Biofilm formation among methicillin-resistant Staphylococcus aureus isolates from patients with urinary tract infection.

Staphylococci have been confirmed to form biofilms on various biomaterials. The purpose of this study was to investigate biofilm formation among methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with urinary tract infection (UTI) and to assess the relationship between biofilm-forming capacities and virulence determinants/clinical background. Over a 12-year period from 1990 through 2001, a total of 109 MRSA isolates were collected from patients (one isolate per patient) with UTI at the urology ward of Okayama University Hospital. We used the in vitro microtiter plate assay to quantify biofilm formation. We then investigated the presence of several virulence determinants by polymerase chain reaction assay and found eight determinants (tst, sec, hla, hlb, fnbA, clfA, icaA, and agrII) to be predominant among these isolates. Enhanced biofilm formation was confirmed in hla-, hlb-, and fnbA-positive MRSA isolates, both individually and in combination. Upon review of the associated medical records, we concluded that the biofilm-forming capacities of MRSA isolates from catheter-related cases were significantly greater than those from catheter-unrelated cases. The percentage of hla-, hlb-, and fnbA-positive isolates was higher among MRSA isolates from catheter-related cases than those from catheter-unrelated cases. Our studies suggest that MRSA colonization and infection of the urinary tract may be promoted by hla, hlb, and fnbA gene products.</p

Clinical implications of biofilm formation by Enterococcus faecalis in the urinary tract.

The potential relationships between biofilm formation and pathogenicity of Enterococcus faecalis in urinary tract infections (UTI) were investigated. Over a 12-year period from 1991 through 2002, a total of 352 E.faecalis isolates were collected from patients with complicated UTI (one isolate per patient) at the urology ward of Okayama University Hospital. We analyzed the prevalence and transferability of genes encoding virulence factors(asa1, esp, cylA, gelE /sprE )and antimicrobial resistance(aac(6') /aph(2'')). The production of biofilm, hemolysin and gelatinase by these isolates was also examined and the associated medical records of patients were retrospectively reviewed. Of 352 E. faecalis isolates, 315 possessed and/or genes. Of the 63 hemolysin- and 167 gelatinase-producing isolates, 59 and 94 isolates, respectively, possessed both asa1 and esp genes. E. faecalis isolates with both asa1 and esp genes formed biofilms at significantly higher rates than those with neither gene (P=0.038). The genes encoding asa1, cylA , and aac(6') /(aph(2'') were transferable and appeared to have accumulated in these isolates. The E. faecalis isolates possessing asa1 and/or esp genes were found from both catheter-related or -unrelated UTI. Our study indicates that E. faecalis isolates that have accumulated virulence genes are apt to form persistent biofilms in the urinary tracts.</p

Synergistic effect of fosfomycin and fluoroquinolones against Pseudomonas aeruginosa growing in a biofilm.

Ulifloxacin is the active form of the prodrug prulifloxacin and shows a highly potent antipseudomonal activity. In this study, we examined the combined effect of fosfomycin and ulifloxacin against Pseudomonas aeruginosa (P. aeruginosa) growing in a biofilm using a modified Robbins device with artificial urine, and compared it to that of the combination of fosfomycin and ciprofloxacin or levofloxacin. An ATP bioluminescence assay was used to evaluate the antibacterial activity of the agents against sessile cells in a mature biofilm developed on a silicon disk. The total bioactivity of P. aeruginosa growing in a biofilm that had not been fully eradicated by fosfomycin or any of the fluoroquinolones alone at 10 times the MIC decreased after combination treatment with fosfomycin and fluoroquinolones. Morphological changes occurred in a time-dependent fashion; namely, swollen and/or rounding cells emerged within a couple of hours after combination treatment, marking the initial stage in the process leading to the destruction of the biofilms. We could not find any difference among the 3 fluoroquinolones with regard to their synergistic effects when administered with fosfomycin. The combination treatment of fosfomycin and fluoroquinolones with highly potent antipseudomonal activities was effective in eradicating sessile cells of P. aeruginosa in the biofilm and promises to be beneficial against biofilm-associated infectious diseases.</p

Experimental and Clinical Studies on Fluoroquinolone-insusceptible Escherichia coli Isolated from Patients with Urinary Tract Infections from 1994 to 2007

Urinary tract infections (UTIs) due to fluoroquinolone-insusceptible Escherichia coli have become increasingly common in recent years. We investigated the potential relationships between clinical measures to combat fluoroquinolone-insusceptible E. coli and experimental analyses on E. coli isolates. Over a 14-year period from 1994 through 2007, a total of 828 E. coli isolates were collected from patients (one isolate per patient) with UTI at the urology ward of Okayama University Hospital. We analyzed the mutations in quinolone resistance-determining regions of DNA gyrase (gyrA) and topoisomerase IV (parC). The production of biofilm by these isolates was also examined and the associated medical records were retrospectively reviewed. Seven of 189 (3.7%) strains from uncomplicated UTIs and 82 of 639 (12.8%) strains from complicated UTIs were insusceptible to fluoroquinolones. Amino acid replacements of type II topoisomerases were frequently observed at positions 83 and 87 in GyrA and at positions 80 and 84 in ParC. No significant difference in the biofilm-forming capabilities was observed between fluoroquinolone-susceptible and -insusceptible E. coli. Our study suggests that biofilm formation of fluoroquinolone-insusceptible E. coli isolates is not a major mechanism of resistance in patients with UTI.</p

Epidemiology of Chlamydophila caviae-like Chlamydia Isolated from Urethra and Uterine Cervix

In 2000, chlamydial strains OK133 and OK135 were isolated from 2 female patients with cervicitis. These strains were unresponsive to commercially available PCR and LCR test kits for the diagnosis of Chlamydia trachomatis infection, and their phenotypic characteristics were very similar. The OK135 nucleotide sequence in MOMP-VD2 gene closely resembled that of Chlamydophila caviae GPIC. A similar strain was isolated in 2003 from a male patient OKM2 with urethritis, from which the strain SC10-6 was cloned by the plaque purification method. The nucleotide sequence of the entire MOMP gene of SC10-6 was exactly the same as that of OK135. Thus, the strains OK135 and SC10-6, together with OK133, have been called C. caviae-like Chlamydia. We designed primers for nested PCR assay, the product of which showed a single-band 311-bp fragment, to detect C. caviae-like Chlamydia. Of swab specimens obtained from 202 patients from 2003 to 2006 (119 male and 83 female patients), 18 specimens (8.9%) from 14 male and 4 female patients were positive, suggesting that C. caviae-like Chlamydia infection is rather common. Thus far, it has not been determined whether C. caviae-like Chlamydia is pathogenic for humans.</p

Study on the method to quantify viable bacterial cellson the surface of endotracheal suction catheters

気管内吸引カテーテルに付着した一般細菌の生菌数測定方法について,超音波法およびチューブミキサーによ攪拌法を用いて検討した｡まず,人工的に緑膿菌を付着させた気管内吸引カテーテルを超音波処理することにより生菌数を測定した｡その結果,処理時間が1分を経過すると生菌数は減少をはじめ経時的に減少傾向を示した｡一方,攪拌法では0.5分の処理をピーク値としてその後の減少傾向は認められなかった｡次に,在宅療養患者に使用したカテーテルをチューブミキサーで0.5分攪拌後,生菌数の測定を行った｡また,同じカテーテルを用いて走査型電子顕微鏡による観察を行った結果,画面上の細菌数の印象と生菌数の測定結果に矛盾はなかった｡これらのことから気管内吸引カテーテルに付着した一般細菌の生菌数測定方法として,生理食塩水に入れたカテーテルをチューブミキサーで攪拌する方法が有用であると考えられた｡As a method for the detection of viable bacteria attached to endotracheal suction catheters, we evaluated sonication and dissociation using a tube mixer. The catheter fragments with Pseudomonas aeruginosa PAO1 were treated by each of the two methods, and viable cells in the elutions were counted. The highest number of viable cells was observed at 0.5 min by either method. The viable cell count decreased when the sonication time exceeded 1 min, while only a slight decrease of viable cells was observed by using a tube mixer. The catheters used for patients receiving care at home were fragmented and treated by a tube mixer to detach bacteria, and viable cells were counted. Electron microscopy observation showed an association between the viable cell count and morphology of surfaces of the catheters. These results suggest that adequate removal of bacteria attached to endotracheal suction catheters is possible by agitating catheter fragments for 0.5 min in physiological saline using a tube mixer

Molecular Epidemiology and Clinical Implications of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Isolated from Urine

We conducted a study on molecular epidemiology and clinical implications of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa isolated from urine. Over a 10-year period from 2001 through 2010, a total of 92 MBL-producing P. aeruginosa urine isolates were collected from patients (one isolate per patient) who were admitted to 5 hospitals in Okayama Prefecture, Japan. When cross-infection was suspected in the hospital, pulsed-field gel electrophoresis was performed. In the resulting dendrogram of 79 MBL-producing P. aeruginosa urine isolates, no identical isolates and 7 pairs of isolates with ≥80% similarity were found. The biofilm-forming capabilities of 92 MBL-producing P. aeruginosa urine isolates were significantly greater than those of 92 non-MBL-producing urine isolates in a medium of modified artificial urine. The imipenem resistance transferred in 16 of 18 isolates tested, and these frequencies were in the range of 10－3 to 10－9. All of 18 isolates tested belonged to internationally spread sequence type 235 and had 3 gene cassettes of antimicrobial resistance genes in the class 1 integron. The strong biofilm-forming capabilities of MBL-producing P. aeruginosa urine isolates could be seriously implicated in nosocomial infections. To prevent spread of the organism and transferable genes, effective strategies to inhibit biofilm formation in medical settings are needed

Enhancement activity of QS autoinducer analog

In this study, we have investigated the effects of the newly synthesized analog of Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 (autoinducer analog) against antibiotic-resistant bacteria. In vitro susceptibility and killing assays for P. aeruginosa PAO1ΔoprD mutant and clinical isolates were performed by using antibiotics and AIA-1. In an in vivo assay, a luminescent carbapenem-resistant strain derived from PAO1ΔoprD was injected into neutropenic ICR mice and bioluminescence images were acquired after the treatment with antibiotics and AIA-1. Additionally, we investigated the effects of the combination use against carbapenem- resistant Enterobacteriaceae (CRE). Using killing assays in P. aeruginosa, the survival rates in the presence of antibiotics and AIA-1 significantly decreased in comparison with those with antibiotics alone. Furthermore, dual treatment of biapenem and AIA-1 was more effective than biapenem alone in a mouse infection model. AIA-1 did not change the MICs in P. aeruginosa, suggesting that AIA-1 acts on the mechanism of antibiotic tolerance. Conversely, the MICs of antibiotics decreased in the presence of AIA-1 in some CRE strains, indicating that AIA-1 may require additional mechanism to act on CRE. In conclusion, AIA-1 may be a potent drug for clinical treatment of infections caused by antibiotic-resistant bacteria

Model studies on the alteration of the phospholipid composition of Staphyloccous aureus in response to the lack of the cell wall

In order to elucidate why cardiolipin increases markedly in Staphylococcus aureus cells which lack cell walls, the phase transition temperature of cardiolipin (CL) was determined and compared with that of a major phospholipid, phosphatidylglycerol (PG). CL composed of a fatty acid with a given length was synthesized from dimyristoyl PG and dipalmitoyl PG with the aid of phospholipase D prepared from cabbages and was purified by chromatography. Analysis by differential scanning calorimetry showed that the phase transition temperatures of dimyristoyl PG, tetramyristoyl CL, dipalmitoyl PG and tetrapalmitoyl CL were 25.0, 47.0, 40.5 and 62.2℃, respectively. A mixture of the two phospholipids showed a higher phase transition temperature than PG alone, but lower than CL alone. In the presence of divalent cations, especially Ca(2+), the phase transition temperature of CL increased more than that of PG. These results clearly indicate that cardiolipin can increase the membrane rigidity and suggest that S. aureus may increase cardiolipin content of the membrane to compensate for the loss of mechanical protection due to the lack of the cell wall

Description of a 23.9-Kilobase Chromosomal Deletion Containing a Region Encoding fsr Genes Which Mainly Determines the Gelatinase-Negative Phenotype of Clinical Isolates of Enterococcus faecalis in Urine

Expression of virulence-related extracellular proteases, gelatinase, and serine protease of Enterococcus faecalis is regulated by a quorum-sensing system encoded by the fsr gene cluster. In this study, a 23.9-kb chromosomal deletion containing the fsr gene cluster region was found to be present in the majority (79%) of gelatinase-negative clinical isolates of E. faecalis from urine