Hosmin Anwar

Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy.

Few discoveries in the history of medicine have had such a profound impact on human life and society as the discovery of antibiotics to combat bacterial infections. In the last few decades, there has been an observed exponential increase in the number of antibacterial compounds with diverse chemical structures introduced clinically to control infectious diseases. The most significant development in antibiotic research has been the ability to extend and improve the action of naturally occurring compounds by chemical modification. An example of this is the chemical modification of the penicillin molecule such that the new derivatives are immune to attack by bacterial enzymes (e.g., ,B-lactamases). Despite efforts in the search for new antibiotics as well as the improvement of existing antibiotic performance, bacterial resistance to antimicrobial agents remains a problem in the treatment of infections. Before a new antibiotic is introduced into clinical practice, extensive laboratory studies are performed to prove that the new compound is superior to existing compounds in killing the many species of pathogens for which the antibiotic is intended. These species of pathogens, which are grown planktonically in test tubes, are often used to test the effectiveness of the compound in their eradication. If the results are promising, then carefully designed in vivo animal models and clinical trials are used to obtain data regarding the efficacy of the compound. Undoubtedly, this approach has been extremely successful, and many new antibiotics are introduced into clinical practice annually. However, resistance to the antibiotic may be detected soon after its release for clinical use. We believe that this is partly due to the inadvertent use of sublethal concentrations of antibiotics in the treatment of chronic infections such as medical device-related infections. It is therefore of great importance to consider the strategies that are being used to deal with medical device-related infections. It is hoped that this minireview will help to stimulate the formulation of effective strategies to combat these chronic infections.

TESTING THE SUSCEPTIBILITY OF BACTERIA IN BIOFILMS TO ANTIBACTERIAL AGENTS

In this minireview, we summarize recent studies of the interaction of bacteria in biofilms with antibacterial agents and we discuss the parameters that should be considered in the development of experimental protocols for study of the efficacy of antibacterial agents against pathogenic bacteria in biofilm.

Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system

Pseudomonas aeruginosa was cultivated at low growth rates under iron-limiting conditions on acrylic tiles. Biofilm cells exhibited increased tobramycin resistance compared with that of planktonic cells, and in old biofilms were more resistant than were cells in young biofilms. However, on suspension of the biofilm bacteria, glycocalyx-mediated resistance was lost.

Dynamic interactions of biofilms of mucoid Pseudomonas aeruginosa with tobramycin and piperacillin.

The dynamic interaction of planktonic and biofilm cells of mucoid Pseudomonas aeruginosa with tobramycin and piperacillin was investigated in a chemostat system. The results indicated that planktonic and young biofilm cells of the 2-day-old chemostat culture of P. aeruginosa were susceptible to killing by chemostat-controlled doses of either 250 micrograms of piperacillin per ml plus 5 micrograms of tobramycin per ml or 500 micrograms of piperacillin per ml plus 5 micrograms of tobramycin per ml. Complete eradication of the planktonic and young biofilm cells was observed after exposure of the cells to six chemostat-controlled doses of these antibiotic at 8-h intervals for 7 days. Regrowth of the organism was not observed after the termination of antibiotic therapy on day 7. A different picture was observed when antibiotic treatment was initiated on day 10 after inoculation. Viable old biofilm cells were reduced to approximately 20% after exposure to the chemostat-controlled doses of 500 micrograms of piperacillin per ml plus 5 micrograms of tobramycin per ml. Complete eradication of old biofilm cells could not be achieved, and regrowth of the organism occurred after the termination of antibiotic therapy. These data suggest that young biofilm cells of mucoid P. aeruginosa can be effectively eradicated with the combination of piperacillin and tobramycin, while old biofilm cells are very resistant to these antibiotics and eradication of old biofilm cells is not achievable with the chemostat-controlled doses of piperacillin and tobramycin used in this study.

Kinetic interaction of biofilm cells of Staphylococcus aureus with cephalexin and tobramycin in a chemostat system.

Planktonic and young biofilm cells were completely eradicated after exposure of these cells to drug levels representing one loading and two maintenance doses of tobramycin and cephalexin. A very different picture was observed when antibiotic exposure was initiated on day 21. Complete eradication of the old biofilm cells was not observed even when the antibiotic exposure was continued for an extra 6 days. Regrowth of the organism was observed when the antibiotic exposure was terminated.

Effectiveness of ciprofloxacin microspheres in eradicating bacterial biofilm

The treatment of cultures of planktonic cells and aged biofilm cells of P. aeruginosa and S. aureus with ciprofloxacin (CFX) microspheres of poly(l-lactic acid) (PLA) has been investigated using a modified, open in vitro chemostat system. The kinetics of release of CFX as a function of drug loading and the dose of microspheres were correlated with the rate and extent of killing and eradication of the planktonic cells and biofilm cells cultured on pieces of silicone tubing in the chemostat, respectively. At 71% w/w drug loading, a minimum dose of 7 mg of CFX in microspheres was required to sustain CFX concentration for about 10 h above the minimum inhibitory concentration (MIC) in order to completely eradicate the cells of either bacteria. In contrast, peak concentrations obtained from an equivalent dose of free CFX decreased to low values within 30 min and the bacteria cells were not eradicated. Lower microsphere loadings of CFX were ineffective at the same dose. Thus, this study has identified the formulation requirements for PLA microspheres to sustain CFX concentrations above the MIC for several days in order to eradicate bacteria, particularly aged biofilm cells, in the open chemostat in which the drug is being continually diluted, or similarly at a site of administration, for example, in the peritoneal cavity.

Changing characteristics of aging biofilms

Abstract The use of an in-vitro chemostat system in the study of the interaction of young and aging biofilms with antibiotics is discussed. The study reveals that young biofilms are susceptible while aging biofilms are resistant to antibiotics. Possible physiological responses of bacteria in biofilms to the physical and chemical microenvironmental niches are used to illustrate the mechanisms of resistance of aging biofilms to antibiotics. It is the hope that this minireview will stimulate the development of effective strategies to eradicate aging biofilms.

Method of evaluation of sustained release microsphere formulations using the open chemostat system

Abstract The sustained release of cephalexin (CPX) as the monohydrate and ciprofloxacin (CFX) as the hydrochloride from separate 100000 Mol. Wt poly( l -lactic acid) microspheres (250–425 gmm sieve size range) was evaluated in an open chemostat system. Drug concentrations in pH 7.4 phosphate buffer solution (PBS) reached peak levels which were sustained for different periods of time, depending on the flow rate of PBS at a constant volume of 120 ml in the chemostat. At 0.46 ml/min flow rate CPX microspheres (33% w/w loading) sustained CPX concentrations for approx. 90 min (solubility = 40 mg/ml at pH 7.4, 37°C) whereas CFX (solubility = 5 mg/ml) was sustained for at least 6 h. Increasing drug loading increased peak levels of either antibiotic but decreased the sustained period of CPX only. Decreasing microsphere size to 125–250 gmm increased CPX or CFX concentration levels and decreased the sustained period to about 45 min and 2 h, respectively. At higher doses, CFX was sustained at higher concentrations over the 6 h period. Decreasing the flow rate in the chemostat increased sustained levels of CFX while increasing the flow rate decreased sustained levels. Thus, the chemostat system is convenient for testing the sustained release of drugs as a function of formulation parameters and to obtain information about the optimum doses of sustained release medication for in vivo administration.

Association of a 38 kDa bovine serum protein with the outer membrane of Bordetella pertussis

Upon cultivation of Bordetella pertussis in bovine serum, a 38 kDa protein was found to be tightly associated with the outer membrane. The intensity of the 40 kDa porin was reduced under these growth conditions. Exposure of Bordetella pertussis, grown in Stainer and Scholte medium, to bovine serum for 1 h did not result in the appearance of the 38 kDa protein. Unlike the 40 kDa porin however, the electrophoretic mobility of this protein was affected neither by temperature of denaturation nor by the presence of 2-mercaptoethanol. Amino acid sequence analysis of the N-terminal of the 38 kDa protein revealed that his protein had 87% homology to both the mouse and human complement C3 precursors.