Rational Mutational Analysis of a Multidrug MFS Transporter CaMdr1p of Candida albicans by Employing a Membrane Environment Based Computational Approach

CaMdr1p is a multidrug MFS transporter of pathogenic Candida albicans. An over-expression of the gene encoding this protein is linked to clinically encountered azole resistance. In-depth knowledge of the structure and function of CaMdr1p is necessary for an effective design of modulators or inhibitors of this efflux transporter. Towards this goal, in this study, we have employed a membrane environment based computational approach to predict the functionally critical residues of CaMdr1p. For this, information theoretic scores which are variants of Relative Entropy (Modified Relative Entropy REM) were calculated from Multiple Sequence Alignment (MSA) by separately considering distinct physico-chemical properties of transmembrane (TM) and inter-TM regions. The residues of CaMdr1p with high REM which were predicted to be significantly important were subjected to site-directed mutational analysis. Interestingly, heterologous host Saccharomyces cerevisiae, over-expressing these mutant variants of CaMdr1p wherein these high REM residues were replaced by either alanine or leucine, demonstrated increased susceptibility to tested drugs. The hypersensitivity to drugs was supported by abrogated substrate efflux mediated by mutant variant proteins and was not attributed to their poor expression or surface localization. Additionally, by employing a distance plot from a 3D deduced model of CaMdr1p, we could also predict the role of these functionally critical residues in maintaining apparent inter-helical interactions to provide the desired fold for the proper functioning of CaMdr1p. Residues predicted to be critical for function across the family were also found to be vital from other previously published studies, implying its wider application to other membrane protein families

The Ecology of Antibiotic Use in the ICU: Homogeneous Prescribing of Cefepime but Not Tazocin Selects for Antibiotic Resistant Infection

Background: Antibiotic homogeneity is thought to drive resistance but in vivo data are lacking. In this study, we determined the impact of antibiotic homogeneity per se, and of cefepime versus antipseudomonal penicillin/beta-lactamase inhibitor combinations (APP-beta), on the likelihood of infection or colonisation with antibiotic resistant bacteria and/or two commonly resistant nosocomial pathogens (methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa). A secondary question was whether antibiotic cycling was associated with adverse outcomes including mortality, length of stay, and antibiotic resistance

Segmented flow generator for serial crystallography at the European X-ray free electron laser

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported

pEl1573 carrying blaIMP-4, from Sydney, Australia, is closely related to other IncL/M plasmids

Complete sequencing of pEl1573, a representative IncL/M plasmid carrying blaIMP-4 from Sydney, Australia, revealed an ∼60-kb backbone almost identical to those of IncL/M plasmids pCTX-M3, from Poland, and pCTX-M360, from China, and less closely related to pNDM-HK, pOXA-48a, and pEL60, suggesting different lineages. The ∼28-kb Tn2-derived multiresistance region in pEl1573 is inserted in the same location as those in pCTX-M3 and pNDM-HK and shares some of the same components but has undergone rearrangements.4 page(s

MALDI-TOF Mass Spectrometry for Multilocus Sequence Typing of <i>Escherichia coli</i> Reveals Diversity among Isolates Carrying <i>bla</i>_CMY-2-Like Genes

<div><p>Effective surveillance and management of pathogenic <i>Escherichia coli</i> relies on robust and reproducible typing methods such as multilocus sequence typing (MLST). Typing of <i>E</i>. <i>coli</i> by MLST enables tracking of pathogenic clones that are known to carry virulence factors or spread resistance, such as the globally-prevalent ST131 lineage. Standard MLST for <i>E</i>. <i>coli</i> requires sequencing of seven alleles, or a whole genome, and can take several days. Here, we have developed and validated a nucleic-acid-based MALDI-TOF mass spectrometry (MS) method for MLST as a rapid alternative to sequencing that requires minimal operator expertise. Identification of alleles was 99.6% concordant with sequencing. We employed MLST by MALDI-TOF MS to investigate diversity among 62 <i>E</i>. <i>coli</i> isolates from Sydney, Australia, carrying a <i>bla</i>_CMY-2-like gene on an IncI1 plasmid to determine whether any dominant clonal lineages are associated with the spread of this globally-disseminated resistance gene. Thirty-four known sequence types were identified, including lineages associated with human disease, animal and environmental sources. This suggests that the dissemination of <i>bla</i>_CMY-2-like-genes is more complex than the simple spread of successful pathogenic clones. <i>E</i>. <i>coli</i> MLST by MALDI-TOF MS, employed here for the first time, can be utilised as an automated tool for large-scale population analyses or for targeted screening for known high-risk clones in a diagnostic setting.</p></div

Examples of discrepancies between MLST by MALDI-TOF MS and sequencing.

<p>^a These alleles differ by the presence/absence of a 10,290 Da peak that is often not detected by the software but is visible upon manual inspection. Other alleles that differ by the presence/absence of this peak are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143446#pone.0143446.s005" target="_blank">S3 Table</a>.</p><p>^b Indistinguishable alleles have identical spectral patterns for all four cleavage reactions. Other indistinguishable alleles can be determined by the simulation software.</p><p>^c Workflow for discrepant alleles detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143446#pone.0143446.s001" target="_blank">S1 Fig</a>.</p><p>Examples of discrepancies between MLST by MALDI-TOF MS and sequencing.</p

Locally Acquired mcr-1 in Escherichia coli, Australia, 2011 and 2013

We identified discrete importation events of the mcr-1 gene on incompatibility group IncI2 plasmids in Escherichia coli isolated from patients in New South Wales, Australia, in 2011 and 2013. mcr-1 is present in a small minority of colistin-resistant Enterobacteriaceae and appears not to be established locally

Sequence types of <i>E</i>. <i>coli</i> strains carrying IncI1-<i>bla</i>_CMY-2 plasmids.

<p>^a Sequence type</p><p>^b Sequence type complex based on <a href="http://mlst.warwick.ac.uk/mlst/dbs/Ecoli" target="_blank">http://mlst.warwick.ac.uk/mlst/dbs/Ecoli</a>;–, no STC</p><p>^c One isolate has a new combination of alleles and one has a new <i>gyrB</i> variant.</p><p>Sequence types of <i>E</i>. <i>coli</i> strains carrying IncI1-<i>bla</i>_CMY-2 plasmids.</p

A Novel Gene Cassette, aacA43, in a Plasmid-Borne Class 1 Integron▿

A novel gene cassette, aacA43, was identified in the aadB-aacA43-oxa10-smr2 cassette array in a class 1 integron. Like related aminoglycoside-(6′)-acetyltransferases, AacA43 confers clinically relevant resistance to kanamycin, tobramycin, and some less-used aminoglycosides but not to gentamicin. Although transferable on an IncL/M plasmid, aacA43 was identified in only two different Klebsiella pneumoniae strains (14 isolates), one Escherichia coli strain (2 isolates), and one Enterobacter cloacae strain in a survey of patients in a Sydney intensive care unit in 2004-2005