Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence

Enterotoxigenic Escherichia coli (ETEC) produces a variety of colonization factors necessary for attachment to the host cell, among which CS6 is one of the most prevalent in ETEC isolates from developing countries. The CS6 operon is composed of 4 genes, cssA, cssB, cssC, and cssD. The molecular mechanism of CS6 assembly and cell surface presentation, and the contribution of each protein to the attachment of the bacterium to intestinal cells remain unclear. In the present study, a series of css gene-deletion mutants of the CS6 operon were constructed in the ETEC genetic background, and their effect on adhesion to host cells and CS6 assembly was studied. Each subunit deletion resulted in a reduction in the adhesion to intestinal cells to the same level of laboratory E. coli strains, and this effect was restored by complementary plasmids, suggesting that the 4 proteins are necessary for CS6 expression. Bacterial cell fractionation and western blotting of the mutant strains suggested that the formation of a CssA–CssB–CssC complex is necessary for recognition by CssD and transport of CssA–CssB to the outer membrane as a colonization factor

Oldenlandia diffusa Extract Inhibits Biofilm Formation by Haemophilus influenzae Clinical Isolates.

Oldenlandia diffusa has been empirically used as a therapeutic adjunct for the treatment of respiratory infections. To establish the basic evidence of its clinical usefulness, antimicrobial and biofilm inhibitory activities of an O. diffusa extract were examined against clinical isolates of Haemophilus influenzae, a major causative pathogen of respiratory and sensory organ infections. No significant growth inhibitory activity was observed during incubation for more than 6 h after the extract addition into a culture of H. influenzae. On the other hand, biofilm formation by H. influenzae, evaluated by a crystal violet method, was significantly and dose-dependently inhibited by the O. diffusa extract. Furthermore, the mRNA level of the biofilm-associated gene luxS of H. influenzae significantly decreased soon after the extract addition, and the suppressive effect continued for at least 2 h. At 2 h after the addition of the O. diffusa extract, the autoinducer in the culture supernatant was also significantly reduced by the O. diffusa extract in a dose-dependent manner. These results revealed that O. diffusa extract shows inhibitory activity against luxS-dependent biofilm formation but has no antimicrobial activity against planktonic cells of H. influenzae. Thus, O. diffusa extract might be useful as an adjunctive therapy for the treatment of respiratory infections caused by H. influenzae

In vitro anti-biofilm effect of anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) agents against the USA300 clone

Objectives: Infection with a typical community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), the USA300 clone, has become a worldwide epidemic. Biofilm formation at the site of infection is one of the reasons for the development of intractable infectious diseases resulting from this clone. Here we evaluated the in vitro antibiofilm effects of anti-MRSA agents to identify the most effective agent against the USA300 clone embedded in biofilms. Methods: Vancomycin, linezolid, teicoplanin, daptomycin, arbekacin and tigecycline were used as anti-MRSA agents. The biofilm permeability of the anti-MRSA agents was assessed using a biofilm-coated Transwell®. Morphological and compositional effects of anti-MRSA agents against biofilms were analysed based on the distribution of fluorescence intensity using confocal laser microscopy. Bactericidal activities of the anti-MRSA agents against biofilm-embedded S. aureus were compared. Results: The permeability rates of linezolid (93.1%), daptomycin (91.3%), arbekacin (87.1%) and tigecycline (99.7%) for biofilms formed by the USA300 clone were found to be significantly higher than those of vancomycin (64.9%) and teicoplanin (62.3%) (P < 0.01). Confocal microscopic analysis showed that daptomycin greatly altered the biofilm morphology (decreased thickness and increased roughness) and markedly reduced the area occupied by the biofilm. Furthermore, daptomycin effectively reduced the extracellular DNA of biofilms and showed the highest bactericidal activity against biofilm-embedded USA300 clone among the anti-MRSA agents. Conclusion: The findings from this study demonstrate that, of the tested anti-MRSA agents, daptomycin is the most effective against biofilm-embedded USA300 clone in vitro

Changes in Capsule and Drug Resistance of Pneumococci after Introduction of PCV7, Japan, 2010–2013

We aimed to clarify changes in serotypes and genotypes mediating β-lactam and macrolide resistance in Streptococcus pneumoniae isolates from Japanese children who had invasive pneumococcal disease (IPD) after the 7-valent pneumococcal conjugate vaccine (PCV7) was introduced into Japan; 341 participating general hospitals conducted IPD surveillance during April 2010–March 2013. A total of 300 pneumococcal isolates were collected in 2010, 146 in 2011, and 156 in 2012. The proportion of vaccine serotypes in infectious isolates decreased from 73.3% to 54.8% to 14.7% during the 3 years. Among vaccine serotype strains, genotypic penicillin-resistant S. pneumoniae strains also declined each year. Among nonvaccine serotype strains, 19A, 15A, 15B, 15C, and 24 increased in 2012. Increases were noted especially in genotypic penicillin-resistant S. pneumoniae isolates of serotypes 15A and 35B, as well as macrolide resistance mediated by the erm(B) gene in 15A, 15B, 15C, and 24

Serotype Changes and Drug Resistance in Invasive Pneumococcal Diseases in Adults after Vaccinations in Children, Japan, 2010–2013

After 7-valent pneumococcal conjugate vaccine (PCV) for children was introduced in Japan in November 2010, we examined changes in Streptococcus pneumoniae serotypes and in genetic antimicrobial drug resistance of isolates from adults with invasive pneumococcal diseases. During April 2010–March 2013, a total of 715 isolates were collected from adults with invasive pneumococcal diseases. Seven-valent PCV serotypes in adults decreased from 43.3% to 23.8%, most noticeably for serotype 6B. Concomitantly, 23-valent pneumococcal polysaccharide vaccine (PPSV23) serotypes decreased from 82.2% to 72.2%; non-PPSV23 serotypes increased from 13.8% to 25.1%. Parallel with serotype changes, genotypic penicillin-resistant S. pneumoniae decreased from 32.4% to 21.1%, and 6 non-PPSV23 serotypes emerged (6D, 15A, 15C, 16F, 23A, and 35B). Respective vaccine coverage rates for 13-valent PCV and PPSV23 differed by disease: 73.9% and 84.3% for patients with pneumonia, 56.4% and 69.2% for patients with bacteremia and sepsis, and 45.7% and 69.3% for patients with meningitis

Phase-contrast light microscopic images of <i>H</i>. <i>influenzae</i> 2013–86 (magnification ×1,000).

<p><i>H</i>. <i>influenzae</i> 2013–86 was cultured in a 24-well plate, and each well was washed with PBS three times to remove planktonic bacteria. (A) without OdiE (B) with 20 mg/mL of OdiE.</p

Growth of <i>H</i>. <i>influenzae</i> with or without <i>Oldenlandia diffusa</i> extract.

<p>Each experiment was performed three times on separate occasions, and the data are shown as the mean ± standard deviation (SD). The <i>P</i> value was calculated by the Welch's <i>t</i>-test. OdiE, <i>Oldenlandia diffusa</i> extract</p

Antimicrobial susceptibility and biofilm formation ability of <i>Haemophilus influenzae</i> clinical isolates.

<p>Antimicrobial susceptibility and biofilm formation ability of <i>Haemophilus influenzae</i> clinical isolates.</p

Biofilm formation assay.

<p>Biofilm formation was evaluated by the crystal violet assay. (A), Screening for biofilm formation. <i>H</i>. <i>influenzae</i> ATCC 49247 and 2013–72 represented non-biofilm-forming bacteria. (B), Biofilm formation with or without OdiE. The upper panel shows a photograph of each well after adding 95% ethanol. Each experiment was performed three times on separate occasions, and the data are shown as the mean ± SD. <i>P</i> values were calculated by the Student's <i>t</i>-test. Dotted line shows the cutoff value for biofilm formation in this study.</p