Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori

The contribution of pyocyanin to the virulence of Pseudomonas aeruginosa against the silkworm Bombyx mori was studied. First, purified pyocyanin was injected into the hemocoel of B. mori. Acute toxicity was observed only when a high dose of pyocyanin was injected. The lethal dose 50% value of pyocyanin was found to be 9.52 microg per larva. Next, mutant strains of phzM and phzS, which encode putative phenazine-specific methytransferase and flavin-containing monooxygenase, respectively, were created, and their virulence was compared with that of the PAO1 parent strain. Although the ability to produce pyocyanin was completely lost in the phz-mutant strains, they maintained the same level of virulence as the PAO1 parent strain. In addition, the complementation of the corresponding gene in trans in the mutant strains did not have any effect on the virulence of those mutant strains. These results indicated that pyocyanin does not act as a virulence factor in B. mori after invasion, which was different from the results obtained in other Lepidopteran host models.     

Pyocyanin reactions with Pseudomonas aeruginosa cells and their fragments

Pyocyanin added to suspensions of Pseudomonas aeruginosa P changes the structural organization of the cells depending on their physiological characteristics, in particular, the capability to liberate pyocyanin into the cultural broth. Exogenous pyocyanin does not interact with the cells of the parent strain producing the pigment. However, the structure of the isolated cell walls deformed after the fractionation becomes similar, upon contact with pyocyanin, to the structure of the freshly isolated cell walls. The fraction of the cytoplasmic membranes of the parent strain also slightly changes its spatial organization after addition of pyocyanin. The cells of the mutant which do not produce pyocyanin display the ability for structural interaction with exogenous pyocyanin. In contrast, the fraction of their cell walls does not react to addition of the pigment. The cytoplasmic membranes of the mutant interact with pyocyanin in the same manner as the whole cells. These changes caused by pyocyanin always involve certain parts of the molecules. As a result, the following features are observed in their spectra: the absorption at 1475 cm-1 becomes more intensive; the "knee" at the band Amide II is more pronounced in the range of 1500 cm-1; the absorption at 1520 cm-1 decreases as well as the intensity of the band Amide II as a whole. The interaction of pyocyanin with biological objects stems, apparently, from the presence of certain proteins in them since peroxidase was found to respond to pyocyanin in a manner similar to that described for the cells and their fractions.     

Reduced expression of virulence factors in multidrug-resistant Pseudomonas aeruginosa strains

MDR Pseudomonas aeruginosa strains are isolated from clinical specimens with increasing frequency. It seems that acquiring genes which determine antibiotic resistance usually comes at a biological cost of impaired bacterial physiology. There is no information on investigations comparing phenotypic differences in MDR and MDS P. aeruginosa strains in literature. The study included 150 clinical P. aeruginosa isolates (75 classified as MDS and 75 as MDR). PFGE analysis revealed five pairs of identical isolates in the group of MDR strains and the results obtained for these strains were not included in the statistical analyses. MDR strains adhered to polystyrene to a lesser extent than MDS strains. The growth rate in the liquid medium was significantly lower for MDR strains. Detectable amounts of alginate were present in the culture supernatants of seven MDS and six MDR strains. The MDR P. aeruginosa strains which were investigated produced significantly lower amounts of extracellular material binding Congo Red, lower lipolytic, elastase, LasA protease, phospholipase C activity and pyocyanin quantity in culture supernatants when compared with MDS strains. No significant differences were observed between MDR and MDS strains in proteolytic activity. In conclusion, the MDR P. aeruginosa strains have impaired virulence when compared to MDS strains.     

Coexistence of antibiotic-producing and antibiotic-sensitive bacteria in biofilms is mediated by resistant bacteria

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus Brevibacillus from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer, Pseudomonas aeruginosa strain P1. In addition, pyocyanin-resistant strains belonging to the genus Raoultella were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1, Brevibacillus strain S1, and Raoultella strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.     

Metabolic engineering of E. coli for pyocyanin production

Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.     

Rapid identification using pyrolysis mass spectrometry and artificial neural networks of Propionibacterium acnes isolated from dogs

Curie-point pyrolysis mass spectra were obtained from reference Propionibacterium strains and canine isolates. Artificial neural networks (ANNs) were trained by supervised learning (with the back-propagation algorithm) to recognize these strains from their pyrolysis mass spectra; all the strains isolated from dogs were identified as human wild type P. acnes. This is an important nosological discovery, and demonstrates that the combination of pyrolysis mass spectrometry and ANNs provides an objective, rapid and accurate identification technique. Bacteria isolated from different biopsy specimens from the same dog were found to be separate strains of P. acnes , demonstrating a within-animal variation in microflora. The classification of the canine isolates by Kohonen artificial neural networks (KANNs) was compared with the classical multivariate techniques of canonical variates analysis and hierarchical cluster analysis, and found to give similar results. This is the first demonstration, within microbiology, of KANNs as an unsupervised clustering technique which has the potential to group pyrolysis mass spectra both automatically and relatively objectively.     

Enterotoxigenic Bacteroides fragilis in Hungary

Bacteroides fragilis, which constitutes about 1% of the colonic microflora in humans, is the most frequent anaerobic species involved in abscesses, soft-tissue infections and bacteraemias. Additionally, enterotoxigenic strains of B. fragilis have been demonstrated to be associated with diarrhoea in domestic animals and humans. Enterotoxigenic strains of B. fragilis derived from stool specimens and from infectious processes produce a toxin which induces a cytotoxic response in HT-29 colon carcinoma cells. These findings prompted us to investigate the prevalence of enterotoxigenic strains of B. fragilis isolated from various clinical specimens in Hungary. A total of 134 strains were collected from different clinical settings: 74 from infectious processes, 20 from stools of healthy subjects and 40 from the faeces of patients with diarrhoea where no other enteric pathogen could be isolated. Cell culture assays with HT-29 cells were performed on the filtered culture supernatants of the isolated strains. Of the 134 strains, 34 (25.3%) proved toxin-positive. The presence of free toxin was also observed in 20 of 50 (40%) of the faeces of adults with diarrhoea.     

A new method of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity.

Pyocyanin was prepared in 60% yield from phenazine methoxysulfate by a photooxidation procedure and purification by silica gel chromatography. Monitoring was performed by thin-layer chromatography. Approximately 50% of clinical Pseudomonas aeruginosa isolates were found to produce pyocyanin at 37°C. Among Proteus strains, P. morganii strains were sensitive to concentrations of pyocyanin 16 to 64 times lower than concentrations that inhibited the growth of P. mirabilis and P. vulgaris strains.     

Range of action and genetic bacteriocin codification of Pseudomonas aeruginosa isolated from three different ecological niches

Strains of Pseudomonas aeruginosa isolated from sediments in wells showed a greater bacteriocinogenic activity in those isolated from river and clinical specimens. More than half of all the strains examined had extrachromosomal DNA. Plasmid DNA was extracted from all the strains and in only 24/120, all from different origins, was the curing achieved; all these strains coded their bacteriocins in the chromosomal DNA.     

Sensitivity of Staphylococcus aureus strains isolated from the patients of the SP ZOZ Hospital in Nidzica to bacteriocidal agents

The aim of this study was to evaluate a frequency of isolation and antimicrobial susceptibility testing of Staphylococcus aureus strains isolated from clinical specimens obtained from patients hospitalized in different hospital wards (SP ZOZ) in Nidzica from 01. 09. 2000 to 31. 12. 2003. During over three years 716 Staphylococcus aureus strains were cultured out of 15517 clinical specimens supplied to the Bacteriological Laboratory of SP ZOZ in Nidzica. S. aureus strains were isolated from 4.6% of examined samples. Samples were collected from patients hospitalized in all wards (five wards). Analysis of susceptibility to antimicrobial agents of identified S. aureus strains was performed. Seventy strains (9.8%) were metihicillin-resistant (MRSA). One hundred twenty four strains (17.3%) revealed inducible resistance to macrolides, linkosamides and streptogramins B (MLS, mechanism). The greatest activity in vitro against clinical S. aureus strains showed glycopeptide antibiotic--vancomycin (100% of susceptible strains). Clinical S. aureus strains isolated from patients of hospital in Nidzica are in the majority susceptible to antibiotics/chemotherapeutics, except of penicillin. Percentage of methicillin-resistant strains (MRSA) is not high (<10). Nevertheless, constant monitoring of a drug susceptibility of nosocomial S. aureus strains is important, considering the necessity of control of current epidemiological and therapeutic situation.     

Range of action and genetic bacteriocin codification of Pseudomonas aeruginosa isolated from three different ecological niches.

Strains of Pseudomonas aeruginosa isolated from sediments in wells showed a greater bacteriocinogenic activity in those isolated from river and clinical specimens. More than half of all the strains examined had extrachromosomal DNA. Plasmid DNA was extracted from all the strains and in only 24/120, all from different origins, was the curing achieved; all these strains coded their bacteriocins in the chromosomal DNA.     

Adherence of Abiotrophia defectiva and Granulicatella species to fibronectin: is there a link with endovascular infections?

During a 6-year period, we isolated three Abiotrophia defectiva, six Granulicatella adiacens and two G. 'para-adiacens' strains from clinical specimens. All A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas the Granulicatella spp. strains were isolated from immunosuppressed patients with primary bacteremia. As the capacity of bacteria to adhere to the host extracellular matrix (ECM) has been implicated in the pathogenesis of endovascular infection, we investigated the ability of A. defectiva and Granulicatella spp. isolates to bind different ECM components immobilized in microtiter plates. Adherence tests showed a strong attachment of A. defectiva strains to fibronectin, whereas Granulicatella spp. strains were not adherent. The poor adherence of Granulicatella spp. strains to the ECM could be correlated with a lower propensity to induce endocarditis.     

Evaluation of interactions between strains of S. aureus isolated from different clinical specimens with peripheral blood phagocytic cells

The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal elutriation were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocyanate (FITC) and the percentage of cells containing FITC-labelled bacteria was analysed by flow cytometry. The data obtained show that strains of S. aureus originated from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from respiratory tract show the lowest sensitivity for killing. These strains differ too in their ability to trigger monocyte CL response. Contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.     

Drug susceptibility of Pseudomonas aeruginosa strains isolated from patients of a hospital and specialistic outpatients clinics of the SP ZOZ in Nidzica

The aim of this study was to evaluate a frequency of isolation and antimicrobial susceptibility testing (AST) of Pseudomonas aeruginosa strains cultured from clinical specimens collected from patients hospitalized in wards and specialistic outpatients clinics of a hospital in Nidzica (01. 09. 2000 -31. 12. 2003). During over three years 392 Pseudomonas aeruginosa strains were cultured from 16346 clinical samples provided to bacteriological laboratory. P. aeruginosa strains were isolated from 2.5% of examined specimens. Susceptibility of Pseudomonas aeruginosa strains to antimicrobial agents was tested. The highest in vitro activity against clinical P. aeruginosa strains demonstrated imipenem. One strain was resistant to imipenem. This strain was isolated from a patient of a surgical department. Metalo-beta-lactamase was not detected (MBL-negative strain).Twenty nine strains were ESBL producer (7.4% of all strains). The contribution of Pseudomonas aeruginosa strains to the etiology of nosoconial and ambulatory infections increases. In vitro activity of antibacterial agents against P. aeruginosa strains should be monitored during therapy of infections. Resistance to antibiotics/chemothe-rapeutics may be acquired during treatment with antibacterial agent to which P. aeruginosa strain was susceptible according to the antibiogram.     

Evaluation of the interactions between strains of S. aureus and peripheral blood phagocytic cells

The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal evaluation these cells were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate the efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocynate (FITC) and the percentage of cells containing FITC-labelled bacteria were analysed by flow cytometry. The data obtained show the strains of S. aureus derived from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from the respiratory tract have the lowest sensitivity to killing. These strains differ too in their ability to trigger monocyte CL response. On the contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed a significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.     

利用rpoB基因芯片技术进行快速分枝杆菌菌种鉴定

利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因, 用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后, 分枝杆菌标准株均扩增出360 bp DNA片段, 在其它细菌中, 除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外, 其它细菌均未见扩增。21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外, 其余均为特异性杂交。对126株临床分离株进行鉴定, 89株为结核分枝杆菌, 占70.6％(89/126), 非结核分枝杆菌(NTM)占9.2％(9/98)。应用rpoB基因芯片技术鉴定分枝杆菌菌种, 是一种快速、准确的方法, 具有较高的临床应用价值。     

Comparison of frequency and susceptibility to antimicrobials of bacterial strains of the family Enterobacteriaceae isolated from patients hospitalised in intensive care units

The aim of the study was estimation of frequency and susceptibility to antimicrobial agents of gram-negative rods isolated from clinical specimens obtained from patients requiring intensive care, with emphasis on profile of the unit. The analysis comprised strains of gram-negative rods isolated from patients of two intensive care units (ICUs) of a tertiary care hospital (1200 beds). Identification of cultured isolates was done using automated VITEK and API systems (bioMerieux, France). Susceptibility to antimicrobial agents was tested by a disk-diffusion method according to the NCCLS recommendations. In total the analysis comprised 722 strains of gram-negative rods. In blood cultures predominated strains of Enterobacter spp. (42.5%) and Klebsiella pneumoniae (37.5%). In cultures of clinical specimens other than blood 41.6% comprised strains of Klebsiella pneumoniae, 14.8% Escherichia coli and 14.4% Proteus mirabilis. Frequency of multi-drug resistant strains of bacteria of the family Enterobacteriaceae was much higher among blood isolates in comparison to strains cultured from other clinical specimens. There was a relatively high percentage of strains of Enterobacteriaceae susceptible to piperacillin and tazobactam (69.0%) and ceftazidime (54.6%). Conclusions: 1. All strains were susceptible to carbapenems. 2. There was a relatively high percentage of strains of gram-negative rods susceptible to piperacillin/tazobactam and ceftazidime. 3. Bacteria isolated from blood cultures were characterised by a much higher percentage of resistant strains in comparison to other specimens. 4. Longer stay in ICU promoted selection of strains resistant to antimicrobials.     

利用rpoB基因芯片技术进行快速分枝杆菌菌种鉴定

利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定.以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株.分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp DNA片段,在其它细菌中,除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它细菌均未见扩增.21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外,其余均为特异性杂交.对126株临床分离株进行鉴定,89株为结核分枝杆菌,占70.6%(89/126),非结核分枝杆菌(NTM)占9.2%(9/98).应用rpoB基因芯片技术鉴定分枝杆菌菌种,是一种快速、准确的方法,具有较高的临床应用价值.     

Possibility of detecting pyocyanine in Pseudomonas aeruginosa cells

Pyocyanin can be detected in the cells of Pseudomonas aeruginosa using UV and IR spectroscopy of disturbed complete inner reflection (DCIR). Intact cells of the parent strain liberating the pigment into the cultural broth and mutant cells lacking the ability contain pyocyanin within the cells. Occasionally, pyocyanin can be detected in the outer layers of the cells, which is more typical of the parent strain. In the freshly isolated fractions of the parent strain cellular walls, pyocyanin seems to be pesent in the bound state that has changed significantly its structural organization. In due course, the hypothetical complex pyocyanin--cellular wall decomposes to yield an "oxidized" pigment similar to that liberated into the cultural broth. the cell wall of the mutant possesses the properties of "oxidized" pyocyanin immediately after isolation of the fraction. The pigment cannot be identified in the fractions of cytoplasmic membranes; pyocyanin is present in the "oxidized" state in the fractions of cytoplasm for the cells of both types. The paper discusses the role of the permeability of cytoplasmic membranes in the transport of pyocyanin from the cytoplasm into the cellular wall of the bacterium and then into the surrounding medium.     

Identification and susceptibility to antimicrobial agents of strictly anaerobic bacteria isolated from hospitalized patients

The aim of this study was to identify anaerobic strains isolated in 2001 from clinical specimens obtained from patients of Warsaw hospital and to evaluate a susceptibility of these strains to antimicrobial agents. In 2001 two hundred and twenty five clinical strains of obligate anaerobes were cultured, which were identified in the automatic ATB system (bioMérieux, France) using biochemical tests API 20 A. Drug-susceptibility of strains was determined also in ATB system with the use of ATB ANA strips. C. difficile strains were isolated on selective CCCA medium. Toxins A/B of C. difficile directly in stool specimens were detected by means of ELISA test (TechLab, USA). Fifty four strains of Gram-negative anaerobes (B. fragilis strains dominated) and 171 strains of Gram-positive anaerobes (the greatest number of strains belonged to genus Peptostreptococcus) were cultured from clinical specimens. In the cases of antibiotic-associated diarrhea 28 C. difficile strains were isolated and C. difficile toxins A/B were detected in 39 stool samples. The most active in vitro antimicrobials against Gram-negative anaerobes were metronidazole, imipenem, ticarcillin combined with clavulanic acid and piperacillin with tazobactam. Gram-positive, clinical strains of anaerobes were the most susceptible in vitro to beta-lactam antibiotics combined with beta-lactamase inhibitors (amoxicillin/clavulanate, piperacillin/tazobactam, ticarcillin/clavulanate) and imipenem.