Ampd, Essential for Both Beta-Lactamase Regulation and Cell Wall Recycling, Is a Novel Cytosolic N-Acetylmuramyl-L-Alanine Amidase

In enterobacteria, the ampD gene encodes a cytosolic protein which acts as a negative regulator of beta-lactamase expression. It is shown here that the AmpD protein is a novel N-acetylmuramyl-L-alanine amidase (E.C.3.5.1.28) participating in the intracellular recycling of peptidoglycan fragments. Surprisingly, AmpD exhibits an exclusive specificity for substrates containing anhydro muramic acid. This anhydro bond is mainly found in the peptidoglycan degradation products formed by the periplasmic lytic transglycosylases and thus might behave as a 'recycling tag' allowing the enzyme to distinguish these fragments from the newly synthesized peptidoglycan precursors. The AmpD substrate (or substrates) which accumulates in the absence of the corresponding enzymatic activity acts as an intracellular positive effector for beta-lactamase expression and might represent an element of a communication network between the chromosome and the cell wall peptidoglycan

Antimicrobial Effect of Fluoroquinolones for the Eradication of Nontypeable Haemophilus Influenzae Isolates within Biofilms

Biofilms can be defined as communities of microorganisms attached to a surface. Those bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in respiratory infections, as it forms biofilms both in vitro and in vivo such as human middle ear. Recent reports indicate that otitis media, paranasal sinusitis and lower respiratory tract infections caused by Haemophilus influenzae have become more difficult to treat with oral antibiotic therapy. However, there has been no attention given to antibiotic eradication of NTHi biofilm. To investigate the antimicrobial effect of various antibiotics against NTHi biofilm formation, we conducted the following comparative study using both β-lactamase-negative ampicillin (AMP)-susceptible (BLNAS) and AMP-resistant (BLNAR) NTHi strains. In a microtiter biofilm assay, both levofloxacin and gatifloxacin, of the fluoroquinolone antibiotic group, significantly inhibited biofilm formation by BLNAS and BLNAR NTHi in a dose-dependent fashion compared to ampicillin of the penicillin antibiotic group, cefotaxime of the cephalosporin antibiotic group, and both erythromycin and clarithromycin of the macrolide antibiotic group. Furthermore, in flow cell chamber studies, confocal laser scanning microscopy counted survival bacteria in mature biofilm had been treated with gatifloxacin, ampicillin, cefotaxime and erythromycin. Only gatifloxacin completely killed the BLNAR NTHi isolates within biofilms without regard to the thickness of biofilm formation. The results of this study suggest that fluoroquinolones potentially have a role in therapy against diseases caused by both BLNAS and BLNAR NTHi isolates within biofilms