Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, andStaphylococcus aureus ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (P > 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.

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In this study, the adhesive exopolysaccharides of strains of Pseudomonas putida and P. fluorescens, both isolated from freshwater epilithic communities, were examined with regard to their chemical composition, biosynthesis, and their role in adhesion. Electron microscopy showed that both strains were enrobed in fibrous glycocalyces and that these structures were involved in attachment of the cells to a solid surface and as structural matrices in the microcolony mode of growth. In batch culture experiments most of the extracellular polysaccharide of both strains was found to be soluble in the growth medium rather than being associated with bacterial cells. Exopolysaccharide was synthesized during all phases of growth, but when growth was limited by exhaustion of the carbon source, exopolysaccharide synthesis ceased whereas exopolysaccharide synthesis continued for some time after cessation of growth in nitrogen-limited cultures. Exopolysaccharide from both strains was isolated and purified. Pseudomonas putida synthesized an exopolysaccharide composed of glucose, galactose, and pyruvate in a ratio of 1:1:1; the P. fluorescens polymer contained glucose, galactose, and pyruvate in a ratio of 1:1:0.5, respectively. Polymers from both strains were acetylated to a variable degree.

BackgroundEnvironmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA.ResultsThe concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments.ConclusionThe abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.

Xanthomonas maltophilia isolates were collected from 63 patients in three acute-care hospitals in Calgary, Alberta, Canada. On the basis of Centers for Disease Control and Prevention definitions, 48 patients had nosocomial and 15 had community-acquired X. maltophilia. Thirty-eight of the patients were colonized and 25 were infected. Sixty-four percent of patients who acquired X. maltophilia in the intensive care unit (ICU) became infected, whereas 32% of patients in a non-ICU setting became infected. ICU patients tended to be hospitalized for a shorter period of time than non-ICU patients before the onset of X. maltophilia infection. Regardless of being colonized or infected, all patients had debilitating conditions, with respiratory disease being the most common underlying illness (35%). Forty-two patients (88%) with hospitalacquired X. maltophilia received prior antibiotic therapy which included gentamicin, tobramycin, ceftazidime, piperacillin, and imipenem. Agar dilution MICs showed that patient isolates were resistant to these antimicrobial agents that patients had received. Pulsed-field gel electrophoresis of SpeI-digested genomic DNA revealed that six epidemiologically linked patient isolates from the ICU of one acute-care hospital had identical DNA profiles. In contrast, isolates from patients from the other two hospitals had unique genotype profiles (n ‫؍‬ 57) regardless of the presence or absence of an epidemiologic association. In these patients there was genetic evidence against the acquisition of a resident hospital clone. These results indicate that pulsed-field gel electrophoresis can resolve genotypically distinct strains of X. maltophilia and, consequently, is a useful tool for evaluating nosocomial infections caused by X. maltophilia.

Analysis of a clinical isolate ofAcinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the SalmoneUla typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extraceliular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(M) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 ,uM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate ofA. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.Acinetobacter baumannii, formerly known asAcinetobacter calcoaceticus subsp. anitratus (15), belongs to a bacterial species that is widely distributed throughout the environment (6). This microorganism can be isolated from the skin (3, 43) and respiratory tract (59) of healthy ambulatory adults as well as from hospital personnel and equipment (36,39,46,67).Lately, numerous outbreaks of nosocomial infections caused by A. baumannii have been reported (7-10, 16, 42, 47, 56, 57, 73), which are of particular concern because of the widespread and increasing antibiotic resistance of the isolated strains (17,30,32,33,40,41,48). Nevertheless, most infections have occurred in patients compromised by either antibiotic therapy, respiratory instrumentation and manipulations, dialysis, or surgery (18,19,39,70). Thus, it was proposed that reduced host defenses and interaction with other bacterial species, rather than the expression of specific bacterial virulence factors (49), are the factors responsible for opportunistic infections caused by Acinetobacter species.Acinetobacter species are able to survive under restricted nutrient conditions, such as those imposed by the host. Iron is one of the essential bacterial nutrients that is tightly controlled by the host, through its chelation by the highaffinity binding glycoproteins transferrin (TF) and lactoferrin (23). Thus, bacteria have developed efficient iron uptake mechanisms that allow them to scavenge iron from these host proteins, either by direct interaction with the ironprotein complexes (34) or by the synthesis an...

We have used modern techniques of direct microscopic examination and quantitative bacterial recovery to show the existence of a route of bacterial colonization along the external and internal surfaces of Tenckhoff catheters implanted in experimental animals. The external route of progressive bacterial colonization extends from the cutaneous exit site through the dacron cuff and into the peritoneum. Bacterial growth along this route consists primarily of glycocalyx enclosed bacterial biofilms adherent to catheter and tissue surfaces, and this surface colonization may or may not give rise to peritoneal infection in which free-living bacteria are found in the peritoneal fluid. The rate of this progressive bacterial colonization depends on the degree of bacterial contamination of the exit site at the time of implantation. Exit site sterilization (hibitane) delays the process while inoculation with rabbit skin strains of Staphylococcus epidermidis accelerates it. Even with optimal implantation techniques, bacterial colonization proceeds via this subcutaneous route so that most Tenckhoff catheter surfaces are covered with a bacterial biofilm, consisting predominantly of gram positive cocci, within three weeks after the implantation of these devices. The rate of bacterial biofilm development on both surfaces of these Tenckhoff catheters, the bacterial colonization of peritoneal tissues, and the dissemination of bacteria into the peritoneal fluid are all significantly accelerated by dialysis in this experimental animal model of continuous ambulatory peritoneal dialysis (CAPD).