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Objectives: Antibiotic tolerance causes chronic, refractory and persistent infections. In order to advance the development of a new type of drug for the treatment of infectious diseases, we herein investigated the effects of a newly synthesized analogue of the Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 ( a uto i nducer a nalogue) on antibiotic tolerance in P. aeruginosa ., Methods: A P. aeruginosa luminescent strain derived from PAO1 was injected into neutropenic ICR mice and bioluminescence images were acquired for a period of time after treatments with antibiotics and AIA-1. In vitro susceptibility testing and killing assays for the planktonic and biofilm cells of PAO1 were performed using antibiotics and AIA-1. The expression of quorum-sensing-related genes was examined using real-time PCR., Results: In vivo and in vitro assays showed that AIA-1 alone did not exert any bactericidal effects and also did not affect the MICs of antibiotics. However, the combined use of AIA-1 and antibiotics exerted markedly stronger therapeutic effects against experimental infection than antibiotics alone. The presence of AIA-1 also enhanced the killing effects of antibiotics in planktonic and biofilm cells. Although AIA-1 did not inhibit the expression of lasB and rhlA genes, which are directly regulated by quorum sensing, it clearly suppressed expression of the rpoS gene., Conclusions: The new compound, AIA-1, did not alter the antibiotic susceptibility of P. aeruginosa by itself; however, its addition enhanced the antibacterial activity of antibiotics. AIA-1 did not inhibit quorum sensing, but reduced the antibiotic tolerance of P. aeruginosa by suppressing rpoS gene expression., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Bacteria attached to a surface are generally more tolerant to antibiotics than their planktonic counterparts, even without the formation of a biofilm. The mechanism of antibiotic tolerance in biofilm communities is multifactorial, and the genetic background underlying this antibiotic tolerance has not yet been fully elucidated. Using transposon mutagenesis, we isolated a mutant with reduced tolerance to biapenem (relative to that of the wild type) from adherent cells. Sequencing analysis revealed a mutation in the pslL gene, which is part of the polysaccharide biosynthesis operon. The Pseudomonas aeruginosa PAO1Δ pslBCD mutant demonstrated a 100-fold-lower survival rate during the exposure of planktonic and biofilm cells to biapenem; a similar phenotype was observed in a mouse infection model and in clinical strains. Transcriptional analysis of adherent cells revealed increased expression of both pslA and pelA , which are directly regulated by bis-(3',5')-cyclic dimeric GMP (c-di-GMP). Inactivation of wspF resulted in significantly increased tolerance to biapenem due to increased production of c-di-GMP. The loss of pslBCD in the Δ wspF mutant background abolished the biapenem-tolerant phenotype of the Δ wspF mutant, underscoring the importance of psl in biapenem tolerance. Overexpression of PA2133, which can catalyze the degradation of c-di-GMP, led to a significant reduction in biapenem tolerance in adherent cells, indicating that c-di-GMP is essential in mediating the tolerance effect. The effect of pslBCD on antibiotic tolerance was evident, with 50- and 200-fold-lower survival in the presence of ofloxacin and tobramycin, respectively. We speculate that the psl genes, which are activated by surface adherence through elevated intracellular c-di-GMP levels, confer tolerance to antimicrobials., (Copyright © 2017 American Society for Microbiology.)

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. J. Med. Invest. 64: 101-109, February, 2017.

We conducted a study on molecular epidemiology and clinical implications of metallo-beta-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.

Recently, there have been reports concerning an increased frequency of isolation of multi-drug resistant Pseudomonas aeruginosa (MDRP) strains in hospitals and other clinical settings as well as the associated risk of their hospital-acquired infections; in such a situation, it has been a major challenge to establish methods of managing and treating the infections. In order to investigate the trend of P. aeruginosa, the Infection Forum in the Chugoku Region has conducted to a multi-center collaborative study to isolate P. aeruginosa strains from sputum and urine samples collected between October 2006 and September 2008, analyzed the drug susceptibility and the pulsed-field gel electrophoresis (PFGE) patterns of each strain, and assessed epidemiologic characteristics. Of the 738 P. aeruginosa strains collected in this study, 152 (20.6%), 179 (24.3%), 47 (6.4%), and 39 (5.3%) were found to be ciprofloxacin-resistant, imipenem-resistant, amikacin-resistant, and MDRP, respectively. Among the various antimicrobial agents tested, arbekacin (ABK) revealed the strongest inhibitory effects on each drug-resistant and MDRP strain; therefore, ABK was considered as a potential candidate for future treatment of diseases caused by P. aeruginosa. The study also showed that the detection rates of MDRP varied a lot from hospital to hospital. In addition, PFGE-based cluster analyses revealed several strains isolated in the same hospital exhibited a similar PFGE pattern and the same drug susceptibility, suggesting the presence of "unique" hospital-specific strains.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is one of the most refractory to therapy when it forms biofilms in the airways of cystic fibrosis patients. To date, studies regarding the production of an immunogenic and protective antigen to inhibit biofilm formation by P. aeruginosa have been superficial. The previously uncharacterized outer membrane protein (OMP) Opr86 (PA3648) of P. aeruginosa is a member of the Omp85 family, of which homologs have been found in all gram-negative bacteria. Here we verify the availability of Opr86 as a protective antigen to inhibit biofilm formation by P. aeruginosa PAO1 and several other isolates. A mutant was constructed in which Opr86 expression could be switched on or off through a tac promoter-controlled opr86 gene. The result, consistent with previous Omp85 studies, showed that Opr86 is essential for viability and plays a role in OMP assembly. Depletion of Opr86 resulted in streptococci-like morphological changes and liberation of excess membrane vesicles. A polyclonal antibody against Opr86 which showed reactivity to PAO1 cells was obtained. The antibody inhibited biofilm formation by PAO1 and the other clinical strains tested. Closer examination of early attachment revealed that cells treated with the antibody were unable to attach to the surface. Our data suggest that Opr86 is a critical OMP and a potential candidate as a protective antigen against biofilm formation by P. aeruginosa.

Foreign body-associated infectious disease is currently one of the most problematic hospital-acquired infections. Patients with placement of urinary catheters are especially susceptible to such infection, that is biofilm infection. In this study, we focused on the therapeutic efficacy of prulifloxacin (PUFX) against Pseudomonas aeruginosa OP 14-210, isolated from a patient with complicated urinary tract infection. This microbe formed a biofilm on the surface of a polyethylene tube (PT) placed in a rat bladder without surgical manipulation. In addition, we attempted to eradicate the biofilm by treatment with a combination of PUFX and fosfomycin (FOM). A single oral administration of PUFX at a dose of 20 mg/kg was effective against P. aeruginosa as a biofilm, yielding a significant reduction in CFU per PT of approximately 1 log(10) CFU/PT compared with that in untreated controls. A similar therapeutic effect was also observed in levofloxacin-treated rats, and albeit slightly weaker, in ciprofloxacin-treated animals as well. Because 3 days' consecutive treatment with each fluoroquinolone did not further decrease the viable cell counts on the PT, we tested the efficacy of combining PUFX and FOM. These two drugs, administered once a day for 3 days, at doses of 20 and 100 mg/kg, respectively, resulted in significant decreases of viable cell counts on the PT of more than 1.5 log(10) CFU/PT compared with PUFX alone (P < 0.05). As seen by scanning electron microscopy, destruction and disappearance of multilayer biofilms occurred after treatment with this drug combination. Such combination therapy with PUFX and FOM may be advantageous for treating biofilm-related infectious diseases.

The bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa) isolated from patients diagnosed as urinary tract infections (UTIs) in 14 institutions in Japan were collected between August 2004 and July 2005. The susceptibilities of these bacteria to various antimicrobial agents were measured. The bacteria were divided into 2 groups consisting of uncomplicated UTIs and complicated UTIs (with and without indwelling catheter) based on their isolation origins. The results were compared with those obtained between 1995 and 2003. The drug sensitivity of S. aureus in this year was similar to those in up to the previous year and S. aureus showed the best susceptibility to vancomycin (VCM) and arbekacin (ABK). The drug sensitivity of E. faecalis in this year also was similar to those in up to the previous year. The susceptibility of E. coli to cephems in this year was generally good and was similar to those in up to the previous year. MIC90 of cefozopran (CZOP) was the most stable and 0.125 microg/mL or less since 1995. The susceptibility of E. coli to cefpirome (CPR) and cefotiam (CTM) also was good but to cefaclor (CCL), cefixime (CFIX), and cefpodoxime (CPDX) was largely decreased in complicated UTI groups. The sensitivity of E. coli to carbapenems also was good but to carumonam (CRMN) tended to decrease. The susceptibility of E. coli to quinolones, however, has largely changed and has decreased since 2003 in uncomplicated UTIs and 2000 in complicated UTIs. That was suggested the development of the resistance to the drug. The susceptibility of Klebsiella spp. to cefazolin (CEZ), CTM, CCL, CPDX, and cefditoren (CDTR) decreased in the previous year and recovered to the year before the previous year in this year. The susceptibility of Klebsiella spp. to other cephems was stable since 1995, especially against CZOP, the highest sensitivity (MIC90: < or = 0.125 microg/mL) was maintained. The susceptibility of Klebsiella spp. to carbapenems and CRMN also was good. The susceptibility of Klebsiella spp. to aminoglycosides was lower than to CZOP but was stable since 1995. The susceptibility of P. aeruginosa was generally low and has largely changed against the majority of the agents since 1995. The susceptibility of P. aeruginosa isolated from uncomplicated UTIs has largely changed against ceftazidime (CAZ), cefsulodin (CFS), CZOP, imipenem (IPM), meropenem (MEPM), aztreonam (AZT), CRMN, gentamicin (GM), and tobramycin (TOB). The susceptibility of P. aeruginosa isolated from complicated UTIs has largely changed against CSF, CZOP, MEPM, GM, and ciprofloxacin (CPFX). The susceptibility of P. aeruginosa isolated from complicated UTIs has been stable against amikacin (AMK). For annual changes in MIC50, TOB and IPM had a relatively stable and high activity (MIC50: 0.5-2 microg/mL).

The bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa) isolated from 565 patients diagnosed as urinary tract infections (UTIs) in 14 institutions in Japan were collected between August 2003 and July 2004. The susceptibilities of these bacteria to various antimicrobial agents were examined. The bacteria were divided into 2 groups consisting of uncomplicated UTIs and complicated UTIs (with and without indwelling catheter) based on their isolation origins. The results were compared with those obtained between 1994 and 2002. The drug sensitivity of S. aureus in this year was similar to those in up to the previous years and S. aureus showed the best susceptibility to vancomycin. The drug sensitivity of E. faecalis in this year also was similar to those in up to the previous years. The drug sensitivity of E. coli in this year was generally good except penicillins and was similar to those in up to the previous years. Among cephems, cefozopran (CZOP) and cefpirome (CPR) showed the highest potency activity (MIC90: < or = 0.125 microg/mL). An antibacterial activity of cefotiam (CTM) was stable for 10 years and was fine (MIC0: < or = 0.5 microg/mL). The sensitivity of E. coli to carbapenems and carumonam (CRMN) also was good like to CZOP. The sensitivity of the complicated UTIs group to quinolones, however, has decreased after 2000 and it was suggested that the resistance to the drug has developed. Kiebsiella spp. showed a decrease in the susceptibility to some of cephems. The drugs indicating a big decrease in the sensitivity were cefazolin, CTM, cefaclor, and cefpodoxime. Imipenem, carbapenems, also indicated a decrease in the sensitivity. The susceptibility of the strain to the other drugs was similar to that in up to the previous years. Among them, CZOP maintained good susceptibility (MIC90: > or = 0.125 microg/mL against uncomplicated UTIs, 0.25 microg/mL against complicated UTIs) like meropenem. The drug sensitivity of P. aeruginosa was generally low and was not much different from that in up to the previous years.

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.

The bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa) isolated from patients diagnosed as urinary tract infections (UTIs) in 13 institutions in Japan were supplied between August 2002 and July 2003. The susceptibilities of these bacteria to various antimicrobial agents were examined. The bacteria were divided into 2 groups consisting of uncomplicated UTIs and complicated UTIs (with and without indwelling catheter) based on their isolation origins. The results were compared with those obtained between 1993 and 2001. The drug sensitivity of S. aureus in this year was similar to those in up to the previous year and S. aureus showed the best susceptibility to vancomycin. The drug sensitivity of E. faecalis in this year also was similar to those in up to the previous year. The drug sensitivity of E. coli in this year was generally good except penicillins and was similar to those in up to the previous year. Among cephems, cefozopran (CZOP) and cefpirome (CPR) showed the highest potency (MIC90: < or = 0.125 microg/mL). An antibacterial activity of cefotiam (CTM) was similar to it in 10 years ago and was fine (MIC90: < or = 1 microg/mL). The sensitivity of E. coli to carbapenems and carumonam (CRMN) also was good like to CZOP. However, the sensitivity of the complicated UTIs group to quinolones decreased after 2000 and was suggested to develop the resistance to the drug. The drug sensitivity of Klebsiella spp. in this year also was similar to those in up to the previous year. The bacteria showed good susceptibility (MIC: < or = 0.125 microg/mL) to cefmenoxime (CMX), CPR, cefixime (CFIX), flomoxef (FMOX), and CZOP among cephems. The drug sensitivity of P. aeruginosa was generally low. Most of the bacteria were little sensitive to cephems except CZOP and ceftazidime (CAZ). The sensitive bacteria to CZOP and ceftazidime (CAZ) were observed to be 26.8% (15/56 strains) and 39.3% (22/56 strains) in complicated UTIs group, respectively. The sensitivity profile of P. aeruginosa to the other tested drugs was not much different from that in up to the previous year. However, the sensitivity of the bacteria to carbapenems tended to decrease after 2000, and the low sensitive strains (MIC: > or = 256 microg/mL) were detected at 22.2% (2/9 strains) in the uncomplicated UTIs group and 3.6% (2/56 strains) in the complicated UTIs group.

We examined the combined effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa biofilms of four clinical isolates with different susceptibilities to ofloxacin. A clear synergistic effect was detected in all four strains in accordance with their susceptibilities to ofloxacin. To clarify the mechanism of this synergistic action, changes in cellular accumulation of ofloxacin into fosfomycin-pretreated cells and morphological changes in cells treated with fosfomycin, ofloxacin, or fosfomycin plus ofloxacin were investigated. Pretreatment with fosfomycin significantly enhanced cellular uptake of labeled or unlabeled ofloxacin in biofilm cells as well as in floating cells. The accumulation of ofloxacin into fosfomycin-pretreated biofilm cells was further enhanced by treating cells simultaneously with ofloxacin and fosfomycin. Morphological studies using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM) demonstrated that fosfomycin induced dramatic changes in cell shape and the outer membrane structure responsible for the altered membrane permeability of both surface and embedded biofilm cells. The resulting increased accumulation of ofloxacin in multilayers of biofilm cells was correlated with the kinetics of biofilm cell eradication, and this synergistic killing effect was confirmed by a combined study using SEM, TEM, and CLSM.

We examined the combined effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa in biofilms by using an in vitro experimental system with a modified Robbins device. Sessile cells in a mature or immature biofilm, developed on a silicon disk, were used, and an ATP bioluminescence assay was employed to assess antibacterial effects. A synergistic effect of fosfomycin and ofloxacin was clearly detected when concentrations at which each drug independently produced no detectable decrease in the bioactivity of sessile cells were used. Exposure of the cells in a mature biofilm to fosfomycin at concentrations of one-eighth of the MIC to 10 times the MIC (6.25 to 500 micrograms/ml) and ofloxacin at three or 10 times the MIC (18.75 or 62.5 micrograms/ml) resulted in reduction of the bioactivity to 1.5 to 4.5% after 72 h. Young sessile cells in an immature biofilm were more susceptible to this combination therapy. With a combination of fosfomycin at three times the MIC and ofloxacin at three times the MIC, complete eradication was confirmed by both ATP assay and scanning electron microscopy.

We developed new sandwich cup method to assay the penetration of various antimicrobial agents through Pseudomonas exopolysaccharides. Using alginate extracted from mucoid-type Pseudomonas aeruginosa and gellan gum from Pseudomonas elodea, the role of exopolysaccharides as a barrier against drug penetration was examined. The penetration of positively charged hydrophilic drugs such as aminoglycosides and polypeptides was markedly inhibited by the gels tested, but that of beta-lactams, quinolones, and macrolides was not inhibited. The penetration of gentamicin was strongly influenced by the gel concentration, the solution to be used, and the presence of Ca2+. These results suggest that the microenvironment at the infection site could greatly influence drug penetration through biofilms in vivo.