Development of Multiplex Nested PCR for Simultaneous Detection of Ectoparasitic Fungi Laboulbeniopsis termitarius and Antennopsis gallica on Reticulitermes speratus (Blattodea: Rhinotermitidae).

Laboulbeniopsis termitarius (Thaxt) and Antennopsis gallica (Buchli and Heim) are two of the most common ectoparasitic fungi found on the body surface of termites. While visual observation under a dissecting microscope is a common method used to screen for such fungi, it generally requires a large number of termites and is thus very time consuming. In this study, we develop a fast, efficient protocol to detect fungal infection on the termite Reticulitermes speratus (Kolbe). Species-specific primers were designed based on sequence data and amplified using a number of universal fungus primer pairs that target partial sequences of the 18s rRNA gene of the two fungi. To detect these fungi in a robust yet economic manner, we then developed a multiplex nested polymerase chain reaction assay using species-specific primers. Results suggested that both fungi could be successfully detected, even in cases where L. termitarius was at low titer (e.g., a single thallus per termite). The new method described here is recommended for future surveys of these two fungi, as it is more sensitive, species specific, and faster than visual observation, and is likely to facilitate a better understanding of these fungi and their dynamics in host populations.

Toluidine blue O photodynamic inactivation on multidrug-resistant Pseudomonas aeruginosa.

BACKGROUND AND OBJECTIVES: Multidrug-resistant (MDR) Pseudomonas aeruginosa infection is becoming a critical problem worldwide. Currently, only limited therapeutic options are available for the treatment of infections caused by MDR P. aeruginosa, therefore, the development of new alternative treatments is needed. Toluidine blue O (TBO) is an effective antibacterial photosensitizing agent against various bacteria. However, reports on antibacterial photosensitization of MDR bacteria are limited. This study aims to determine the in vitro photobactericidal activity of TBO against MDR P. aeruginosa. STUDY DESIGN/MATERIALS AND METHODS: The efficacy of antibacterial photodynamic inactivation, DNA fragmentation and protein carbonylation of three MDR P. aeruginosa strains and one susceptible strain was compared using TBO as the photosensitizer followed by red light irradiation (630 nm, 90 J/cm(2)) from a light-emitting diode light source. Subsequently, the efficacy of TBO photodynamic inactivation (TBO-PDI) on 60 MDR strains, including 11 with the efflux pump phenotype and 49 with no pump activity, was tested using the minimum lethal drug concentration (MLC) assay. RESULTS: TBO-PDI caused similar bactericidal effect (6-7 logs of killing effect), DNA fragmentation and protein carbonylation in three MDR and one susceptible P. aeruginosa strains. Although the TBO accumulation assay indicated that TBO is a substrate for the efflux pump, TBO-PDI produce similar photobactericidal activity against 60 MDR P. aeruginosa strains, either with or without efflux-pump phenotype, and 19 susceptible strains. CONCLUSION: MDR did not affect the susceptibility of P. aeruginosa strains to TBO-PDI. The efflux pump played an insignificant role in TBO-PDI of MDR P. aeruginosa.

Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital.

The staphylococcal chromosome cassette (SCC)mec types of 382 hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates in Taiwan were analysed over a 7-year period (1999-2005). There was an abrupt increase in SCCmec type IV in HA-MRSA during 2005. The molecular epidemiology of a subset (n = 69) of HA-MRSA isolates with SCCmec types III, IV or V was characterised and compared with that of community-acquired MRSA (CA-MRSA) (n = 26, collected during 2005). Pulsed-field gel electrophoresis revealed three major pulsotypes (A, B and C) and 15 minor clones. Pulsotypes B and C, which contained isolates carrying SCCmec types IV and V, respectively, included both CA-MRSA and HA-MRSA isolates. Among 24 toxin genes analysed, five genes had significant differential distribution between CA-MRSA and SCCmec type III HA-MRSA. Furthermore, among SCCmec type IV isolates, the seb gene was detected more commonly in HA-MRSA. Analysis of representative members of the three major pulsotypes by multilocus sequence typing revealed two sequence types (STs), namely ST239 (SCCmecIII) and ST59 (SCCmecIV or SCCmecV). This suggests that ST59:SCCmecIV, which is usually community-acquired, has become an important nosocomial pathogen in the hospital studied.

Typhoid fever and typhoid hepatitis in Taiwan.

The annual incidence of typhoid fever in Taiwan was 2.1-3.6 cases per 1,000,000 population from 1995 to 2002. More than 80% of 45 patients with typhoid fever treated at National Taiwan University Hospital from 1996 to 2002 had elevated serum aminotransferase levels at presentation. Ten of these patients were treated during an outbreak in Taipei County in 2002, and seven of them who did not have pre-existing liver disease developed hepatitis, which was unrelated to other aetiologies. All Salmonella typhi isolates were susceptible to extended-spectrum cephalosporins and fluoroquinolones. Multidrug resistance (intermediate resistance to ampicillin, trimethoprim-sulphamethoxazole, and chloramphenicol) was found in one (2.5%) of the 40 isolates studied. Pulsed-field gel electrophoresis analysis demonstrated a high genetic diversity among S. typhi isolates and identified a novel clone associated with the 2002 outbreak. Physicians should be alert to the possibility of typhoid fever when patients, without other gastrointestinal symptoms, present with sustained fever and hepatitis.

Pan-drug-resistant Pseudomonas aeruginosa causing nosocomial infection at a university hospital in Taiwan.

This study evaluated the clinical and microbiological characteristics of 16 patients who were colonised or infected with 26 isolates of pan-drug-resistant Pseudomonas aeruginosa (PDRPA; intermediately-resistant or resistant to all cephalosporins, piperacillin-tazobactam, aztreonam, carbapenems, ciprofloxacin and aminoglycosides) in a university hospital during 1999-2002. All the isolates had colistin MICs < or = 4 mg/L, 19 (73%) isolates had bla(VIM-3), and 25 (96%) isolates had class I integrons (intI). Time-kill studies for two PDRPA blood isolates demonstrated synergism for cefepime-amikacin after 24 h. Pulsed-field gel electrophoresis analysis of the isolates revealed a polyclonal nature (12 pulsotypes), although clonal dissemination of PDRPA isolates among these patients was also present.