Identification of causative pathogens in eyes with bacterial conjunctivitis by bacterial cell count and microbiota analysis.

PURPOSE: To determine the causative pathogens in eyes with bacterial conjunctivitis. DESIGN: Evaluation of diagnostic test or technology. PARTICIPANTS: Thirteen eyes diagnosed clinically with bacterial conjunctivitis and 12 eyes with normal conjunctival sac were studied. METHODS: The bacterial cell numbers were counted in the samples stained by ethidium bromide (EtBr). The microbiota was determined by the clone library method using polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA (rRNA) gene with universal primers. In addition, examinations of smears and cultures of samples were performed. MAIN OUTCOME MEASURES: Bacterial cell numbers determined by the EtBr staining method and microbiota analysis based on 16S rRNA gene of samples from eyes with bacterial conjunctivitis. RESULTS: The bacterial cell numbers in the eyes with bacterial conjunctivitis were significantly higher than those in the normal conjunctival sacs. Ten of 13 samples from the eyes with bacterial conjunctivitis had positive PCR results. The remaining 3 samples and all 12 samples from the normal conjunctiva had negative PCR results. In 5 of the 10 PCR-positive samples, the predominant species accounted for 84.5% or more of each clone library. In the remaining 5 samples, the predominant and the second dominant species accounted for 27.4% to 56.3% and 19.0% to 26.8%, respectively, of each clone library. The number of detected species in the clone libraries was between 8 and 20 (average ± standard deviation, 7.5 ± 5.8). Bacteria were detected in 8 of the 10 bacterial conjunctivitis samples and in 5 of the 12 normal samples in the cultures. The number of species detected by cultures was 1 in the eyes with bacterial conjunctivitis and between 1 and 3 (mean ± standard deviation, 1.6 ± 0.9) in the normal conjunctiva. CONCLUSIONS: These results showed that the bacterial cell number in a sample is a good method of determining bacterial conjunctivitis. The microbiota analysis detected a diverse group of microbiota in the eyes with bacterial conjunctivitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The combination of bacterial cell count and microbiota analysis is a good method for identifying the causative pathogens in cases of monomicrobial and polymicrobial conjunctivitis.

Bacterial hazards of sludge brought ashore by the tsunami after the great East Japan earthquake of 2011.

OBJECTIVES: The aim of this study was to identify bacteria in sludge brought by the 2011 tsunami in Japan to determine the necessary precautions for workers who handle the sludge. METHODS: Two sludge samples and one water sample were collected from each of two sites in Miyagi Prefecture in June 2011. We also obtained control samples from a paddy field and a dry beach in Fukuoka, Japan. The samples were subjected to physicochemical analyses, conventional cultivation methods, and molecular methods for bacterial flora analysis. The bacterial floras were analyzed using a clone library method employing fragments of the 16S ribosomal RNA gene (rDNA) amplified with universal primers. RESULTS: We detected 51-61 genera in sludge samples and 14 and 17 genera in water samples collected in the tsunami-affected areas. In sludge samples collected in the tsunami-affected areas, more genera belonged to Proteobacteria than to Bacteroidetes, but in water samples collected in these areas, more genera belonged to Bacteroidetes than to Proteobacteria. Non-O1, non-O139 V. cholerae (non-agglutinable vibrio) was found at approximately 10(4) cells/m/ near the coast of the tsunami affected area. Sulfate-reducing bacteria were detected in sludge collected from the paddy field, and a relatively high concentration of sulfate ions was found in the water sample (258 mg/l). CONCLUSIONS: Sludge brought by the tsunami contained some pathogens; therefore, frequent hand washing is recommended for workers who have direct contact with the sludge to minimize their risk of infection. Under the anaerobic conditions of paddy fields, hydrogen sulfide could be produced by sulfate-reducing bacteria metabolizing sulfate ions.

Synergism of Staphylococcus aureus colonization and allergic reaction in the nasal cavity in mice.

BACKGROUND: The aim of this study is to investigate the reciprocal effect of Staphylococcus aureus colonization and allergic rhinitis in an allergy model of mice. METHODS: BALB/c mice with intraperitoneal ovalbumin (OVA) sensitization and/or intranasal S. aureus inoculation were prepared. The following 4 groups were designed: an OVA-sensitized S. aureus-inoculated (AR-SA) group, an OVA-sensitized uninoculated (AR) group, a nonsensitized S. aureus-inoculated (SA) group, and a nonsensitized uninoculated (control) group. After intranasal OVA challenge, nasal lavage fluid, peripheral blood, and nasal mucosa were collected. Polymorphonuclear cells in the nasal lavage fluid were counted, serum OVA-specific IgE and IgG1 were measured by enzyme immunoassays, and IL-4, IL-5, and IFN-γ mRNAs in the nasal mucosa were assessed by quantitative real-time reverse transcription-PCR. The number of S. aureus in the nasal mucosa and lavage fluid was counted. RESULTS: Both eosinophil and neutrophil counts were larger in the AR-SA group than in the other groups. Both IgE and IgG1 levels were higher in the AR and AR-SA groups than in the SA and control groups, and the IgG1 level was higher in the AR-SA group than in the AR group. The expression of IL-4 mRNA was higher in the AR-SA group than in the other groups, and the expression of IL-5 mRNA was higher in the AR-SA group than in the SA group. The AR-SA group showed higher counts of S. aureus in the nasal mucosa than the SA group. CONCLUSION: These results indicate the mutually potentiating effect of S. aureus colonization and allergic rhinitis.

Small and rough colony pseudomonas aeruginosa with elevated biofilm formation ability isolated in hospitalized patients.

Pseudomonas aeruginosa is a key pathogen of nosocomial infection, and causes persistent infection in patients with specific diseases like cystic fibrosis (CF). It has been reported that patients affected with CF discharge, at a high frequency, small colony variants with high adherence ability. In routine laboratory testing, we found atypical small and rough type (SR) colony variants of P. aeruginosa. The SRs and the counterpart wild type (WT) colonies showed similar biochemical features, antimicrobial susceptibilities, pulsed-field gel electrophoresis (PFGE) profiles, serotypes, and twitching motilities. The biofilm formation abilities of all the SR colonies, however, were extremely elevated as compared to those of the counterpart WT colonies. The frequency of SR-positive patients was 3.1% of the P. aeruginosa-positive inpatients (5/160), and that of the SR isolates was 0.6% of the P. aeruginosa strains (6/970) isolated in our laboratory over a period of 6 months. The SR-positive patients did not have any common disease or particular antibiotics treatment. The PFGE profiles showed that the SRs and the counterpart WTs were identical to each other, and also that three of the five SR/WT pairs were clonally similar. The three pairs were recovered from the feces, urine, and endotracheal secretion, respectively, of three patients hospitalized in two distinct wards. The results suggest that P. aeruginosa spontaneously produced highly adherent SR colonies in hospitalized patients, and these colonies may tend to spread in a hospital.

[A bacteriological study of multiple-drug-resistant Pseudomonas aeruginosa isolates derived from 2 patients].

We investigated the isolation circumstances of multiple-drug-resistant Pseudomonas aeruginosa (MDRP) in the UOEH hospital and the bacterial analysis of isolated MDRP. From January to October 2003, MDRP was isolated from 2 patients. During this period, the isolation frequency of MDRP was 0.57% (2/350). Case 1 had 2 MDRP isolates from catheter urine, and case 2 had 5 MDRP isolates from pus. Regarding serotype, 2 isolates from case 1 were B type and the other 16 isolates from case 2 were E type. Pyomelanin was produced by 9 isolates of 16 E type isolates. The same PFGE patterns were observed in 2 isolates from case 1; that is, 9 pyomelanin producers from case 2 and the other 7 isolates from case 2, respectively. Metallo-beta-lactamase was produced by 2 isolates from case 1. bla(IMP) was detected from the 2 isolates by PCR, and the clones from case 1 were quite different from the clones from case 2. Regarding the pyomelanin producing isolates from case 2, although the clones were the same genetically, the MICs of imipenem and meropenem increased from 8 to > 32 microg/ml with the progress of time. In the UOEH hospital, 6 patients with MDRP isolates have been isolated so far, but these 6 patients are not correlated with each other. It is important that we detect and report MDRP as early as possible to prevent nosocomial infection.

[Control measures against Serratia marcescens colonization at the neonatal intensive care unit of UOEH hospital].

In September 2001, twelve neonatal intensive care unit (NICU) patients were found to be colonized with pigment-producing strains of Serratia marcescens. The UOEH Infection Control Group (ICG) committee investigated the source of this epidemic and carried out several remedial measures. Immediate investigation of both the environment and the hands of health care workers were enforced. The most likely means of transmission was thought to be from the hands contaminated with S. marcescens that was found on antiseptic cotton, kept in shared stainless steel canisters, used for wiping the patients' buttocks. Therefore, we suggested the following interventions: 1) abolish the stainless steel canisters, and prepare antiseptic cottons for each patient, 2) monitor cultures with some specimens for all patients in the NICU, 3) periodically investigate the environment, 4) enforce workers to wash and disinfect their hands before and after patient care, 5) use new gloves for each treatment, 6) re-examine and modify the caring procedures for inpatients by the nursing staff. In January 2002, this nosocomial colonization came to an end without any serious infection. One of the key points of this success was the quick response by the clinical staff and ICG committee members to the laboratory results of bacteriological examinations. Furthermore, the early investigation of reservoir and good communication between the clinical staff and ICG committee members mostly prevented this nosocomial colonization from becoming worse.