Campylobacter Express Resistance Array for detecting the presence of fluoroquinolone- and macrolide-resistant Campylobacter jejuni and Campylobacter coli in broiler farms.

AIMS: The aim of the study was to develop a microarray-based method for the detection of antibiotic-resistant Campylobacter in broiler farms to decrease the risk of contamination of chicken meat. METHODS AND RESULTS: A combination of DNA microarray and primer extension for rapid and simultaneous detection of fluoroquinolone- and macrolide-resistant Campylobacter jejuni/Campylobacter coli, termed Campylobacter Express Resistance Array (CAMERA), was used to analyse chicken caecal droppings. CAMERA assays could detect at least 105 colony forming units of C. jejuni/C. coli g-1 of chicken caecal contents spiked with C. jejuni/C. coli. To compare the CAMERA method and direct culturing method for screening antibiotic-resistant C. jejuni/C. coli in poultry farms, chicken caecal droppings obtained from 42 poultry houses were analysed using both methods. In total, 95.2% of the results (40/42 poultry houses) obtained using the CAMERA and culturing method were identical. In the remaining two poultry houses, the CAMERA could detect the prevalent strain of C. jejuni/C. coli based on results of the culturing method. CONCLUSIONS: The culturing method required >3 days to isolate and identify antibiotic-resistant C. jejuni/C. coli. In contrast, the CAMERA required only 6 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This method can facilitate quick screening and control of fluoroquinolone- and macrolide-resistant C. jejuni/C. coli in broiler farms.

The first case of human protothecosis caused by Prototheca zopfii in Japan.

This report describes a fatal case Prototheca zopfii genotype 2 infection in an immunosuppressed patient. The patient was a 62-year-old housewife who presented general malaise in April 2011. Hairy cell leukemia was highly suspected. Chemotherapy was started because the patient developed severe pancytopenia in October 2011. Itraconazole capsules (100 mg/day) and trimethoprim (320 mg/day) plus sulfamethoxazole (1600 mg/day) combinations were orally administered for prophylaxis of fungal infections. Of BacT/ALERT 3D FA aerobic culture bottles and FN anaerobic culture bottles, only FA aerobic blood culture bottles produced positive reactions when the patient developed fever in January 2012. Gram-staining of blood culture bottles revealed Gram-negative elliptical sporangia. Culturing on Sabouraud dextrose agar produced smooth and creamy white, yeast-like colonies. Partial DNA sequences of the nuclear 18S rDNA and 28S rDNA D1/D2 domains of the isolated strain were identical to those of P. zopfii genotype 2. The MICs and minimal lethal concentrations of antifungals revealed that it was susceptible to amphotericin B and itraconazole. The patient died, at which time plasma (1 â†’ 3)-ß-D-glucan was positive (131 pg/mL).

In vitro susceptibility of Prototheca zopfii genotypes 1 and 2.

Prototheca zopfii causes bovine mastitis that leads to reduced milk production. Since P. zopfii isolates from mastitis have been assigned P. zopfii genotype 2, it suggests that this genotype is the etiologic agent of the infection. However, isolates of P. zopfii have not been investigated with regard to their in vitro drug susceptibility. In this study, we examine the susceptibility of genotype 2 strains from bovine mastitis and genotype 1 isolates recovered from cow-barn surroundings. The in vitro susceptibility of ten isolates and the type strain (SAG2063(T)) of P. zopfii genotype 1, and equal number of genotype 2 isolates and the type strain (SAG2021(T)) were assessed by E-test against amphotericin B (AMB), gentamicin (GM), kanamycin (KM) and itraconazole (ITZ). Results showed that P. zopfii genotype 1 isolates are more susceptible in vitro to AMB, GM and KM than those of genotype 2. Moreover, genotype 2 isolates and seven isolates of genotype 1, including the type strain, are not susceptible to ITZ (>10 µg/ml). Thus, drug susceptibility of P. zopfii differs between these two genotypes.

26S rDNA-based phylogenetic investigation of Japanese cattle-associated Prototheca zopfii isolates.

The study has been conducted to assess the applicability of partial 26S ribosomal DNA sequence for the phylogenic analysis of P.zopfii and to arrive relationships between specific genotype of P.zopfii and clinical mastitis. The phylogenetic analysis indicated that all genotype 2 isolates were grouped into the cluster of type strain of P.zopfii. Thus, all mastitis isolates in Japan were identified as P.zopfii genotype 2 and were independent of the cluster of the genotype 1 isolates. In one clinical case, a fecal isolate could not be identified by the 18S rDNA-based genotype specific PCR-assay and appeared to belong to a same cluster with the type strain of P. blaschkeae by 26S rDNA analysis. The isolate was the first isolation in Japan.

[Clostridium difficile TOX A/B II TEST evaluation].

We compared the performance of two commercial toxin detection kits, C. difficile toxin A/B (C. difficile TOX A/B II test; TOX A/B II) and C. difficile toxin A (Uniquick), for (i) detection using highly purified toxin A solution; (ii) cross-reactivity using culture supernatants of toxin A-positive and B-positive C. difficile, toxin A-negative and B-positive C. difficile, and toxin A-negative and B-negative C. difficile strains and other bacteria; and (iii) sensitivity and specificity using clinical specimens. Results indicated that TOX A/B II detected toxin A at concentrations of 0.35 ng/mL and Uniquick at concentrations of 0.7 ng/mL. Uniquick performance was specific for detecting toxin A alone, while TOX A/B II detected toxin A/B specifically. Kit performance was then evaluated using 99 fecal specimens--43 specimens from patients with toxin B-positive C. difficile and 56 from those without. Sensitivity of TOX A/B II vs Uniquick was 95.3% vs 76.7%, specificity 98.2% vs 98.2%, positive predictive 97.6% vs 97.1%, and negative predictive value 96.5% vs 84.6%. Findings thus indicate that TOX A/B II is a more suitable diagnostic aid for CDAD than Uniquick because it correlates well with toxin B-positive C. difficile culture results. Stool culture for C. difficile is also required, however.

Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia.

Loop-mediated isothermal amplification (LAMP) is a novel, rapid nucleic acid amplification method with high specificity and sensitivity under isothermal conditions. In this study a LAMP assay for diagnosing Pneumocystis pneumonia (PCP) was developed. Oligonucleotide primers specific for Pneumocystis species were designed corresponding to 18S rRNA gene sequences. The assay, performed for 30 min at 61 degrees C, was capable of detecting 50 copies per tube (2 x 10(3) copies ml(-1)) in 30 min and did not show cross-reactivity to other species of fungi, including the genera Candida, Aspergillus and Cryptococcus. A total of 21 of 24 clinical specimens (sputum and bronchoalveolar lavage fluid) from patients with suspected PCP tested positive using the LAMP assay by real-time fluorescence detection. The results of the LAMP reaction were also observed by real-time turbidity detection and end-point visual turbidity or fluorescence detection. With real-time fluorescence detection, melting curves of the products were effective at distinguishing specific amplification from non-specific amplification or self-amplification. Visual detection was also possible as a rapid and easy assay using only a heat block and a black light.

Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan.

This study is the first investigation on Japanese isolates of Prototheca zopfii from bovine mastitis and the cow-barn surroundings by molecular characterization to clarify routes of infection for bovine protothecal mastitis. We performed isolation of Prototheca from cow-barn surroundings (drinking water, sewage and feces) and milk samples from cases of bovine mastitis. Genotypes of the 32 isolates of P. zopfii from cow-barn surroundings and 67 isolates from mastitis were analyzed by genotype-specific PCR assays and restriction fragment length polymorphism (RFLP) assays. All mastitis isolates were identified as P. zopfii genotype 2. Conversely, 29 isolates from cow-barn surroundings were identified as P. zopfii genotypes 1 and 3 isolates as genotype 2, respectively. Given these results, both genotypes of P. zopfii could exist in cow-barn surroundings, but no sites were identified as frequent sources of P. zopfii genotype 2. P. zopfii isolates should thus be further explored with regard to genotype to clarify the reservoir of etiological agents in bovine Prototheca mastitis.

Comparative study of ability for growth support and species differentiation by colony features on commercial chromogenic agar, Pourmedia Vi Candida and CHROMagar Candida.

Ability for growth support and species differentiation by colony features were compared on two commercial chromogenic agars, CHROMagar Candida and newly developed Pourmedia Vi Candida. Eleven strains (ten species) of standard strains and twenty-four isolates (five species) of clinical strains were tested. All isolates were grown on both agar plates in 48 hours at 35 degrees C, however, several species had not matured in 22 hours. Color of the colonies for each strain were stable on both agars. The results show that Pourmedia Vi Candida is equivalent to CHROMagar Candida in its ability to differentiate species as a primary culture plate.

Development of safety culture tube for molds and proposed procedure for collecting conidia or fixing strains to control fungal infection and allergy.

The conidia of filamentous fungi can be easily blown into the air and tend to be contaminants in the laboratory environment. We developed a new "safety culture tube for fungi" to prevent biohazards and a procedure for collecting conidia for passage or fixing strains was proposed.

[Evaluation of a new chromogenic medium for isolation of MRSA].

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections, and accurate methods to detect such strains are needed. We tested the susceptibility of 3 kinds of MRSA isolation medium, MRSA Screen Agar, Oxacillin Resistance Screening Agar and CHROMagar MRSA. Both sensitivity and specificity of CHROMagar MRSA were 100%. The sensitivity and specificity of both MRSA Screen Agar and Oxacillin Resistance Screening Agar were 100% and 91.5%, respectively. It is suggested that CHROMagar MRSA is a useful medium to detect MRSA including mecA positive and oxacillin susceptible strains.

Detection and identification of genotypes of Prototheca zopfii in clinical samples by quantitative PCR analysis.

In this study, a specific quantitative PCR system for the detection and identification of Prototheca zopfii genotypes was developed using a TaqMan(®) MGB probe and ResoLight dye. The P. zopfii-specific primers 18PZF1 and 18PZR1 were generated on the basis of the alignment of the small subunit ribosomal DNA domain base sequences of the genera Chlorella and Prototheca obtained from DDBJ/EMBL/GenBank, and the TaqMan(®) MGB probe PZP1 was designed corresponding to this amplification region. Analysis of the melting curves of the amplicons using ResoLight dye was able to differentiate between P. zopfii genotypes 1 and 2. The specificity of this detection system was examined using strains from a culture collection (28 strains) and clinical isolates (140 strains). The TaqMan(®) MGB probe amplicon was detected only in reference strains of P. zopfii (n = 12) and clinical isolates (n = 135). Ninety-two clinical specimens from cows with mastitis (36 samples) and healthy controls (56 samples) were also tested. All isolates from milk samples (n = 92) and clinical isolates (n = 135) were identified as P. zopfii genotype 2.

In vitro algaecide effect of disinfectants on Prototheca zopfii genotypes 1 and 2.

Bovine mastitis due to Prototheca zopfii leads to reduced milk production and is difficult to cure. Therefore, prevention is the best approach and this is best achieved through the use of effective disinfectants. The aim of this study was to evaluate the in vitro algaecide efficacy of conventional disinfectants against strains of P. zopfii genotype 1 and 2. The minimal algaecide concentration (MAC) of alkyldiaminoethylglycine hydrochloride, chlorhexidine, dioxide chlorine, povidone iodine and sodium hypochlorous acid against 10 isolates and the type strain (SAG2063(T)) of P. zopfii genotype 1 as well as 10 isolates and the type strain (SAG2021(T)) of P. zopfii genotype 2 were examined using the micro dilution method. This in vitro study indicated that alkyldiaminoethylglycine hydrochloride, chlorhexidine, povidone iodine and sodium hypochlorous acid, but not dioxide chlorine, are effective against both genotypes of P. zopfii.

Vital growth factors of Malassezia species on modified CHROMagar Candida.

A comparison of several media, i.e., potato dextrose agar with olive oil (Oil-PDA), modified Dixon agar (mDIX) and variations of Leeming and Notman agar (LNA) for the isolation and growth of Malassezia and Candida species was examined. Since LNA supported the highest growth of Malassezia species its key components, i.e., ox bile, glycerol monostearate, glycerol and Tween 60, were added to CHROMagar Candida. All 7 species of Malassezia grew well on this modified medium (LN-CHROM) after incubation for 4 days at 30 degrees C and development was equal to that observed on LNA. Colonies on LN-CHROM were smooth and from pink to dark purple in color. Furthermore, the use of LN-CHROM did not alter the colony characteristics of Candida species as compared to that found on CHROMagar Candida. The results of the present investigation indicate that the use of LN-CHROM would make possible the simultaneous isolation and identification of Malassezia and Candida species.