Healthcare personnel intestinal colonization with multidrug-resistant organisms.

OBJECTIVES: This study aims to assess the association between patient contact and intestinal carriage of multidrug-resistant organisms (MDRO) by sampling healthcare personnel (HCP) and staff without patient contact. METHODS: For this observational study, we recruited 400 HCP who worked in our 200-bed research hospital and 400 individuals without patient contact between November 2013 and February 2015. Participants submitted two self-collected perirectal swabs and a questionnaire. Swabs were processed for multidrug-resistant Gram-negative bacteria and vancomycin-resistant enterococci (VRE). Questionnaires explored occupational and personal risk factors for MDRO carriage. RESULTS: Among 800 participants, 94.4% (755/800) submitted at least one swab, and 91.4% (731/800) also submitted questionnaires. Extended spectrum ß-lactamase-producing organisms were recovered from 3.4% (26/755) of participants, and only one carbapenemase-producing organism was recovered. No VRE were detected. The potential exposure of 68.9% (250/363) of HCP who reported caring for MDRO-colonized patients did not result in a rate of MDRO carriage among HCP (4.0%; 15/379) significantly higher than that of staff without patient contact (3.2%; 12/376; p 0.55). CONCLUSIONS: This is the largest US study of HCP intestinal MDRO carriage. The low colonization rate is probably reflective of local community background rates, suggesting that HCP intestinal colonization plays a minor role in nosocomial spread of MDROs in a non-outbreak setting. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01952158.

Horizontal Transfer of Carbapenemase-Encoding Plasmids and Comparison with Hospital Epidemiology Data.

Carbapenemase-producing organisms have spread worldwide, and infections with these bacteria cause significant morbidity. Horizontal transfer of plasmids carrying genes that encode carbapenemases plays an important role in the spread of multidrug-resistant Gram-negative bacteria. Here we investigate parameters regulating conjugation using an Escherichia coli laboratory strain that lacks plasmids or restriction enzyme modification systems as a recipient and also using patient isolates as donors and recipients. Because conjugation is tightly regulated, we performed a systematic analysis of the transfer of Klebsiella pneumoniae carbapenemase (blaKPC)-encoding plasmids into multiple strains under different environmental conditions to investigate critical variables. We used four blaKPC-carrying plasmids isolated from patient strains obtained from two hospitals: pKpQIL and pKPC-47e from the National Institutes of Health, and pKPC_UVA01 and pKPC_UVA02 from the University of Virginia. Plasmid transfer frequency differed substantially between different donor and recipient pairs, and the frequency was influenced by plasmid content, temperature, and substrate, in addition to donor and recipient strain. pKPC-47e was attenuated in conjugation efficiency across all conditions tested. Despite its presence in multiple clinical species, pKPC_UVA01 had lower conjugation efficiencies than pKpQIL into recipient strains. The conjugation frequency of these plasmids into K. pneumoniae and E. coli patient isolates ranged widely without a clear correlation with clinical epidemiological data. Our results highlight the importance of each variable examined in these controlled experiments. The in vitro models did not reliably predict plasmid mobilization observed in a patient population, indicating that further studies are needed to understand the most important variables affecting horizontal transfer in vivo.

Genetic analysis of Israel acute paralysis virus: distinct clusters are circulating in the United States.

Israel acute paralysis virus (IAPV) is associated with colony collapse disorder of honey bees. Nonetheless, its role in the pathogenesis of the disorder and its geographic distribution are unclear. Here, we report phylogenetic analysis of IAPV obtained from bees in the United States, Canada, Australia, and Israel and the establishment of diagnostic real-time PCR assays for IAPV detection. Our data indicate the existence of at least three distinct IAPV lineages, two of them circulating in the United States. Analysis of representatives from each proposed lineage suggested the possibility of recombination events and revealed differences in coding sequences that may have implications for virulence.

Biochemical and biophysical characterization of OmpG: A monomeric porin.

A recombinant form of the porin OmpG, OmpGm, lacking the signal sequence, has been expressed in Escherichia coli. After purification under denaturing conditions, the protein was refolded in the detergent Genapol X-080, where it gained a structure rich in beta sheet as evidenced by a CD spectrum similar to that of the native form. Electrophoretic analysis and limited proteolysis experiments suggested that refolded OmpGm exists in at least three forms. Nevertheless, the recombinant protein formed uniform channels in planar bilayers with a conductance of 0.81 nS (1 M NaCl, pH 7.5). Previous biochemical studies had suggested that OmpG is a monomeric porin, rather than the usual trimer. Bilayer recordings substantiated this proposal; voltage-induced closures occurred consistently in a single step, and channel block by Gd(3+) lacked the cooperativity seen with the trimeric porin OmpF. The availability of milligram amounts of a monomeric porin will be useful both for basic studies of porin function and for membrane protein engineering.

A functional protein pore with a "retro" transmembrane domain.

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.

Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter.

The detection of organic molecules is important in many areas, including medicine, environmental monitoring and defence. Stochastic sensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. Engineered transmembrane protein pores are promising sensor elements for stochastic detection, and in their simplest manifestation they produce a fluctuating binary ('on/off') response in the transmembrane electrical current. The frequency of occurrence of the fluctuations reveals the concentration of the analyte, and its identity can be deduced from the characteristic magnitude and/or duration of the fluctuations. Genetically engineered versions of the bacterial pore-forming protein alpha-haemolysin have been used to identify and quantify divalent metal ions in solution. But it is not immediately obvious how versatile binding sites for organic ligands might be obtained by engineering of the pore structure. Here we show that stochastic sensing of organic molecules can be procured from alpha-haemolysin by equipping the channel with an internal, non-covalently bound molecular 'adapter' which mediates channel blocking by the analyte. We use cyclodextrins as the adapters because these fit comfortably inside the pore and present a hydrophobic cavity suitable for binding a variety of organic analytes. Moreover, a single sensing element of this sort can be used to analyse a mixture of organic molecules with different binding characteristics. We envisage the use of other adapters, so that the pore could be 'programmed' for a range of sensing functions.

The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes.

The conserved bifactorial endosperm box found in the promoter of wheat storage protein genes comprises two different cis elements that are thought to be involved in regulating endosperm-specific gene expression. Endosperm nuclear extracts contain binding activities. One is called ESBF-I, which binds to the endosperm motif (EM), and the other is called ESBF-II, which binds to the GCN4-like motif(GLM). Here, we present a functional analysis of the endosperm box of a low-molecular-weight glutenin gene found on the 1D1 chromosome of hexaploid wheat (LMWG-1D1) in transgenic tobacco plants. Our analysis demonstrates the necessity of the EM and GLM for endosperm-specific gene expression and suggests the presence in tobacco of functional counterparts of wheat ESBF-I and ESBF-II. Furthermore, we describe the isolation and characterization of cDNA clones encoding SPA, a seed-specific basic leucine zipper protein from wheat that can activate transcription from the GLMs of the -326-bp LMWG-1D1 promoter in both maize and tobacco leaf protoplasts. This activation is also partially dependent on the presence of functional EMs, suggesting interactions between SPA with ESBF-I-like activities.

An audit of ANCA in routine clinical practice.

We have reviewed the medical records of 301/327 consecutive patients in whom anti-neutrophil cytoplasmic antibodies (ANCA) were detected by the Regional Immunology Laboratory in Northern Ireland between January 1988 and October 1991 (45 months). We have collected data for each patient regarding age, sex, smoking habit, area of residence, and details of any other autoantibody activity. Clinical diagnosis was established, with the number of organ systems involved and the evidence for that involvement (symptomatic, biochemical, radiological, and histological). Diagnoses were divided into four groups according to their recognised vasculitic features and these were related to the pattern of immunofluorescence and maximum ANCA titre detected. The most frequent diagnosis was rheumatoid arthritis (18.2% of patients) and the connective tissue disorders as a whole accounted for 27.9% of patients. ANCA were also detected in a wide range of clinical conditions which are not associated with vasculitis and these patients were an important source of 'false-positives'. The positive predictive value (PPV) of ANCA of all patterns and titres for vasculitic conditions was 27%, however, the detection of a classical ANCA pattern at high titre (> or = 1:640) was associated with an increased PPV of 75%. The coexistence of an antinuclear antibody (ANA) reduces the PPV of both classical and perinuclear ANCA, although perinuclear ANCA with antimyeloperoxidase specificity had an improved PPV. We conclude that ANCA testing should not be used as the only screening investigation for vasculitis but should be included in a rational investigative scheme. The interpretation of a positive ANCA result must take into account the presence of other autoantibodies and the full range of non-vasculitic conditions when the clinical situation is not typical of vasculitis.

Comparison of immunofluorescence and an immunohistochemical technique for the detection of perinuclear anti-neutrophil cytoplasmic antibody.

Using the standard indirect immunofluorescent (IIF) technique two types of autoantibodies are detected in the sera of patients with vasculitic disorders. These are cytoplasmic or classical antineutrophil cytoplasmic antibody (cANCA) and perinuclear anti-neutrophil cytoplasmic antibody (pANCA). In order to resolve the problems associated with the detection of pANCA an immunocytochemical technique-alkaline phosphatase anti-alkaline phosphatase (APAAP) was developed and used to detect ANCA in various groups of patients. Comparison with the standard immunofluorescence method showed that the results correlated only when immunostaining patterns were of the cANCA type. Detection of pANCA by APAAP was more uncertain than by immunofluorescence because of the greater number of staining patterns seen. Interference from antinuclear antibodies (ANA) appeared to cause more problems with the APAAP technique and sera containing ANA were not distinguished from pANCA using either immunofluorescence or APAAP. In conclusion, it appears that the APPAP technique is not a reliable method of the screening of ANCA and that pANCA has several antigenic specificities.