Resistance ofPseudomonas aeruginosa to β-lactam antibiotics

A series of experiments were performed withP. aeruginosa to demonstrate which of the biochemical mechanisms are responsible for the resistance to the β-lactam antibiotics. The constitutive β-lactamase was isolated and characterized for the strain used as type OXA whose pI was 7.1, with a molar mass of 49 kg/mol. The strain was also submitted to a series of treatments with Tris and Tris -EDTA to disrupt the outer membrane. The treated cells demonstrated a ten-fold reduction in the MIC with cloxacillin, six-fold with penicillin, and five-fold with oxacillin. At least two different biochemical mechanisms were responsible for the resistance to the β-lactams studied which could be important in the prevalence ofP. aeruginosa in nosocomial infections.     

Postantibiotic effects of subinhibitory concentrations of some antibiotics and their influence onPseudomonas aeruginosa enzymic activity

The postantibiotic effects of subinhibitory concentrations (PA SMEs) and virulence factor alterations induced by ciprofloxacin, tobramycin and netilmicin inPseudomonas aeruginosa were studied. After induction of the postantibiotic phase (PA) (2x or 4x MIC) the cultures were exposed to subinhibitory concentrations (0.1, 0.2 and 0.3x MIC) of the same antibiotic PA SME). The regrowth of treated as well as control cultures was followed for 24 or 45 h. In the sterile culture filtrates obtained from these bacterial cultures, elastase and proteinase were determined. Ciprofloxacin and aminoglycosides exhibited PA SMEs of 3.5–35 h for certain combinations of supra-subinhibitory antibiotic concentrations. Longer PA SMEs were observed after treatment with higher sub-MICs. Tobramycin at 0.2 and 0.3x MIC (postantibiotic phase induced by 2x MIC) and at all sub-MICs added to the bacteria previously exposed to 4x MIC do not allow any regrowth of bacterial culture. PA SMEs of tested antibiotics affected virulence factors ofP. aeruginosa. Elastase compared to proteinase was suppressed more effectively. Ciprofloxacin at 0.3x MIC reduced elastase and proteinase activity most significantly (to 14.2 and 60 % of the control values).     

In vitro PYOCIN activity ofPseudomonas aeruginosa strains pretreated with antibiotics

Fifty-six selected strains of Pseudomonas aeruginosa belonging to 8 different pyocin types (H, I, 15, 6, PTI-1, PTI-2, PTI-3, PTI-4) were treated with subinhibitory concentrations (MIC/2) of either gentamicin or carbenicillin. Both treatments induced changes in pyocin patterns for all types but at different levels. The percentage of strains that retained their pyocin pattern were more or less equal in both treatments. In treated and untreated producers, the growth inhibition ability for 5 different strains of Enterobacteriaceae (Escherichia coli K12, E. coli EB, Proteus vulgaris, Salmonella typhi, Shigella flexneri) was also investigated. In all pyocin patterns the number of producers that inhibit the growth of these strains was lower after treatment with gentamicin or with carbenicillin, a smaller decrease was detected in the latter treatment. It appeared that the subinhibitory concentrations of these antibiotics are capable of protecting the Enterobacteriaceae strains from the action of the pyocins.     

Effects of subinhibitory concentrations of antibiotics on biological properties ofSalmonella typhimurium

We followed the effects of subinhibitory concentrations (sub-MICs) of 7 antibiotics (ticarcilin, cefotaxim, streptomycin, gentamicin, ciprofloxacin, pefloxacin, mitomycin C) on the sensitivity of aSalmonella typhimurium strain to standard bacteriophages, on the phage DNA as well as on the factors of virulence (permeability and cytotoxic activity). The phage type was not changed by the sub-MICs of the tested antibiotics. However, differences were found in culture filtrates prepared from the bacterial suspensions of the strain cultivated with the sub-MICs. Marked inducing effects on phage DNA were exhibited by mitomycin C (1/2, 1/4, 1/8 of the MIC), pefloxacin (1/2, 1/4, 1/8 of the MIC) and ciprofloxacin (1/2, 1/4, weakly also 1/8 of the MIC). Ticarcilin (1/2 of the MIC), like the aminoglycosides streptomycin and gentamicin (1/2, 1/4, 1/8 of the MIC), had a weak effect. Sub-MICs of the studied antibiotics (with the exception of 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of ticarcilin) decreased the permeability reaction in rabbit skin. Most effective was streptomycin (1/2 of the MIC). Sub-MICs of the tested antibiotics (with the exception of 1/4 and 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of pefloxacin) caused also an inhibition of the factor responsible for morphological changes on Vero cells. Gentamicin and streptomycin were effective at all the sub-MICs tested.     

The effect of subinhibitory concentrations of antibiotics on the adhesion of enterobacteria

It is impossible to determine rigidly a net result of the influence of antibiotics on the interaction between parasite and host cells, as many factors participating in this process are not studied. Adhesion of microorganisms is one of the essential mechanisms of the above interaction. Antibiotics with a different mechanism of action in the subinhibitory concentrations affecting viability of microbes either slightly or nowise have been studied for their effect on adhesion on a model of the intestine section of human embryos and experimental animals. Most of antibiotics influenced differently adhesion of the microorganisms, that also depended on the species attribution of the latter. The accelerated selection of resistance during a successive passage via the suggested adhesion system was observed. The data obtained elucidated certain mechanisms of the effect of antibiotics on the microbial populations at the initial phase of the infectious process and under the primary contamination of mucosa.     

Biosurfactant production by two isolates ofPseudomonas aeruginosa

Two strains of biosurfactant-producing bacteria, identified asPseudomonas aeruginosa, were isolated from injection water and crude oil-associated water in Venezuelan oil fields. Both biosurfactants resembled rhamnolipids and produced stable emulsions of heavy and extra-heavy crude oils, reducing the surface tension of water from 72 to 28 dynes/cm. Tenso-active properties of the biosurfactants were not affected by pH, temperature, salinity or Ca²⁺ or Mg²⁺ at concentrations in excess of those found in many oil reservoirs in Venezuela.     

Effect of subinhibitory concentrations of antibiotics on catalase activity of plague microbes]

It was shown that the presence of subinhibitory concentrations of ampicillin, cefotaxime or gentamicin in the cultivation medium had a marked inhibitory effect on the catalase activity of plague microbe. The effect depended on the characteristic features of plague microbe strains and the incubation temperature. When the cells of a virulent strain of the plague microbe Y. pestis 1300 were cultivated at a temperature of 37 degrees C on a medium containing the subinhibitory concentrations of ampicillin or cefotaxime, the pathogen virulence for albino mice significantly decreased.     

Resistance ofPseudomonas aeruginosa to sodium dimethyldithiocarbamate by adaptation

Resistance and the development thereof inPseudomonas aeruginosa to the bactericide sodium dimethyldithiocarbamate (SMT) was investigated.P. aeruginosa was cultured in nutrient-poor broth in the presence of subinhibitory concentrations of SMT. It adapted over 21 days of exposure from 250 g·ml^–1 to 490 g·ml^–1. The initial high MIC was ascribed to exclusion of SMT by the lipopolysaccharide layer, since removal thereof by EDTA rendered cells highly susceptible. The alginate-producing mutant PAO 579 was much more susceptible to SMT than was its parent PAO 381, indicating that extracellular polysaccharide does not act as an exclusion barrier to SMT. Following 24 h of exposure to SMT,P. aeruginosa had an altered profile of outer membrane proteins as determined by SDS-PAGE. Resistant cells had a further altered profile. Resistance ofP. aeruginosa is ascribed to a change in the outer membrane protein profile, leading to improved exclusion of SMT.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Pseudomonas aeruginosa is one of the major nosocomial pathogen that can causes a wide variety of acute and chronic infections P. aeruginosa is a dreaded bacteria not just because of the high intrinsic and acquired antibiotic resistance rates but also the biofilm formation and production of multiple virulence factors. We investigated the in vitro activities of antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin and colistin) and antimicrobial cationic peptides (AMPs; LL-37, CAMA: cecropin(1–7)-melittin A(2–9) amide, melittin, defensin and magainin-II) alone or in combination against biofilms of laboratory strain ATCC 27853 and 4 clinical strains of P. aeruginosa. The minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentrations (MBEC) were determined by microbroth dilution technique. The MBEC values of antibiotics and AMPs were 80–>5120 and 640–>640 mg/L, respectively. When combined with the LL-37 or CAMA at 1/10× MBEC, the MBEC values of antibiotics that active against biofilms, were decreased up to 8-fold. All of the antibiotics, and AMPs were able to inhibit the attachment of bacteria at the 1/10× MIC and biofilm formation at 1× or 1/10× MIC concentrations. Time killing curve studies showed 3-log₁₀ killing against biofilms in 24 h with almost all studied antibiotics and AMPs. Synergism were seen in most of the studied combinations especially CAMA/LL-37 + ciprofloxacin against at least one or two strains’ biofilms. Since biofilms are not affected the antibiotics at therapeutic concentrations, using a combination of antimicrobial agents including AMPs, or inhibition of biofilm formation by blocking the attachment of bacteria to surfaces might be alternative methods to fight with biofilm associated infections.     

The mechanism of pathogenicity ofPseudomonas aeruginosa

Extra-cellular proteinases ofPseudomonas aeruginosa toxic for larvae of the greater wax moth were separated from the culture filtrate by means of ammonium sulphate precipitation, chromatography on DEAE cellulose, gel filtration on Sephadex G-75 and preparative disc-electrophoresis respectively. The five proteinases isolated were characterized by pH optima, molecular weights, elastase and collagenase activities.     

The mechanism of pathogenicity ofPseudomonas aeruginosa

Proteinases produced byPseudomonas aeruginosa are toxic for galleria larvae and their LD₅₀ differ from one enzyme to the other. The most toxic enzyme has a specific affinity to some protein fractions of insect haemolymph as shown by disc electrophoresis.     

Bacterial alginate lyase inactive on alginate biosynthesized by Pseudomonas aeruginosa

A bacterium (strain Al) isolated from a ditch produces three kinds of intracellular alginate lyases [Al-I (molecular weight: M.W. 60,000), Al-II-1 (M.W. 60,000) and Al-II-2]; the former two lyases have been purified and characterized (Yonemoto et al., J. Ferment. Bioeng., 72, 152–157, 1991). As part of a series of studies, Al-II-2 lyase was purified from cell-free extract of the bacterium. The lyase, with a M.W. of 25,000, depolymerized sodium-, potassium- and propyleneglycol alginates most efficiently at pH 8.0, 70°C, but it was inactive toward bacterial alginates with O-acetyl groups.     

Inactivation of allantoinase ofPseudomonas aeruginosa in vivo

The total and specific activity of allantoinase decreased in the stationary growth phase whenPseudomonas aeruginosa was cultured in a medium containing allantoin or allantoate as the sole source of carbon, nitrogen and energy. The enzyme was not instableper se. Inactivation was proven to be due to the synthesis of a protein, which exhibited no proteolytic activity toward allantoinase. The synthesis of a specific binding protein is postulated.     

Production of biosurfactants by the resting cells ofPseudomonas aeruginosa CFTR-6

A resting cell system was used for the production of glycolipids byPseudomonas aeruginosa CFTR-6. In this, the growth phase was separated from the production phase to overcome the inhibition of glycolipid production by inorganic phosphate. It was shown that when the cells were transferred after the growth phase into a medium devoid of phosphate, glycolipid production was increased nearly twofold. The maximum glycolipid concentration was attained much more rapidly than the conventional batch fermentation system, thus increasing the productivity.     

Substrate specificity of the paraffin hydroxylase ofPseudomonas aeruginosa

The paraffin hydroxylating enzyme system isolated fromPseudomonas aeruginosa (strain 473) grown onn-heptane has been studied with respect to its substrate specificity. It has been found that cell-free extracts of this organism can hydroxylate a wide variety of hydrocarbons. In order to function as substrates, molecules should be able to assume a more or less planar conformation. The compounds used in this study can be divided into three classes: (a) alkanes, (b) alkylbenzenes and (c) (alkyl)cycloalkanes. In each of these groups hydroxylation occurs at a specific site in the molecule. Noteworthy is the hydroxylation of isopropyl groups at a methyl carbon and that of monoalkylcyclohexanes at the 4-transposition.From the geometry of the substrate and non-substrate molecules the conclusion was drawn that the active centre of the hydroxylase is a cleft in the enzyme surface of about 5 Å wide and 8 Å deep.We gratefully acknowledge the skillfull assistance of Miss W. T. Hempel.     

Sensitivity of bacteria exposed to subinhibitory concentrations of antibiotics to the action of serum and liver phagocytes

Incubation of E.coli and S. aureus with subinhibitory concentration (1/5 MIC) of cefamandole modified bacterial morphology and resistance to host defence mechanisms. In fact, cefamandole induced filamentous forms of E. coli, when added to the perfusing medium of the isolated rat liver system, were phagocytized at a much fortes rate then control bacteria, but appeared less sensitive to serum bactericidal activity. In contrast, S.aureus, after exposure to the antibiotic, was more sensitive to the bactericidal activity of serum then controls, while treated and untreated cells were phagocytized at the some rate. The data suggest that even at low doses some antibiotics may alter bacterial structure and increase their susceptibility to host factors.     

In vitro effect of subinhibitory concentrations of ceftazidime and meropenem on the serum sensitivity of Pseudomonas aeruginosa strains

The aim of this study was to determine the effect of subminimal inhibitory concentrations (subMICs) of ceftazidime, meropenem and gentamicin on the in vitro serum sensitivity of Pseudomonas aeruginosa strains isolated from a variety of isolation sites at two medical wards and an intensive care unit in a government university hospital in Croatia. A total of 20 serum-resistant P aeruginosa strains isolated from different clinical specimens were selected. Bacteria were exposed to 1/2, 1/4, 1/8, 1/16, and 1/32 x MIC of each antibiotic tested. Sensitivity of P. aeruginosa strains to bactericidal activity of normal human serum before and after bacterial exposure to subMICs was determined. Significant difference in serum sensitivity of the strains was observed after the bacteria were exposed to subMICs of ceftazidime and meropenem (p < 0.01), while the exposure to subMICs of gentamicin did not affect significantly the resistance of tested strains to the serum bactericidal activity. Comparing the number of serum-resistant strains before and after exposure to subMICs of antibiotics, statistically significant differences were determined (p < 0.01) after exposure of the strains to 1/2, 1/4, 1/8 and 1/16 x MIC of meropenem, and after exposure to 1/2, 1/4 and 1/8 x MIC of ceftazidime. SubMICs of ceftazidime and meropenem affected not only the resistance to serum bactericidal activity of bacteria, but also their morphology. The alterations in bacterial morphology caused by subMICs of ceftazidime and meropenem could be connected with consecutive bacterial serum sensitivity.     

Effect of mucoid property on antibiotic susceptibility ofPseudomonas aeruginosa

Clinical isolates ofPseudomonas aeruginosa from patients with cystic fibrosis (CF) were examined for susceptibility to the antibiotics carbenicillin, ticarcillin, tobramycin, gentamicin, and tetracycline. Minimal inhibitory concentrations of the antibiotics were determined for mucoid and nonmucoid isolates from the same patient by a single-colony replica plating method. This method allows the rapid determination of antibiotic susceptibility of a single cell’s progeny and the individual screening of each colony against all antibiotics. Twenty of 34 (58%) cystic fibrosis patients had a mucoid isolate which was more susceptible to antibiotics than their nonmucoid isolate of the same serotype. Nonmucoid revertant segregants of mucoid strains isolated from 50% of the patients demonstrated greater resistance to at least one antibiotic than the original mucoid strain. Multiple isolates from 25 patients were serotyped by Difco (Liu) or Homma antiserum; only 2 patients harbored multiple strains with no common serotyping antigens. Serotypes of the nonmucoid revertants were the same as the original mucoid isolate even if the susceptibilities of the two strains were not similar.