A drug-repositioning screening identifies pentetic acid as a potential therapeutic agent for suppressing the elastase-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 µM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.

Vitamin B12-mediated restoration of defective anaerobic growth leads to reduced biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa undergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO(3)(-)) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments against P. aeruginosa biofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B(12) to be a molecule that can reinstate defective anaerobic growth of P. aeruginosa. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B(12), an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B(12), the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth of P. aeruginosa with normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B(12), further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth of P. aeruginosa and illustrates the ability of vitamin B(12) to modulate the biofilm-forming capacity of P. aeruginosa under such condition.

Faecal microbiota transplantation reduces amounts of antibiotic resistance genes in patients with multidrug-resistant organisms.

BACKGROUND: Multidrug-resistant organisms (MDROs) such as vancomycin-resistant enterococci (VRE) and carbapenemase-producing Enterobacteriaceae (CPE) are associated with prolonged hospitalisation, increased medical costs, and severe infections. Faecal microbiota transplantation (FMT) has emerged as an important strategy for decolonisation. This study aimed to evaluate the genetic response of MDROs to FMT. METHODS: A single-centre prospective study was conducted on patients infected with VRE, CPE, or VRE/CPE who underwent FMT between May 2018 and April 2019. Genetic response was assessed as the change in the expression of the resistance genes VanA, blaKPC, blaNDM, and blaOXA on days 1, 7, 14, and 28 by real-time reverse-transcription polymerase chain reaction. RESULTS: Twenty-nine patients received FMT, of which 26 (59.3%) were infected with VRE, 5 (11.1%) with CPE, and 8 (29.6%) with VRE/CPE. The mean duration of MDRO carriage before FMT was 71 days. Seventeen patients (63.0%) used antibiotics within a week of FMT. In a culture-dependent method, the expression of VanA and overall genes significantly decreased (p = 0.011 and p = 0.003 respectively). In a culture-independent method, VanA, blaNDM, and overall gene expression significantly decreased over time after FMT (p = 0.047, p = 0.048, p = 0.002, respectively). Similar results were confirmed following comparison between each time point in both the culture-dependent and -independent methods. Regression analysis did not reveal important factors underlying the genetic response after FMT. No adverse events were observed. CONCLUSION: FMT in patients infected with MDROs downregulates the expression of resistance genes, especially VanA, and facilitates MDRO decolonisation.

Fecal Microbiota Transplantation for multidrug-resistant organism: Efficacy and Response prediction.

OBJECTIVES: The increasing prevalence of multidrug-resistant microorganisms (MDRO) is increasing the frequency of poor clinical outcomes, prolonging hospitalizations, and raising healthcare costs. This study evaluated the eradication efficacy of fecal microbiota transplantation (FMT) and identified microbial and functional biomarkers of MDRO decolonization. METHODS: Fecal solution obtained from healthy unrelated donors was infused in the participants' guts which had been colonized with carbapenemase-producing enterobacteriacea (CPE), vancomycin-resistant enterococci (VRE), or both CPE and VRE. Fecal samples from recipients were collected and microbiome changes before and after FMT were assessed. RESULTS: Twenty-four (68.6%) out of 35 patients were decolonized within one year of receiving FMT. Multivariate analysis showed that FMT (FMT: hazard ratio (HR) = 5.343, 95% confidence interval (CI) = 1.877-15.212, p = 0.002) and MDRO types (CPE: HR = 11.146, 95% CI = 2.420-51.340, p = 0.002; CPE/VRE: HR = 2.948, 95% CI = 1.200-7.246, p = 0.018; VRE served as the reference) were significant independent factors associated with time to decolonization. Microbiota analysis showed higher richness and biodiversity before FMT resulted in VRE decolonization. The species Clostridium ramosum and the genuses Anaerostipes and Eisenbergiella could serve as taxonomic biomarkers and K02017 could serve as a functional biomarker for VRE clearance. CONCLUSION: FMT is an effective way to decolonize MDRO and its effectiveness may be predicted by microbiome analysis.

Antibiotic susceptibility of conjunctival bacterial isolates from refractive surgery patients.

PURPOSE: To determine the in vitro antibiotic susceptibility patterns of conjunctival bacterial flora isolated before surgery from patients undergoing refractive surgery. DESIGN: In vitro laboratory investigation. PARTICIPANTS: One hundred five eyes from 105 patients scheduled for refractive surgery at Balgensesang Ophthalmology Clinic between September 2005 and January 2006 were studied. Among 105 patients, 71 (67.6%) underwent LASIK using a femtosecond laser, 24 (22.9%) underwent LASIK using an automated microkeratome, 8 (7.6%) underwent LASEK, and 2 (1.9%) patients underwent phakic intraocular lens implantation. METHODS: Preoperative conjunctival swab samples were inoculated directly in culture media at the bedside before topical anesthetic or antibiotic application. Blood agar, chocolate agar, thioglycolate broth, Sabouraud dextrose agar, and Ogawa media were used for bacterial, fungal, and mycobacterial cultures. MAIN OUTCOME MEASURES: Minimum inhibitory concentrations (MICs) of ofloxacin (OFX), levofloxacin (LEV), gatifloxacin (GAT), moxifloxacin (MOX), gemifloxacin (GEM), and other commonly used antibiotics were determined using an E test. RESULTS: From 105 patients, 73 (85%) coagulase-negative staphylococci (CNS), 2 (2.3%) Staphylococcus aureus, 1 (1.2%) Streptococcus pneumoniae, and 5 (4.8%) gram-negative bacilli were isolated. No fungi or mycobacteria were isolated. The MIC that would inhibit the growth of 90% of the tested bacterial isolates (MIC(90)) of OFX, LEV, GAT, MOX, and GEM for methicillin-susceptible CNS (n = 46) were 0.5 microg/ml, 0.19 microg/ml, 0.094 microg/ml, 0.047 microg/ml, and 0.023 microg/ml, respectively. The MIC(90) values for methicillin-resistant CNS (n = 27) were 32 microg/ml, 4 microg/ml, 1 microg/ml, 0.5 microg/ml, and 0.25 microg/ml, respectively (P<0.001). CONCLUSIONS: The most effective against conjunctival bacteria isolated from refractive surgery patients were GEM, MOX, and GAT; however, resistance to earlier-generation fluoroquinolones (OFX and LEV) is increasing among methicillin-resistant CNS. It may be a therapeutic option to use newer fluoroquinolones in patients undergoing refractive eye surgery to reduce such infections as methicillin-resistant CNS. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Dissemination of CTX-M-15 beta-lactamase genes carried on Inc FI and FII plasmids among clinical isolates of Escherichia coli in a university hospital in Istanbul, Turkey.

The CTX-M-1 group was found in 86.8% of the Escherichia coli isolates from Istanbul. A subset study revealed all isolates carrying bla(CTX-M-15) genes flanked by the insertion element ISEcp1. Plasmid typing of transconjugates carrying bla(CTX-M-15) showed that most isolates belonged to the Inc/rep FII group but that one isolate also belonged to the FI group.

Survey of childhood empyema in Asia: implications for detecting the unmeasured burden of culture-negative bacterial disease.

BACKGROUND: Parapneumonic empyema continues to be a disease of significant morbidity and mortality among children despite recent advances in medical management. To date, only a limited number of studies have assessed the burden of empyema in Asia. METHODS: We surveyed medical records of four representative large pediatric hospitals in China, Korea, Taiwan and Vietnam using ICD-10 diagnostic codes to identify children <16 years of age hospitalized with empyema or pleural effusion from 1995 to 2005. We also accessed microbiology records of cultured empyema and pleural effusion specimens to describe the trends in the epidemiology and microbiology of empyema. RESULTS: During the study period, we identified 1,379 children diagnosed with empyema or pleural effusion (China, n = 461; Korea, n = 134; Taiwan, n = 119; Vietnam, n = 665). Diagnoses of pleural effusion (n = 1,074) were 3.5 times more common than of empyema (n = 305), although the relative proportions of empyema and pleural effusion noted in hospital records varied widely between the four sites, most likely because of marked differences in coding practices. Although pleural effusions were reported more often than empyema, children with empyema were more likely to have a cultured pathogen. In addition, we found that median age and gender distribution of children with these conditions were similar across the four countries. Among 1,379 empyema and pleural effusion specimens, 401 (29%) were culture positive. Staphylococcus aureus (n = 126) was the most common organism isolated, followed by Streptococcus pneumoniae (n = 83), Pseudomonas aeruginosa (n = 37) and Klebsiella (n = 35) and Acinetobacter species (n = 34). CONCLUSION: The age and gender distribution of empyema and pleural effusion in children in these countries are similar to the US and Western Europe. S. pneumoniae was the second leading bacterial cause of empyema and pleural effusion among Asian children. The high proportion of culture-negative specimens among patients with pleural effusion or empyema suggests that culture may not be a sufficiently sensitive diagnostic method to determine etiology in the majority of cases. Future prospective studies in different countries would benefit from standardized case definitions and coding practices for empyema. In addition, more sensitive diagnostic methods would improve detection of pathogens and could result in better prevention, treatment and outcomes of this severe disease.

Delayed onset Mycobacterium intracellulare keratitis after laser in situ keratomileusis: A case report and literature review.

RATIONALE: Infectious keratitis is a relatively uncommon but potentially sight-threatening complication of laser in situ keratomileusis (LASIK). Mycobacterial keratitis is usually regarded as late onset keratitis among post-LASIK keratitis. There has been no documented case of Mycobacterium intracellulare post-LASIK keratitis of a long-latent period. PATIENT CONCERNS: A 36-year-old man was referred to our out-patient clinic, for persistent corneal epithelial defect with intrastromal infiltration. He had undergone uneventful bilateral LASIK procedure 4 years before. He complained decreased vision, accompanied by ocular pain, photophobia, and redness in his left eye for 7 months. DIAGNOSIS: Lamellar keratectomy was taken using femtosecond laser. Bacterial culture with sequenced bacterial 16s ribosomal DNA confirmed the organism to be M intracellulare. INTERVENTIONS: After 3 months of administration of topical clarithromycin, amikacin, and moxifloxacin, the corneal epithelial defect was resolved and the infiltration was much improved. However, newly developed diffuse haziness with surrounding granular infiltration in the central cornea was noted. Drug toxicity was suspected and topical moxifloxacin was discontinued, resulting in resolution of the diffuse haze with infiltration. OUTCOME: The patient was followed up regularly without medication thereafter and recurrence was not found for 7 years. LESSONS: This case presents the first case of M intracellulare keratitis after LASIK. LASIK surgeons should aware that post-LASIK keratitis can develop long after the operation and careful suspicion of infectious disease with meticulous diagnostic test is needed.

Outbreaks of Serratia marcescens bacteriuria in a neurosurgical intensive care unit of a tertiary care teaching hospital: a clinical, epidemiologic, and laboratory perspective.

BACKGROUND: Serratia marcescens is an aerobic gram-negative bacillus belonging to the family Enterobacteriacea. Infections caused by S marcescens may be difficult to treat because of their resistance to a variety of antibiotics, including beta-lactams and aminoglycosides. METHODS: This study aimed to (1) identify the risk factors associated with the development of Serratia marcescens bacteriuria in neurosurgical intensive care units (NSICU); (2) genotype the pathogens to determine the source of infection; (3) compare these results with antibiograms; and (4) determine and implement appropriate control measures. A retrospective case-control study of the epidemiologic data, the surveillance of environmental cultures, and the genotyping of strains using arbitrarily primed polymerase chain reaction (AP-PCR) were performed at a 750-bed, tertiary care teaching hospital. Seventy-four bacteriuria patients were compared with 74 age/sex-matched control patients in the NSICU between March 2002 and March 2004. The factors assessed were patient demographics; duration of hospital stay; duration of indwelling catheter use before and during stay in the NSICU; chronic underlying illnesses (diabetes mellitus, cardiovascular disease, malignancy); other sites of infection; history of trauma; exposure to a nasogastric tube; mechanical ventilation; urinary catheterization; central venous catheterization; surgical drainage; tracheostomy; brain or spine surgery; and receipt of total parenteral nutrition (TPN), antimicrobials (beta-lactams, aminoglycosides, quinolones, carbapenems, vancomycins), or steroids. RESULTS: Patients with S marcescens bacteriuria were more likely to have a longer NSICU stay and other sites of infection. Environmental surveillance showed the handling of urine jugs to be the point source of contamination. Genotyping and antibiograms of 14 patients were the same except for those of 2 patients. CONCLUSION: The patient-related risk factors were identified, and a rapid identification of the organism was made. Heightened surveillance, infection control measures, and empiric therapy led to improved methods for handling urine jugs, which terminated the outbreak.

Loop-mediated isothermal amplification of vanA gene enables a rapid and naked-eye detection of vancomycin-resistant enterococci infection.

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infection at intensive care unit (ICU). A rapid and sensitive detection of VRE infection is in high demand for timely and suitable antibiotic treatment. Here, we optimized a distinct DNA-based diagnostic technique, loop-mediated isothermal amplification (LAMP) for a rapid detection of the presence of vanA gene, a critical component of the gene cluster required for vancomycin resistance. Amplification efficiency was optimal at 62°C and with 2mM MgSO4. The detection limit of the DNA template was 80pg and LAMP amplicons were detected within 40min; thereby suggesting a potential applicability of LAMP as a sensitive and urgent diagnostic method. Furthermore, positive LAMP reaction was directly detected with the naked-eye by monitoring the formation of a white precipitate or the color change induced by hydroxy naphthol blue (HNB) dye. Finally, 56 clinical isolates were successfully tested for the presence of vanA gene by LAMP, which was determined to be more sensitive than PCR. Together, our results clearly demonstrate the usefulness of LAMP for the diagnosis of VRE infection.

Evaluation of humoral immune response to nosocomial pathogen and functional status in elderly patients with sepsis.

The clinical significance of humoral immune response to nosocomial pathogens and functional status in elderly patients with sepsis is not clear. We evaluated the humoral immune to nosocomial pathogens and the effect of functional dependencies on clinical outcomes among elderly patients with sepsis. This study prospectively enrolled patients aged ≥65 years with sepsis from September 2011 to May 2012 at a 2000-bed university hospital. The data including CD4 and CD8 T-cell count, functional status by measuring basic activities of daily living (ADL) and instrumental activities of daily living (IADL) were collected for all patients. In addition, the collected blood samples were analyzed for serum antibody levels against nosocomial pathogens using an ELISA. During the study period, 72 patients (38 males) treated with sepsis were enrolled. The all-cause in-hospital mortality rate was 16.7% (12/72). The mean CD4/CD8 T-cell ratio was significantly lower in nonsurvivors than in survivors (1.08 ± 0.72 vs. 1.93 ± 1.42, P=0.003). Serum antibody titers to Acinetobacter baumannii, Klebsiella pneumonia, Stenotrophomonas maltophilia, and Enterococcus faecalis were statistically higher in nonsurvivors than in survivors. On multivariate analysis, the IADL score was independently predictive of mortality in elderly patients with sepsis (odds ratio 1.410, 95% confidence interval 1.007-1.975, P=0.046). These results suggest that IADL scores could be used as predictors to identify elderly patients with a poor prognosis of nosocomial infections.

In vitro activity of gemifloxacin against recent clinical isolates of bacteria in Korea.

Gemifloxacin is an enhanced-affinity fluoroquinolone with broad-spectrum antibacterial activity. In Korea, resistant bacteria are relatively more prevalent than in other industrialized countries. In this study, we studied the in vitro activities of gemifloxacin, gatifloxacin, moxifloxacin, levofloxacin, ciprofloxacin, and other commonly used antimicrobial agents against 1,689 bacterial strains isolated at four Korean university hospitals during 1999-2000. Minimum inhibitory concentrations (MICs) were determined using the agar dilution method of National Committee for Clinical Laboratory Standards. Gemifloxacin had the lowest MICs for the respiratory pathogens: 90% of Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae were inhibited by 0.06, 0.03, and 0.03 mg/L, respectively. Gemifloxacin was more active than the other fluoroquinolones against methicillin-susceptible Staphylococcus aureus, coagulase-negative staphylococci, streptococci, and Enterococcus faecalis. The MIC90s of gemifloxacin for Klebsiella oxytoca, Proteus vulgaris, and non-typhoidal Salmonella spp. were 0.25, 1.0, and 0.12 mg/L, respectively, while those for other Gram-negative bacilli were 4-64 mg/L. In conclusion, gemifloxacin was the most active among the comparative agents against Gram-positive species, including respiratory pathogens isolated in Korea.