Impact of clonal lineages on susceptibility of Staphylococcus lugdunensis to chlorhexidine digluconate and chloride benzalkonium.

BACKGROUND: Little is known about susceptibility of Staphylococcus lugdunensis to antiseptics. The objective of this study was to evaluate, at the molecular and phenotypic level, the susceptibility of 49 clinical S. lugdunensis strains (belonging to the seven clonal complexes [CCs] defined by multilocus sequence typing) to two antiseptics frequently used in healthcare settings (chlorhexidine digluconate [CHX] and chloride benzalkonium [BAC]). RESULTS: The minimum inhibitory concentrations (MICs), by broth microdilution method, varied for BAC from 0.25 mg/L to 8 mg/L (MIC50 = 1 mg/L, MIC90 = 2 mg/L) and for CHX from 0.5 mg/L to 2 mg/L (MIC50 = 1 mg/L, MIC90 = 2 mg/L). The BAC and CHX minimum bactericidal concentrations (MBCs) varied from 2 mg/L to 8 mg/L (MBC50 = 4 mg/L, MBC90 = 8 mg/L) and from 2 mg/L to 4 mg/L (MBC50 and MBC90 = 4 mg/L), respectively. A reduced susceptibility to CHX (MIC = 2 mg/L) was observed for 12.2% of the strains and that to BAC (MIC ≥ 4 mg/L) for 4.1%. The norA resistance gene was detected in all the 49 isolates, whereas the qacA gene was rarely encountered (two strains; 4.1%). The qacC, qacG, qacH, and qacJ genes were not detected. The two strains harboring the qacA gene had reduced susceptibility to both antiseptics and belonged to CC3. CONCLUSION: The norA gene was detected in all the strains, suggesting that it could belong to the core genome of S. lugdunensis. S. lugdunensis is highly susceptible to both antiseptics tested. Reduced susceptibility to BAC and CHX was a rare phenomenon. Of note, a tendency to higher MICs of BAC was detected for CC3 isolates. These results should be confirmed on a larger collection of strains.

Are Escherichia coli causing recurrent cystitis just ordinary Uropathogenic E. coli (UPEC) strains?

Specific determinants associated with Uropathogenic Escherichia coli (UPEC) causing recurrent cystitis are still poorly characterized. The aims of this study were (i) to describe genomic and phenotypic traits associated with recurrence using a large collection of recurrent and paired sporadic UPEC isolates, and (ii) to explore within-host genomic adaptation associated with recurrence using series of 2 to 5 sequential UPEC isolates. Whole genome comparative analyses between 24 recurrent cystitis isolates (RCIs) and 24 phylogenetically paired sporadic cystitis isolates (SCIs) suggested a lower prevalence of putative mobile genetic elements (MGE) in RCIs, such as plasmids and prophages. The intra-patient evolution of the 24 RCI series over time was characterized by SNP occurrence in genes involved in metabolism or membrane transport, and by plasmid loss in 5 out of the 24 RCI series. Genomic evolution occurred early in the course of recurrence, suggesting rapid adaptation to strong selection pressure in the urinary tract. However, RCIs did not exhibit specific virulence factor determinants and could not be distinguished from SCIs by their fitness, biofilm formation, or ability to invade HTB-9 bladder epithelial cells. Taken together, these results suggest a rapid but not convergent adaptation of RCIs that involves both strain- and host-specific characteristics.

Enterococcus faecalis Maltodextrin Gene Regulation by Combined Action of Maltose Gene Regulator MalR and Pleiotropic Regulator CcpA.

Enterococci are Gram-positive bacteria present in the healthy human microbiota, but they are also a leading cause of nosocomial infections. Maltodextrin utilization by Enterococcus faecalis has been identified as an important factor for colonization of mammalians hosts. Here, we show that the LacI/GalR transcriptional regulator MalR, the maltose gene regulator, is also the main regulator of the operons encoding an ABC transporter (mdxEFG) and three metabolic enzymes (mmdH-gmdH-mmgT) required for the uptake and catabolism of maltotetraose and longer maltodextrins. The utilization of maltose and maltodextrins is consequently coordinated and induced by the disaccharide maltose, which binds to MalR. Carbon catabolite repression of the mdxEFG and mmdH-gmdH-mmgT operons is mediated by both P-Ser-HPr/MalR and P-Ser-HPr/CcpA. The latter complex exerts only moderate catabolite repression, which became visible when comparing maltodextrin operon expression levels of a malR- mutant (with a mutant allele for the malR gene) and a malR- ΔccpA double mutant grown in the presence of maltose, which is transported via a phosphotransferase system and, thus, favors the formation of P-Ser-HPr. Moreover, maltodextrin transport via MdxEFG slows rapidly when glucose is added, suggesting an additional regulation via inducer exclusion. This complex regulation of metabolic operons likely allows E. faecalis to fine-tune gene expression in response to changing environmental conditions.IMPORTANCEEnterococcus faecalis represents a leading cause of hospital-acquired infections worldwide. Several studies highlighted the importance of carbohydrate metabolism in the infection process of this bacterium. The genes required for maltodextrin metabolism are particularly induced during mouse infection and, therefore, should play an important role for pathogenesis. Since no data were hitherto available concerning the regulation of expression of the maltodextrin operons, we have conducted experiments to study the underlying mechanisms.

Within-Host Microevolution of Pseudomonas aeruginosa Urinary Isolates: A Seven-Patient Longitudinal Genomic and Phenotypic Study.

BACKGROUND: Pseudomonas aeruginosa is responsible for up to 10% of healthcare associated urinary tract infections (UTI), which can be difficult to treat and can lead to bacterial persistence. While numerous whole genome sequencing (WGS) analyses have explored within-host genomic adaptation and microevolution of P. aeruginosa during cystic fibrosis (CF) infections, little is known about P. aeruginosa adaptation to the urinary tract. RESULTS: Whole genome sequencing was performed on 108 P. aeruginosa urinary isolates, representing up to five isolates collected from 2 to 5 successive urine samples from seven patients hospitalized in a French hospital over 48-488 days. Clone type single nucleotide polymorphisms (ctSNPs) analysis revealed that each patient was colonized by a single clone type (<6000 SNPs between two isolates) at a given time and over time. However, 0-126 SNPs/genome/year were detected over time. Furthermore, large genomic deletions (1-5% of the genome) were identified in late isolates from three patients. For 2 of them, a convergent deletion of 70 genes was observed. Genomic adaptation (SNPs and deletion) occurred preferentially in genes encoding transcriptional regulators, two-component systems, and carbon compound catabolism. This genomic adaptation was significantly associated with a reduced fitness, particularly in artificial urine medium, but no strict correlation was identified between genomic adaptation and biofilm formation. CONCLUSION: This study provides the first insight into P. aeruginosa within-host evolution in the urinary tract. It was driven by mutational mechanisms and genomic deletions and could lead to phenotypic changes in terms of fitness and biofilm production. Further metabolomic and phenotypic analyses are needed to describe in-depth genotype-phenotype associations in this complex and dynamic host-environment.

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl-phosphorylated-HPr.

Enterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions.

Characterization of the gen locus involved in ß-1,6-oligosaccharide utilization by Enterococcus faecalis.

The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-ß-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of ß-1,6- rather than ß-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6'-P with a Kd of 0.04 mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6'-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.

A Lab-on-Chip Analyzer for in Situ Measurement of Soluble Reactive Phosphate: Improved Phosphate Blue Assay and Application to Fluvial Monitoring.

Here, we present a new in situ microfluidic phosphate sensor that features an improved "phosphate blue" assay which includes polyvinylpyrrolidone in place of traditional surfactants-improving sensitivity and reducing temperature effects. The sensor features greater power economy and analytical performance relative to commercially available alternatives, with a mean power consumption of 1.8 W, a detection limit of 40 nM, a dynamic range of 0.14-10 µM, and an infield accuracy of 4 ± 4.5%. During field testing, the sensor was continuously deployed for 9 weeks in a chalk stream, revealing complex relations between flow rates and phosphate concentration that suggest changing dominance in phosphate sources. A distinct diel phosphorus signal was observed under low flow conditions, highlighting the ability of the sensor to decouple geochemical and biotic effects on phosphate dynamics in fluvial environments. This paper highlights the importance of high resolution in situ sensors in addressing the current gross under-sampling of aquatic environments.

Determination of trace zinc in seawater by coupling solid phase extraction and fluorescence detection in the Lab-On-Valve format.

By virtue of their compactness, long-term stability, minimal reagent consumption and robustness, miniaturized sequential injection instruments are well suited for automation of assays onboard research ships. However, in order to reach the sensitivity and limit of detection required for open-ocean determinations of trace elements, it is necessary to preconcentrate the analyte prior its derivatization and subsequent detection by fluorescence. In this work, a novel method for the determination of dissolved zinc (Zn) at subnanomolar levels in seawater is described. The proposed method combines, for the first time, automated matrix removal, extraction of the target element, and fluorescence detection within a miniaturized flow manifold, based on the Lab-On-Valve (LOV) concept. The key feature of the microfluidic manipulation of the sample is flow programming, designed to pass sample through a mini-column where the target analyte and other complexable cations are retained, while the seawater matrix is washed out. Next, zinc is eluted and merged with a Zn selective fluorescent probe (FluoZin-3) at the confluence point of the LOV central channel using two high-precision stepper motor driven pumps that are operated in concert. Finally, the thus formed Zn complex is transported to the LOV flow cell for selective fluorescence measurement. This work describes the characterization and optimization of the method including Solid Phase Extraction using the Toyopearl AF-Chelate-650M resin, and detailed assay protocol controlled by a commercially available software and instrument. The proposed method features a LOD of 0.02 nM, high precision (<3% at 0.1 and 2 nM Zn levels), an assay cycle of 13 min and a reagent consumption of 150 µL FluoZin-3 per sample, which makes the method highly suitable for oceanographic shipboard analysis. The accuracy of the method has been validated through the analysis of seawater reference standards and comparison with ICP-MS determinations on seawater samples collected in the upper 1300 m of the subtropical south Indian Ocean. This work confirms that integration of sample pretreatment with optical detection in the LOV format offers a widely applicable approach to trace analysis of seawater.

Towards chemiluminescence detection in micro-sequential injection lab-on-valve format: a proof of concept based on the reaction between Fe(II) and luminol in seawater.

Micro-sequential injection lab-on-valve (µSI-LOV) is a well-established analytical platform for absorbance and fluorescence based assays but its applicability to chemiluminescence detection remains largely unexplored. In this work, we describe a novel fluidic protocol and two distinct strategies for photon collection that enable chemiluminescence detection using µSI-LOV for the first time. To illustrate this proof of concept, we selected the reaction between Fe(II) and luminol and developed a preliminary protocol for Fe(II) determinations in acidified seawater. The optimized fluidic strategy consists of holding 100 µL of the luminol reagent in a confined zone of the LOV and then displacing it with 50 µL of sample while monitoring the chemiluminescent product. Detection is achieved using two strategies: one based on a bifurcated optical fiber and the other based on a customized detection window created by mounting a photomultiplier tube atop of the LOV device. We show that detection is possible using both strategies but that the window strategy yields significantly enhanced sensitivity (355×) due to the larger detection area. In our final experimental conditions and using window detection, it was possible to achieve a limit of detection (LOD) of 1 nmol L(-1) and to quantify Fe(II) in acidified seawater samples up to 20.00 nmol L(-1) with high precision (RSD<6%). These analytical features combined with the long-term stability of luminol solution and the full automation and low reagent consumption make this approach a promising analytical tool for shipboard analysis of Fe(II). The intrinsic capacity of the LOV to operate at a low microliter level and to handle solid phases also opens up a new avenue for chemiluminescence applications. Moreover, this contribution shows that LOV can be a universal platform for optical detection, capable of absorbance, fluorescence and luminescence measurements in a single instrument setup.

Determination of dissolved zinc in seawater using micro-Sequential Injection lab-on-valve with fluorescence detection.

This paper introduces the preliminary design and optimization of a micro-Sequential Injection lab-on-valve system (µSI-LOV) with fluorescence detection for the direct determination of trace Zn(2+) in an unacidified seawater matrix. The method capitalizes on the sensitivity and selectivity of FluoZin-3, which was originally designed to measure zinc in living cells. The optimum reaction conditions, sources of blank signal and physical parameters of the µSIA-LOV are evaluated with the requirements of trace metal analysis in mind, namely high sensitivity and low background signals. A detailed investigation of the effect of sample and reagent sequencing on sensitivity is presented for the first time using µSIA-LOV. We find that the order of sequencing greatly influences peak shape and analytical sensitivity with the highest and smoothest peaks obtained when a large volume of sample (75 µL) is aspirated last in the sequence prior to flow reversal and detection. The optimized reaction conditions and reagent/sample sequencing protocol yield a detection limit of 0.3 nM Zn(2+), high precision (RSD < 2.5%), a linear quantification range up to 40 nM and an analytical cycle of ∼1 min per sample. This work demonstrates that µSI-LOV is capable of attaining detection limits that are close to those needed for open ocean determinations of Zn(2+) without preconcentration or separation of the analyte from the seawater matrix. The low reagent consumption (50 µL per sample), full automation and minimal maintenance requirements of µSI-LOV make it well suited for shipboard analysis and, eventually, for development to meet the pressing need for trace element measurements in unattended locations.