Frequency of resistance to methicillin and other antimicrobial agents among Staphylococcus aureus strains isolated from pigs and their human handlers in Trinidad

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has emerged recently worldwide in production animals, particularly pigs and veal calves, which act as reservoirs for MRSA strains for human infection. The study determined the prevalence of MRSA and other resistant strains of S. aureus isolated from the anterior nares of pigs and human handlers on pig farms in Trinidad. Methods: Isolation of S. aureus was done by concurrently inoculating Baird-Parker agar (BPA) and Chromagar MRSA (CHROM) with swab samples and isolates were identified using standard methods. Suspect MRSA isolates from Chromagar and BPA were subjected to confirmatory test using Oxoid PBP2 latex agglutination test. The disc diffusion method was used to determine resistance to antimicrobial agents. Results: The frequency of isolation of MRSA was 2.1% (15 of 723) for pigs but 0.0% (0 of 72) for humans. Generally, for isolates of S. aureus from humans there was a high frequency of resistance compared with those from pigs, which had moderate resistance to the following antimicrobials: penicillin G (54.5%, 51.5%), ampicillin (59.1%, 49.5%), and streptomycin (59.1%, 37.1%), respectively. There was moderate resistance to tetracycline (36.4%, 41.2%) and gentamycin (27.2%, 23.7%) for human and pig S. aureus isolates, respectively, and low resistance to sulfamethoxazole-trimethoprim (4.5%, 6.2%) and norfloxacin (9.1%, 12.4%), respectively. The frequency of resistance to oxacillin by the disc method was 36.4 and 34.0% from S. aureus isolates from humans and pigs, respectively. Out of a total of 78 isolates of S. aureus from both human and pig sources that were resistant to oxacillin by the disc diffusion method, only 15 (19.2%) were confirmed as MRSA by the PBP'2 latex test kit. Conclusions: The detection of MRSA strains in pigs, albeit at a low frequency, coupled with a high frequency of resistance to commonly used antimicrobial agents in pig and humans could have zoonotic and therapeutic implications. Finally, the diagnostic limitation of using CHROMagar and testing for oxacillin resistance by the disc diffusion method alone to determine MRSA strains without performing confirmatory tests cannot be overemphasized because the possibility of overdiagnosis of MRSA infections cannot be ignored

Conformational Targeting of Fibrillar Polyglutamine Proteins in Live Cells Escalates Aggregation and Cytotoxicity

Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases

The utility of molecular diagnostics to predict recurrence of head and neck carcinoma

BACKGROUND: Locoregional recurrence is the major cause of treatment failure after surgery for oral squamous cell carcinoma. Molecular diagnostics have the potential to improve on clinicopathological parameters to predict this recurrence and plan adjuvant treatment. The test most frequently applied is based on detecting TP53 mutations, but alternative methodology is required for cases that harbour the wild-type gene. METHODS: One hundred and two cases with tumour-adjacent margins, considered to be clear margins by microscopy, were examined using carefully optimised molecular diagnostics based on detection of the TP53 and Ly-6D markers. The markers were also combined to provide a dual approach. RESULTS: The dual molecular diagnostic identified cases with a significant increase in the probablility of developing locoregional recurrence when tumour-adjacent positive and clear margins were compared (P=0.0001). These tests were most useful when the clearance at the resection margins was 5 mm or less. The TP53-based diagnostic was a better predictor of locoregional recurrence than established clinicopathological parameters. CONCLUSION: The optimised TP53-based diagnostic rapidly identifies an important subgroup of cases with close margins that will benefit from new treatment modalities to reduce the risk of recurrence

Molecular diagnosis of minimal residual disease in head and neck cancer patients

Aim Locoregional recurrences and distant metastases in adequately treated head and neck squamous cell carcinoma (HNSCC) patients have a dismal effect on survival. Tumor cells that escape histopathological detection might be the prime cause of this effect. We evaluated whether minimal residual cancer (MRC) in deep surgical margins and disseminated tumor cells (DTCs) in bone marrow aspirates are associated with clinicohistopathological parameters and outcome. Methods Submucosal samples of deep resection margins of 105 HNSCC patients with histopathologically tumor-free surgical margins were analysed for the presence of MRC using hLy-6D qRT-PCR. Bone-marrow aspirates of 76 of these patients were analysed for DTCs by immunocytochemical staining. Presence of molecular-positive deep surgical margins, presence of DTC in bone marrow aspirates, and clinicohistopathological parameters were tested for associations with survival parameters by univariate and multivariate analyses. Results In addition to lymph node stage, it appeared that vasoinvasive growth and particularly infiltrative growth pattern are significant predictors for locoregional recurrence (p = 0.041 and p = 0.006, respectively) and disease-free survival (p = 0.014 and p = 0.008, respectively). Remarkably, neither the presence of molecular-positive deep surgical margins nor that of DTC in bone marrow aspirates were significantly related to outcome. Conclusions The presence of vasoinvasive and infiltrative growth in HNSCC tumor specimens are significant risk-factors for locoregional recurrence and disease-free survival. At present there seems no role for molecular analysis of deep surgical margins and bone marrow aspirates in predicting outcome with the methods used