Antimicrobial susceptibility changes and T-OMP shifts in pyrithione-passaged planktonic cultures of Pseudomonas aeruginosa PAO1

AIMS: The aim of this study was to determine whether passaging Pseudomonas aeruginosa PAO1 with sub-MICs of the pyrithione biocides results in both the induction of decreased susceptibility towards these antimicrobials and associated outer membrane profile changes. METHODS AND RESULTS: Previous work by this group has shown that it is possible to induce susceptibility changes towards the isothiazolone biocides in Ps. aeruginosa PAO1 by successive passages in the presence of increasing sub-MICs of biocide. This procedure was accompanied by the loss of a 35 kDa outer membrane protein, T-OMP. In this experiment, this process was repeated with the biocides sodium pyrithione (NaPT), zinc pyrithione (ZnPT) and cetrimide. The pattern of susceptibility was similar to that observed with the isothiazolone biocides. Upon removal of biocide, the observed MIC did not return to the original pre-exposure value. The onset and development of resistance was accompanied by the loss of T-OMP from outer membrane profiles, which suggests that this is a non-specific membrane channel whose production within the cell is sensitive to biocide presence. The T-OMP reappeared when the cells were passaged in the absence of pyrithione. Cross-resistance studies indicated that induced resistance to one biocide yields partial resistance towards other members of the group and the positive control. CONCLUSIONS: These results indicate that the pyrithione biocides have similar susceptibility profiles in Ps. aeruginosa to those exhibited by the isothiazolones, but that the acquired changes in susceptibility to the pyrithiones is largely irreversible. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that acquired susceptibility changes towards sub-MICs of selected biocides are multifactorial in nature.     

Growth inhibitory and biocidal activity of some isothiazolone biocides

Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizosaccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1-0.5 micrograms/ml for CMIT, 15-20 micrograms/ml for BIT and 40-250 micrograms/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this compound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.     

Growth inhibitory and biocidal activity of some isothiazolone biocides

C ollier , P.J., R amsey , A.J., A ustin , P. & G ilbert , P. 1990. Growth inhibitory and biocidal activity of some isothiazolone biocides. Journal of Applied Bacteriology 69 , 569–577.
Similar patterns of growth inhibition were observed for the three biocides, benzisothiazol-3-one (BIT), 5-chloro-N-methylisothiazol-3-one (CMIT) and N-methylisothiazol-3-one (MIT) against Escherichia coli ATCC 8739 and Schizo-saccharomyces pombe NCYC 1354. After periods of induced stasis, proportional to biocide concentration, growth proceeded at an inhibited rate. Extrapolation of the static periods and inhibited growth rates against biocide concentration gave minimum growth inhibitory concentration estimates of 0.1–0.5 μg/ml for CMIT, 15–20 μg/ml for BIT and 40–250 μg/ml for MIT. Patterns of growth inhibition by CMIT and induced morphological changes in inhibited cultures suggested this com-pound to also inhibit initiation of DNA replication. Growth inhibitory activity was rapidly quenched by the addition of thiol-containing materials such as glutathione and cysteine. The activity of CMIT was additionally quenched by the presence of the non-thiol amino acids valine and/or histidine. These results suggest that the chlorinated isothiazolones can react with amines as well as with essential thiol groups.     

Chemical reactivity of some isothiazolone biocides

Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7-10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Resistance of Pseudomonas aeruginosa to isothiazolone

This investigation was to determine whether Pseudomonas aeruginosa could acquire resistance to the bactericide isothiazolone, and what the nature of such a resistance mechanism would be. The Pseudomonas was cultured in nutrient-limited broth in the presence of sub-inhibitory concentrations of isothiazolone (a mixture of 1.15% 5-chloro- N -methylisothiazolone (CMIT) and 0.35% N -methylisothiazolone (MIT)). Three cultures tested in parallel adapted gradually during exposure for 15 d from an initial minimum inhibitory concentration (MIC) of 300 μl 1^-1 to 607 μl ^-1. The three parallel cultures adapted at similar rates, so the adaptation was not ascribed to mutation but to a specific mechanism. Resistant cells did not produce any extracellular isothiazolone-quenching compounds nor undergo detectable alterations in their lipopolysaccharide layer. In wild cells, a 35 kDa outer membrane protein (protein T) was detectable, whereas resistant cells lacked this protein. Production of protein T was suppressed within 24 h of exposure to isothiazolone. It was still suppressed after 72 h of growth in isothiazolone-free medium. It is proposed that Ps. aeruginosa acquires resistance to isothiazolone by a process of adaptation where the outer membrane protein T is suppressed.     

The effects of some formulation factors used in ophthalmic preparations on thiomersal activity against Pseudomonas aeruginosa and Staphylococcus aureus

AIMS: To investigate the effects of formulation ingredients used in ophthalmic preparations on thiomersal activity against Staphylococcus aureus and Pseudomonas aeruginosa. METHODS AND RESULTS: Minimum inhibition concentrations (MICs) of the tested ingredients and their combinations were studied using partial broth dilution checkerboard method. Complex formation was determined using differential scanning calorimetry (DSC) and u.v. scan. Isotonic agents showed insignificant difference in thiomersal activity. Low concentrations of propylene glycol and glycerol (2 to about 6.5%) significantly reduced the activity of thiomersal against P. aeruginosa. Higher concentrations up to about 40%, of the two cosolvents did not affect the antibacterial activity. Viscosity increasing agents significantly reduced the antibacterial activity of thiomersal. Low concentrations of 0.05% and 0.05-0.1% of ethylenediaminetetraacetic acid resulted in a significant decrease in thiomersal activity against S. aureus and P. aeruginosa, respectively. However, concentrations above 0.25 and 0.5 up to 4% caused significant increase on the antibacterial activity against the two later micro-organisms, respectively. CONCLUSIONS: Results showed that thiomersal formed complexes with ingredients containing polyhydroxy groups and chelating agents, thus thiomersal is not recommended to be used with such ingredients. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the importance of proper selection of ingredients and preservatives for ophthalmic preparations.     

Chemical reactivity of some isothiazolone biocides

C ollier , P.J., R amsey , A., W aigh , R.D., D ouglas , K.T., A ustin , P. & G ilbert , P. 1990. Chemical reactivity of some isothiazolone biocides. Journal of Applied Bacteriology 69 , 578–584.
Chemical reactions between the isothiazolone biocides, N-methylisothiazol-3-one (MIT), benzisothiazol-3-one (BIT) and 5-chloro-N-methylisothiazol-3-one (CMIT) with cysteine have been investigated by u.v. and NMR spectroscopy. At physiological pH all three agents interacted oxidatively with thiols to form disulphides. Further interaction with thiols caused the release of cystine and formation of a reduced, ring-opened form of the biocide (mercaptoacrylamide). In an analogous fashion to the initial reaction the mercaptoacrylamide reacted with another molecule of biocide to give biocide dimers. NMR spectral studies indicated that for CMIT the mercaptoacrylamide form is capable of tautomerization to a highly reactive thio-acyl chloride. Formation of mercaptoacrylamide was in all cases highly pH-dependent. Alcohol dehydrogenase was insensitive to all three agents but was highly sensitive to CMIT when co-administered with dithiothreitol. Capacity to form a thioacyl chloride from the mercaptoacrylamide is suggested to account for much of this enhanced activity. Stopped-flow spectroscopic studies showed rates of reaction with glutathione (GSH) to directly parallel antimicrobial activity. Additionally, CMIT was able to react directly with both ionization states of GSH (pH 7–10) whilst BIT and MIT appeared only to interact when the glutamyl-nitrogen of GSH was charged (pH 8.5).     

Adaptive resistance to benzalkonium chloride,amikacin and tobramycin: the effect on susceptibility to other antimicrobials

AIMS: To produce strains of antimicrobial-resistant Pseudomonas aeruginosa via adaptation to benzalkonium chloride, amikacin and tobramycin and to then examine the incidence, or otherwise, of cross-resistance between antibiotics and between antibiotics and benzalkonium chloride. METHODS AND RESULTS: Adaptation was obtained by progressive subculturing in subinhibitory concentrations of the antimicrobials. Pseudomonas aeruginosa NCIMB 10421 adapted to grow in high concentrations of benzalkonium chloride (BC) had lower MIC to antibiotics than the wild type, whereas Ps. aeruginosa adapted to grow in antibiotics had greater MIC to benzalkonium by a small degree. CONCLUSIONS: Adaptive resistance to BC of Ps. aeruginosa generally produced cultures with a decrease in resistance to several antibiotics. Adaptive resistance to the aminoglycosides Ak and Tm produced a low-level increase in tolerance to BC. The adaptive mechanisms of resistance appear to be different for the different types of antimicrobials used. SIGNIFICANCE AND IMPACT OF THE STUDY: The relationships between biocide and antibiotic resistance are complex. It appears, from this study, that an organism resistant to a common biocide can become sensitive to antibiotics, but the converse was not true. Could this observation be used in a strategy to alleviate antibiotic resistance?     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000

AIMS: To analyse population minimum inhibitory concentrations (MICs) data from clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa for changes over a 10-year period and to look for correlations between the antimicrobials tested. METHODS AND RESULTS: Data from the MIC study of 256 clinical isolates of Staph. aureus [169 methicillin-sensitive Staph. aureus (MSSA), 87 methicillin-resistant Staph. aureus (MRSA)] and 111 clinical isolates of Ps. aeruginosa against eight antimicrobial biocides and several clinically relevant antibiotics was analysed using anova, Spearman-Rho correlation and principal component analysis. Comparisons suggest that alterations in the mean susceptibility of Staph. aureus to antimicrobial biocides have occurred between 1989 and 2000, but that these changes were mirrored in MSSA and MRSA suggests that methicillin resistance has little to do with these changes. Between 1989 and 2000 a sub-population of MRSA has acquired a higher resistance to biocides, but this has not altered the antibiotic susceptibility of that group. In both Staph. aureus and Ps. aeruginosa several correlations (both positive and negative) between antibiotics and antimicrobial biocides were found. CONCLUSIONS: From the analyses of these clinical isolates it is very difficult to support a hypothesis that increased biocide resistance is a cause of increased antibiotic resistance either in Staph. aureus or in Ps. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation of negative correlations between antibiotics and biocides may be a useful reason for the continued use of biocides promoting hygiene in the hospital environment.     

Antimicrobial activity of chlorhexidine diacetate and benzalkonium chloride against Pseudomonas aeruginosa and its response to biocide residues

AIMS: The aims of this study were to evaluate the antimicrobial activity of chlorhexidine diacetate (CHX) and benzalkonium chloride (BZK) for strains of Pseudomonas aeruginosa exhibiting increased minimum inhibitory concentrations (MIC) for CHX, and to determine whether residues of chlorhexidine digluconate (CHG) and Hibiscrub (Hib, a formulation containing CHG) affect the susceptibility of P. aeruginosa to these biocides and a number of antibiotics. METHODS AND RESULTS: The bactericidal activity of CHX and BZK was evaluated for strains of P. aeruginosa exhibiting increased MIC for CHX with established suspension and surface disinfection tests. None of the strains of P. aeruginosa exhibiting raised MIC for CHX was less sensitive than the parent strain to CHX or BZK in either method. A test was designed to investigate the effects of dried CHG and Hib residues on P. aeruginosa cells. Exposure of P. aeruginosa to dried residues of CHG or Hib did not result in the organism becoming less sensitive to either biocide or a number of antibiotics. CONCLUSIONS: Pseudomonas aeruginosa strains with raised MIC to CHX were no less sensitive than the parent strain to CHX and BZK in bactericidal investigations. Exposure to dried residues of CHG and Hib did not render P. aeruginosa less sensitive to either of these agents or a number of antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: An increase in the MIC for a biocide in a micro-organism does not necessarily result in a failure of the biocide to effectively kill the organism. The residue that remains after the use of an antimicrobial agent can be at a far lower concentration than that initially applied and this study highlights the necessity for further investigations into the effect of residues, at low concentration, on bacterial populations and their role, if any, in the continued problem of antibiotic resistance.     

A study of the effect of isothiazolones on the performance and characteristics of a laboratory-scale rotating biological contactor

AIMS: To study the effect of the isothiazolone biocide (Kathon WT) on the performance of laboratory-scale rotating biological contactors (RBCs) and their component biofilms. METHODS AND RESULTS: Biofilms were established on the RBCs and then exposed to 0.7-15 p.p.m. isothiazolones. Young, 1-week-old, biofilms were found to attain treatment efficiency equal to that of mature, 2-month-old, biofilms. Isothiazolone concentrations at 3 p.p.m. and above caused a progressive decline in treatment efficiency and 15 p.p.m. isothiazolones inhibited all microbial activity and resulted in the death of the biofilms. Bio-oxidation and the biodegradation of isothiazolones within the biofilms ontinued unhindered at concentrations which caused the total inhibition of planktonic bacteria. CONCLUSION: There was at least a 10-fold difference in susceptibility of planktonic and biofilm bacteria to isothiazolones. The chemical oxygen demand (COD) test was shown to be a reliable tool for investigating the efficiency of wastewater treatment units when the influent contains isothiazolones, while the biochemical oxygen demand (BOD) was unreliable due to the inhibition of bio-oxidation by the biocide. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that RBCs can be used to treat effluents containing isothiazolones at concentrations up to 1.5 p.p.m.     

Microemulsions are highly effective anti-biofilm agents

AIMS: The demonstration of the antibiofilm effects of pharmaceutical microemulsions. METHODS AND RESULTS: Microemulsions were prepared as physically stable oil/water systems. Previous work by this group has shown that microemulsions are highly effective antimembrane agents that result in rapid losses of viability in planktonic populations of Pseudomonas aeruginosa and Staphylococcus aureus. In this experiment a microemulsion preparation was used upon established biofilm cultures of Ps. aeruginosa PA01 for a period of 4 h. The planktonic MIC of sodium pyrithione and the planktonic and biofilm MICs of cetrimide were used as positive controls and a biofilm was exposed to a volume of normal sterile saline as a treatment (negative) control. Results indicate three log-cycle reductions in viability within the microemulsion treated biofilm, as compared to those observed in control treatments of similar biofilms (one log-cycle reduction in viabilities). CONCLUSIONS: The results indicate that the microemulsions are highly effective antibiofilm agents. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that microemulsions may have a role in the treatment of industrial and environmental biofilms.     

A review on biocide reduced susceptibility due to plasmid-borne antiseptic-resistant genes—special notes on pharmaceutical environmental isolates

Biocides (antiseptics and disinfectants) are widely used in hospitals and pharmaceutical industries for contamination control. The emergence of reduced susceptibility to biocides is the major concern and this is caused by various factors, among which plasmid-mediated resistance is common. Many publications describe the antibiotic resistance and mechanisms in a clinical setting. However, there are only limited studies available worldwide addressing the molecular mechanisms of biocide resistance in the pharmaceutical sector. In addition, there is a considerable lack of scientific reports regarding minimum inhibitory concentration (MIC) values of typical biocides against pharmaceutical cleanroom environmental isolates. This review analyses the plasmid-mediated resistance in typical pharmaceutical micro-organisms and prevalence of biocide-resistant genes among common clinical and pharmaceutical isolates. This review discusses the MIC values of biocides in pharmaceutical environmental isolates, indicating the importance of the correlation between the presence or absence of biocide-resistant genes and reduced susceptibility of MIC values. This review recommends that pharmaceutical organizations adopt policies and test methodologies to examine the MICs of common cleanroom biocides against the most common types of cleanroom environmental isolates.     

Adaptation of Pseudomonas aeruginosa ATCC 15442 to didecyldimethylammonium bromide induces changes in membrane fatty acid composition and in resistance of cells

The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of didecyldimethyl ammonium bromide (DDAB) was investigated. In this study, the strain was sub-cultured in a medium with increasing DDAB concentrations. After adaptation, Ps. aeruginosa was able to grow until the DDAB concentration in the medium was about five times greater than the Minimal Inhibitory Concentration. Resistance of cells to the bactericidal activity of DDAB also increased gradually during adaptation. This resistance was dependent on the presence of the biocide, as it quickly decreased when the cells were transferred to medium without biocide. Adapted cells showed changes in membrane fatty acid composition. The modifications mainly affected lauric, beta-hydroxylauric and palmitic acids, and they underlined the implication of the membranes in the cell response to the presence of the biocide. Simple linear regression analysis showed that the membrane fatty acid composition of Ps. aeruginosa played an important part in the resistance mechanisms of cells to the bactericidal activity of DDAB.     

Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for its intrinsic drug resistance. We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the addition of phosphate to the inner core region of P. aeruginosa lipopolysaccharide. The first gene is a waaP homologue, whereas the other two (wapP and wapQ) are unique to P. aeruginosa. Repeated attempts using a variety of membrane-stabilizing conditions to generate waaP:Gm (Gm, gentamicin) or wapP:Gm mutants were unsuccessful. We were able to generate a chromosomal waaP mutant that had a wild-type copy of either waaPPa or waaPEc in trans, but were unable to cure this plasmid-borne copy of the gene. These results are consistent with the fact that P. aeruginosa mutants lacking inner core heptose (Hep) or phosphate have never been isolated and demonstrate the requirement of Hep-linked phosphate for P. aeruginosa viability. A wapQ:Gm mutant was isolated and it had an unaltered minimum inhibitory concentration (MIC) for novobiocin and only a small decrease in the MIC for sodium dodecyl sulphate (SDS), suggesting that the loss of a phosphate group transferred by WapQ may only be having a small impact on outer-membrane permeability. Nuclear magnetic resonance and methylation linkage analysis showed that WaaPPa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant. The expression of WaaPPa increased the outer-membrane integrity of these complemented mutants, as evidenced by 35-fold and 75-fold increases in the MIC for novobiocin and SDS respectively. The S. typhimurium waaP mutant transformed with both waaP and wapP had over 250-fold and 1000-fold increases, respectively, in these MICs. The inner core phosphates of P. aeruginosa appear to be playing a key role in the intrinsic drug resistance of this bacterium.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 adapted to 2-phenoxyethanol shows cross-resistance to dissimilar biocides and increased susceptibility to antibiotics

The growth adaptability to increasing concentration of the biocide 2-phenoxyethanol (PE) was determined in Pseudomonas aeruginosa PAO1 (P.a.) as part of efforts to understand and control the biocide tolerance and its effect on cross-resistance to other biocides and resistance to antibiotics. After repeated subculturing in media containing increasing sub-minimum-inhibitory PE concentration, P.a. exhibited an adaptive resistance indicated by two-fold increase in MIC at the 10th passage. The resistance was stable and remained after passaging the strain in further 7 successive passages in PE-free growth media. The strain showed cross-resistance towards dissimilar biocides and displayed increased susceptibility to antibiotics, which was not influenced by the presence of the efflux inhibitor ‘carbonyl cyanide m-chlorophenyl hydrazone’. Outer membranes of adapted strain showed altered protein profile when examined by SDS-PAGE.     

Comparative ultrastructure of a mucoid strain of Pseudomonas aeruginosa isolated from cystic fibrosis patient and its spontaneous non-mucoid mutant

The ultrastructure of a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin and its spontaneous non-mucoid variant was compared by transmission electron microscopy. Negatively-stained preparations of the mucoid strain obtained from plate cultures demonstrated dense, fibrous material projecting from the cell. No such material was observed in thin-sections or in negatively-strained preparations from liquid cultures. Thin-sections of ethanol-precipitated extracellular material from liquid cultures of the mucoid-strain revealed a cottony mesh of thin electron dense fibres. The non-mucoid strain did not produce such material. When prefixed with glutaraldehyde/malachite green mixture, cells of both strains demonstrated electron dense intracellular and extracellular malachite green-stainable structures. The internal complexes were frequently associated with the nucleoid or cell membrane and were replaced by electron transparent areas in cells prefixed with glutaraldehyde alone. Aeruginocins of the R-type were observed in mitomycin C induced cultures of both strains. Bacteriophages with 'claw-shaped' tail-tips were observed in the mucoid strain. Crystalline material was produced by the mucoid strain but only when plated on certain media.     

High rates of macrolide resistance among clinical isolates of Streptococcus agalactiae in Tunisia

One hundred sixty non duplicate erythromycin resistant Streptococcus agalactiae isolates were collected in Tunisia from January 2005 to December 2007 They were investigated to determine their resistance level to different macrolides and the mechanisms involved. Most erythromycin resistant S. agalactiae isolates were isolated from urinary specimens (38.75%, 62/160). The constitutive MLSB phenotype (cMLS) showed in 84.3% (135/160) with high MICs of macrolides and lincosamides (MIC90>256 microg/mL) and 8.2% (13/160) inducible MLSB phenotype (iMLS) with high MICs of macrolides (MIC90>256 microg/mL) and moderately increased MICs of lincosamides (MIC90=8 microg/mL). The M phenotype showed in 7.5% (12/160) with moderately increased MICs of macrolides (MIC90=32 microg/mL) and low MICs of lincosamides (MIC90=0.75 microg/mL). All strains were susceptible to quinupristun-dalfopristin association and linezolid (MIC90: 05 and 0.38 microg/mL respectively). Strains with MLSB phenotype harboured erm(B) gene with 825% (n=132), erm(TR) gene with 8.12% (n=13) and erm(B) plus mef (A) with 1.88% (n=3). All strains categorized as M phenotype carried the mef(A) gene (75%, n=12). cMLSB phenotype conferring cross resistance to macrolides, lincosamides and streptogramins B with high level of resistance was the most prevalent.