Molecular Epidemiology of Tuberculosis in Kaohsiung City Located at Southern Taiwan, 2000-2008

Background

We present the first comprehensive analysis of Mycobacterium tuberculosis (MTB) isolates circulating in southern Taiwan. In this 9-year population-based study, the TB situation in the Kaohsiung region was characterized by genotypic analysis of 421 MTB isolates.

Methods

All 421 isolates of MTB were analyzed by spoligotyping and MIRU-VNTR typing. Drug-resistance patterns were also analyzed.

Results

The percentage of EAI (East African-Indian) strains increased across sampling years (2000–2008) in southern Taiwan, whereas the proportion of Beijing lineages remained unchanged. Clustering was more frequent with EAI genotype infections (odds ratio = 3.6, p<0.0001) when compared to Beijing genotypes. Notably, MTB resistance to streptomycin (STR) had significantly increased over time, but resistance to other antibiotics, including multidrug resistance, had not. Three major genes (gidB, rpsL and rrs) implicated in STR resistance were sequenced and specific mutations identified.

Conclusions

This study revealed that EAI strains were highly transmissible and that STR resistance has increased between 2000 and 2008 in Kaohsiung, Taiwan.     

Common Oncogenic Mutations Are Infrequent in Oral Squamous Cell Carcinoma of Asian Origin

Objectives

The frequency of common oncogenic mutations and TP53 was determined in Asian oral squamous cell carcinoma (OSCC).

Materials and Methods

The OncoCarta^™ panel v1.0 assay was used to characterize oncogenic mutations. In addition, exons 4-11 of the TP53 gene were sequenced. Statistical analyses were conducted to identify associations between mutations and selected clinico-pathological characteristics and risk habits.

Results

Oncogenic mutations were detected in PIK3CA (5.7%) and HRAS (2.4%). Mutations in TP53 were observed in 27.7% (31/112) of the OSCC specimens. Oncogenic mutations were found more frequently in non-smokers (p = 0.049) and TP53 truncating mutations were more common in patients with no risk habits (p = 0.019). Patients with mutations had worse overall survival compared to those with absence of mutations; and patients who harbored DNA binding domain (DBD) and L2/L3/LSH mutations showed a worse survival probability compared to those patients with wild type TP53. The majority of the oncogenic and TP53 mutations were G:C > A:T and A:T > G:C base transitions, regardless of the different risk habits.

Conclusion

Hotspot oncogenic mutations which are frequently present in common solid tumors are exceedingly rare in OSCC. Despite differences in risk habit exposure, the mutation frequency of PIK3CA and HRAS in Asian OSCC were similar to that reported in OSCC among Caucasians, whereas TP53 mutations rates were significantly lower. The lack of actionable hotspot mutations argue strongly for the need to comprehensively characterize gene mutations associated with OSCC for the development of new diagnostic and therapeutic tools.     

Carrier Status for the Common R501X and 2282del4 Filaggrin Mutations Is Not Associated with Hearing Phenotypes in 5377 Children from the ALSPAC Cohort

Background

Filaggrin is a major protein in the epidermis. Several mutations in the filaggrin gene (FLG) have been associated with a number of conditions. Filaggrin is expressed in the tympanic membrane and could alter its mechanical properties, but the relationship between genetic variation in FLG and hearing has not yet been tested.

Methodology/Principal Findings

We examined whether loss-of function mutations R501X and 2282del4 in the FLG gene affected hearing in children. Twenty eight hearing variables representing five different aspects of hearing at age nine years in 5,377 children from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort were tested for association with these mutations. No evidence of association was found between R501X or 2282del4 (or overall FLG mutation carrier status) and any of the hearing phenotypes analysed.

Conclusions/Significance

In conclusion, carrier status for common filaggrin mutations does not affect hearing in children.     

Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients

Background

Oncogenic mutations are powerful predictive biomarkers for molecularly targeted cancer therapies. For mutation detection patients have to undergo invasive tumor biopsies. Alternatively, archival samples are used which may no longer reflect the actual tumor status. Circulating tumor cells (CTC) could serve as an alternative platform to detect somatic mutations in cancer patients. We sought to develop a sensitive and specific assay to detect mutations in the EGFR gene in CTC from lung cancer patients.

Methods

We developed a novel assay based on real-time polymerase chain reaction (PCR) and melting curve analysis to detect activating EGFR mutations in blood cell fractions enriched in CTC. Non-small-cell lung cancer (NSCLC) was chosen as disease model with reportedly very low CTC counts. The assay was prospectively validated in samples from patients with EGFR-mutant and EGFR-wild type NSCLC treated within a randomized clinical trial. Sequential analyses were conducted to monitor CTC signals during therapy and correlate mutation detection in CTC with treatment outcome.

Results

Assay sensitivity was optimized to enable detection of a single EGFR-mutant CTC/mL peripheral blood. CTC were detected in pretreatment blood samples from all 8 EGFR-mutant lung cancer patients studied. Loss of EGFR-mutant CTC signals correlated with treatment response, and its reoccurrence preceded relapse.

Conclusions

Despite low abundance of CTC in NSCLC oncogenic mutations can be reproducibly detected by applying an unbiased CTC enrichment strategy and highly sensitive PCR and melting curve analysis. This strategy may enable non-invasive, specific biomarker diagnostics and monitoring in patients undergoing targeted cancer therapies.     

Serotype Distribution and Antibiotic Susceptibility of Streptococcus pneumoniae Strains Carried by Children Infected with Human Immunodeficiency Virus

Background

We studied the serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae isolates carried by children infected with HIV in Jakarta, Indonesia.

Methods

Nasopharyngeal swabs were collected from 90 HIV infected children aged 4 to 144 months. S. pneumoniae was identified by conventional and molecular methods. Serotyping was performed with sequential multiplex PCR and antibiotic susceptibility with the disk diffusion method.

Results

We identified S. pneumoniae carriage in 41 children (46%). Serotype 19F was most common among 42 cultured strains (19%) followed by 19A and 6A/B (10% each), and 23F (7%). Most isolates were susceptible to chloramphenicol (86%), followed by clindamycin (79%), erythromycin (76%), tetracycline (43%), and sulphamethoxazole/trimethoprim (41%). Resistance to penicillin was most common with only 33% of strains being susceptible. Strains of serotypes targeted by the 13-valent pneumococcal conjugate polysaccharide vaccine (PCV13) were more likely to be multidrug resistant (13 of 25 or 52%) compared to non-PCV13 serotype isolates (3 of 17 or 18%; Fisher exact test p = 0.05).

Conclusion

Our study provides insight into the epidemiology of pneumococcal carriage in young HIV patients in Indonesia. These findings may facilitate potential preventive strategies that target invasive pneumococcal disease in Indonesia.     

Whole-Exome Sequencing to Identify a Novel LMNA Gene Mutation Associated with Inherited Cardiac Conduction Disease

Background

Inherited cardiac conduction diseases (CCD) are rare but are caused by mutations in a myriad of genes. Recently, whole-exome sequencing has successfully led to the identification of causal mutations for rare monogenic Mendelian diseases.

Objective

To investigate the genetic background of a family affected by inherited CCD.

Methods and Results

We used whole-exome sequencing to study a Chinese family with multiple family members affected by CCD. Using the pedigree information, we proposed a heterozygous missense mutation (c.G695T, Gly232Val) in the lamin A/C (LMNA) gene as a candidate mutation for susceptibility to CCD in this family. The mutation is novel and is expected to affect the conformation of the coiled-coil rod domain of LMNA according to a structural model prediction. Its pathogenicity in lamina instability was further verified by expressing the mutation in a cellular model.

Conclusions

Our results suggest that whole-exome sequencing is a feasible approach to identifying the candidate genes underlying inherited conduction diseases.     

Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

Background

Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.

Methodology/Principal Findings

We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.

Conclusions/Significance

This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.     

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Objective

To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential.

Results

Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain.

Conclusion

A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

     

Subtype Distribution of Blastocystis Isolates in Sebha,Libya

Background

Blastocystis is a genetically diverse and a common intestinal parasite of humans with a controversial pathogenic potential. This study was carried out to identify the Blastocystis subtypes and their association with demographic and socioeconomic factors among outpatients living in Sebha city, Libya.

Methods/Findings

Blastocystis in stool samples were cultured followed by isolation, PCR amplification of a partial SSU rDNA gene, cloning, and sequencing. The DNA sequences of isolated clones showed 98.3% to 100% identity with the reference Blastocystis isolates from the Genbank. Multiple sequence alignment showed polymorphism from one to seven base substitution and/or insertion/deletion in several groups of non-identical nucleotides clones. Phylogenetic analysis revealed three assemblage subtypes (ST) with ST1 as the most prevalent (51.1%) followed by ST2 (24.4%), ST3 (17.8%) and mixed infections of two concurrent subtypes (6.7%).

Blastocystis

ST1 infection was significantly associated with female (P = 0.009) and low educational level (P = 0.034). ST2 was also significantly associated with low educational level (P= 0.008) and ST3 with diarrhoea (P = 0.008).

Conclusion

Phylogenetic analysis of Libyan Blastocystis isolates identified three different subtypes; with ST1 being the predominant subtype and its infection was significantly associated with female gender and low educational level. More extensive studies are needed in order to relate each Blastocystis subtype with clinical symptoms and potential transmission sources in this community.     

Genetic Diversity of Staphylococcus aureus in Buruli Ulcer

Background

Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Previous studies have shown that wounds of BU patients are colonized with M. ulcerans and several other microorganisms, including Staphylococcus aureus, which may interfere with wound healing. The present study was therefore aimed at investigating the diversity and topography of S. aureus colonizing BU patients during treatment.

Methodology

We investigated the presence, diversity, and spatio-temporal distribution of S. aureus in 30 confirmed BU patients from Ghana during treatment. S. aureus was isolated from nose and wound swabs, and by replica plating of wound dressings collected bi-weekly from patients. S. aureus isolates were characterized by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa-typing, and antibiotic susceptibility was tested.

Principal Findings

Nineteen (63%) of the 30 BU patients tested positive for S. aureus at least once during the sampling period, yielding 407 S. aureus isolates. Detailed analysis of 91 isolates grouped these isolates into 13 MLVF clusters and 13 spa-types. Five (26%) S. aureus-positive BU patients carried the same S. aureus genotype in their anterior nares and wounds. S. aureus isolates from the wounds of seven (37%) patients were distributed over two different MLVF clusters. Wounds of three (16%) patients were colonized with isolates belonging to two different genotypes at the same time, and five (26%) patients were colonized with different S. aureus types over time. Five (17%) of the 30 included BU patients tested positive for methicillin-resistant S. aureus (MRSA).

Conclusion/Significance

The present study showed that the wounds of many BU patients were contaminated with S. aureus, and that many BU patients from the different communities carried the same S. aureus genotype during treatment. This calls for improved wound care and hygiene.     

No Association between Loss-of-Function Mutations in filaggrin and Diabetes,Cardiovascular Disease,and All-Cause Mortality

Background

Common loss-of-function mutations in the filaggrin gene (FLG) are a major predisposing risk factor for atopic disease due to reduced epidermal filaggrin protein levels. We previously observed an association between these mutations and type 2 diabetes and hypothesized that an inherited impairment of skin barrier functions could facilitate low-grade inflammation and hence increase the risk of diabetes and cardiovascular disease. We examined the association between loss-of-function mutations in FLG and diabetes, stroke, ischemic heart disease (IHD), and all-cause mortality in the general population.

Methods

The R501X and 2282del4 loss-of function mutations in FLG were genotyped in four Danish study populations including a total of 13373 adults aged 15-77 years. Two of the studies also genotyped the R2447X mutation. By linkage to Danish national central registers we obtained information for all participants on dates of diagnoses of diabetes, stroke, and IHD, as well as all-cause mortality. Data were analyzed by Cox proportional hazard models and combined by fixed effect meta-analyses.

Results

In meta-analyses combining the results from the four individual studies, carriage of loss-of-function mutations in FLG was not associated with incident diabetes (hazard ratio (HR) (95% confidence intervals (CI)) = 0.95 (0.73, 1.23), stroke (HR (95% CI) = 1.27 (0.97, 1.65), ischemic heart disease (HR (95%CI) = 0.92 (0.71, 1.19), and all-cause mortality (HR (95%CI) = 1.02 (0.83, 1.25)). Similar results were obtained when including prevalent cases in logistic regression models.

Conclusion

Our results suggest that loss-of-function mutations in FLG are not associated with type 2 diabetes, cardiovascular disease, and all-cause mortality. However, larger studies with longer follow-up are needed to exclude any associations.     

Comparative evaluation of <Emphasis Type="Italic">Populus</Emphasis> variants total sugar release and structural features following pretreatment and digestion by two distinct biological systems

Background

Populus natural variants have been shown to realize a broad range of sugar yields during saccharification, however, the structural features responsible for higher sugar release from natural variants are not clear. In addition, the sugar release patterns resulting from digestion with two distinct biological systems, fungal enzymes and Clostridium thermocellum, have yet to be evaluated and compared. This study evaluates the effect of structural features of three natural variant Populus lines, which includes the line BESC standard, with respect to the overall process of sugar release for two different biological systems.

Results

Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar release from hydrothermal pretreatment combined with either enzymatic hydrolysis or Clostridium thermocellum fermentation compared to the Populus natural variant, BESC standard. However, C. thermocellum outperformed the fungal cellulases yielding 96.0, 95.5, and 85.9% glucan plus xylan release from SKWE 24-2, BESC 876, and BESC standard, respectively. Among the feedstock properties evaluated, cellulose accessibility and glycome profiling provided insights into factors that govern differences in sugar release between the low recalcitrant lines and the BESC standard line. However, because this distinction was more apparent in the solids after pretreatment than in the untreated biomass, pretreatment was necessary to differentiate recalcitrance among Populus lines. Glycome profiling analysis showed that SKWE 24-2 contained the most loosely bound cell wall glycans, followed by BESC 876, and BESC standard. Additionally, lower molecular weight lignin may be favorable for effective hydrolysis, since C. thermocellum reduced lignin molecular weight more than fungal enzymes across all Populus lines.

Conclusions

Low recalcitrant Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar yields than BESC standard when hydrothermal pretreatment was combined with biological digestion. However, C. thermocellum was determined to be a more robust and effective biological catalyst than a commercial fungal cellulase cocktail. As anticipated, recalcitrance was not readily predicted through analytical methods that determined structural properties alone. However, combining structural analysis with pretreatment enabled the identification of attributes that govern recalcitrance, namely cellulose accessibility, xylan content in the pretreated solids, and non-cellulosic glycan extractability.

     

Complete Genomic Sequence and Comparative Analysis of the Genome Segments of Sweet Potato Chlorotic Stunt Virus in China

Background

Sweet potato chlorotic stunt virus (family Closteroviridae, genus Crinivirus) features a large bipartite, single-stranded, positive-sense RNA genome. To date, only three complete genomic sequences of SPCSV can be accessed through GenBank. SPCSV was first detected from China in 2011, only partial genomic sequences have been determined in the country. No report on the complete genomic sequence and genome structure of Chinese SPCSV isolates or the genetic relation between isolates from China and other countries is available.

Methodology/Principal Findings

The complete genomic sequences of five isolates from different areas in China were characterized. This study is the first to report the complete genome sequences of SPCSV from whitefly vectors. Genome structure analysis showed that isolates of WA and EA strains from China have the same coding protein as isolates Can181-9 and m2-47, respectively. Twenty cp genes and four RNA1 partial segments were sequenced and analyzed, and the nucleotide identities of complete genomic, cp, and RNA1 partial sequences were determined. Results indicated high conservation among strains and significant differences between WA and EA strains. Genetic analysis demonstrated that, except for isolates from Guangdong Province, SPCSVs from other areas belong to the WA strain. Genome organization analysis showed that the isolates in this study lack the p22 gene.

Conclusions/Significance

We presented the complete genome sequences of SPCSV in China. Comparison of nucleotide identities and genome structures between these isolates and previously reported isolates showed slight differences. The nucleotide identities of different SPCSV isolates showed high conservation among strains and significant differences between strains. All nine isolates in this study lacked p22 gene. WA strains were more extensively distributed than EA strains in China. These data provide important insights into the molecular variation and genomic structure of SPCSV in China as well as genetic relationships among isolates from China and other countries.     

Nasal Carriage of Methicillin-Resistant Staphylococcus aureus among Pediatricians in Taiwan

Background

Health care workers (HCWs) are at the interface between hospitals and communities. The survey for methicillin-resistant Staphylococcus aureus (MRSA) carriage among HCWs has mostly been conducted to investigate outbreaks or endemics. Community-associated MRSA are prevalent among children in Taiwan. We conducted this study to better understand the carriage rate of MRSA among pediatricians in non-outbreak situations in Taiwan,.

Methods

A total of 220 pediatricians from Taiwan who attended the annual meeting of Taiwan Pediatric Association in April, 2010 were recruited to participate in this study and were sampled from the nares for the detection of MRSA by polymerase chain reaction (PCR) and further by culture. The following molecular analyses were performed, including pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), typing of staphylococcal cassette chromosome mec (SCCmec) and the presence of Panton-Valentine leukocidin (PVL) genes.

Results

MRSA was detected from 15 attendees (6.8%) by PCR. MRSA-colonized attendees had a significantly lower rate (0.041) of working in the medical center, while borderline significantly higher rate of working in the Regional Hospital (p=0.056), than those without MRSA colonization. From those 15 samples, 12 MRSA isolates were identified by culture and molecularly characterized. Three PFGE patterns, two sequence types (ST 59, ST 508), and two SCCmec types (IV and V_T) were identified, respectively. Five isolates, including three carrying SCCmec types V_T, were PVL-positive. All 12 isolates were susceptible to vancomycin, teicoplanin, linezolid, fusidic acid, trimethoprim/sulfamethoxazole, and doxycyclin, and resistant to penicillin.

Conclusion/significance

Around seven percent of pediatricians in Taiwan harbored CA-MRSA in their nares.     

Can K-ras Gene Mutation Be Utilized as Prognostic Biomarker for Colorectal Cancer Patients Receiving Chemotherapy? A Meta-Analysis and Systematic Review

Introduction

K-ras gene mutations were common in colorectal patients, but their relationship with prognosis was unclear.

Objective

Verify prognostic differences between patient with and without mutant K-ras genes by reviewing the published evidence.

Method

Systematic reviews and data bases were searched for cohort/case-control studies of prognosis of colorectal cancer patients with detected K-ras mutations versus those without mutant K-ras genes, both of whom received chemotherapy. Number of patients, regimens of chemotherapy, and short-term or long-term survival rate (disease-free or overall) were extracted. Quality of studies was also evaluated.

Principal Findings

7 studies of comparisons with a control group were identified. No association between K-ras gene status with neither short-term disease free-survival (OR=1.01, 95% CI, 0.73-1.38, P=0.97) nor overall survival (OR=1.06, 95% CI, 0.82-1.36, P=0.66) in CRC patients who received chemotherapy was indicated. Comparison of long-term survival between two groups also indicated no significant difference after heterogeneity was eliminated (OR=1.09, 95% CI, 0.85-1.40, P=0.49).

Conclusions

K-ras gene mutations may not be a prognostic index for colorectal cancer patients who received chemotherapy.     

High Postoperative Serum Cortisol Level Is Associated with Increased Risk of Cognitive Dysfunction Early after Coronary Artery Bypass Graft Surgery: A Prospective Cohort Study

Context

Stress response induced by surgery is proposed to play an important role in the pathogenesis of postoperative cognitive dysfunction.

Objective

To investigate the association between postoperative serum cortisol level and occurrence of cognitive dysfunction early after coronary artery bypass graft surgery.

Design

Prospective cohort study.

Setting

Two teaching hospitals.

Patients

One hundred and sixth-six adult patients who were referred to elective coronary artery bypass graft surgery from March 2008 to December 2009.

Intervention

None.

Main Outcome Measures

Neuropsychological tests were completed one day before and seven days after surgery. Cognitive dysfunction was defined using the same definition as used in the ISPOCD1-study. Blood samples were obtained in the first postoperative morning for measurement of serum cortisol concentration. Multivariate Logistic regression analyses were performed to assess the relationship between serum cortisol level and occurrence of postoperative cognitive dysfunction.

Results

Cognitive dysfunction occurred in 39.8% (66 of 166) of patients seven days after surgery. Multivariate Logistic regression analysis showed that high serum cortisol level was significantly associated with the occurrence of postoperative cognitive dysfunction (odds ratio [OR] 2.603, 95% confidence interval [CI] 1.371-4.944, P = 0.003). Other independent predictors of early postoperative cognitive dysfunction included high preoperative New York Heart Association functional class (OR 0.402, 95% CI 0.207-0.782, P = 0.007), poor preoperative Grooved Pegboard test score of nondominant hand (OR 1.022, 95% CI 1.003-1.040, P = 0.020), use of penehyclidine as premedication (OR 2.565, 95% CI 1.109-5.933, P = 0.028), and occurrence of complications within seven days after surgery (OR 2.677, 95% CI 1.201-5.963, P = 0.016).

Conclusions

High serum cortisol level in the first postoperative morning was associated with increased risk of cognitive dysfunction seven days after coronary artery bypass graft surgery.     

Genotyping of Human Brucella melitensis Biovar 3 Isolated from Shanxi Province in China by MLVA16 and HOOF

Background

Brucellosis presents a significant economic burden for China because it causes reproductive failure in host species and chronic health problems in humans. These problems can involve multiple organs. Brucellosis is highly endemic in Shanxi Province China. Molecular typing would be very useful to epidemiological surveillance. The purpose of this study was to assess the diversity of Brucella melitensis strains for epidemiological surveillance. Historical monitoring data suggest that Brucella melitensis biovar 3 is the predominant strain associated with the epidemic of brucellosis in Shanxi Province.

Methods/Principal Findings

Multiple-locus variable-number repeat analysis (MLVA-16) and hypervariable octameric oligonucleotide fingerprinting (HOOF-print) were used to type a human-hosted Brucella melitensis population (81 strains). Sixty-two MLVA genotypes (discriminatory index: 0.99) were detected, and they had a genetic similarity coefficient ranging from 84.9% to 100%. Eighty strains of the population belonged to the eastern Mediterranean group with panel 1 genotypes 42 (79 strains) and 43 (1 strain). A new panel 1 genotype was found in this study. It was named 114 MLVAorsay genotype and it showed similarity to the two isolates from Guangdong in a previous study. Brucella melitensis is distributed throughout Shanxi Province, and like samples from Inner Mongolia, the eastern Mediterranean genotype 42 was the main epidemic strain (97%). The HOOF-printing showed a higher diversity than MLVA-16 with a genetic similarity coefficient ranging from 56.8% to 100%.

Conclusions

According to the MLVA-16 and HOOF-printing results, both methods could be used for the epidemiological surveillance of brucellosis. A new genotype was found in both Shanxi and Guangdong Provinces. In areas with brucellosis, the MLVA-16 scheme is very important for tracing cases back to their origins during outbreak investigations. It may facilitate the expansion and eradication of the disease.     

Selected Lactic Acid-Producing Bacterial Isolates with the Capacity to Reduce Salmonella Translocation and Virulence Gene Expression in Chickens

Background

Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB) isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control.

Methodology/Principal Findings

In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0) and high bile salt (0.3–1.5%) and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10^6–7 CFU/chick) or phosphate-buffered saline (PBS) at 1 day of age followed by Salmonella challenge (10⁴ CFU/chick) next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1). These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures) were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10) in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression.

Conclusions/Significance

The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in vivo can be one of the strategies for controlling Salmonella infection in chickens.     

Nontuberculous Mycobacterial Ocular Infections—Comparing the Clinical and Microbiological Characteristics between Mycobacterium abscessus and Mycobacterium massiliense

Purpose

To analyze the clinical characteristics of nontuberculous mycobacterial (NTM) ocular infections and the species-specific in vitro antimicrobial susceptibility.

Material and Methods

In 2000 to 2011 at the National Taiwan University Hospital, multilocus sequencing of rpoB, hsp65 and secA was used to identify NTM isolates from ocular infections. The clinical presentation and treatment outcomes were retrospectively compared between species. Broth microdilution method was used to determine the minimum inhibitory concentrations of amikacin (AMK), clarithromycin (CLA), ciprofloxacin (CPF), levofloxacin (LVF), moxifloxacin (MXF) and gatifloxacin (GAF) against all strains. The activities of antimicrobial combinations were assessed by the checkerboard titration method.

Results

A total of 24 NTM strains (13 Mycobacterium abscessus and 11 Mycobacterium massiliense) were isolated from 13 keratitis, 10 buckle infections, and 1 canaliculitis cases. Clinically, manifestations and outcomes caused by these two species were similar and surgical intervention was necessary for medically unresponsive NTM infection. Microbiologically, 100% of M. abscessus and 90.9% of M. massiliense ocular isolates were susceptible to amikacin but all were resistant to fluoroquinolones. Inducible clarithromycin resistance existed in 69.3% of M. abscessus but not in M. massiliense isolates. None of the AMK-CLA, AMK-MXF, AMK-GAF, CLA-MXF and CLA-GAF combinations showed synergistic or antagonistic effect against both species in vitro.

Conclusions

M. abscessus and M. massiliense are the most commonly identified species for NTM ocular infections in Taiwan. Both species were resistant to fluoroquinolones, susceptible to amikacin, and differ in clarithromycin resistance. Combined antimicrobial treatments showed no interaction in vitro but could be considered in combination with surgical interventions for eradication of this devastating ocular infection.     

Molecular Typing and Virulence Characteristic of Methicillin-Resistant Staphylococcus Aureus Isolates from Pediatric Patients in Bucaramanga,Colombia

Background

Staphylococcus aureus is among the most common global nosocomial pathogens. The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) is a public health problem worldwide that causes nosocomial and community infections. The goals of this study were to establish the clonal complexes (CC) of the isolates of MRSA obtained from pediatric patients in a university hospital in Colombia and to investigate its molecular characteristics based on the virulence genes and the genes of staphylococcal toxins and adhesins.

Methods

A total of 53 MRSA isolates from pediatric patients with local or systemic infections were collected. The MRSA isolates were typed based on the SCCmec, MLST, spa and agr genes. The molecular characterization included the detection of Panton-Valentine Leukocidin, superantigenic and exfoliative toxins, and adhesin genes. The correlation between the molecular types identified and the profile of virulence factors was determined for all isolates.

Results

Four CC were identified, including CC8, CC5, CC80 and CC78. The ST8-MRSA-IVc-agrI was the predominant clone among the isolates, followed by the ST5-MRSA-I-agrII and ST5-MRSA-IVc-agrII clones. Twelve spa types were identified, of which t10796 and t10799 were new repeat sequences. The isolates were carriers of toxin genes, and hlg (100%), sek (92%) and pvl (88%) were the most frequent. Ten toxin gene profiles were observed, and the most frequent were seq-sek-hlg (22.6%), sek-hlg (22.6%), seb-seq-sek-hlg (18.9%) and seb-sek-hlg (15.1%). The adhesion genes were present in most of the MRSA isolates, including the following: clf-A (89%), clf-B (87%), fnb-A (83%) and ica (83%). The majority of the strains carried SCCmec-IVc and were identified as causing nosocomial infection. No significant association between a molecular type and the virulence factors was found.

Conclusion

Four major MRSA clone complexes were identified among the isolates. ST8-MRSA-IVc-agrI pvl+ (USA300-LV) was the most frequent, confirming the presence of community-associated MRSA in Colombian hospitals.