update on resistance rates globally, (iii) surveillance of antimicrobial resistance in campylobacters originating from animals, particularly poultry, (iv) the role of the environment in the acquisition and transmission of antibiotic-resistant campylobacters, as well as (v) issues of biocide resistance in campylobacters.

An Update on Global Tuberculosis (TB).

Tuberculosis globally results in almost 2 million human deaths annually, with 1 in 4 deaths from tuberculosis being human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS)-related. Primarily a pathogen of the respiratory system, aerobic Mycobacterium tuberculosis complex (MTBC) infects the lungs via the inhalation of infected aerosol droplets generated by people with pulmonary disease through coughing. This review focuses on M. tuberculosis transmission, epidemiology, detection methods and technologies.

Comparison of minimum inhibitory concentration by broth microdilution testing versus standard disc diffusion testing in the detection of penicillin, erythromycin and ciprofloxacin resistance in viridans group streptococci.

The aim of this study was to investigate the reliability of disc diffusion testing with penicillin, erythromycin and ciprofloxacin within the viridans group streptococci (VGS). In total, the antibiotic susceptibilities of 167 VGS isolates were compared by standard disc diffusion and broth microdilution methods, and these phenotypic data were compared to the carriage of the respective gene resistance determinants [ermB and mefA/E (macrolides); QRDR, gyrA, gyrB, parC and parE (quinolones)]. Overall, there were 35 discrepancies [resistant by MIC and susceptible by zone diameter (21.0%)] between MIC and disc diameter when penicillin susceptibility was interpreted by Clinical and Laboratory Standards Institute criteria. Scattergrams showed a bimodal distribution between non-susceptible and susceptible strains when erythromycin susceptibility was tested by both methods. Thirty-four (20.4%) isolates were categorized as resistant by MIC breakpoints, while disc diameter defined these as having intermediate resistance. With ciprofloxacin, three isolates (1.8%) showed minor discrepancies between MIC breakpoints and disc diameter. Isolates non-susceptible to all three antimicrobial agents tested were reliably distinguished from susceptible isolates by disc diffusion testing, except for the detection of low-level resistance to penicillin, where broth microdilution or an alternative quantitative MIC method should be used. Otherwise, we conclude that disc diffusion testing is a reliable method to detect strains of VGS non-susceptible to penicillin, erythromycin and ciprofloxacin, as demonstrated with their concordance to their gene resistance characteristics.

Population structure and characterization of viridans group streptococci (VGS) including Streptococcus pneumoniae isolated from adult patients with cystic fibrosis (CF).

A study was undertaken to examine the population structure of viridans group streptococci (VGS) in the sputum of adult patients with cystic fibrosis (CF). Freshly expectorated sputa (n=58) from 45 adult CF patients were examined by selective conventional culture on Mitis-Salivarius agar and yielded 190 isolates of VGS. Sequence analyses of the rpnB and 16-23S rRNA ITS genes identified these isolates to belong to 12 species of VGS and included S. anginosus, S. australis, S. cristatus, S. gordonii, S. infantis, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pneumoniae, S. salivarius and S. sanguinis. The most frequently VGS organism isolated was S. salivarius (47/190; 24.7%), followed by S. mitis (36/190; 19%), S. sanguinis (25/190; 13.2%), S. oralis (20/190; 11.0%), S. pneumoniae (19/190; 10.0%), S. parasanguinis (16/190; 8.4%), S. infantis (11/190; 5.8%), S. gordonii (7/190; 3.7%), S. anginosus (4/190; 2.1%), S. cristatus (2/190; 1.1%), S. australis (1/190; 0.5%), S. mutans (1/190; 0.5%) and S. agalactiae (1/190; 0.5%). All, but four, patients harboured at least one VGS species, which ranged from one to five streptococcal species, with a mean of 2.85 species per patient. There was no clonality at the subspecies level employing ERIC RAPD PCR. Antibiotic susceptibility was determined by Minimum Inhibitory Concentration (MIC) testing against penicillin, erythromycin and ciprofloxacin. Overall, resistance to penicillin with all VGS was 73/190 (38.4%) and 167/190 (87.9%) for erythromycin. With regard to ciprofloxacin, 27/190 (14.2%) were fully resistant, whilst a further 21/190 (11.1%) showed intermediate resistance, which equated to approximately three quarters (74.7%) of isolates being fully sensitive to this agent. In addition, as a comparator control population, we examined antibiotic susceptibility, as above, in a non-CF population comprising 12 individuals (50 VGS isolates), who were not receiving chronic antibiotics. In comparison, 8% and 38% of VGS isolates from non-CF individuals were resistant by disk susceptibility testing to penicillin and erythromycin, respectively. None of the non-CF VGS organisms were resistant to ciprofloxacin, but 42% showed intermediate resistance.

Colonization of broilers by Campylobacter jejuni internalized within Acanthamoeba castellanii.

Although Campylobacter survives within amoeba in-vitro, it is unknown if intra-amoeba Campylobacter jejuni can colonize broilers. Five groups of 28 day-of-hatch chicks were placed into separate isolators. Groups (1) and (2) were challenged with page's amoeba saline (PAS), and disinfected planktonic C. jejuni NCTC 11168, respectively. Groups (3), (4) and (5) were challenged with a C. jejuni positive control, C. jejuni in PAS, and intra-amoeba C. jejuni, respectively. After 1, 3, 7 and 14 days post challenge, seven birds from each unit were examined for C. jejuni colonization. For the first time we report that intra-amoeba C. jejuni colonized broilers.

Genotypes and subtypes of Cryptosporidium spp. in neonatal calves in Northern Ireland.

Cryptosporidium spp. in diarrheic calves less than 30 days old from farms across Northern Ireland were examined over a year period by microscopic, genotyping, and subtyping techniques to characterize the transmission dynamics. Cryptosporidium oocysts were detected in 291 of 779 (37.4%) animals. The prevalence rates of rotavirus, coronavirus, and Escherichia coli K99+ were lower as seen in 242 of 806 (30.0%), 46/806 (5.7%), and 16/421 (3.8%) of animals, respectively. Of the 224 Cryptosporidium-positive specimens available for molecular analysis, Cryptosporidium parvum was identified in 213 (95.1%) specimens, Cryptosporidium bovis in eight (3.6%), and Cryptosporidium deer-like genotype in three (1.3%). Sequence analysis of the 60-kDa glycoprotein gene identified 16 IIa subtypes and a new subtype family, with 120 of the 216 (55.6%) positive specimens having the subtype IIaA18G3R1. Eight of the IIa subtypes were previously seen in humans in Northern Ireland. Several subtypes were temporally or geographically unique. The genetic diversity in calves in Northern Ireland was much greater than that reported from other areas. This work demonstrates the utility of genotyping and subtyping tools in characterizing the transmission of Cryptosporidium spp. in calves and humans.

Proteomics analysis and protein expression during sporozoite excystation of Cryptosporidium parvum (Coccidia, Apicomplexa).

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.

Cryptosporidiosis in developing countries.

Globally, Cryptosporidium infection continues to be a significant health problem where it is recognized as an important cause of diarrhoea in both immunocompromised and immunocompetent people. In developing countries persistent diarrhoea is the leading cause of death in children younger than five years of age, where it accounts for 30 to 50 percent of those deaths. Encouragingly an increasing number of investigations in developing countries employ molecular tools, significantly improving the quality of epidemiological information. This improved Cryptosporidium monitoring, with appropriate molecular methods, in surface water, livestock, wildlife and humans, will increase current knowledge of infection and transmission patterns, and ultimately help to control Cryptosporidium via improved risk assessments in the future.