Gui-Xin He

Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens

Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations.

Evaluation of impact of exposure of Sudan azo dyes and their metabolites on human intestinal bacteria.

Sudan azo dyes are banned for food usage in most countries, but they are illegally used to maintain or enhance the color of food products due to low cost, bright staining, and wide availability of the dyes. In this report, we examined the toxic effects of these azo dyes and their potential reduction metabolites on 11 prevalent human intestinal bacterial strains. Among the tested bacteria, cell growth of 2, 3, 5, 5, and 1 strains was inhibited by Sudan I, II, III, IV, and Para Red, respectively. At the tested concentration of 100 μM, Sudan I and II inhibited growth of Clostridium perfringens and Lactobacillus rhamnosus with decrease of growth rates from 14 to 47%. Sudan II also affected growth of Enterococcus faecalis. Growth of Bifidobacterium catenulatum, C. perfringens, E. faecalis, Escherichia coli, and Peptostreptococcus magnus was affected by Sudan III and IV with decrease in growth rates from 11 to 67%. C. perfringens was the only strain in which growth was affected by Para Red with 47 and 26% growth decreases at 6 and 10 h, respectively. 1-Amino-2-naphthol, a common metabolite of the dyes, was capable of inhibiting growth of most of the tested bacteria with inhibition rates from 8 to 46%. However, the other metabolites of the dyes had no effect on growth of the bacterial strains. The dyes and their metabolites had less effect on cell viability than on cell growth of the tested bacterial strains. Clostridium indolis and Clostridium ramosum were the only two strains with about a 10 % decrease in cell viability in the presence of Sudan azo dyes. The present results suggested that Sudan azo dyes and their metabolites potentially affect the human intestinal bacterial ecology by selectively inhibiting some bacterial species, which may have an adverse effect on human health.

SugE, a New Member of the SMR Family of Transporters, Contributes to Antimicrobial Resistance in Enterobacter cloacae

ABSTRACT We cloned a gene, sugE, from the chromosome of Enterobacter cloacae ATCC 13047. Analysis of the susceptibilities of the sugE-containing strain (Escherichia coli KAM32/pSUGE28) and sugE-deficient E. cloacae (EcΔsugE) showed that SugE confers resistance to cetyltrimethylammonium bromide, cetylpyridinium chloride, tetraphenylphosphonium, benzalkonium chloride, ethidium bromide, and sodium dodecyl sulfate. We also investigated expression of sugE. We confirm here that SugE from E. cloacae is an SMR family transporter as determined by observing its energy-dependent drug efflux activity.

EmmdR, a new member of the MATE family of multidrug transporters, extrudes quinolones from Enterobacter cloacae

We cloned a gene, ECL_03329, from the chromosome of Enterobacter cloacae ATCC13047, using a drug-hypersensitive Escherichia coli KAM32 cell as the host. We show here that this gene, designated as emmdR, is responsible for multidrug resistance in E. cloacae.E. coli KAM32 host cells containing the cloned emmdR gene (KAM32/pEMMDR28) showed decreased susceptibilities to benzalkonium chloride, norfloxacin, ciprofloxacin, levofloxacin, ethidium bromide, acriflavine, rhodamine6G, and trimethoprim. emmdR-deficient E. cloacae cells (EcΔemmdR) showed increased susceptibilities to several of the antimicrobial agents tested. EmmdR has twelve predicted transmembrane segments and some shared identity with members of the multidrug and toxic compound extrusion (MATE) family of transporters. Study of the antimicrobial agent efflux activities revealed that EmmdR is an H+-drug antiporter but not a Na+ driven efflux pump. These results indicate that EmmdR is responsible for multidrug resistance and pumps out quinolones from E. cloacae.

A comparison of conventional methods for the quantification of bacterial cells after exposure to metal oxide nanoparticles

BackgroundDue to potential interference of nanoparticles on bacterial quantification, there is a challenge to develop a fast, accurate and reproducible method for bacterial quantification. Currently various bacterial quantification methods are used by researchers performing nanoparticles study, but there has been no efficacy evaluation of these methods. Here we study interference of nanoparticles on three most commonly used conventional bacterial quantification methods, including colony counting to determine the colony-forming units (CFU), spectrophotometer method of optical density (OD) measurement, and flow cytometry (FCM).ResultsThree oxide nanoparticles including ZnO, TiO2, and SiO2 and four bacterial species including Salmonella enterica serovar Newport, Staphylococcus epidermidis, Enterococcus faecalis, and Escherichia coli were included in the test. Results showed that there is no apparent interference of the oxide nanoparticles on quantifications of all four bacterial species by FCM measurement; CFU counting is time consuming, less accurate and not suitable for automation; and the spectrophotometer method using OD measurement was the most unreliable method to quantify and detect the bacteria in the presence of the nanoparticles.ConclusionIn summary, FCM measurement proved to be the best method, which is suitable for rapid, accurate and automatic detection of bacteria in the presence of the nanoparticles.

Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation.

Causative agents of infectious disease that are multidrug resistant bacterial pathogens represent a serious public health concern due to the increasingly difficult nature of achieving efficacious clinical treatments. Of the various acquired and intrinsic antimicrobial agent resistance determinants, integral-membrane multidrug efflux pumps of the major facilitator superfamily constitute a major mechanism of bacterial resistance. The major facilitator superfamily (MFS) encompasses thousands of known related secondary active and passive solute transporters, including multidrug efflux pumps, from bacteria to humans. This review article addresses recent developments involving the targeting by various modulators of bacterial multidrug efflux pumps from the major facilitator superfamily. It is currently of tremendous interest to modulate bacterial multidrug efflux pumps in order to eventually restore the clinical efficacy of therapeutic agents against recalcitrant bacterial infections. Such MFS multidrug efflux pumps are good targets for modulation.

Detection of benzalkonium chloride resistance in community environmental isolates of staphylococci.

We isolated a total of 653 strains from 64 community environmental samples in Massachusetts, USA. Among these isolates, 9.65 % (63 strains) were benzalkonium chloride (BC)-resistant staphylococci. All BC-resistant strains were collected from surfaces upon which antibacterial wipes or antibacterial sprays containing 0.02-0.12 % BC had frequently been used in the fitness centres. However, isolates from surfaces upon which antibacterial wipes or antibacterial sprays had not been used were all sensitive to BC. All BC-resistant strains were also resistant to erythromycin, penicillin and ampicillin. In addition, 51 strains showed resistance to cetyltrimethylammonium bromide (CTAB), 15 strains showed resistance to chloramphenicol, 12 strains showed resistance to ciprofloxacin and four strains showed resistance to meticillin. Resistance gene analysis demonstrated that 41 strains contained qacA/B, 30 strains had qacC, 25 strains contained qacG, 16 strains had qacH and eight strains contained qacJ. These data indicate that application of BC is associated with environmental staphylococcal antimicrobial resistance.

Genome Sequence of Non-O1 Vibrio cholerae PS15

ABSTRACT The draft genome sequence of a non-O1 Vibrio cholerae strain, PS15, organized into 3,512 open reading frames within a 3.9-Mb genome, was determined. The PS15 genome sequence will allow for the study of the evolution of virulence and environmental adaptation in V. cholerae.

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae.

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence–related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.

Mutation network-based understanding of pleiotropic and epistatic mutational behavior of Enterococcus faecalis FMN-dependent azoreductase

We previously identified a highly active homodimeric FMN-dependent NADH-preferred azoreductase (AzoA) from Enterococcus faecalis, which cleaves the azo bonds (R-N˭N-R) of diverse azo dyes, and determined its crystal structure. The preliminary network-based mutational analysis suggested that the two residues, Arg-21 and Asn-121, have an apparent mutational potential for fine-tuning of AzoA, based on their beneficial pleiotropic feedbacks. However, epistasis between the two promising mutational spots in AzoA has not been obtained in terms of substrate binding and azoreductase activity. In this study, we further quantified, visualized, and described the pleiotropic and/or epistatic behavior of six single or double mutations at the positions, Arg-21 and Asn-121, as a further research endeavor for beneficial fine-tuning of AzoA. Based on this network-based mutational analysis, we showed that pleiotropy and epistasis are common, sensitive, and complex mutational behaviors, depending mainly on the structural and functional responsibility and the physicochemical properties of the residue(s) in AzoA.