Retrospective study of necrotizing fasciitis and characterization of its associated methicillin-resistant Staphylococcus aureus in Taiwan.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a prevalent pathogen of necrotizing fasciitis (NF) in Taiwan. A four-year NF cases and clinical and genetic differences between hospital acquired (HA)- and community-acquired (CA)-MRSA infection and isolates were investigated. METHODS: A retrospective study of 247 NF cases in 2004-2008 and antimicrobial susceptibilities, staphylococcal chromosomal cassette mec (SCCmec) types, pulsed field gel electrophoresis (PFGE) patterns, virulence factors, and multilocus sequence typing (MLST) of 16 NF-associated MRSA in 2008 were also evaluated. RESULTS: In 247 cases, 42 microbial species were identified. S. aureus was the major prevalent pathogen and MRSA accounted for 19.8% of NF cases. Most patients had many coexisting medical conditions, including diabetes mellitus, followed by hypertension, chronic azotemia and chronic hepatic disease in order of decreasing prevalence. Patients with MRSA infection tended to have more severe clinical outcomes in terms of amputation rate (p < 0.05) and reconstruction rate (p = 0.001) than those with methicillin-sensitive S. aureus or non-S. aureus infection. NF patients infected by HA-MRSA had a significantly higher amputation rate, comorbidity, C-reactive protein level, and involvement of lower extremity than those infected by CA-MRSA. In addition to over 90% of MRSA resistant to erythromycin and clindamycin, HA-MRSA was more resistant than CA-MRSA to trimethoprim-sulfamethoxazole (45.8% vs. 4%). ST59/pulsotype C/SCCmec IV and ST239/pulsotype A/SCCmec III isolates were the most prevalent CA- and HA-MRSA, respectively in 16 isolates obtained in 2008. In contrast to the gene for γ-hemolysin found in all MRSA, the gene for Panton-Valentine leukocidin was only identified in ST59 MRSA isolates. Other three virulence factors TSST-1, ETA, and ETB were occasionally identified in MRSA isolates tested. CONCLUSION: NF patients with MRSA infection, especially HA-MRSA infection, had more severe clinical outcomes than those infected by other microbial. The prevalent NF-associated MRSA clones in Taiwan differed distinctly from the most predominant NF-associated USA300 CA-MRSA clone in the USA. Initial empiric antimicrobials with a broad coverage for MRSA should be considered in the treatment of NF patients in an endemic area.

Clonal dissemination of the multi-drug resistant Salmonella enterica serovar Braenderup, but not the serovar Bareilly, of prevalent serogroup C1 Salmonella from Taiwan.

BACKGROUND: Nontyphoidal Salmonella is the main cause of human salmonellosis. In order to study the prevalent serogroups and serovars of clinical isolates in Taiwan, 8931 Salmonellae isolates were collected from 19 medical centers and district hospitals throughout the country from 2004 to 2007. The pulsed-field eletrophoresis types (PFGE) and antibiotic resistance profiles of Salmonella enterica serovars Bareilly (S. Bareilly) and Braenderup (S. Braenderup) were compared, and multi-drug resistance (MDR) plasmids were characterized. RESULTS: Over 95% of human salmonellosis in Taiwan was caused by five Salmonella serogroups: B, C1, C2-C3, D1, and E1. S. Typhymurium, S. Enteritidis, S. Stanley and S. Newport were the four most prevalent serovars, accounting for about 64% of isolates. While only one or two major serovars from four of the most prevalent serogroups were represented, four predominant serovars were found in serogroup C1 Salmonellae. The prevalence was decreasing for S. Choleraeuis and S. Braenderup, and S. Virchow and increasing for S. Bareilly. S. Braenderup mainly caused gastroenteritis in children; in contrast, S. Bareiley infected children and elderly people. Both serovars differed by XbaI-PFGE patterns. Almost all S. Bareilly isolates were susceptible to antibiotics of interest, while all lacked plasmids and belonged to one clone. Two distinct major clones in S. Braenderup were cluster A, mainly including MDR isolates with large MDR plasmid from North Taiwan, and cluster B, mainly containing susceptible isolates without R plasmid from South Taiwan. In cluster A, there were two types of conjugative R plasmids with sizes ranging from 75 to 130 kb. Type 1 plasmids consisted of replicons F1A/F1B, blaTEM, IS26, and a class 1 integron with the genes dfrA12-orfF-aadA2-qacEDelta1-sulI. Type 2 plasmids belonged to incompatibility group IncI, contained tnpA-blaCMY-2-blc-sugE genetic structures and lacked both IS26 and class 1 integrons. Although type 2 plasmids showed higher conjugation capability, type 1 plasmids were the predominant plasmid. CONCLUSIONS: Serogroups B, C1, C2-C3, D1, and E1 of Salmonella caused over 95% of human salmonellosis. Two prevalent serovars within serogroup C1, S. Bareilly and cluster B of S. Braenderup, were clonal and drug-susceptible. However, cluster A of S. Braenderup was MDR and probably derived from susceptible isolates by acquiring one of two distinct conjugative R plasmids.

Genomic diversity and molecular differentiation of Riemerella anatipestifer associated with eight outbreaks in five farms.

Riemerella anatipestifer causes infectious serositis of ducks and geese. The genomic diversity of R. anatipestifer associated with outbreaks in waterfowls was studied using 24 multidrug-resistant R. anatipestifer isolates collected from the visceral organs of ducks and geese from seven outbreaks in four goose farms and one outbreak in one duck farm. Seven methods were used to differentiate these isolates. Plasmid patterns differed in plasmid number and size, ranging from 2.9 kb to 20 kb, and provided seven profiles. Divergent nucleotide sequences (predominant in 670 to 830 base pairs) of the ompA gene categorized the 24 isolates into three groups based on cluster analysis and polymerase chain reaction-restriction fragment length polymorphism. Repetitive-sequence polymerase chain reaction and pulsed-field gel electrophoresis analysis revealed the highest genotypic variations among the isolates. Genotypes and serotypes differed among farms and within the same farm and even within a single goose. In conclusion, a difference in R. anatipestifer genotypes and serotypes was observed for multiple outbreaks in waterfowls.

Hepatoprotective effects of litchi (Litchi chinensis) procyanidin A2 on carbon tetrachloride-induced liver injury in ICR mice.

Drug tolerance, lacking liver regenerative activity and inconclusive inhibition of steatosis and cirrhosis by silymarin treatment during chronic liver injury have increased the demand for novel alternative or synergistic treatments for liver damage. Litchi fruit is abundant in polyphenolic compounds and is used in traditional Chinese medicine for treatments that include the strengthening of hepatic and pancreatic functions. Unique polyphenolic compounds obtained from litchi pericarp extract (LPE) were studied in vitro and in vivo for hepatoprotection. Epicatechin (EC) and procyanidin A2 (PA2) of LPE were obtained by fractionated-extraction from pulverized litchi pericarps. All fractions, including LPE, were screened against silymarin in carbon tetrachloride (CCl4)-treated murine embryonic liver cell line (BNL). The effects of daily gavage-feeding of LPE, silymarin (200 mg/kg body weight) or H2O in CCl4-intoxicated male ICR mice were evaluated by studying serum chemicals, liver pathology and glutathione antioxidative enzymes. The effects of EC and PA2 on liver cell regenerative activity were investigated using a scratch wound healing assay and flow cytometric cell cycle analysis; the results of which demonstrated that LPE protected BNL from CCl4-intoxication. Gavage-feeding of LPE decreased serum glutamic oxaloacetate transaminase and glutamic pyruvic transaminase levels, and exhibited superior retention of the hexagonal structure of hepatocytes and reduced necrotic cells following liver histopathological examinations in CCl4-intoxicated ICR mice. Glutathione peroxidise and glutathione reductase activities were preserved as the normal control level in LPE groups. EC and PA2 were principle components of LPE. PA2 demonstrated liver cell regenerative activity in scratch wound healing assays and alcohol-induced liver cell injury in vitro. The present findings suggest that litchi pericarp polyphenolic extracts, including EC and PA2, may be a synergistic alternative to silymarin in hepatoprotection and liver cell regeneration.

Polymerase chain reaction-mediated characterization of molds belonging to the Aspergillus flavus group and detection of Aspergillus parasiticus in peanut kernels by a multiplex polymerase chain reaction.

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.

Calvatia lilacina protein-extract induces apoptosis through glutathione depletion in human colorectal carcinoma cells.

This paper reports that a novel protein extract isolated from Calvatia lilacina (CL) can induce cell death against four types of human colorectal cancer cells. Importantly, CL was shown to be free of apoptotic effects against normal rat liver cells. We have also identified that CL-induced glutathione (GSH) depletion is the major contributor responsible for the apoptotic cell death induction of SW 480 cells, as evidenced by the observation that exogenously added N-acetylcysteine (NAC), or GSH, but not vitamin C, could offer a near complete protection of CL-treated cells against apoptotic cell death. Furthermore, the participation of reactive oxygen species (ROS) evoked a drop in the transmembrane potential (Delta Psi(m)) in the CL-induced apoptotic cell death. This observation can only be deemed as a minor pathway due to the fact that cyclosporine A (CyA) could only partially rescue the CL-treated cells from apoptotic cell death. Likewise, despite the fact that CL could induce the upregulation of Bax, its knockdown via siRNA (48 h) failed to completely mitigate apoptotic cell death, indicating that its role in this apoptotic process was insignificant. To further explore the possible underlying mechanism associated with CL-induced GSH depletion, we proceeded to determine the effect of CL on the cellular gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme responsible for GSH biosynthesis, and demonstrated that indeed gamma-GCS could be repressed by CL. Taken together, we report here for the first time that the anticancer effect of CL on human colorectal cancer cells is mediated through GSH depletion mechanism rather than a ROS-mediated killing process. This functional attribute of CL can thus provide the basis for the strategic design of a treatment of colorectal cancer.