Synergistic effects of starch nanoparticles and chitin nanofibers on the stability of oil-in-water Pickering emulsions.

Starch nanoparticles (SNPs) and Chitin nanofibers (ChFs) have been recognized to be effective for emulsion stabilization. Hence, the use of multiple solid nanoparticles seems to be a promising approach to improve emulsion stability. This work aims to studyemulsions stabilized by a combination of SNPs and ChFs at different concentrations over storage time and different environmental conditions. Sonicated emulsions were found to have a significantly higher stability compared to non-sonicated emulsions. Furthermore, SNP/ChF-stabilized emulsions showed smaller droplet sizes and higher stability within a wide range of temperatures and pH, suggesting a synergistic effect between both particles as stabilizers. The addition of NaCl showed limited impact, particularly in concentrations up to 200 mM, on the improvement of the stability of emulsions. The combined use of SNPs and ChFs allowed emulsion stabilization at lower solid nanoparticles concentrations than when only either SNPs or ChFs were used.

Correction: Curcumin encapsulation in Pickering emulsions co-stabilized by starch nanoparticles and chitin nanofibers.

[This corrects the article DOI: 10.1039/D1RA01622A.].

Curcumin encapsulation in Pickering emulsions co-stabilized by starch nanoparticles and chitin nanofibers.

This study examined the stability and release of curcumin encapsulated in Pickering emulsions stabilized by starch nanoparticles and chitin nanofibers under different conditions. Curcumin stability under UV exposure and the release of curcumin from Pickering emulsions in excess water were evaluated over 24 h; while the storage stability of curcumin was assessed over 16 d. The amount of curcumin remaining in the emulsions was quantified spectrophotometrically to characterize its stability and kinetics of release. The progress of lipid oxidation was also monitored by determining peroxide (PV) and p-anisidine (AV) values. The results of passive release measurements indicated over 60% of curcumin was retained after 24 h. SNP/ChF-stabilized Pickering emulsions showed approximately 50% and 45% of curcumin retention upon 16 d of storage and under UV exposure, respectively. Moreover, significant improvement in the curcumin retention was found when higher concentrations of both solid nanoparticles were used. The degradation kinetics of curcumin over storage time and under UV exposure were found to follow first order kinetics. When both emulsifiers were doubled (C4S2), shelf-life was extended to longer than 60 d (AV < 10). This study provides a promising approach to protect encapsulated curcumin, which could potentially be used in functional food products with extended shelf-life.

First case of neurognathostomiasis in Taiwan--A Thai laborer presenting with eosinophilic meningitis and intracranial hemorrhage.

We report a case of neurognathostomiasis in a Thai laborer for the first time in Taiwan. For patients with eosinophilic meningitis, neurognathostomiasis should be considered when brain image discloses subarachnoid or intracranial hemorrhage and when an appropriate exposure risk is available, especially a history of raw freshwater fish consumption in endemic areas, even a long time ago.

Infrequent cross-transmission of Shigella flexneri 2a strains among villages of a mountainous township in Taiwan with endemic shigellosis.

BACKGROUND: Shigellosis is rare in Taiwan, with an average annual incidence rate of 1.68 cases per 100,000 persons in 2000-2007. However, the incidence rate for a mountainous township in eastern Taiwan, Zhuoxi, is 60.2 times the average rate for the entire country. Traveling between Zhuoxi's 6 villages (V1-V6) is inconvenient. Disease transmission among the villages/tribes with endemic shigellosis was investigated in this study. METHODS: Demographic data were collected in 2000-2010 for epidemiological investigation. Thirty-eight Shigella flexneri 2a isolates were subjected to pulsed-field gel electrophoresis (PFGE) genotyping and antimicrobial susceptibility testing (AST). RESULTS: Fifty-five shigellosis cases were identified in 2000-2007, of which 38 were caused by S. flexneri 2a from 2000-2007, 16 cases were caused by S. sonnei from 2000-2003, and 1 case was caused by S. flexneri 3b in 2006. S. flexneri 2a caused infections in 4 of the 6 villages of Zhuoxi Township, showing the highest prevalence in villages V2 and V5. PFGE genotyping categorized the 38 S. flexneri 2a isolates into 2 distinct clusters (clones), 1 and 2. AST results indicated that most isolates in cluster 1 were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and trimethoprim-sulfamethoxazole (ACSSuX); all isolates in cluster 2 were resistant to ACSSuX and tetracycline. Genotypes were primarily unique to different villages or tribes. Tribe V2-1 showed the highest endemic rates. Eighteen isolates recovered from V2-1 tribe members fell into 6 genotypes, where 5 were the same clone (cluster 1). An outbreak (OB2) in 2004 in village V2 was caused by different clonal strains; cases in tribe V2-1 were caused by 2 strains of clone 1, and those in tribe V2-2 were infected by a strain of clone 2. CONCLUSIONS: From 2000-2007, 2 S. flexneri 2a clones circulated among 4 villages/tribes in the eastern mountainous township of Zhuoxi. Genotyping data showed restricted disease transmission between the villages and tribes, which may be associated with difficulties in traveling between villages and limited contact between different ethnic aborigines. Transmission of shigellosis in this township likely occurred via person-to-person contact. The endemic disease was controlled by successful public health intervention.

Molecular characterization of clinical and environmental Vibrio parahaemolyticus isolates in Taiwan.

Vibrio parahaemolyticus is the most prevalent foodborne pathogen in Taiwan and it is frequently recovered from seafood. In this study, V. parahaemolyticus that was isolated in recent years from aquacultural environments and clinical specimens were comparatively analyzed by NotI-restricted pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction, targeting common toxin genes (tdh, trh, ureC), MTase gene, toxR regulator, markers for pandemic strains (ORF8, group-specific toxRS) and representative genes of type three secretion systems T3SS1 (vcrD1, VP1680, vopD) and T3SS2α (vcrD2, vopD2, vopB2, vopP, vopC, vopT). Among the 48 clinical isolates and 93 environmental isolates that were analyzed by PFGE, a total of 26 and 76 pulsetypes were identified and grouped into six and nine clusters, respectively, at 80% similarity. The pandemic O3:K6 clones and other clinical and environmental isolates were further characterized according to the distribution of these examined target genes. The MTase gene and the vopB2, vopP, vopC and vopT genes of T3SS2α were present at a significantly higher frequency (>90%) in the pandemic clones than in other clinical isolates. The MTase gene and some other virulence-associated genes were also present in a few of the environmental isolates, and these results suggest the horizontal transfer of these genes in the clinical and environmental isolates of this species.

Effective interventions and decline of antituberculosis drug resistance in Eastern Taiwan, 2004-2008.

BACKGROUND: The Taiwan health authority recently launched several tuberculosis (TB) control interventions, which may have an impact on the epidemic of drug-resistant TB. We conducted a population-based antituberculosis drug resistance surveillance program in Eastern Taiwan to measure the proportions of notified TB patients with anti-TB drug resistance and the trend from 2004 to 2008. METHODS AND FINDINGS: All culture-positive TB patients were enrolled. Drug susceptibility testing results of the first isolate of each TB patient in each treatment course were analyzed. In total, 2688 patients were included, of which 2176 (81.0%) were new TB cases and 512 (19.0%) were previously treated cases. Among the 2176 new TB cases, 97 (4.5%) were retreated after the first episode of TB treatment within the study period. The proportion of new patients with any resistance, isoniazid resistance but not multidrug-resistant TB (resistant to at least isoniazid and rifampin, MDR-TB), and MDR-TB was 16.4%, 7.5%, and 4.0%, respectively, and that among previously treated cases was 30.9%, 7.9%, and 17.6%, respectively. The combined proportion of any resistance decreased from 23.3% in 2004 to 14.3% in 2008, and that of MDR-TB from 11.5% to 2.4%. CONCLUSIONS: The proportion of TB patients with drug-resistant TB in Eastern Taiwan remains substantial. However, an effective TB control program has successfully driven the proportion of drug resistance among TB patients downward.

Pilot study of twice-weekly therapy for pulmonary tuberculosis in Taiwan.

BACKGROUND/PURPOSE: Taiwan has never implemented supervised intermittent chemotherapy for pulmonary tuberculosis, and its application and results are unknown. The purpose of this study was to evaluate the efficacy and feasibility of a 62-dose, four-drug, 6-month, twice-weekly regimen administered by directly observed therapy for the treatment of pulmonary tuberculosis. METHODS: From January 2004 through December 2006, a total of 116 patients with suspected or confirmed pulmonary tuberculosis were enrolled. Thirty-two of these patients were excluded from the analysis because of drug resistance, negative culture, self withdrawal and drug reactions. Isoniazid, rifampin, pyrazinamide, and ethambutol were administered daily for 2 weeks, followed by twice weekly at a higher dose for 6 weeks. Thereafter, isoniazid, ethambutol and rifampin were administered twice weekly for 18 weeks. Treatment was directly observed by nurses or outreach workers. RESULTS: Of the 84 evaluable patients, 75 [89.3%, 95% confidence interval (CI): 80.6s-95.0%] patients were considered as successfully treated, three (3.6%, 95% CI: 0.7-10.1%) transferred out, five (6.0%, 95% CI: 2.0-13.3%) were unsuccessful and one (1.2%, 95% CI: 0.03-6.5%) died. Five patients were treatment failure (6.0%, 95% CI: 2.0-13.3%). Three patients had recurrences of pulmonary tuberculosis, and the recurrences were 4, 6 and 15 months after the completion of therapy. Sixpatients (5.2%) had severe adverse drug reactions and had their regimen modified. CONCLUSION: Prior to this study, intermittent therapy has not been attempted in Taiwan. This four-drug mostly twice-weeldy pulmonary tuberculosis treatment regimen is efficacious and relatively nontoxic, with a higher treatment success rate and a lower death rate. It considerably simplifies treatment and facilitates the execution of directly observed therapy. The reasons for the relatively high failure rate require further studies.

An outbreak of human metapneumovirus infection in hospitalized psychiatric adult patients in Taiwan.

Human metapneumovirus (hMPV) is a paramyxovirus that is associated with respiratory tract infection (RTI) mostly in children, but these outbreaks have rarely been reported in adults. We encountered an outbreak of this disease involving 10 adults in a psychiatric ward in eastern Taiwan. The nasopharyngeal swab specimens from 13 patients with symptoms of RTI were obtained and analyzed. The RT-PCR tests were negative to influenza virus A/B, adenovirus, RSV, parainfluenza virus, coronavirus, Nipah virus and Legionella. The antigen tests were negative to Legionella, Chlamydia, and Mycoplasma. Blood culture was negative in all except patient no. 1, who was found positive for coagulase-negative staphylococci. The hMPV was identified in 10 of 13 adults (77%), but negative for the other virus. Cough was present in all (100%), fever in 90%, and X-ray evidence of pneumonia in 7 patients. One patient died of respiratory failure. We report this outbreak in a mental hospital to alert the medical profession that this unusual infection of hMPV can occur as an outbreak in an adult setting and is an occupational hazard for healthcare personnel.

Multilocus variable-number tandem repeat analysis for molecular typing and phylogenetic analysis of Shigella flexneri.

BACKGROUND: Shigella flexneri is one of the causative agents of shigellosis, a major cause of childhood mortality in developing countries. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related bacterial isolates for investigation of disease outbreaks and provide information for establishing phylogenetic patterns among isolates. The present study aimed to develop an MLVA method for S. flexneri and the VNTR loci identified were tested on 242 S. flexneri isolates to evaluate their variability in various serotypes. The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) to compare the discriminatory power and to evaluate the usefulness of MLVA as a tool for phylogenetic analysis of S. flexneri. RESULTS: Thirty-six VNTR loci were identified by exploring the repeat sequence loci in genomic sequences of Shigella species and by testing the loci on nine isolates of different subserotypes. The VNTR loci in different serotype groups differed greatly in their variability. The discriminatory power of an MLVA assay based on four most variable VNTR loci was higher, though not significantly, than PFGE for the total isolates, a panel of 2a isolates, which were relatively diverse, and a panel of 4a/Y isolates, which were closely-related. Phylogenetic groupings based on PFGE patterns and MLVA profiles were considerably concordant. The genetic relationships among the isolates were correlated with serotypes. The phylogenetic trees constructed using PFGE patterns and MLVA profiles presented two distinct clusters for the isolates of serotype 3 and one distinct cluster for each of the serotype groups, 1a/1b/NT, 2a/2b/X/NT, 4a/Y, and 6. Isolates that had different serotypes but had closer genetic relatedness than those with the same serotype were observed between serotype Y and subserotype 4a, serotype X and subserotype 2b, subserotype 1a and 1b, and subserotype 3a and 3b. CONCLUSIONS: The 36 VNTR loci identified exhibited considerably different degrees of variability among S. flexneri serotype groups. VNTR locus could be highly variable in a serotype but invariable in others. MLVA assay based on four highly variable loci could display a comparable resolving power to PFGE in discriminating isolates. MLVA is also a prominent molecular tool for phylogenetic analysis of S. flexneri; the resulting data are beneficial to establish clear clonal patterns among different serotype groups and to discern clonal groups among isolates within the same serotype. As highly variable VNTR loci could be serotype-specific, a common MLVA protocol that consists of only a small set of loci, for example four to eight loci, and that provides high resolving power to all S. flexneri serotypes may not be obtainable.

Immunoproteomic identification of the hypothetical protein NMB1468 as a novel lipoprotein ubiquitous in Neisseria meningitidis with vaccine potential.

Many potential vaccine candidates for serogroup B Neisseria meningitidis (NMB) have been identified by reverse vaccinology, a genome-based approach. However, some candidates may be unseen owing to uncertain annotation or their peculiar properties. In this study, we describe the preparation and identification of a novel lipoprotein expressed in all meningococcal strains tested. mAb were first prepared from mice immunized with a meningococcal B strain isolated in Taiwan. Total proteins from the immunizing strain were separated by 2-DE in duplicate. Clone 4-7-3, which crossreacted to 174 tested meningococcal isolates, was used as the primary antibody for Western blotting. The immunoreactive spot was identified by LC-mass spectrometric analysis of the corresponding spot from the silver-stained gel and confirmed by molecular biology approach to be a novel lipoprotein encoded by the hypothetical NMB1468 gene. The potential use of this protein, designated Ag473/NMB1468, as a vaccine component was evaluated using the recombinant protein produced in Escherichia coli. Immunized mice were found to be protected from developing meningococcal disease after intraperitoneal inoculation with a lethal dose of meningococcal strain Nm22209, suggesting that Ag473/NMB1468 may be a promising vaccine candidate. This study also demonstrates the usefulness of the immunoproteomic approach in identification of novel vaccine candidates.

Treatment outcome of pulmonary tuberculosis in eastern Taiwan - experience at a medical center.

BACKGROUND/PURPOSE: The World Health Organization (WHO) has proposed that treatment outcome is an important indicator of tuberculosis control. This study investigated the outcome of tuberculosis treatment at a medical center in eastern Taiwan. METHODS: A total of 166 pulmonary tuberculosis patients notified by Tzu Chi Hospital in 2002 were included in this study. Treatment outcome data were collected at the local level and categorized according to WHO recommendations as cured, treatment completed, failed, died, defaulted, or transferred. Outcomes of the 166 patients, as reported by the National Tuberculosis Program were obtained from the Taiwan Center for Disease Control (Taiwan CDC) for comparison. RESULTS: Of the 166 patients, outcome was classified as cured in 46 (27.7%), treatment completed in 73 (44.0%), died in 27 (16.3%), treatment failed in five (3.0%), and defaulted in 15 (9.0%). Males were more likely to die or to default than females, and the elderly were more likely to die than younger patients. Patients with comorbidities were significantly more likely to die than patients without (p = 0.025). Patients with a history of tuberculosis were more likely to default (p = 0.050). Smear-positive patients were more likely to fail, and patients without cavitation on chest radiograph were more likely to have successful treatment. Outcomes of 26 (15.7%) cases in this cohort were unavailable (18 cases) or inaccurate (8 cases) on the Taiwan CDC website. CONCLUSION: The unsatisfactory outcome of pulmonary tuberculosis treatment in this medical center in eastern Taiwan highlights the importance of implementing directly observed treatment short course strategy. Improvement in the quality of data reported by the National Tuberculosis Program is urgently needed.

Epidemiology of scrub typhus in eastern Taiwan, 2000-2004.

The epidemiology of scrub typhus in eastern Taiwan was studied by analyzing the data from the CDC Web reporting system. A total of 1,396 cases with 403 confirmed cases were reported in the period of 2000 to 2004. The cases were commonly found in all counties with the highest number in Yuli Township, Hualien County (53 cases) and Taitung City, Taitung County (40 cases). Monthly changes in the number of cases showed epidemic periods in the spring with a peak in May, and again in the fall, with an October-November peak. The occurrence of disease varied with age, gender, and occupation. Our results showed that the infection rates in the elderly (50-69 years old), males (62.8%), and farmers (25.6%) were higher than those in other age groups, females, and other occupations. Five major clinical symptoms, fever, headache, eschar, rash, and lymphadenopathy, were observed in 90.1, 61.9, 23.1, 21.6, and 10.7% of the cases, respectively. Almost 90% (89.3%) of the cases showed 1-3 clinical symptoms and some showed 4-5 symptoms (10%). Only one patient with no symptoms (0.8%) was found. This paper reports the status of scrub typhus in eastern Taiwan, and suggests that a health education program could train individuals to self-recognize the disease symptoms.

The suitable restriction enzymes for pulsed-field gel electrophoresis analysis of Bordetella pertussis.

We searched the restriction enzymes with rare cutting sites on the genome of Bordetella pertussis strain Tohama I using the Restriction Digest Tool software provided in the Institute for Genomic Research web site. The usefulness of 5 enzymes for pulsed-field gel electrophoresis (PFGE) analysis was evaluated with 68 B. pertussis isolates. The results indicated that AflII, DraI, SpeI, and XbaI were useful enzymes, and AflII was the best one for PFGE analysis of B. pertussis isolates.

Antigenic diversity of Neisseria meningitidis isolated in Taiwan between 1995 and 2002.

The geographic distribution of N. meningitidis is diverse. Information on the antigenic variation of N. meningitidis is important for the development of an outer membrane protein-based vaccine. As a first step towards vaccine development, serological typing was performed to determine the antigenic properties of 127 invasive N. meningitidis isolates collected in Taiwan between 1995 and 2002. With 31.5% non-serotypeable and 32.3% non-serosubtypeable, the 127 isolates fell into 51 phenotypes, with W135:NT:P1.5,2:L3,7,9, Y:14P1.5,2, and B:1:NST:L3,7,9 being the 3 most prevalent. Among the 37 serogroup B isolates, 15 serosubtypes were found, with P1.5,2 and P1.12,13 being the most prevalent. The high diversity of Por A among serogroup B isolates circulating in Taiwan poses a great challenge for the development of a PorA-based vaccine. Because 85% of the serogroup B isolates had the L3,7,9 immunotype, inclusion of L3,7,9 lipooligosaccharides in a PorA-based vaccine may be a promising approach. In addition, based on the phenotypic characterization, we suggest that both serogroup B and W135 isolates were endemic and that serogroup A, C, and Y isolates were imported, which may reflect increased international travel.

Molecular epidemiology and emergence of worldwide epidemic clones of Neisseria meningitidis in Taiwan.

BACKGROUND: Meningococcal disease is infrequently found in Taiwan, a country with 23 million people. Between 1996 and 2002, 17 to 81 clinical cases of the disease were reported annually. Reported cases dramatically increased in 2001-2002. Our record shows that only serogroup B and W135 meningococci have been isolated from patients with meningococcal disease until 2000. However, serogroup A, C and Y meningococci were detected for the first time in 2001 and continued to cause disease through 2002. Most of serogroup Y meningococcus infections localized in Central Taiwan in 2001, indicating that a small-scale outbreak of meningococcal disease had occurred. The occurrence of a meningococcal disease outbreak and the emergence of new meningococcal strains are of public health concern. METHODS: Neisseria meningitidis isolates from patients with meningococcal disease from 1996 to 2002 were collected and characterized by serogrouping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genetic relatedness and clonal relationship between the isolates were analyzed by using the PFGE patterns and the allelic profiles of the sequence types (STs). RESULTS: Serogroups A, B, C, W135, Y, and non-serogroupable Neisseria meningitidis were, respectively, responsible for 2%, 50%, 2%, 35%, 9%, and 2% of 158 culture-confirmed cases of meningococcal disease in 1996-2002. Among 100 N. meningitidis isolates available for PFGE and MLST analyses, 51 different PFGE patterns and 30 STs were identified with discriminatory indices of 0.95 and 0.87, respectively. Of the 30 STs, 21 were newly identified and of which 19 were found in serogroup B isolates. A total of 40 PFGE patterns were identified in 52 serogroup B isolates with the patterns distributed over several distinct clusters. In contrast, the isolates within each of the serogroups A, C, W135, and Y shared high levels of PFGE pattern similarity. Analysis of the allelic profile of the 30 STs suggested the serogroup B isolates be assigned into 5 clonally related groups/ clonal complexes and 7 unique clones. The ST-41/44 complex/Lineage 3, and the ST-3439 and ST-3200 groups represented 79% of the serogroup B meningococci. In contrast, isolates within serogroups A, serogroup W135 (and C), and serogroup Y, respectively, simply belonged to ST-7, ST-11, and ST-23 clones. CONCLUSION: Our data suggested that serogroup B isolates were derived from several distinct lineages, most of which could either be indigenous or were introduced into Taiwan a long time ago. The serogroup A, W135 (and C), and Y isolates, respectively, belonged to the ST-7, ST-11, and ST-23, and the represented clones that are currently the major circulating clones in the world and are introduced into Taiwan more recently. The emergence of serogroup A, C and Y strains contributed partly to the increase in cases of meningococcal disease in 2001-2002.

Molecular epidemiology of Shigella flexneri in a long-stay psychiatric nursing center during 2001 to 2003.

With six separate wards accommodating more than 1,600 patients, V Nursing Center (VNC) is a long-stay psychiatric nursing center in eastern Taiwan. During 2001 to 2003, 39 shigellosis cases occurred in VNC. Different from the notion that most cases of shigellosis are caused by Shigella sonnei, all except one of these cases were caused by S. flexneri, with the remaining one caused by an S. sonnei isolate. O-antigen serotyping showed that the 38 S. flexneri strains were of either type 1a (n = 20) or 4a (n = 18), two less prevalent serotypes in Taiwan. NotI-based pulsed-field gel electrophoresis analyses performed with 8 type 1a non-VNC strains and 9 type 4a non-VNC strains isolated from 1996 to 2003 for comparison divided the 28 type 1a strains and the 27 type 4a strains into 7 and 10 subtypes, designated subtypes P1A to P1G and subtypes P4A to P4J, respectively. Subtypes P1A and P4A, which appeared in three consecutive years in VNC as well as outside of VNC, are the most prevalent subtypes. Analyses of the relatedness of the VNC strains on the basis of the banding patterns grouped the type 1a and 4a strains into four and five clusters, respectively. All except one of the type 1a strains had 95% similarity, indicating that they had a common parent, whereas the type 4a strains had similarities that ranged from 77 to 93%, suggesting that they were of diverse origins. In two of the outbreaks, less related subtypes of the type 4a strains were found in the same VNC wards in consecutive years, suggesting the possible existence of different subtypes in VNC all the time. Antibiotic susceptibility testing showed that all except one of the S. flexneri strains were sensitive to at least seven antibiotics; the remaining isolate was sensitive to three antibiotics. The data from the latter tests should be helpful for selection of proper treatments for S. flexneri infections in Taiwan.

Biochemical and virulence characterization of viable but nonculturable cells of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer. Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist. This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V. parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt-0.5% NaCl medium incubated at 4 degrees C. The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed. The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state. Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells. Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells. Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation. Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V. parahaemolyticus enters into the VBNC state.

Cloning and characterization of the lipase and lipase activator protein from Vibrio vulnificus CKM-1.

The gene (lipA) encoding the extracellular lipase and its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned and sequenced. Nucleotide sequence analysis and alignments of amino acid sequences suggest that Lip Ais a member of bacterial lipase family I.1 and that LipB is a lipase activator of LipA. The active LipA was produced in recombinant Escherichia coli cells only in the presence of the lipB. In the hydrolysis of p-nitrophenyl esters and triacylglycerols, using the reactivated LipA, the optimum chain lengths for the acyl moiety on the substrate were C14 for ester hydrolysis and C10 to C12 for triacylglycerol hydrolysis.

Molecular epidemiology of Bordetella pertussis isolated in Taiwan, 1992-1997.

In Taiwan, the number of pertussis cases including various types of infection has been increasing in recent years, especially in 1997. Since 71% of the reported cases concentrated in the densely populated Taipei metropolitan area, concerns have been raised that a highly contagious strain of Bordetella pertussis might have appeared in Taipei. In this study, 114 strains of B. pertussis including those isolated in 1992-1996 (n = 53) and 1997 (n = 61) were subjected to pulsed-field gel electrophoresis (PFGE) of the Xba I digests from their chromosomes. Based on the band patterns, they were divided into 21 subtypes, P1 to P21. The strains isolated in 1997 consist of 17 subtypes including 9 new subtypes which did not appear in the previous years, indicating that the outbreaks in 1997 were not caused by a sole specific virulent strain. Dendrogram analysis indicated that the 21 subtypes can be grouped into five clusters, with the first four subtypes possessing 60 to 95% relatedness to one another, whereas relatedness between cluster 5 (containing P21 only) and the other clusters is less than 50%. Notably, all the subtypes except P12 and P21 appeared at least once in Taipei and the majority of the strains (54%) belong to two clusters, 3 and 4. These results suggest that highly dense population may facilitate spread and accelerate genetic divergence of this pathogen. This is the first report on pertussis molecular epidemiology in Taiwan.