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.     

Molecular characterization of Pseudomonas aeruginosa 2NR degrading naphthalene

Three naphthalene-degrading strains were isolated from compost, characterized by morphological and physiological properties and differentiated by 16S rDNA RFLP. During growth on naphthalene, Pseudomonas aeruginosa 2NR produced ortho catechol pathway intermediates and gentisic acid. The ability to accumulate and degrade gentisic acid shows that Ps. aeruginosa 2NR has a different salicylate pathway to that of the intensely studied Ps. putida NCIB 9816. Molecular analysis showed the presence both of genes of the upper naphthalene pathway and genes of the ortho and meta catechol pathways. The insertion of nagH and nagG, coding for salicylate 5-hydroxylase in Pseudomonas sp. U2, was absent in Ps. aeruginosa 2NR, as in Ps. putida NCIMB 9816.     

哈尔滨市112株绿脓杆菌的血清学分型和耐药性测定

对从临床分离的112株绿脓杆菌进行系统鉴定后,血清学分型表明:6、2和3型分别占32.14％、15.18％、15.18％,为主要流行型,共占总分离株的62.50％。耐药性测定结果为:对10种抗生素5耐以上者占69.6％。其中对多粘菌素、妥布霉素、丁胺卡那霉素三种抗生素最为敏感,敏感率分别为100％、70.6％、86.5％。     

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?     

Comparative activity of tobramycin and gentamicin against Pseudomonas,Proteus and Providencia species.

Tobramycin is a new antibiotic resembling gentamicin. We measured the minimal inhibitory concentrations of these two antibiotics against five bacterial species that cause hospital-acquired infections and are resistant to many presently available antibiotics. The organisms tested were 500 strains of Pseudomones aeruginosa, 100 strains of each of Proteus rettgeri and Pr. morganii, 50 strains of Pr. vulgaris and 250 strains of Providencia stuartii. Tobramycin was 2 to 4 times more active than gentamicin against Ps. aeruginosa; all except 6 of 70 strains resistant to 4 mug/ml of gentamicin were sensitive to 4 mug/ml of tobramycin. The two antibiotics showed a similar degree of activity against the other four species. Tobramycin promises to be of particular value in the treatment of Pseudomonas infections.     

Polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas stutzeri 1317 had different substrate specificities

The whole polyhydroxyalkanoate (PHA) synthesis gene locus of Pseudomonas stutzeri strain 1317 containing PHA synthase genes phaC1Ps, phaC2Ps and PHA depolymerase gene phaZPs was cloned using a PCR cloning strategy. The sequence analysis results of the phaC1Ps, phaC2Ps and phaZPs showed high homology to the corresponding pha loci of the known Pseudomonas strains, respectively. PhaC1Ps and PhaC2Ps were functionally expressed in recombinant Escherichia coli strains and their substrate specificity was compared. The results demonstrated that PhaC1Ps and PhaC2Ps from P. stutzeri 1317 had different substrate specificities when expressed in E. coli. In details, PhaC2Ps could incorporate both short-chain-length 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates (mcl 3HA) into PHA, while PhaC1Ps only favored mcl 3HA for polymerization.     

Comparison of dentrification by Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa.

The course of denitrification of nitrate, nitrite and both compounds together by static cultures of Paracoccus denitrificans, Pseudomonas stutzeri and Pseudomonas aeruginosa was studied. These strains represent three different types of denitrification: 1. reduction of nitrate to gaseous nitrogen without accumulation of nitrite (P. denitrificans); 2. partial accumulation of nitrite in growing cultures during reduction of nitrate to gaseous nitrogen (P. aeruginosa) and 3. two-phase denitrification that includes reduction of nitrates at the very beginning of the process, and then, after depletion of the former, the reduction of nitrates to gaseous nitrogen (P. stutzeri). These observations differ from the results reported in the literature and possible reasons are discussed.     

Quantitative real-time PCR study of the influence of probiotic culture soluble fraction on the expression of Pseudomonas aeruginosa quorum sensing genes

The opportunistic pathogen Pseudomonas (Ps.) aeruginosa causes severe infections, particularly in immunocompromised individuals and patients with cystic fibrosis (CF). A serious side effect of antibiotic therapy in Ps. aeruginosa infections is the development of resistance to antibiotics. During the infection process Ps. aeruginosa forms biofilms, rendering bacterial cells more resistant to disinfectants, antibiotics and the action of host immune defense effectors. Pseudomonas aeruginosa employs the intercellular communication system, known as quorum sensing (QS) to coordinate the expression of tissue-damaging factors. Since the QS systems controls the production of different virulence factors, it is possible that the inhibition of its regulatory activity to severely compromise the ability of Ps. aeruginosa to cause infections in humans. Many studies have shown that some probiotic strains exhibit inhibitory activity on different virulence properties of pathogenic bacteria (adherence to cellular or inert substrate, soluble virulence factors expression). The aim of the present study was to investigate by real-time RT-qPCR the influence of probiotic culture soluble factors on the QS genes expression in 30 Ps. aeruginosa strains isolated from patients hospitalized in the National Institute for Cardiovascular Infections, Prof. C.C. Iliescu Fundeni Hospital, Bucharest. The results of the real time RT-qPCR have shown that in all Ps. aeruginosa strains grown in the presence of probiotic culture sterile filtrates, the level of QS genes expression was reduced comparatively with those from control cultures. In conclusion, these results proved that the inhibition of virulence factors regulation mechanisms by soluble molecules secreted by probiotics could represent an interesting way pathogenicity and virulence attenuation in Ps. aeruginosa nosocomial strains.     

Identification and distribution of Pseudomonas stutzeri in clinical material

During the 19 year period ending December 1984, 114 (2.4%) of 4840 strains of Gram-negative non-fermentative bacteria submitted, mainly by laboratories in the UK, to the National Collection of Type Cultures for computer-assisted identification were strains of Pseudomonas stutzeri. These figures suggest that Ps. stutzeri is a relatively uncommon species in clinical material in the UK but that when it does occur laboratories have difficulty in identifying it. The sources from which the strains were isolated and also characteristics of the species by which it may be recognized are reported. The clinical significance of Ps. stutzeri is discussed and also the susceptibility of this species to antimicrobial agents.     

Identification and distribution of Pseudomonas stutzeri in clinical material

During the 19 year period ending December 1984, 114 (2.4%) of 4840 strains of Gram-negative non-fermentative bacteria submitted, mainly by laboratories in the UK, to the National Collection of Type Cultures for computer-assisted identification were strains of Pseudomonas stutzeri. These figures suggest that Ps. stutzeri is a relatively uncommon species in clinical material in the UK but that when it does occur laboratories have difficulty in identifying it. The sources from which the strains were isolated and also characteristics of the species by which it may be recognized are reported. The clinical significance of Ps. stutzeri is discussed and also the susceptibility of this species to antimicrobial agents.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Genetic relationships among Pseudomonas stutzeri strains based on molecular typing methods

Detailed characterization of the genetic variability among strains belonging to Pseudomonas stutzeri was achieved using different rapid molecular typing methods based on polymerase chain reaction (PCR), Southern blot and Western blot. Consensus motifs complementary to fragments of repetitive elements dispersed throughout the genomes of bacteria were used as primers & allowed differentiation at subspecies levels. Further & simple differentiation was also achieved based on the direct amplification of spacer regions between 16S & 23S rRNA, combined with single-strand conformation polymorphism (SSCP) analysis of the generated fragments. These methods are fast, sensitive, reliable for determining relationships & have demonstrated a great genetic diversity among the strains of Ps. stutzeri studied in agreement with the heterogeneous phenotypic traits of the species.     

Identification of siderophores of Pseudomonas stutzeri

We have identified two types of siderophores produced by Pseudomonas, one of which has never before been found in the genus. Twelve strains of Pseudomonas stutzeri belonging to genomovars 1, 2, 3, 4, 5, and 9 produced proferrioxamines, the hydroxamate-type siderophores. Pseudomonas stutzeri JM 300 (genomovar 7) and DSM 50238 (genomovar 8) and Pseudomonas balearica DSM 6082 produced amonabactins, catecholate-type siderophores. The major proferrioxamines detected were the cyclic proferrioxamines E and D2. Pseudomonas stutzeri KC also produced cyclic (X1 and X2) and linear (G1 and G2a-c) proferrioxamines. Our data indicate that the catecholate-type siderophores belong to amonabactins P 750, P 693, T 789, and T 732. A mutant of P. stutzeri KC (strain CTN1) that no longer produced the secondary siderophore pyridine-2,6-dithiocarboxylic acid continued to produce all other siderophores in its normal spectrum. Siderophore profiles suggest that strain KC (genomovar 9) belongs to the proferrioxamine-producing P. stuzeri. Moreover, a putative ferrioxamine outer membrane receptor gene foxA was identified in strain KC, and colony hybridization showed the presence of homologous receptor genes in all P. stutzeri and P. balearica strains tested.     

Effect of anionic detergents on wild-type and envelope mutants of Escherichia coli and Pseudomonas aeruginosa

Escherichia coli UB1005 (DCO), an envelope mutant (DC2), Pseudomonas aeruginosa 799 and an envelope mutant (799/61) were exposed to sodium deoxycholate (DOC), sarkosyl and sodium lauryl sulphate (SLS). DOC was the least effective lytic agent, but the two Ps. aeruginosa strains, especially 799/61, were more susceptible to DOC and sarkosyl than the E. coli ones. SLS was an efficient lysing agent, although Ps. aeruginosa 799 was the least susceptible of the four strains. DC2 was lysed more rapidly and to a greater extent than UB1005 by all three agents. The mutant strains, especially DC2, were more sensitive to selective media than the wild-type ones.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

Gentamycin activity in combination with etonium in relation to Pseudomonas aeruginosa]

Gentamicin is one of the most effective drugs for treatment of infections caused by Ps. aeruginosa. However, isolation of the strains resistant to the antibiotics was not infrequent. It was shown in the experiments with 40 cultures that the activity of gentamicin against Ps. aeruginosa increased approximately 20 times when it was used in combination with ethonium, a derivative of bis-quaternary ammonium compounds.     

Antibiotic sensitivity and pyocin dependence of clinical strains of Ps. aeruginosa]

The effect of antibiotics on clinical strains of Ps. aeruginosa was analyzed. It shown that 92 per cent of the strains had multiple resistance to the most of the antibiotics used in the clinic. All the strains were sensitive to gentamicin and polymyxin. The apyocinogenic strains of Ps. aeruginosa were more sensitive to the effect of the antibiotics than the pyocinogenic ones.     

The Action of Ethylenediaminetetra-acetic Acid on Pseudomonas aeruginosa

It has been shown that ethylenediaminetetra-acetic acid (EDTA) has a direct bactericidal action against strains of Pseudomonas aeruginosa and Alcaligenes faecalis . The action against Ps. aeruginosa is considered to take the form of competition for a metal essential to the integrity of the cells: some of the factors influencing this antibacterial action have been discussed. From a study of the release of solutes from cells of Ps. aeruginosa , it was concluded that the action of EDTA takes place at the cell wall of the organism: an effect by EDTA on the isolated walls of sensitive organisms has been demonstrated.     

Characterization of Nonfermentative Nonfastidious Gram Negative Bacteria encountered in Medical Bacteriology

More than 90 morphological and physiological characters of 546 strains of nonfermentative, nonfastidious, Gram negative bacteria isolated from clinical specimens were examined to determine those features most useful for the identification of these bacteria. The species examined included Moraxella osloensis, Mor. lacunata, Acinetobacter anitratum, Ac. haemolyticus subsp. haemolyticus, Ac. haemolyticus subsp. alcaligenes, Ac. Iwoffi, Alcaligenes faecalis, Alc. odorans var. viridans, Pseudomonas fluorescens, Ps. putida, Ps. pseudomallei, Ps. maltophilia, Ps. stutzeri, Ps. acidovorans, Ps. alcaligenes , and atypical strains of Ps. aeruginosa.