Staphylococcal cassette chromosome mec (SCCmec) and characterization of the attachment site (attB) of methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible Staphylococcus aureus (MSSA) isolates.

This study was designed to screen for SCCmec types and to characterize the attachment site (attB) and universal insertion site (orfX) of SCCmec in a collection of 27 isolates (n = 11) methicillin resistant S. aureus and (n = 16) methicillin susceptible S. aureus isolates in Malaysia. Screening of SCCmec types and characterization of the attachment site was carried out using PCR amplification and Sanger's sequencing method. The result showed that a large proportion of the MRSA isolates carried SCCmec type III 7/11 (63%). Three isolates 3/11 (27%) and 1/11 (9.0%) carried SCCmec type II and IVd respectively. Amplification of the universal insertion site of the SCCmec (orfX) and attachment site (attB) showed that all 16 S. aureus isolates were positive for the orfX gene, while only 7 were positive for the attB gene. Phylogenetic diversity showed that the isolates clustered around strains with features similar to a community acquired MRSA. In conclusion, a high carriage rate of SCCmec type III was observed. The result also showed that all the S. aureus isolates have the orfX structure; however, not all isolates possesses the attB site on the 3' end of the orfX region.

Lipopolysaccharide-binding alkylpolyamine DS-96 inhibits Chlamydia trachomatis infection by blocking attachment and entry.

Vaginally delivered microbicides are being developed to offer women self-initiated protection against transmission of sexually transmitted infections such as Chlamydia trachomatis. A small molecule, DS-96, rationally designed for high affinity to Escherichia coli lipid A, was previously demonstrated to bind and neutralize lipopolysaccharide (LPS) from a wide variety of Gram-negative bacteria (D. Sil et al., Antimicrob. Agents Chemother. 51: 2811-2819, 2007, doi:10.1128/AAC.00200-07). Aside from the lack of the repeating O antigen, chlamydial lipooligosaccharide (LOS) shares general molecular architecture features with E. coli LPS. Importantly, the portion of lipid A where the interaction with DS-96 is expected to take place is well conserved between the two organisms, leading to the hypothesis that DS-96 inhibits Chlamydia infection by binding to LOS and compromising the function. In this study, antichlamydial activity of DS-96 was examined in cell culture. DS-96 inhibited the intercellular growth of Chlamydia in a dose-dependent manner and offered a high level of inhibition at a relatively low concentration (8 µM). The data also revealed that infectious elementary bodies (EBs) were predominantly blocked at the attachment step, as indicated by the reduced number of EBs associated with the host cell surface following pretreatment. Of those EBs that were capable of attachment, the vast majority was unable to gain entry into the host cell. Inhibition of EB attachment and entry by DS-96 suggests that Chlamydia LOS is critical to these processes during the developmental cycle. Importantly, given the low association of host toxicity previously reported by Sil et al., DS-96 is expected to perform well in animal studies as an active antichlamydial compound in a vaginal microbicide.

Towards fabrication of 3D printed medical devices to prevent biofilm formation.

The use of three-dimensional (3D) printing technologies is transforming the way that materials are turned into functional devices. We demonstrate in the current study the incorporation of anti-microbial nitrofurantoin in a polymer carrier material and subsequent 3D printing of a model structure, which resulted in an inhibition of biofilm colonization. The approach taken is very promising and can open up new avenues to manufacture functional medical devices in the future.

Anti-biofilm performance of three natural products against initial bacterial attachment.

Marine bacteria contribute significantly towards the fouling consortium, both directly (modern foul release coatings fail to prevent "slime" attachment) and indirectly (biofilms often excrete chemical cues that attract macrofouling settlement). This study assessed the natural product anti-biofilm performance of an extract of the seaweed, Chondrus crispus, and two isolated compounds from terrestrial sources, (+)-usnic acid and juglone, against two marine biofilm forming bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. Bioassays were developed using quantitative imaging and fluorescent labelling to test the natural products over a range of concentrations against initial bacterial attachment. All natural products affected bacterial attachment; however, juglone demonstrated the best anti-biofilm performance against both bacterial species at a concentration range between 5-20 ppm. In addition, for the first time, a dose-dependent inhibition (hormetic) response was observed for natural products against marine biofilm forming bacteria.

Pretreatment of epithelial cells with rifaximin alters bacterial attachment and internalization profiles.

Rifaximin is a poorly absorbed semisynthetic antibiotic derivative of rifampin licensed for use in the treatment of traveler's diarrhea. Rifaximin reduces the symptoms of enteric infection, often without pathogen eradication and with limited effects on intestinal flora. Epithelial cells (HEp-2 [laryngeal], HCT-8 [ileocecal], A549 [lung], and HeLa [cervical]) were pretreated with rifaximin (or control antibiotics) prior to the addition of enteroaggregative Escherichia coli (EAEC). EAEC adherence was significantly reduced following rifaximin pretreatment compared to pretreatment with rifampin or doxycycline for three of the four cell lines tested. The rifaximin-mediated changes to epithelial cells were explored further by testing the attachment and internalization of either Bacillus anthracis or Shigella sonnei into A549 or HeLa cells, respectively. The attachment and internalization of B. anthracis were significantly reduced following rifaximin pretreatment. In contrast, neither the attachment nor the internalization of S. sonnei was affected by rifaximin pretreatment of HeLa cells, suggesting that rifaximin-mediated modulation of host cell physiology affected bacteria utilizing distinct attachment/internalization mechanisms differently. In addition, rifaximin pretreatment of HEp-2 cells led to reduced concentrations of inflammatory cytokines from uninfected cells. The study provides evidence that rifaximin-mediated changes in epithelial cell physiology are associated with changes in bacterial attachment/internalization and reduced inflammatory cytokine release.

High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.

Pseudomonas aeruginosa is both a model biofilm-forming organism and an opportunistic pathogen responsible for chronic lung infections in cystic fibrosis (CF) patients and infections in burn patients, among other maladies. Here we describe the development of an efficient high-throughput screen to identify small-molecule modulators of biofilm formation. This screen has been run with 66,095 compounds to identify those that prevent biofilm formation without affecting planktonic bacterial growth. The screen is a luminescence-based attachment assay that has been validated with several strains of P. aeruginosa and compared to a well-established but low-throughput crystal violet staining biofilm assay. P. aeruginosa strain PAO1 was selected for use in the screen both because it forms robust biofilms and because genetic information and tools are available for the organism. The attachment-inhibited mutant, strain PAO1 DeltafliC, was used as a screening-positive control. We have also developed and validated a complementary biofilm detachment assay that can be used as an alternative primary screen or secondary screen for the attachment screening-positive compounds. We have determined the potencies of 61 compounds against biofilm attachment and have identified 30 compounds that fall into different structural classes as biofilm attachment inhibitors with 50% effective concentrations of less than 20 microM. These small-molecule inhibitors could lead to the identification of their relevant biofilm targets or potential therapeutics for P. aeruginosa infections.

Inoculation of Chlamydia pneumoniae or Chlamydia trachomatis with ligands that inhibit attachment to host cells reduces infectivity in the mouse model of lung infection: implication for anti-adhesive therapy.

Previous studies have shown that the chlamydial glycan contains a high-mannose oligosaccharide, which mediates attachment and infectivity of the organism. Removal of the glycan decreases infectivity in vitro and in vivo. The present study demonstrates that simultaneous inoculation of chlamydial organisms and a ligand that prevents glycan binding reduces lung burden in infected animals.

Emerging drug targets for antiretroviral therapy.

Current targets for antiretroviral therapy (ART) include the viral enzymes reverse transcriptase and protease. The use of a combination of inhibitors targeting these enzymes can reduce viral load for a prolonged period and delay disease progression. However, complications of ART, including the emergence of viruses resistant to current drugs, are driving the development of new antiretroviral agents targeting not only the reverse transcriptase and protease enzymes but novel targets as well. Indeed, enfuvirtide, an inhibitor targeting the viral envelope protein (Env) was recently approved for use in combination therapy in individuals not responding to current antiretroviral regimens. Emerging drug targets for ART include: (i) inhibitors that directly or indirectly target Env; (ii) the HIV enzyme integrase; and (iii) inhibitors of maturation that target the substrate of the protease enzyme. Env mediates entry of HIV into target cells via a multistep process that presents three distinct targets for inhibition by viral and cellular-specific agents. First, attachment of virions to the cell surface via nonspecific interactions and CD4 binding can be blocked by inhibitors that include cyanovirin-N, cyclotriazadisulfonamide analogues, PRO 2000, TNX 355 and PRO 542. In addition, BMS 806 can block CD4-induced conformational changes. Secondly, Env interactions with the co-receptor molecules can be targeted by CCR5 antagonists including SCH-D, maraviroc (UK 427857) and aplaviroc (GW 873140), and the CXCR4 antagonist AMD 070. Thirdly, fusion of viral and cellular membranes can be inhibited by peptides such as enfuvirtide and tifuvirtide (T 1249). The development of entry inhibitors has been rapid, with an increasing number entering clinical trials. Moreover, some entry inhibitors are also being evaluated as candidate microbicides to prevent mucosal transmission of HIV. The integrase enzyme facilitates the integration of viral DNA into the host cell genome. The uniqueness and specificity of this reaction makes integrase an attractive drug target. However, integrase inhibitors have been slow to reach clinical development, although recent contenders, including L 870810, show promise. Inhibitors that target viral maturation via a unique mode of action, such as PA 457, also have potential. In addition, recent advances in our understanding of cellular pathways involved in the life cycle of HIV have also identified novel targets that may have potential for future antiretroviral intervention, including interactions between the cellular proteins APOBEC3G and TSG101, and the viral proteins Vif and p6, respectively. In summary, a number of antiretroviral agents in development make HIV entry, integration and maturation emerging drug targets. A multifaceted approach to ART, using combinations of inhibitors that target different steps of the viral life cycle, has the best potential for long-term control of HIV infection. Furthermore, the development of microbicides targeting HIV holds promise for reducing HIV transmission events.

In vitro and in vivo effects of soluble, monovalent globotriose on bacterial attachment and colonization.

Epithelial cells lining the urinary tract are rich in globo series glycolipids, structurally defined by a Galalpha1,4Gal motif in the oligosaccharide moiety of this glycolipid family. This Galalpha1,4Gal motif is the attachment target for the P-fimbrial adhesin of uropathogenic Escherichia coli. We investigated the ability of a trisaccharide analog of this core motif, globotriose (Galalpha1,4Galbeta1,4Glc), to interfere with uropathogen attachment and colonization in vitro and in vivo. We assessed the ability of globotriose to inhibit and reverse the binding and agglutination of a P-fimbriated strain of E. coli (JR1) using human erythrocytes and immortalized human colonic epithelial cells as targets. Globotriose (5 mg/ml) completely inhibited and reversed cell agglutination and caused a 10- to 100-fold reduction in JR1 binding to target cells, as determined by flow cytometry. In preparation for an in vivo efficacy study, we investigated the distribution and pharmacokinetics of globotriose in the BALB/c mouse. Globotriose was administered via the tail vein, targeting an instantaneous plasma concentration of 5 mg/ml, and in a different experiment, animals were gavaged at 10 times the intravenous (i.v.) dose. Globotriose was rapidly cleared from plasma (half-life [t1/2], 6 min) and slowly excreted via the kidney (t1/2, 4 h). Urine levels of >5 mg/ml were maintained from 4 to 12 h after the i.v. bolus dose, which resulted in a 1-log reduction in established bladder colonization by JR1. These results suggest that free, soluble globotriose is a feasible alternative therapy for urinary tract infections.

HIV entry inhibitors in clinical development.

Despite recent advances, present therapies for human immunodeficiency virus type 1 (HIV-1) infection are limited by their failure to eradicate HIV-1, by the emergence of multidrug-resistant variants and by significant toxicities. Current therapies collectively target two viral enzymes involved in intracellular viral replication processes, and there is an urgent need for new treatment modalities. HIV-1 entry is a multistep process that comprises viral attachment, co-receptor interactions and fusion. This cascade of events offers opportunities for therapeutic intervention, and clinical proof-of-principle has been obtained for inhibitors of each step. These agents are referred to broadly as entry inhibitors.

Opportunities and challenges in targeting HIV entry.

Characterization of the mechanisms by which HIV-1 enters cells has allowed for an increased understanding of not only tropism and pathogenesis, but also the identification of new targets for rational drug design. Several classes of HIV-1 entry inhibitors have been developed. Antagonists targeting the interaction of the viral envelope protein and receptors on the cell surface, as well as peptides that target an intermediate in the fusion process, have shown promise in vitro and are currently being evaluated in clinical trials. The addition of entry inhibitors to current drug regimens has the potential to significantly improve the therapeutic options for infected individuals, which is crucial for those resistant to or failing currently available therapies.

Neutralization of influenza virus by low concentrations of hemagglutinin-specific polymeric immunoglobulin A inhibits viral fusion activity, but activation of the ribonucleoprotein is also inhibited.

High concentrations of hemagglutinin-specific neutralizing polymeric monoclonal immunoglobulin A (IgA) inhibit attachment of the majority of type A influenza virus virions to cell monolayers and tracheal epithelium (H. P. Taylor and N. J. Dimmock, J. Exp. Med. 161:198-209, 1985; M. C. Outlaw and N. J. Dimmock, J. Gen. Virol. 71:69-76, 1990). A minority of virions attaches but is not infectious. Here, we report that a different mechanism operates when influenza virus A/Puerto Rico/8/34 (H1N1) is neutralized by low concentrations of monoclonal polymeric IgA or when A/fowl plague virus/Rostock/34 (H7N1) is neutralized by low concentrations of polyclonal rat secretory IgA. Under these conditions, neutralized virus attaches to cells and is taken up by them. However, upon entering the cell, the nucleoprotein (NP) of neutralized virus is found in the perinuclear cytoplasm, whereas NP from nonneutralized virus is concentrated in the nucleus itself. Further data show that the low-pH-mediated cell fusion activity of virions is inhibited by IgA in proportion to loss of infectivity. The possibilities that neutralization by low amounts of polymeric IgA is caused by inhibition of the virion fusion activity and that the aberrant distribution of NP from neutralized virus results from its failure to escape from the endosomal system were investigated by using A/PR/8/34 and the fusogenic agent polyethylene glycol (PEG) at pH 5.4. A/PR/8/34 attached to cells at 4 degrees C, with minimal internalization of the virus; treatment with PEG at pH 5.4 and 4 degrees C for 1 min led to infectious fusion of nonneutralized virus with the plasma membrane and, under these conditions, was more efficient than PEG at pH 7 or medium at pH 5.4. Neutralized virus which was attached to cells and treated with acidified PEG appeared to undergo primary and secondary uncoating, with its NP protein becoming concentrated in the nucleus and M1 becoming concentrated in the perinuclear cytoplasm. Although the distribution of NP and M1 was indistinguishable from infectious virus, infectivity was not restored. Thus, even when IgA-induced inhibition of fusion is reversed, virus is still neutralized. We suggest that infectious influenza virus undergoes an activation stage which may be the relaxation of the ribonucleoprotein structure needed to permit transcription or may be the removal of M1 bound to the ribonucleoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple effects of Fis on integration and the control of lysogeny in phage lambda.

Fis is a small, basic, site-specific DNA-binding protein present in Escherichia coli. A Fis-binding site (F) has been previously identified in the attP recombination site of phage lambda (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). The present study demonstrates that in the absence of the phage-encoded Xis protein, the binding of Fis to F can stimulate integrative recombination and therefore increase the frequency of lambda lysogeny in vivo. Additionally, Fis exerts a stimulatory effect on both integration and lysogeny that is independent of binding to the attP F site. Maintenance of the lysogenic state also appears to be enhanced in the presence of Fis, as shown by the increased sensitivity of lambda prophages encoding temperature-sensitive repressors to partial thermoinduction in a fis mutant. In the presence of Xis, however, Fis binding to F interferes with integration by stimulating excision, the competing back-reaction. Since Fis stimulates both excision and integration, depending on the presence or absence of Xis, respectively, we conclude that Xis binding to X1 is the key determinant directing the formation of an excisive complex.

Integration host factor (IHF) stimulates binding of the gpNu1 subunit of lambda terminase to cos DNA.

The lambda terminase enzyme binds to the cohesive end sites (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12 bp single-stranded cohesive ends of the mature phage genome. In vitro this endonucleolytic cleavage requires spermidine, magnesium ions, ATP and a host factor. One of the E. coli proteins which can fulfill this latter requirement is Integration Host Factor (IHF). IHF and the gpNu1 subunit of terminase can bind simultaneously to their own specific binding sites at cos. DNase I footprinting experiments suggest that IHF may promote gpNu1 binding. Although no specific gpNu1 binding to the left side of cos can be detected, this DNA segment does play a specific role since a cos fragment that does not include the left side or whose left side is replaced by non-cos sequences, is unable to bind gpNu1 unless either spermidine or IHF is present. Binding studies on the right side of cos using individual or combinations of gpNu1 binding sites I, II and III indicate that binding at sites I and II is not optimal unless site III is present.

[Restriction map of the genetic transfer factor pAP42]. / Restriktsionnaia karta faktora geneticheskogo perenosa pAP42.

Based on the calculated molecular weights of EcoR1, HindIII, and SalI fragments of the genetic transfer factor pAP42 the restriction map of this plasmid was designed. Sites recognizing restrictases are mostly located in the plasmid fragment with a molecular weight of 5.7 MD.

Effect of glucose and amino acids on expression of K99 antigen in Escherichia coli.

K99 antigen production by enterotoxigenic Escherichia coli strains of bovine origin was investigated by slide agglutination and in vitro attachment to intestinal villi. Work with two strains (B41 and B44) showed that on minimal medium M2, K99 antigen was not repressed by a high concentration of glucose (2%, w/v). Growth on synthetic or complex medium did not affect K99 antigen detection, which was independent of capsular antigens, and its synthesis was not repressed by Casamino acids or glucose. A survey of 12 strains revealed two groups: in one group K99 antigen production was constitutive on basal medium without glucose, and in the second group K99 antigen was produced only in the presence of glucose. Immunoelectrophoresis patterns, and the results of slide agglutination and attachment tests, were dependent upon K99 type, whereas haemagglutination patterns were not.

Failure of iron to promote attachment of gonococci to human spermatozoa under physiological conditions.

The effect of iron on the attachment of gonococci to human spermatozoa was investigated using the three iron salts, ferric chloride, ferric nitrate, and ammonium ferric citrate (AFC). Ferric chloride and ferric nitrate were found to be unsuitable for such studies because they were insoluble at physiological pH values, produced a marked decrease in the pH of unbuffered medium (Ringer's solution), and agglutinated spermatozoa. AFC, in contrast, was soluble at physiological pH, did not affect the pH value of Ringer's solution, and did not agglunate spermatozoa. When gonococci and spermatozoa were mixed together in media with and without AFC, the proportion of spermatozoa with adherent gonococci was approximately the same in each case. Thus, in contrast to previous report, we have found that the addition of iron does not increase the attachment of gonococci to human spermatozoa.

Attachment of Neisseria gonorrhoeae to human sperm. Microscopical study of trypsin and iron.

Pilated Neisseria gonorrhoeae of colony type 1 (T1) and non-pilated bacteria of colony type 4 (T4) were observed by transmission (TEM) and scanning electron microscopy (SEM). No pili were observed on T4 gonogocci, but two types of pili--straight, type a, and bent, type b--were seen on T1 by TEM. When incubated with human sperum and examined by either TEM or SEM, T1 gonococci were seen to attach by individual pili, by several pili wound together as a rope, or by direct contact. Gonococci from T4 colonies attached only by direct contact. Treatment with typsin (1 mg/ml) damaged or removed pili from gonococci. After incubation with trypsin, attachment of pilated gonococci to sperm was decreased significantly, but such treatment did not affect attachment of non-pilated gonococci. Incubation of gonococci from either colony type in 0.1 mmol/l ferric nitrate, followed by incubation with sperm, significantly increased attachment of only T4 bacteria. No pili were seen on T4 gonococci treated with ferric nitrate; thus, it appears that factors other than pili alone are concerned in attachment of these gonococci to sperm.