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Bis-heteroleptic cyclometalated iridium complexes of the form Ir(La)2(acac), where La is a substituted 2-phenylpyridine derivative and acac is an acetylacetonato ligand, are a useful class of luminescent organometallic complexes for a range of applications. Related tris-heteroleptic complexes of the form Ir(La)(Lb)(acac) offer the potential advantage of greater functionality through the use of two different cyclometalated ligands but are, in general, more difficult to obtain. We report the synthesis of divergent bis- and tris-heteroleptic triisopropylsilylethynyl-substituted intermediate complexes that can be diversified using a "chemistry-on-the-complex" approach. We demonstrate the methodology through one-pot deprotection and Sonogashira cross-coupling of the intermediate complexes with para-R-aryliodides (R = H, SMe, and CN). The photophysical and electrochemical behaviors of the resultant bis- and tris-heteroleptic complexes are compared, and it is shown that the tris-heteroleptic complexes exhibit subtly different emission and redox properties to the bis-heteroleptic complexes, such as further red-shifted emission maxima and lower extinction coefficients, which can be attributed to the reduced symmetry. It is demonstrated, supported by DFT and time-dependent DFT calculations, that the charge-transfer character of the emission can be altered via variation of the terminal substituent; the introduction of an electron-withdrawing cyano group in the terminal position leads to a significant red shift, while the introduction of an SMe group can substantially increase the emission quantum yield. Most notably, this convenient synthetic approach reduces the need to perform the often challenging isolation of tris-heteroleptic complexes to a single divergent intermediate, which will simplify access to families of complexes of the form Ir(La)(Lb)(acac).

ObjectivesAntigen-based rapid diagnostic tests (Ag-RDTs) have been widely used for the detection of SARS-CoV-2 during the Covid-19 pandemic. In settings of low disease prevalence, such as asymptomatic community testing, national guidelines recommend molecular confirmation of positive Ag-RDT results. This often requires patients to be recalled for repeat specimen recollection and subsequent testing in reference laboratories. This project assessed the use of a point-of-care molecular method for SARS-CoV-2 detection on-site at a volunteer-led asymptomatic community testing site, using the residual test buffer (RTB) from positive Ag-RDTs.MethodsThe Abbott COVID-19 ID NOW assay was performed on RTB from two Ag-RDTs: the Abbott Panbio COVID-19 Ag Rapid Test Device and the BTNX Rapid Response COVID-19 Antigen Rapid Test Device. All RTBs were tested using real-time RT-PCR at a reference laboratory using the ThermoFisher TaqPath COVID-19 Combo kit which was used to assign positive Ag-RDTs results as true or false positives. Analytical specificity of the ID NOW was assessed with a panel of various respiratory organisms.ResultsOf 419 positive Ag-RDTs from 5148 tests performed, ID NOW testing of the RTB was positive in 100% of the samples characterized as true positives by RT-PCR. No SARS-CoV-2 detections by ID NOW were observed from 10 specimens characterized as false positive Ag-RDTs, or from contrived specimens with various respiratory organisms.ConclusionsThe use of on-site molecular testing on RTB provides a suitable option for rapid confirmatory testing of positive Ag-RDTs, thereby obviating the need for specimen recollection for molecular testing at local reference laboratories.

ObjectivesAntigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for SARS-CoV-2 detection. However, misleading demonstrations of the Abbott Panbio COVID-19 Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside of manufacturer recommendations.MethodsUsing Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested, with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and tween-20), as well as the impact of temperatures (4°C, 20°C, and 45°C) and humidity (90%) on assay performance.ResultsDirect sample testing (without the kit buffer), resulted in false positive signals resembling those obtained with SARS-CoV-2-positive controls tested under proper conditions. The likely explanation of these artifacts is non-specific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside of manufacturer claims.ConclusionsOur data support strict adherence to manufacturer instructions to avoid false positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation.

ObjectivesTo compare the epidemiology and antimicrobial susceptibility patterns of Streptococcus pneumoniae collected from respiratory and blood culture samples in Canada between 2007 and 2016.MethodsS. pneumoniae strains were obtained from Canadian hospitals as part of the ongoing national surveillance study, CANWARD. Isolates were serotyped using the Quellung method. Antimicrobial susceptibility testing was performed using the CLSI broth microdilution method. MDR and XDR were defined as resistance to three or more and five or more classes of antimicrobials, respectively.ResultsOf the 2581 S. pneumoniae isolates collected, 1685 (65.3%) and 896 (34.7%) were obtained from respiratory and blood samples, respectively. Respiratory isolates demonstrated lower rates of antimicrobial susceptibility than blood isolates to penicillin, ceftriaxone, clarithromycin, clindamycin, doxycycline and trimethoprim/sulfamethoxazole (P ≤ 0.03). From 2007 to 2016, invasive isolates demonstrated trends towards increasing penicillin susceptibility and decreasing clarithromycin susceptibility. MDR was significantly higher in respiratory S. pneumoniae compared with blood (9.1% versus 4.5%, P ConclusionsS. pneumoniae from respiratory samples demonstrated lower antimicrobial susceptibilities and higher MDR in a greater diversity of serotypes than isolates obtained from blood. Many serotypes were associated with one specific specimen source, while others were associated with both; genetic characterization is necessary to elucidate the specific factors influencing the ability of these serotypes to commonly cause both invasive and non-invasive disease.

Indium tin oxide (ITO) is an attractive substrate for single-molecule electronics since it is transparent while maintaining electrical conductivity. Although it has been used before as a contacting electrode in single-molecule electrical studies, these studies have been limited to the use of carboxylic acid terminal groups for binding molecular wires to the ITO substrates. There is thus the need to investigate other anchoring groups with potential for binding effectively to ITO. With this aim, we have investigated the single-molecule conductance of a series of eight tolane or “tolane-like” molecular wires with a variety of surface binding groups. We first used gold–molecule–gold junctions to identify promising targets for ITO selectivity. We then assessed the propensity and selectivity of carboxylic acid, cyanoacrylic acid, and pyridinium-squarate to bind to ITO and promote the formation of molecular heterojunctions. We found that pyridinium squarate zwitterions display excellent selectivity for binding to ITO over gold surfaces, with contact resistivity comparable to that of carboxylic acids. These single-molecule experiments are complemented by surface chemical characterization with X-ray photoelectron spectroscopy, quartz crystal microbalance, contact angle determination, and nanolithography using an atomic force miscroscope. Finally, we report the first density-functional theory calculations involving ITO electrodes to model charge transport through ITO–molecule–gold heterojunctions.

Indium tin oxide (ITO) is an attractive substrate for single-molecule electronics since it is transparent while maintaining electrical conductivity. Although it has been used before as a contacting electrode in single-molecule electrical studies, these studies have been limited to the use of carboxylic acid terminal groups for binding molecular wires to the ITO substrates. There is thus the need to investigate other anchoring groups with potential for binding effectively to ITO. With this aim, we have investigated the single-molecule conductance of a series of eight tolane or “tolane-like” molecular wires with a variety of surface binding groups. We first used gold–molecule–gold junctions to identify promising targets for ITO selectivity. We then assessed the propensity and selectivity of carboxylic acid, cyanoacrylic acid, and pyridinium-squarate to bind to ITO and promote the formation of molecular heterojunctions. We found that pyridinium squarate zwitterions display excellent selectivity for binding to ITO over gold surfaces, with contact resistivity comparable to that of carboxylic acids. These single-molecule experiments are complemented by surface chemical characterization with X-ray photoelectron spectroscopy, quartz crystal microbalance, contact angle determination, and nanolithography using an atomic force miscroscope. Finally, we report the first density-functional theory calculations involving ITO electrodes to model charge transport through ITO–molecule–gold heterojunctions., This work was supported by EPSRC under Grants EP/M005046/1 (Single-Molecule Photo-Spintronics, Liverpool), EP/M029522/1 (Single Molecule Plasmoelectronics, Liverpool), EP/M029204/1 (Single Molecule Plasmoelectronics, Durham), EP/N017188/1, EP/M014452/1, EP/P027156/1, and EP/N03337X/1. Support from the European Commission is provided by the FET Open project 767187 – QuIET. A.V. acknowledges funding from the Royal Society (URF\R1\191241) and thanks Dr. Richard J. Brooke and Prof. Walther Schwarzacher for assistance in developing the Python script used for data processing. I.J.P. would like to thank Vivien Walter for his help with coding some of the data analysis procedures. S.S. thanks the Leverhulme Trust for funding (Early Career Fellowship ECF-2018-375). H.S. thanks UKRI for funding (Future Leaders Fellowship MR/S015329/2). S.M. and P.C. acknowledge financial assistance from Ministerio de Ciencia e Innovación from Spain and fondos FEDER in the framework of projects MAT2016-78257-R and PID2019-105881RB-I00 and support from Gobierno de Aragón through the grant numbers LMP33-18 and E31_20R with European Social Fund (Construyendo Europa desde Aragón).

We studied the electrical conductance of single-molecule junctions formed from molecular wires with four anchor groups. Three tetraphenyl-aza-BODIPYs with four or two thiomethyl anchor groups were synthesized, and their single-molecule conductance was measured using break-junction-STM. Using DFT based calculations these compounds were shown to display a combination of a high and low conductance, depending on the molecule's connectivity in the junction. A scissor correction is employed to obtain the corrected HOMO-LUMO gaps and a tight binding model (TBM) is used to highlight the role of transport through the pi system of the tetraphenyl-aza-BODIPY central unit. The three higher-conductance geometries follow the sequence 3 > 4 > 2, which demonstrates that their conductances are correlated with the number of anchors.

A simple and versatile route for modifying picolinate ligands coordinated to iridium is described. Reacting a μ-chloro iridium(C∧N) dimer (where C∧N is a phenylpyridine-based ligand) with bromopicolinic acid (HpicBr) yields the corresponding iridium(C∧N)2(picBr) complexes (1–4 and 11), which were readily modified by a Sonogashira reaction to give eight alkyne-substituted picolinate complexes (5–10, 12, and 13). The luminescent behavior of these complexes shows that the position of substitution about the picolinate ring has an effect on both photophysical behavior as well as the reactivity.

Volume 62, no. 8, e02576-17, 2018, [https://doi.org/10.1128/AAC.02576-17][1]. This article was published on 27 July 2018 with the second author's surname incorrectly indexed as “del Carmen Parquet” instead of “Parquet.” This has been corrected in the current version, posted on 7 December

Together with the more intuitive and commonly recognized conductance mechanisms of charge‐hopping and tunneling, quantum interference (QI) phenomena have been identified as important factors affecting charge transport through molecules. Consequently, establishing simple, flexible molecular design strategies to understand, control and exploit QI in molecular junctions poses an exciting challenge. Here we demonstrate that destructive quantum interference (DQI) in meta‐substituted phenylene ethylene‐type oligomers (m‐OPE) can be tuned by changing the position and conformation of pendant methoxy (OMe) substituents around the central phenylene ring. These substituents play the role of molecular‐scale ‘taps’, which can be switched on or off to control the current flow through a molecule. Our experimental results conclusively verify recently postulated magic ratio and orbital product rules, and highlight a novel chemical design strategy for tuning and gating DQI features, to create single‐molecule devices with desirable electronic functions.

The single molecule conductances of a series of bis-2,2':6',2″-terpyridine complexes featuring Ru(II), Fe(II), and Co(II) metal ions and trimethylsilylethynyl (Me3SiC≡C-) or thiomethyl (MeS-) surface contact groups have been determined. In the absence of electrochemical gating, these complexes behave as tunneling barriers, with conductance properties determined more by the strength of the electrode-molecule contact and the structure of the "linker" than the nature of the metal-ion or redox properties of the complex.

Improving the spatial alignment of emitting molecules has long been a goal of organic-light-emitting-diode development to improve device efficiencies and to generate polarized emission. Herein we describe a simple approach employing Sonogashira coupling with alkyne iridium(phenylpyridine)2(acetylacetone) synthons (2-5) to generate eight linear iridium complexes (6-13) with crystallographically determined lengths of up to 5 nm. By embedding these "long" complexes into a polymer matrix and stretching it, an improvement of the polarization ratio of unstretched and stretched films of up to 7.1 times was achieved. Additionally, through the inclusion of "twists" in the complexes, the electronic coupling between the iridium center and substituent was controlled, giving a system where the emission behavior is independent of the length.

Controlling the orientation of molecular conductors on the electrode surfaces is a critical factor in the development of single-molecule conductors. In the current study, we used the scanning tunnelling microscopy-based break junction (STM-BJ) technique to explore ‘bare-bones’ tripodal molecular wires, employing different anchor groups (AGs) at the ‘top’ and ‘bottom’ of the tripod. The triarylphosphine tris(4-(methylthio)phenyl)phosphine and its corresponding phosphine sulfide showed only a single high conductance feature in the resulting 1- and 2-dimensional conductance histograms, whereas analogous molecules with fewer than three thiomethyl AGs did not show clear conductance features. Thus, by systematic molecular modifications and with the aid of supporting DFT calculations, the binding geometry, with respect to the surface, was elucidated.

Controlling the orientation of molecular conductors on the electrode surfaces is a critical factor in the development of single-molecule conductors. In the current study, we used the scanning tunnelling microscopy-based break junction (STM-BJ) technique to explore 'bare-bones' tripodal molecular wires, employing different anchor groups (AGs) at the 'top' and 'bottom' of the tripod. The triarylphosphine tris(4-(methylthio)phenyl)phosphine and its corresponding phosphine sulfide showed only a single high conductance feature in the resulting 1- and 2-dimensional conductance histograms, whereas analogous molecules with fewer than three thiomethyl AGs did not show clear conductance features. Thus, by systematic molecular modifications and with the aid of supporting DFT calculations, the binding geometry, with respect to the surface, was elucidated.

The ligands 4'-(4-(methylthio)phenyl)-2,2':6',2″-terpyridine (L(1)), 4'-((4-(methylthio)phenyl)ethynyl)- 2,2':6',2″-terpyridine (L(2)), and bis(tridentate) bridging ligand 2,3,5,6-tetra(pyridine-2-yl)pyrazine (tpp) were used to prepare the complexes [Ru(L(1))2][PF6]2 ([1][PF6]2, [Ru(L(2))2][PF6]2 ([2][PF6]2), [{(L(1))Ru}(μ-tpp){Ru(L(1))}][PF6]4 ([3][PF6]4), and [{(L(2))Ru}(μ-tpp){Ru(L(2))}][PF6]4 ([4][PF6]4). Crystallographically determined structures give S···S distances of up to 32.0 Å in [4](4+). On the basis of electrochemical estimates, the highest occupied molecular orbitals of these complexes fall between -5.55 and -5.85 eV, close to the work function of clean gold (5.1-5.3 eV). The decay of conductance with molecular length across this series of molecules is approximately exponential, giving rise to a decay constant (pseudo β-value) of 1.5 nm(-1), falling between decay factors for oligoynes and oligophenylenes. The results are consistent with a tunnelling mechanism for the single-molecule conductance behavior.

Candida albicansis an important opportunistic pathogen causing various human infections that are often treated with azole antifungals. The U.S. CDC now regards developing candidal antifungal resistance as a threat, creating a need for new and more effective antifungal treatments. Iron is an essential nutrient for all living cells, and there is growing evidence that interference with iron homeostasis ofC. albicanscan improve its response to antifungals. This study was aimed at establishing whether withholding iron by currently used medical iron chelators and the novel chelator DIBI could restrict growth and also enhance the activity of azoles against clinical isolates ofC. albicans. DIBI, but not deferoxamine or deferiprone, inhibited the growth ofC. albicansat relatively low concentrationsin vitro, and this inhibition was reversed by iron addition. DIBI in combination with various azoles demonstrated stronger growth inhibition than the azoles alone and greatly prolonged the inhibition of cell multiplication. In addition, the administration of DIBI along with fluconazole (FLC) to mice inoculated with an FLC-sensitive isolate in a model of experimentalC. albicansvaginitis showed a markedly improved clearance of infection. These results suggest that iron chelation by DIBI has the potential to enhance azole efficacy for the treatment of candidiasis.

Carbapenem-resistant Enterobacter cloacae complex isolates submitted to a reference laboratory from 2010 to 2015 were screened by PCR for seven common carbapenemase gene groups, namely, KPC, NDM, OXA-48, VIM, IMP, GES, and NMC-A/IMI. Nineteen of the submitted isolates (1.7%) were found to harbor Ambler class A bla NMC-A or bla IMI -type carbapenemases. All 19 isolates were resistant to at least one carbapenem but susceptible to aminoglycosides, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Most isolates (17/19) gave positive results with the Carba-NP test for phenotypic carbapenemase detection. Isolates were genetically diverse by pulsed-field gel electrophoresis macrorestriction analysis, multilocus sequence typing, and hsp60 gene analysis. The genes were found in various Enterobacter cloacae complex species; however, bla NMC-A was highly associated with Enterobacter ludwigii . Whole-genome sequencing and bioinformatics analysis revealed that all NMC-A ( n = 10), IMI-1 ( n = 5), and IMI-9 ( n = 2) producers harbored the carbapenemase gene on EludIMEX-1-like integrative mobile elements (EcloIMEXs) located in the identical chromosomal locus. Two novel genes, bla IMI-5 and bla IMI-6 , were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring bla NMC-A/IMI -type carbapenemases are relatively rare in Canada. Though mostly found integrated into the chromosome, some variants are located on plasmids that may enhance their mobility potential.

BACKGROUND: In 2007, Atlantic Canada experienced a large outbreak of mumps predominately in university students who had received a single dose of measles, mumps and rubella vaccine. The present study describes the performance characteristics of reverse transcriptase polymerase chain reaction (RT-PCR) on buccal and urine specimens and immunoglobulin M (IgM) serology in this partially immune population.METHODS: Patients presenting with symptoms suspicious for mumps had a serum, urine and a buccal swab collected for diagnostic testing. Persons were classified as a ‘confirmed’ case according to the Public Health Agency of Canada’s definition. Sera were tested using an enzyme-linked immunoassay. Detection of mumps virus in buccal swabs and urine samples was performed by RT-PCR.RESULTS: A subset of 155 cases and 376 non-cases that had all three specimens submitted was used for calculating the performance characteristics. The sensitivity of RT-PCR on buccal swabs, urine specimens and IgM serology were 79%, 43% and 25%, respectively. The specificity of RT-PCR on buccal swabs, urine specimens and IgM serology was 99.5%, 100% and 99.7%, respectively. Only 12 of 134 (9%) patients had positive urine specimens in the presence of negative oral swabs.CONCLUSION: RT-PCR on buccal swabs is the ideal specimen for diagnosis. Testing an additional urine sample in an outbreak setting did not increase the diagnostic yield significantly, but doubled testing volume and cost. In addition, the data suggest that, in this partially immune group, IgM serology has little value in the diagnosis of acute infection.

Real-time PCR is a powerful tool for the detection and typing of herpes simplex virus (HSV). HSV types 1 and 2 can be distinguished by using the differences in the melting temperatures ( T m s) of the hybridization probes. The efficacy of T m profiling with low DNA concentrations was evaluated in this study.

The ethynyl-phenylene substituted 2,2':6',2''-terpyridine (tpy) derivatives, 4-(phenyl-ethynyl)-2,2':6',2''-terpyridine (L(1)), 4-(methoxyphenyl-ethynyl)-2,2':6',2''-terpyridine (L(2)), 4-(tolyl-ethynyl)-2,2':6',2''-terpyridine (L(3)) and 4-(nitrophenyl-ethynyl)-2,2':6',2''-terpyridine (L(4)) have been used to synthesize four new [RuCl(2,2'-bipyridine)(L(n))]PF6 based complexes. Electronic absorption, resonance Raman, cyclic voltammetry and spectroelectrochemistry aided by DFT calculations were used to explore the influence of the alkynyl substituents on the electronic structures, photochemical and redox properties of the complexes. Furthermore, it is shown that the addition of ethynyl phenyl moieties to the 4-position of the tpy ligand does not have a detrimental effect on these complexes, or the analogous aqua complexes, with respect to their ability to photocatalyse the oxidation of 4-methoxybenzyl alcohol to the corresponding benzaldehyde.

Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit strong peroxidatic but weak catalatic activities, suggesting that other enzymes participate in decomposition of hydrogen peroxide (H 2 O 2 ). Comparative genomics revealed that L. pneumophila and its close relative Coxiella burnetii each contain two peroxide-scavenging alkyl hydroperoxide reductase (AhpC) systems: AhpC1, which is similar to the Helicobacter pylori AhpC system, and AhpC2 AhpD (AhpC2D), which is similar to the AhpC AhpD system of Mycobacterium tuberculosis . To establish a catalatic function for these two systems, we expressed L. pneumophila ahpC1 or ahpC2 in a catalase/peroxidase mutant of Escherichia coli and demonstrated restoration of H 2 O 2 resistance by a disk diffusion assay. ahpC1 ::Km and ahpC2D ::Km chromosomal deletion mutants were two- to eightfold more sensitive to H 2 O 2 , tert -butyl hydroperoxide, cumene hydroperoxide, and paraquat than the wild-type L. pneumophila , a phenotype that could be restored by trans -complementation. Reciprocal strategies to construct double mutants were unsuccessful. Mutant strains were not enfeebled for growth in vitro or in a U937 cell infection model. Green fluorescence protein reporter assays revealed expression to be dependent on the stage of growth, with ahpC1 appearing after the exponential phase and ahpC2 appearing during early exponential phase. Quantitative real-time PCR showed that ahpC1 mRNA levels were ∼7- to 10-fold higher than ahpC2D mRNA levels. However, expression of ahpC2D was significantly increased in the ahpC1 mutant, whereas ahpC1 expression was unchanged in the ahpC2D mutant. These results indicate that AhpC1 or AhpC2D (or both) provide an essential hydrogen peroxide-scavenging function to L. pneumophila and that the compensatory activity of the ahpC2D system is most likely induced in response to oxidative stress.

Empirical treatment is best guided by current surveillance of local resistance patterns. The goal of this study is to characterize the prevalence of antimicrobial nonsusceptibility within pneumococcal isolates from Canada. The Canadian Bacterial Surveillance Network is comprised of laboratories from across Canada. Laboratories collected a defined number of consecutive clinical and all sterile site isolates of S. pneumoniae in 2002. In vitro susceptibility testing was performed by broth microdilution with NCCLS guidelines. Rates of nonsusceptibility were compared to previously published reports from the same network. A total of 2,539 isolates were tested. Penicillin nonsusceptibility increased to 15% (8.5% intermediate, 6.5% resistant) compared to 12.4% in 2000 ( P ≤ 0.025, χ 2 ). Only 32 (1.3%) isolates had an amoxicillin MIC of ≥4 μg/ml and only 2 of 32 cerebrospinal fluid isolates had an intermediate susceptibility to ceftriaxone by meningeal interpretive criteria (MIC = 1 μg/ml). A total of 354 (13.9%) isolates were macrolide nonsusceptible (46.3% MLS B , 56.7% M phenotype), increasing from 11.4% in 2000 ( P ≤ 0.0075, χ 2 ). Only 13 (1 μg/ml. Ciprofloxacin nonsusceptibility (defined as an MIC of ≥4 μg/ml) increased to 2.7% compared to 1.4% in 2000 ( P ≤ 0.0025, χ 2 ) and was primarily found in persons ≥18 years old (98.5%). Nonsusceptibility to penicillin, macrolides, and fluoroquinolones is increasing in Canada. Nonsusceptibility to amoxicillin and ceftriaxone remains uncommon. Newer antimicrobials such as telithromycin and respiratory fluoroquinolones have excellent in vitro activity.

Over the course of a 20-month period, in a hospital respiratory ward in which ciprofloxacin was often used as empirical antimicrobial therapy for lower respiratory tract infections (LRTIs), 16 patients with chronic bronchitis developed nosocomial LRTIs caused by penicillin- and ciprofloxacin-resistant Streptococcus pneumoniae (serotype 23 F). The minimum inhibitory concentration (MIC) of ciprofloxacin for all isolates from the first 9 patients was 4 microg/mL, in association with a parC mutation. Isolates from the subsequent 7 patients all had a ciprofloxacin MIC of 16 microg/mL, in association with an additional mutation in gyrA. The MICs for this isolate were 8 microg/mL of levofloxacin (resistant), 2 microg/mL of moxifloxacin and gatifloxacin (intermediately resistant), and 0.12 microg/mL of gemifloxacin. This outbreak demonstrates the ability of S. pneumoniae to acquire multiple mutations that result in increasing levels of resistance to the fluoroquinolones and to be transmitted from person to person.

We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates of S. pneumoniae for which the ciprofloxacin MIC is ≥4 μg/ml and 28 isolates for which the ciprofloxacin MIC is ≤2 μg/ml. The amino acid substitutions in ParC conferring low-level resistance (MICs, 4 to 8 μg/ml) included Phe, Tyr, and Ala for Ser-79; Asn, Ala, Gly, Tyr, and Val for Asp-83; Asn for Asp-78; and Pro for Ala-115. Isolates with intermediate-level (MICs, 16 to 32 μg/ml) and high-level (MICs, 64 μg/ml) resistance harbored substitutions of Phe and Tyr for Ser-79 or Asn and Ala for Asp-83 in ParC and an additional substitution in GyrA which included either Glu-85-Lys (Gly) or Ser-81-Phe (Tyr). Glu-85-Lys was found exclusively in isolates with high-level resistance. Efflux contributed primarily to low-level resistance in isolates with or without an amino acid substitution in ParC. The impact of amino acid substitutions in ParE was minimal, and no substitutions in GyrB were identified.

OBJECTIVE: To determine the prevalence of antimicrobial resistance in clinical isolates ofStreptococcus pneumoniae, Haemophilus influenzaeandMoraxella catarrhalisfrom medical centres across Canada.METHODS: Fifty laboratories from across Canada were asked to collect up to 25 consecutive clinical isolates ofS pneumoniae,H influenzaeandM catarrhalisat some time between September 1994 and May 1995, and then again between September and December of 1996. A total of 2364S pneumoniae, 575H influenzaeand 200M catarrhalissamples were collected.H influenzaeandM catarrhalisisolates were tested for the production of beta-lactamase.S pneumoniaeisolates were characterized as penicillin susceptible, intermediately resistant or high level penicillin-resistant. Minimal inhibitory concentrations (MICs) were determined using a microbroth dilution technique described by the National Committee of Clinical Laboratory Standards.RESULTS: Between the two collection periods, there was a significant increase in highly penicillin-resistantS pneumoniaefrom 2.1% to 4.4% (PS pneumoniaewas noted among paediatric isolates. No significant difference in the susceptibilities of comparator agents was detected. A significant increase in the number of beta-lactamase producingH influenzae, 34% to 43% (PM catarrhalisisolates were beta-lactamase producers in both time periods.CONCLUSIONS: During the course of this study, the incidence of penicillin resistance inS pneumoniaedoubled. As a result of this increase, infections due to this organism in sites where poor penetration of beta-lactam antibiotics occur may become increasingly difficult to manage.

A real-time reverse transcriptase PCR (RT-PCR) assay that targeted both the mumps virus F gene and human RNase P in clinical samples was adapted for use with the LightCycler platform. LightCycler RT-PCR is as sensitive as conventional nested RT-PCR and significantly less expensive and labor-intensive, making it ideal for mumps diagnosis during outbreaks.

Rapid methods for the detection and confirmatory identification of pandemic influenza A virus (also known as pandemic [H1N1] 2009) are of utmost importance. In this study, a conventional reverse transcriptase PCR (RT-PCR) assay for the detection of influenza A virus and the hemagglutinin of swine lineage H1 (swH1) was designed, optimized, and validated. Nucleic acids were extracted from 198 consecutive nasopharyngeal, nasal, or throat swab specimens collected early in the outbreak (127 negative specimens, 66 specimens with pandemic [H1N1] 2009 influenza virus, 3 specimens with seasonal [H1N1] influenza A virus, and 2 specimens with seasonal [H3N2] influenza A virus). The performance characteristics of the duplex RT-PCR assay were assessed and compared to those of various detection methods: a monoplex RT-PCR assay at the National Microbiology Laboratory, a real-time RT-PCR assay using a Centers for Disease Control and Prevention protocol, an in-house multiplex RT-PCR assay (targeting influenza A virus, influenza B virus, and respiratory syncytial virus), and a rapid antigen test (the Binax Now Influenza A & B assay). The sensitivity of the duplex RT-PCR assay for influenza A virus detection was 97.2%, whereas the sensitivities were 74.6%, 71.8%, 47.8%, and 12.7% for the other four assays, respectively. The duplex RT-PCR assay was also able to identify swH1 in 94% of the cases, thereby reducing the number of specimens forwarded to reference laboratories for confirmatory identification. Only a limited number of specimens that contained influenza A virus had amounts of virus that fell below the limit of detection of the assay with the swH1 primers. Overall, the duplex RT-PCR assay is a reliable method for the simultaneous detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus and would be particularly attractive to laboratories without real-time RT-PCR capabilities.

SUMMARYIn Atlantic Canada, the traditional risk factor for acquisition of Q fever infection has been exposure to infected parturient cats or newborn kittens. In this study we describe the first case of Q fever in Nova Scotia acquired as a result of direct exposure to sheep. A serosurvey of the associated flock was undertaken using an indirect immunofluorescence assay (IFA) testing for antibodies to phase I and phase IICoxiella burnetiiantigens. This serosurvey revealed that 23 of 46 sheep (50%) were seropositive for the phase II antibody. Four of these sheep had titres of 1:64 including three nursing ewes, one of which had delivered two lambs that died shortly after delivery. Only one ewe had phase I antibodies but had the study's highest phase II antibody titre (1:128). Molecular studies using polymerase chain reaction (PCR) failed to detectC. burnetiiDNA in any of the milk specimens.

Legionella pneumophila expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the postexponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the ahpC2D operon. The major protein captured was an ortholog of OxyR (OxyR Lp ). Genetic studies indicated that oxyR Lp was an essential gene expressed postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyR Lp to ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyR Lp ; however, OxyR Lp weakly bound to E. coli OxyR-regulated promoters ( katG , oxyR , and ahpCF ). DNase I protection studies showed that the OxyR Lp binding motif spanned the promoter and transcriptional start sequences of ahpC2 and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H 2 O 2 ). Moreover, the OxyR Lp (pBADLp oxyR )-mediated repression of an ahpC2 - gfp reporter construct in E. coli GS077 (the oxyR mutant) was not reversed by H 2 O 2 challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked OxyR Lp in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.

BackgroundBacterial resistance to antibiotics is thought to develop only in the presence of antibiotic pressure. Here we show evidence to suggest that fluoroquinolone resistance in Escherichia coli has developed in the absence of fluoroquinolone use.MethodsOver 4 years, outreach clinic attendees in one moderately remote and five very remote villages in rural Guyana were surveyed for the presence of rectal carriage of ciprofloxacin-resistant gram-negative bacilli (GNB). Drinking water was tested for the presence of resistant GNB by culture, and the presence of antibacterial agents and chloroquine by HPLC. The development of ciprofloxacin resistance in E. coli was examined after serial exposure to chloroquine. Patient and laboratory isolates of E. coli resistant to ciprofloxacin were assessed by PCR-sequencing for quinolone-resistance-determining-region (QRDR) mutations.ResultsIn the very remote villages, 4.8% of patients carried ciprofloxacin-resistant E. coli with QRDR mutations despite no local availability of quinolones. However, there had been extensive local use of chloroquine, with higher prevalence of resistance seen in the villages shortly after a Plasmodium vivax epidemic (pConclusionsIn these remote communities, the heavy use of chloroquine to treat malaria likely selected for ciprofloxacin resistance in E. coli. This may be an important public health problem in malarious areas.

We have identified an unusual group of viridans group streptococci that resemble Streptococcus pneumoniae . DNA-DNA homology studies suggested that a subset of these isolates represent a novel species that may be included in the S. oralis- S. mitis group of viridans group streptococci. We suggest that this novel species be termed Streptococcus pseudopneumoniae . A combination of phenotypic and genetic reactions allows its identification. S. pseudopneumoniae strains do not have pneumococcal capsules, are resistant to optochin (inhibition zones, less than 14 mm) when they are incubated under an atmosphere of increased CO 2 but are susceptible to optochin (inhibition zones, >14 mm) when they are incubated in ambient atmospheres, are not soluble in bile, and are positive by the GenProbe AccuProbe Pneumococcus test. The bile solubility test is more specific than the optochin test for identification of S. pneumoniae . Genetic tests for pneumolysin ( ply ) and manganese-dependent superoxide dismutase ( sodA ) and identification tests with a commercial probe, AccuProbe Pneumococcus, do not discriminate between the new species and S. pneumoniae .

Community-acquired pneumonia (CAP) is common among children and may have viral, bacterial or, occasionally, other causes. The etiology is complex, with age-related trends, and differs from that in adult CAP, necessitating different management guidelines. There is an absence of current guidelines for the management of pediatric CAP (PCAP) that take into account changing etiologies, antimicrobial-resistance issues and the use of newly licensed antimicrobials. The present review does not provide specific guidelines, but it reviews the literature and presents currrent approaches to the treatment of PCAP. To compile the review, an expert panel was convened to provide a consensus. The review discusses the etiology, diagnosis and antimicrobial treatment of PCAP as well as indications for referral to a hospital emergency department. The goal of the review is to provide those involved with treatment of PCAP in the community setting with information that can be used to make effective treatment choices.

Analysis of 71 ciprofloxacin-resistant (MIC ≥ 4 μg/ml) Streptococcus pneumoniae clinical isolates revealed only 1 for which the quinolone resistance-determining regions of the parC , parE , and gyrB genes were genetically related to those of viridans group streptococci. Our findings support the occurrence of interspecies recombination of type II topoisomerase genes; however, its contribution to the emergence of quinolone resistance among pneumococci appears to have been minimal.