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1.  A commentary on the disparate perspectives of clinical microbiologists and surgeons 
Bioengineered  2014;5(4):218-221.
Prosthetic joints and other orthopedic implants have improved quality of life for patients world-wide and the use of such devices is increasing. However, while infection rates subsequent to associated surgery are relatively low (<3%), the consequences of incidence are considerable, encompassing morbidity (including amputation) and mortality in addition to significant social and economic costs. Emphasis, therefore, has been placed on mitigating microbial risk, with clinical microbiologists and surgeons utilizing rapidly evolving molecular laboratory techniques in detection and diagnosis of infection, which still occurs despite sophisticated patient management. Multidisciplinary approaches are regularly adopted to achieve this. In this commentary, we describe an unusual case of Actinomyces infection in total hip arthroplasty and, in that context, describe the perspectives of the clinical microbiology and surgical teams and how they contrasted. More specifically, this case demonstrates an ad hoc approach to structured eradication of biofilms and intracellular bacteria related to biomaterials, as reflected in early usage of linezolid. This is a complex topic and, as described in this case, such accelerated treatment can be effective. This commentary focuses on the merits of such inadvisable use of potent antimicrobials amid the risk of diminishing valuable antimicrobial efficacy, albeit resulting in desirable patient outcomes.
doi:10.4161/bioe.28722
PMCID: PMC4140865  PMID: 25122489
ad hoc; antimicrobial; clinical microbiology; perspectives; surgery
2.  A commentary on the role of molecular technology and automation in clinical diagnostics 
Bioengineered  2014;5(3):155-160.
Historically, the identification of bacterial or yeast isolates has been based on phenotypic characteristics such as growth on defined media, colony morphology, Gram stain, and various biochemical reactions, with significant delay in diagnosis. Clinical microbiology as a medical specialty has embraced advances in molecular technology for rapid species identification with broad-range 16S rDNA polymerase chain reaction (PCR) and matrix-assisted laser desorption and/or ionization time of flight (MALDI-TOF) mass spectrometry demonstrated as accurate, rapid, and cost-effective methods for the identification of most, but not all, bacteria and yeasts. Protracted conventional incubation times previously necessary to identify certain species have been mitigated, affording patients quicker diagnosis with associated reduction in exposure to empiric broad-spectrum antimicrobial therapy and shortened hospital stay. This short commentary details such molecular advances and their implications in the clinical microbiology setting.
doi:10.4161/bioe.28599
PMCID: PMC4101006  PMID: 24658184
clinical microbiology; MALDI; PCR; patient care; impact
3.  Genome analysis of the staphylococcal temperate phage DW2 and functional studies on the endolysin and tail hydrolase 
Bacteriophage  2014;4:e28451.
This study describes the genome of temperate Siphoviridae phage DW2, which is routinely propagated on Staphylococcus aureus DPC5246. The 41941 bp genome revealed an open reading frame (ORF1) which has a high level of homology with members of the resolvase subfamily of site-specific serine recombinase, involved in chromosomal integration and excision. In contrast, the majority of staphylococcal phages reported to date encode tyrosine recombinases. Two putative genes encoded by phage DW2 (ORF15 and ORF24) were highly homologous to the NWMN0273 and NWMN0280 genes encoding virulence factors carried on the genome of ϕNM4, a prophage in the genome of S. aureus Newman. Phage DW2 also encodes proteins highly homologous to two well-characterized Staphylococcus aureus pathogenicity island derepressors encoded by the staphylococcal helper phage 80α indicating that it may similarly act as a helper phage for mobility of pathogenicity islands in S. aureus. This study also focused on the enzybiotic potential of phage DW2. The structure of the putative endolysin and tail hydrolase were investigated and used as the basis for a cloning strategy to create recombinant peptidoglycan hydrolyzing proteins. After overexpression in E. coli, four of these proteins (LysDW2, THDW2, CHAPE1-153, and CHAPE1-163) were demonstrated to have hydrolytic activity against peptidoglycan of S. aureus and thus represent novel candidates for exploitation as enzybiotics.
doi:10.4161/bact.28451
PMCID: PMC4124061  PMID: 25105056
bacteriophage; Staphylococcus; endolysin; virion-associated peptidoglycan hydrolase; virulence; serine recombinase
4.  A survey of management practices on Irish dairy farms with emphasis on risk factors for Johne’s disease transmission 
Irish Veterinary Journal  2014;67(1):27.
Background
Johne’s disease (JD) is a chronic granulomatous enteritis affecting ruminants. A number of farm management practices are associated with increased risk of JD transmission. The aim of the current study was to document JD-related management practices currently employed on Irish dairy farms.
Survey questions focused on calving area (CA), calf and manure management. Independent variables (region, calving-season, enterprise type, herd size and biosecurity status) were used to examine influences on JD associated dependent variables (survey questions). Additionally general biosecurity practices were also examined.
Results
Results showed management practices implemented by Irish dairy farmers pose a high risk of JD transmission. Of the farmers surveyed, 97% used the CA for more than one calving, 73.5% and 87.8% pooled colostrum and milk respectively, 33.7% never cleaned the CA between calving’s, and 56.6% used the CA for isolating sick cows. Survey results also highlighted that larger herds were more likely to engage in high risk practices for JD transmission, such as pooling colostrum (OR 4.8) and overcrowding the CA (OR 7.8). Larger herds were also less likely than smaller herds to clean the CA (OR 0.28), a practice also considered of risk in the transmission of JD.
Conclusion
Many management practices associated with risk of JD transmission were commonly applied on Irish dairy farms. Larger herds were more likely to engage in high risk practices for JD transmission. Control programmes should incorporate educational tools outlining the pathogenesis and transmission of JD to highlight the risks associated with implementing certain management practices with regard to JD transmission.
Electronic supplementary material
The online version of this article (doi:10.1186/s13620-014-0027-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13620-014-0027-9
PMCID: PMC4300563  PMID: 25610611
Johne’s disease; Survey; Management practices; Biosecurity
5.  The role of the Cronobacter sakazakii ProP C-terminal coiled coil domain in osmotolerance 
Gut Pathogens  2014;6:46.
Background
We investigate the role of the C-terminal coiled coil of the secondary proline porter ProP in contributing to Cronobacter sakazakii osmotolerance.
Findings
The extended C-terminal domain of ProP1 (encoded by ESA_02131) was spliced onto the truncated C-terminal end of ProP2 (encoded by ESA_01706); creating a chimeric protein (ProPc) which exhibits increased osmotolerance relative to the wild type.
Conclusions
It appears that the C-terminal coiled coil domain tunes ProP at low osmolality, whereas ProP transporters lacking the coiled coil domain are more active at a higher osmolality range.
doi:10.1186/s13099-014-0046-9
PMCID: PMC4272814  PMID: 25530808
6.  Analysis of the role of the Cronobacter sakazakii ProP homologues in osmotolerance 
Gut Pathogens  2014;6:15.
Bacteria respond to elevated osmolality by the accumulation of a range of low molecular weight molecules, known as compatible solutes (owing to their compatibility with the cells' normal physiology at high internal concentrations). The neonatal pathogen Cronobacter sakazakii is uniquely osmotolerant, surviving in powdered infant formula (PIF) which typically has a water activity (aw) of 0.2 – inhospitable to most micro-organisms. Mortality rates of up to 80% in infected infants have been recorded making C. sakazakii a serious cause for concern. In silico analysis of the C. sakazakii BAA-894 genome revealed seven copies of the osmolyte uptake system ProP. Herein, we test the physiological role of each of these homologues following heterologous expression against an osmosensitive Escherichia coli host.
doi:10.1186/1757-4749-6-15
PMCID: PMC4047261  PMID: 24910715
Osmolytes; Proline; Osmotolerance; Stress; Cronobacter
7.  The Single Intradermal Cervical Comparative Test Interferes with Johne’s Disease ELISA Diagnostics 
Enzyme-linked immunosorbent assays (ELISA) of milk and serum samples are a routinely used method of screening herds for Mycobacterium avium subspecies paratuberculosis (MAP). Infection with MAP causes granulomatous enteritis of ruminants known as Johne’s disease (JD). The sensitivity (Se) and specificity (Sp) of MAP ELISAs leads to difficulties in the identification of both infected and infectious animals. Interference with MAP ELISA Se and Sp has been reported in MAP seronegative cows following administration of purified protein derivative (PPD) as part of intradermal testing for bovine tuberculosis (bTB). The aim of this study is to examine the impact of the single intradermal cervical comparative test (SICCT) for bTB, on both serum and milk MAP ELISA tests, in a herd containing both seropositive and seronegative cows pre-SICCT. A secondary objective is to provide appropriate timing of JD ELISA tests in relation to the SICCT. A herd of 139 cows were serum and milk sampled pre- and post-SICCT administration. Prior to SICCT, 6% of the herd tested seropositive for MAP using milk ELISA, with 8% positive on serum. ID Screen Paratuberculosis Indirect Screening Test (ID Vet) was used to screen the herd. Within 14 days of PPD administration, a significant increase in the prevalence of seropositive cows was recorded. Identical prevalence’s were recorded with both test matrices (39%). ELISA values remained significantly higher until day 43 post-SICCT in milk (P = 0.850), and day 71 in serum (P = 0.602). If the “new” positives detected post-bTB testing are deemed false positives due to generation of cross-reacting antibodies by administration of PPD, milk would appear a more suitable sample for JD ELISA testing within 2 months of SICCT. In summary, sampling for JD utilizing milk ELISA should be avoided in the 43-day period following PPD administration, with serum ELISA sampling avoided for an additional 28 days.
doi:10.3389/fimmu.2014.00564
PMCID: PMC4228858  PMID: 25429289
Mycobacteriacea; Johne’s disease; TB test; ELISA; PPD
8.  VP4 and VP7 Genotyping of Rotavirus Samples Recovered from Infected Children in Ireland over a 3-Year Period 
Journal of Clinical Microbiology  1999;37(6):1699-1703.
Between September 1995 and August 1998, the incidence and diversity of the main human rotavirus genotypes (G1, G2, G3, and G4 and P[8], P[4], P[6], and P[9]) among Irish children were determined by using established and adapted reverse transcriptase PCR-based genotyping methods. From a total of 193 rotavirus-positive specimens collected from nine hospitals we successfully identified the P type in 182 (94%) of the samples and the G type in 165 (85.5%) of the samples. Only four samples could not be assigned a G or P type. Two P types existed in Ireland, P[8] (78%) and P[4] (16%), and their relative incidence varied over the 3 years of this study. No P[6] or P[9] types were detected. G1 was the most predominant G type (55%), and the incidences of G2, G3, and G4 isolates were 15.5, 1, and 11%, respectively. Three percent of the samples tested had a mixed G type. A P and G type was assigned to 158 (81.8%) of samples. Of the typeable samples, G1 P[8] was the most prevalent (65%), whereas G2 P[4] (17%), G3 P[8] (1%), G4 P[8] (12%), and mixed types (all G1/ G4 P[8]) (4%) were detected less frequently. In the third year a significant genotypic shift from G1 P[8] to G2 P[4] and G4 P[8] was observed. During the study, we noticed that the inclusion of random primers during cDNA synthesis greatly increased the specificity of the PCR typing assays. No correlation was seen between the contributing hospitals and a specific genotype. In conclusion, the coverage of infection given by the recently licensed tetravalent vaccine would be significantly high in Ireland, although future monitoring of genotypic changes among Irish isolates should be encouraged.
PMCID: PMC84927  PMID: 10325310

Results 1-8 (8)