A simple method for the study in vivo of bacterial growth and accompanying host response

Day, Sarah E.J.; Vasli, Kjersti K.; Russell, R.J.; Arbuthnott, J. P.

doi:10.1016/s0163-4453(80)91773-9

Cited by 34 publications

(22 citation statements)

References 15 publications

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“…Indeed, this study was designed to stimulate in vivo growth conditions relevant to environmental conditions found during bacterial infections in humans. Our group and other researchers have used similar diffusion chambers implanted in animals for the study of undiscovered cell properties induced by in vivo growth (8,13,17). This cultivation system allows the recovery of relatively large numbers of bacteria sequestered away from host phagocytic cells and therefore suitable for various analyses.…”

Section: Discussionmentioning

confidence: 99%

Effect of beta-lactams on peptidoglycan metabolism of Haemophilus influenzae grown in animals

Rousseau

Dargis

Gourde

et al. 1992

Antimicrob Agents Chemother

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We have examined bacterial determinants that influence 13-lactam activity in HaemophUilus influenzae cells cultivated in a system that reproduces in vivo growth conditions. Bacteria grown in diffusion chambers were recovered from the peritoneal cavities of rats, and their cell properties were compared with those of bacteria grown in broth cultures by various tests performed in vitro. The rate of peptidoglycan synthesis was measured as the incorporation of [14C]alanine into cell wall material in the presence of chloramphenicol. The total incorporation of [14Cjalanine into peptidoglycan was markedly increased in cells grown in rats prior to the assay but was efficiently reduced by the 13-lactams. The extent of cross-linking was lower in the peptidoglycan of in vivo-grown bacteria, as estimated by sodium dodecyl sulfate-to trichloroacetic acid-insoluble radioactive cell wall material ratios. A whole-cell labeling assay with 1211-penicillin was used to characterize the penicillin-binding proteins (PBPs). Four PBPs showed a striking reduction in the binding of the labeled penicillin in cells grown in rats. Such changes resembled the PBP alterations seen in 13-lactamase-negative clinical strains that were resistant to the 13-lactams. Although ampicillln and moxalactam showed delayed inhibitory activities in vitro for cells collected from rats, cells recovered from P-lactam-treated rats showed evidence of antibiotic effectiveness (binding of the 1-lactams to PBPs in vivo and altered morphology), and the killing of cells exposed to antibiotics in broth or in peritoneal fluid was equally good. Finally, the frequencies of spontaneous resistance or tolerance to ampicillin or moxalactam were estimated, and there was no significant difference for in vitro-or in vivo-grown cells. These data demonstrated that the cultivation of H. influenzae in animals created changes in PBPs and the overall peptidoglycan metabolism. Such alterations did not impair the bactericidal activities of the 13-lactams, although they resulted in delayed bacterial inhibition, a phenomenon that may have important consequences in antibiotherapy.,B-Lactam antibiotics act on bacterial cell wall peptidoglycan synthesis through the inhibition of enzymes termed penicillin-binding proteins (PBPs) (34). Some of these antibiotics are currently used for the treatment of serious infections caused by Haemophilus influenzae. In vitro susceptibility tests are used to determine the antibacterial activities of ,B-lactams and to provide the information necessary for the development of new drugs. However, there is now considerable evidence that growth conditions and the physiological state of bacteria influence dramatically the susceptibilities of cells to 1-lactam antibiotics (9), and there is a need to predict more accurately how bacteria will behave during antibiotherapy (15).For example, it is known that slow-growing or nongrowing Escherichia coli cells are more resistant to the bactericidal action of 13-lactams (30), a phenomenon that is called phenotypic tolerance a...

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Section: Discussionmentioning

confidence: 99%

Effect of beta-lactams on peptidoglycan metabolism of Haemophilus influenzae grown in animals

Rousseau

Dargis

Gourde

et al. 1992

Antimicrob Agents Chemother

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“…Mouse serum was obtained from male mice (strain ddY, weighing 20 + 2 g; Japan SLC, Inc., Hamamatsu, Japan). Mouse ascitic fluid was obtained by implanting two polypropylene chambers (7 mm in diameter and 10 mm in length) filled with sterile distilled water in the abdominal cavities of male mice (strain ddY, weighing 28 ± 2 g; Japan SLC) as described by Day et al (3). Ascitic fluid was taken from the chambers by a syringe and pooled at 2 days after implantation.…”

Section: Methodsmentioning

confidence: 99%

Activity of the carbapenem panipenem and role of the OprD (D2) protein in its diffusion through the Pseudomonas aeruginosa outer membrane

Fukuoka¹,

Ohya²,

Narita³

et al. 1993

Antimicrob Agents Chemother

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Evidence of permeation of panipenem through the OprD (D2) channel of Pseudomonas aeruginosa outer membrane was shown by using OprD protein-producing and -nonproducing strains which contained plasmid pHN4, which codes for L-1 13-lactamase of Xanthomonas maltophilia. Permeation by panipenem was determined by measuring hydrolysis of the carbapenem by 13-lactamase in the periplasmic space. Permeation by panipenem was also determined by counting uptake of [14C]panipenem into P. aeruginosa PAO1 and its OprD protein-deficient mutant, and this permeation of PAO1 was inhibited by L-lysine. These results indicate that panipenem, as well as imipenem, uses the OprD channel, which functions as a specific channel for diffusion of basic amino acids. Panipenem and imipenem showed stronger activities against PAO1 and clinical isolates in human serum than in Mueller-Hinton broth, which contains more amino acids than human serum does. The activities of the carbapenems were reduced by addition of L-lysine to human serum. Similar results were obtained with mouse serum and ascitic fluid. In contrast, such a change in the activities of carbapenems was not observed with an OprD protein-deficient mutant, suggesting that the main reason for the strong activities of carbapenems in biological fluids is a decrease in competition between the antibiotics and basic amino acids for permeation through the OprD channel. Panipenem and imipenem showed much stronger therapeutic efficacies against experimental infections with P. aeruginosa in mice than did the reference antibiotics. Their in vivo activities were more consistent with their MICs in biological fluids than with those in Mueller-Hinton broth.Infections by Pseudomonas aeruginosa are serious clinical problems, particularly in compromised hosts. In recent years, many new extended-spectrum penicillins and cephalosporins have been introduced into clinical practice. Some of them show potent antipseudomonal activity in vitro. However, only a few antibiotics show adequate clinical efficacy against pseudomonal infections. Carbapenems, such as panipenem (formerly RS-533 or CS-533) and imipenem, have been shown to have potent antipseudomonal activity both in vitro and in vivo (2,4,8,9,12,14,15,17,22).We reported previously that panipenem and imipenem showed much stronger antipseudomonal activities in lowamino-acid media, such as a minimal medium, than in Mueller-Hinton medium (5). This mechanism was investigated by using P. aeruginosa PAO1 and its OprD (D2) protein-deficient mutants, and it was assumed that the increase in the antipseudomonal activities of the carbapenems in low-amino-acid media relates to lessened competition between the carbapenems and basic amino acids for permeation through the OprD channel of the P. aeruginosa outer membrane. Trias and Nikaido reported that imipenem permeated the outer membrane of P. aeruginosa through the OprD channel (20) and that the permeation was competitively inhibited by basic amino acids (21). Concentrations of free basic amino acids in human serum are ...

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“…Leukocytes were not observed on the inner surfaces of the filters. After prolonged incubation, the chambers in which growth occurred became encased in fibrin and granulation tissue, as described by Day et al (1980) in their work with chambers containing Staphylococcus aureus.…”

Section: Observation Of the Implanted Chambersmentioning

confidence: 91%

“…As it is difficult to determine the importance of the additives included in some animal models, we used a modification of the method of Day et al (1980), in which bacteria are suspended in a salt solution and enclosed in a membrane-filter bound chamber. This initially allows free passage of diffusible host and bacterial factors, but excludes phagocytes.…”

Section: Introductionmentioning

confidence: 99%

The growth and survival of capsulate and non-capsulate Bacteroides fragilis in vivo and in vitro

Patrick

Reid

Larkin³

1984

Journal of Medical Microbiology

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SUMMARY. The growth of capsulate and non-capsulate Bacteroides fragilis in chambers implanted in the mouse peritoneal cavity was compared. Capsulate and essentially non-capsulate ( < 1 % capsulate) populations of B. fragilis strains NCTC9343 and NCTC 10584 consistently grew exponentially to > lo9 cfu/ml within 24 h in vivo, and low numbers of capsulate bacteria were maintained in the essentially non-capsulate population; however, the degree of capsulation of the capsulate population decreased by more than 60%. B. fragilis ATCC23745 differed from strains NCTC9343 and NCTC 10584 in that growth was unpredictable and only occurred in some of the implanted chambers. Capsule production by cells of strain ATCC23745 varied from chamber to chamber: sometimes the proportion of capsulate cells increased after prolonged implantation. This could occur with either an increase or decrease in viable numbers in vivo and also after in-vitro incubation of this strain in chambers. The survival of capsulate and non-capsulate B. fragilis strains NCTC9343 and ATCC23745 was compared in aerobic and anaerobic conditions in vitro. In anaerobic conditions, capsulate and non-capsulate strain NCTC9343 survived equally well, whereas capsulate ATCC23745 survived better than its non-capsulate variants. Capsulate populations of both strains survived better than non-capsulate in aerobic conditions.

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A simple method for the study in vivo of bacterial growth and accompanying host response

Cited by 34 publications

References 15 publications

Effect of beta-lactams on peptidoglycan metabolism of Haemophilus influenzae grown in animals

Effect of beta-lactams on peptidoglycan metabolism of Haemophilus influenzae grown in animals

Activity of the carbapenem panipenem and role of the OprD (D2) protein in its diffusion through the Pseudomonas aeruginosa outer membrane

The growth and survival of capsulate and non-capsulate Bacteroides fragilis in vivo and in vitro

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