The morphological response of Pseudomonas aeruginosa to azthreonam, cefoperazone, ceftazidime and N-formimidoyl thienamycin

The penicillin-binding protein (PBP) patterns of six strains of Biophila wadsworthia were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and subsequent fluorography of membrane preparations labelled with 13H]benzylpenicillin. The PBP profiles among the strains were similar, generally, seven to nine PBP-reactive bands could be visualized; their molecular weights ranged from 31 to 137 kDa. The relative affinities of the PBPs of four strains ofB. wadsworthia for imipenem were examined and correlated with the morphological responses of the cells to imipenem. Morphological changes were examined by light and electron microscopies. Light microscopy revealed that at low concentrations (less than the MIC), imipenem induced the formation of rounded and bulging cells; rarely, elongation without filamentation was observed. In the presence of imipenem at the MIC, spheroplast formation was observed. Scanning and transmission electron microscopies revealed round forms together with larger, multilobate cells in the presence of subinhibitory concentrations of imipenem, suggesting that new growth sites were initiated while cell division was inhibited. Peeling of the outer membrane was also seen. Spheroplasts were very large (up to 30 ,um in diameter) and stable in aqueous solution. Inhibition of the PBPs could be seen in the presence of low imipenem concentrations.

Section: Discussionsupporting

confidence: 50%

Morphological response of Bilophila wadsworthia to imipenem: correlation with properties of penicillin-binding proteins

Summanen

Wexler

Lee

et al. 1993

“…Under these conditions, PBP 4 was somewere consistent with the observations made for the same times detected. The occasional difficulty in PBP 2 elucidacompounds in aerobic gram-negative rod-shaped bacteria (3, tion and the unpredictable appearance of PBP 4 were not due 7,11,12,14,23). to the time (in the bacterial growth cycle) of cell harvest for Affinity of PBPs for I8-lactam compounds.…”

Section: Methodsmentioning

confidence: 99%

Properties of the penicillin-binding proteins of four species of the genus Bacteroides

Piddock¹,

Wise²

1986

The peniciilin-binding proteins (PBPs) of four species of the genus Bacteroides were examined in cell envelope preparations from exponentially growing cultures and intact cells. Upon examination by sodium dodecyl sulfate-polyacrylamide electrophoresis, three major high-molecular-weight PBPs (molecular weight, 58,000 to 82,000) were resolved, and low-molecular-weight PBPs were seen in all strains except Bacteroidesfragilis. The sporadic appearance of PBP 4 in B. fragilis (molecular weight, approximately 45,000) was shown not to be influenced by the concentration of free iron available in the medium or by the stage of growth at which the batch culture was harvested. No PBP that was inhibited by an aerobic environment was demonstrated. The affinity of 35 ,B-lactam antibiotics for the PBPs from envelope preparations was examined and correlated with the morphological response. Most compounds bound initially to PBP 2 and then PBP 1, correlating with a primary response of filamentation and then spheroplasting and lysis. Compounds such as clavulanic acid bound to PBP 3 at concentrations causing round cells. Based on the data from this study, it is proposed that the three high-molecular-weight PBPs of Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, and Bacteroides ovatus correlate to the three high-molecular-weight PBPs of Escherichia coli and that the PBPs of Bacteroides species perform the same enzymic role in cell wall biosynthesis as their counterparts in E. coli. Therefore, the components of PBP 1 are involved in cell elongation, PBP 2 is involved in septum formation, and PBP 3 is involved in maintenance of cell shape (i.e., PBP 2 in Bacteroides spp. = PBP 3 in E. coli, and PBP 3 in Bacteroides spp. = PBP 2 in E. coli).Species of the genus Bacteroides are commonly encountered in infection, and certain of the species are the anaerobes most resistant to antimicrobial agents (8) and in particular the P-lactam class of antibiotics, owing to the presence of potent P-lactamases (17,18). The development of 3-lactamase-stable r-lactams has not led to greatly enhanced activity for Bacteroides species, and the recent emergence of resistance to cefoxitin, which is not attributable to classical P-lactamase activity (2, 6), has renewed interest in the mechanism of action of 1-lactams in Bacteroides species.

“…For piperacillin, these two curves were concentration independent. The lack of rapid bactericidal activity and the long viability of the filamentous bacteria'were previously described for Pseudomonas aeruginosa exposed to ureidopenicillins (1,2,5,6). When the pH of the medium was modified, the good correlation between growth curves and killing curves was preserved.…”

mentioning

confidence: 99%

Correlation between growth curve and killing curve of Escherichia coli after a brief exposure to suprainhibitory concentrations of ampicillin and piperacillin

Yourassowsky¹,

Linden²,

Lismont³

et al. 1985

Escherichia coli strains that were susceptible to multiple antibiotics were exposed to suprainhibitory concentrations of ampicillin and piperacillin. As with the majority of beta-lactam antibiotics, the growth curves showed an increase in optical density (OD) before lysis during the first hours. This increase in OD depended on the concentration of ampicillin and was independent of the concentration of piperacillin. A good correlation was found between the prelytic increase in OD and the killing curve. During the prelytic increase in OD, the number of CFU per milliliter remained constant. The decrease in the number of CFU per milliliter depended on the concentration of ampicillin and was independent of the concentration of piperacillin. pH variations gave rise to similar effects on growth curves and killing curves.The antimicrobial activity of antibiotics is usually defined by the MIC and the MBC. This usually involves a single value obtained after one night of incubation. Growth curves allow an approach which documents the effect of a brief contact of an antibiotic with a bacterial culture (4). However, growth curves must be interpreted with great caution. Indeed, an increase or a decrease in optical density (OD) may be due to very different microbiological effects. Our previous study (12) showed the value of measuring the prelytic increase in OD. During the first 2 or 3 h after the introduction of beta-lactam antibiotics into an Escherichia coli culture, an increase in OD which could be superimposed on the control curve was observed. This increase in OD was due to the formation of cells with deficient cell walls.The aim of the present study was (i) to verify whether bacteria remain viable during the prelytic increase in OD and, if so, in what proportion, and (ii) to determine whether variations in culture media (e.g., of pH) influence growth curves and the number of CFU per milliliter in the same way. Since it was a matter of verifying the agreement between growth curves and killing curves, we limited our study to strains of E. coli that were susceptible to multiple antibiotics. Only the early response (the first 6 h of contact between the antibiotic and the culture) were taken into consideration. We chose ampicillin and piperacillin at three suprainhibitory concentrations, 16, 64, and 256 ,ug/ml. Indeed, our previous study (12) [Oxoid Ltd., London, England]; macrodilution method; inoculum, 106 CFU/ml) was 1.56 ,ug/ml, with a possible variation of one dilution after 24 h of incubation at 35°C.Culture media. Growth curves and killing curves were determined with Iso-Sensitest medium. Modifications of pH were obtained by adding an acid or a base. The osmolarities of the media so modified were 342 mOsm/k at pH 7.3, 441 mOsm/k at pH 6.3, and 433 mOsm/k at pH 8.3. Colony counts were made on the same media containing 1.5% agar.Growth curves. The MS-2 system (Abbott Laboratories, Diagnostics Div., Irving, Tex.) (8) and open multichamber cuvettes were used to determine growth curves. Ampicillin and piperacillin were ...