Antimicrobial resistance and population genomics of emerging multidrug-resistant Salmonella 4,[5],12:i:- in Guangdong, China.

Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as a global cause of multidrug-resistant salmonellosis and has become endemic in many developing and developed countries, especially in China. Here, we have sequenced 352 clinical isolates in Guangdong, China, during 2009-2019 and performed a large-scale collection of Salmonella 4,[5],12:i:- with whole genome sequencing (WGS) data across the globe, to better understand the population structure, antimicrobial resistance (AMR) genomic characterization, and transmission routes of Salmonella 4,[5],12:i:- across Guangdong. Salmonella 4,[5],12:i:- strains showed broad genetic diversity; Guangdong isolates were found to be widely distributed among the global lineages. Of note, we identified the formation of a novel Guangdong clade (Bayesian analysis of population structure lineage 1 [BAPS1]) genetically diversified from the global isolates and likely emerged around 1990s. BAPS1 exhibits unique genomic features, including large pan-genome, decreased ciprofloxacin susceptibility due to mutation in gyrA and carriage of plasmid-mediated quinolone resistance (PMQR) genes, and the multidrug-resistant IncHI2 plasmid. Furthermore, high genetic similarity was found between strains collected from Guangdong, Europe, and North America, indicating the association with multiple introductions from overseas. These results suggested that global dissemination and local clonal expansion simultaneously occurred in Guangdong, China, and horizontally acquired resistance to first-line and last-line antimicrobials at local level, underlying emergences of extensive drug and pan-drug resistance. Our findings have increased the knowledge of global and local epidemics of Salmonella 4,[5],12:i:- in Guangdong, China, and provided a comprehensive baseline data set essential for future molecular surveillance.IMPORTANCESalmonella 4,[5],12:i:- has been regarded as the predominant pandemic serotype causing diarrheal diseases globally, while multidrug resistance (MDR) constitutes great public health concerns. This study provided a detailed and comprehensive genome-scale analysis of this important Salmonella serovar in the past decade in Guangdong, China. Our results revealed the complexity of two distinct transmission modes, namely global transmission and local expansion, circulating in Guangdong over a decade. Using phylogeography models, the origin of Salmonella 4,[5],12:i:- was predicted from two aspects, year and country, that is, Salmonella 4,[5],12:i:- emerged in 1983, and was introduced from the UK, and subsequently differentiated into the local endemic lineage circa 1991. Additionally, based on the pan-genome analysis, it was found that the gene accumulation rate in local endemic BAPS 1 lineage was higher than in other lineages, and the horizontal transmission of MDR IncHI2 plasmid associated with high resistance played a major role, which showed the potential threat to public health.

Epidemiological Surveillance: Genetic Diversity of Rotavirus Group A in the Pearl River Delta, Guangdong, China in 2019.

Objective: This study aimed to understand the epidemic status and phylogenetic relationships of rotavirus group A (RVA) in the Pearl River Delta region of Guangdong Province, China. Methods: This study included individuals aged 28 days-85 years. A total of 706 stool samples from patients with acute gastroenteritis collected between January 2019 and January 2020 were analyzed for 17 causative pathogens, including RVA, using a Gastrointestinal Pathogen Panel, followed by genotyping, virus isolation, and complete sequencing to assess the genetic diversity of RVA. Results: The overall RVA infection rate was 14.59% (103/706), with an irregular epidemiological pattern. The proportion of co-infection with RVA and other pathogens was 39.81% (41/103). Acute gastroenteritis is highly prevalent in young children aged 0-1 year, and RVA is the key pathogen circulating in patients 6-10 months of age with diarrhea. G9P[8] (58.25%, 60/103) was found to be the predominant genotype in the RVA strains, and the 41 RVA-positive strains that were successfully sequenced belonged to three different RVA genotypes in the phylogenetic analysis. Recombination analysis showed that gene reassortment events, selection pressure, codon usage bias, gene polymorphism, and post-translational modifications (PTMs) occurred in the G9P[8] and G3P[8] strains. Conclusion: This study provides molecular evidence of RVA prevalence in the Pearl River Delta region of China, further enriching the existing information on its genetics and evolutionary characteristics and suggesting the emergence of genetic diversity. Strengthening the surveillance of genotypic changes and gene reassortment in RVA strains is essential for further research and a better understanding of strain variations for further vaccine development.

Carriage and Transmission of mcr-1 in Salmonella Typhimurium and Its Monophasic 1,4,[5],12:i:- Variants from Diarrheal Outpatients: a 10-Year Genomic Epidemiology in Guangdong, Southern China.

The banning of colistin as a feed additive for food-producing animals in mainland China in 2017 caused the decline in the prevalence of Escherichia coli-mobilized colistin resistance (mcr-1) in China. Salmonella Typhimurium and its monophasic 1,4,[5],12:i:- variants are also the main species associated with the spread of mcr-1; however, the evidence of the prevalence and transmission of mcr-1 among Salmonella is lacking. Herein, the 5,354 Salmonella isolates recovered from fecal samples of diarrheal patients in Guangdong, Southern China, from 2009 to 2019 were screened for colistin resistance and mcr-1, and mcr-1-positive isolates were characterized based on whole-genome sequencing (WGS) data. Relatively high prevalence rates of colistin resistance and mcr-1 (4.05%/4.50%) were identified, and more importantly, the prevalence trends of colistin-resistant and mcr-1-positive Salmonella isolates had a similar dynamic profile, i.e., both were first detected in 2012 and rapidly increased during 2013 to 2016, followed by a sharp decrease since 2017. WGS and phylogenetic analysis indicate that, whether before or after the ban, the persistence and cross-hospital transmission of mcr-1 are primarily determined by IncHI2 plasmids with similar backbones and sequence type 34 (ST34) Salmonella in specific clades that are associated with a high prevalence of IncHI2 plasmids and clinically important antimicrobial resistance genes, including blaCTX-M-14-fosA3-oqxAB-floR genotypes. Our work reveals the difference in the prevalence rate of mcr-1 in clinical Salmonella before and after the Chinese colistin ban, whereas mcr-1 transmission was closely linked to multidrug-resistant IncHI2 plasmid and ST34 Salmonella across diverse hospitals over 10 years. Continued surveillance is required to explore the factors related to a sharp decrease in mcr-1 after the recent ban and determine whether the ban has affected the carriage of mcr-1 in Salmonella circulating in the health care system. IMPORTANCE Colistin is one of the last-line antibiotics for the clinical treatment of Enterobacteriaceae. However, the emergence of the mobilized colistin resistance (mcr-1) gene has spread throughout the entire human health system and largely threatens the usage of colistin in the clinical setting. In this study, we investigated the existence of mcr-1 in clinical Salmonella from a 10-year continuous surveillance and genomic study. Overall, the colistin resistance rate and mcr-1 carriage of Salmonella in tertiary hospitals in Guangdong (2009 to 2019) were relatively high and, importantly, rapidly increased from 2013 to 2016 and significantly decreased after the Chinese colistin withdrawal. However, before or after the ban, the MDR IncHI2 plasmid with a similar backbone and ST34 Salmonella were the main vectors involved in the spread of mcr-1. Interestingly, these Chinese mcr-1-carrying Salmonella obtain phylogenetically and phylogeographically distinct patterns compared with those from other continents and are frequently associated with clinically important ARGs including the extended-spectrum ß-lactamases. Our data confirmed that the national stewardship intervention seems to be successful in blocking antibiotic resistance determinants and that continued surveillance of colistin resistance in clinical settings, farm animals, and related products is necessary.

Epidemiology of bla CTX-M-Positive Salmonella Typhimurium From Diarrhoeal Outpatients in Guangdong, China, 2010-2017.

Salmonella enterica can lead to intestinal diarrhea, and the emergence and spread of cephalosporin-resistant Salmonella have brought great challenges to clinical treatment. Therefore, this study investigated the prevalence and transmission of bla CTX-M genes among S. Typhimurium from diarrhoeal outpatients in Guangdong, China, from 2010 to 2017. A total of 221 bla CTX-M-positive isolates were recovered from 1,263 S. Typhimurium isolates from the facal samples of diarrhoea patients in 45 general hospitals from 11 cities. The most popular CTX-M gene was bla CTX-M-55 (39.6%, 72/182) in the CTX-M-1 group, followed by bla CTX-M-14 (22.5%, 41/182) and bla CTX-M-65 (19.2%, 35/182) in the CTX-M-9 group. The isolates that carried bla CTX-M-9G had significantly higher resistance rates to multiple antibacterials compared with bla CTX-M-1G (p < 0.01). Meanwhile, PFGE analysis not only showed the clonal transmission of bla CTX-M-55/14/65-positve isolates of diarrhoeal outpatients' origins from different hospitals in Guangdong province, but also the characteristic of bla CTX-M-55/14/65-positve isolates' bacterial persistence. Multilocus sequence typing (MLST) analysis indicated that these S. Typhimurium isolates possessed ST34 and ST19. Furthermore, genomic Beast phylogenomic analysis provided the evidence of a close relationship of bla CTX-M-positive S. Typhimurium isolates between the outpatients and pork. Most bla CTX-M-55/14/65 genes were transmitted by non-typeable or IncI1/IncFII/IncHI2 plasmids with the size of ranging from ~80 to ~280 kb. Moreover, whole-genome sequencing (WGS) analysis further revealed that bla CTX-M-55/14/65 coexisted with other 25 types of ARGs, of which 11 ARGs were highly prevalent with the detection rates >50%, and it first reported the emergence of bla TEM-141 in S. Typhimurium. This study underscores the importance of surveillance for bla CTX-M-positive microbes in diarrhea patients.

Phylogenomic analysis of Salmonella Indiana ST17, an emerging MDR clonal group in China.

OBJECTIVES: To reconstruct the genomic epidemiology and evolution of MDR Salmonella Indiana in China. METHODS: A total of 108 Salmonella Indiana strains were collected from humans and livestock in China. All isolates were subjected to WGS and antimicrobial susceptibility testing. Phylogenetic relationships and evolutionary analyses were conducted using WGS data from this study and the NCBI database. RESULTS: Almost all 108 Salmonella Indiana strains displayed the MDR phenotype. Importantly, 84 isolates possessed concurrent resistance to ciprofloxacin and cefotaxime. WGS analysis revealed that class 1 integrons on the chromosome and IncHI2 plasmids were the key vectors responsible for multiple antibiotic resistance gene (ARG) [including ESBL and plasmid-mediated quinolone resistance (PMQR) genes] transmission among Salmonella Indiana. The 108 Salmonella Indiana dataset displayed a relatively large core genome and ST17 was the predominant ST. Moreover, the global ST17 Salmonella Indiana strains could be divided into five distinct lineages, each of which was significantly associated with a geographical distribution. Genomic analysis revealed multiple antimicrobial resistance determinants and QRDR mutations in Chinese lineages, which almost did not occur in other global lineages. Using molecular clock analysis, we hypothesized that ST17 isolates have existed since 1956 and underwent a major population expansion from the 1980s to the 2000s and the genetic diversity started to decrease around 2011, probably due to geographical barriers, antimicrobial selective pressure and MDR, favouring the establishment of this prevalent multiple antibiotic-resistant lineage and local epidemics. CONCLUSIONS: This study revealed that adaptation to antimicrobial pressure was possibly pivotal in the recent evolutionary trajectory for the clonal spread of ST17 Salmonella Indiana in China.

The immunodominant and neutralization linear epitopes for SARS-CoV-2.

Although vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are under development, the antigen epitopes on the virus and their immunogenicity are poorly understood. Here, we simulate the 3D structures and predict the B cell epitopes on the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins of SARS-CoV-2 using structure-based approaches and validate epitope immunogenicity by immunizing mice. Almost all 33 predicted epitopes effectively induce antibody production, six of these are immunodominant epitopes in individuals, and 23 are conserved within SARS-CoV-2, SARS-CoV, and bat coronavirus RaTG13. We find that the immunodominant epitopes of individuals with domestic (China) SARS-CoV-2 are different from those of individuals with imported (Europe) SARS-CoV-2, which may be caused by mutations on the S (G614D) and N proteins. Importantly, we find several epitopes on the S protein that elicit neutralizing antibodies against D614 and G614 SARS-CoV-2, which can contribute to vaccine design against coronaviruses.

Kinetic Characteristics of Neutralizing Antibody Responses Vary among Patients with COVID-19.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody (NAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: In total, 605 serum samples from 125 COVID-19 patients (from January 1 to March 14, 2020) varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2. RESULTS: NAbs were detectable approximately 10 days post-onset (dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective. CONCLUSION: We demonstrated that NAb levels vary among all categories of COVID-19 patients. Long-term studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.

Global clonal spread of mcr-3-carrying MDR ST34 Salmonella enterica serotype Typhimurium and monophasic 1,4,[5],12:i:- variants from clinical isolates.

OBJECTIVES: To investigate the prevalence and transmission of mcr-3 among Salmonella enterica serotype Typhimurium and 1,4,[5],12:i:-. METHODS: A total of 4724 clinical Salmonella isolates were screened for the presence of mcr-3 in China during 2014-19. The clonal relationship of the mcr-3-positive isolates and their plasmid contents and complete sequence were also characterized based on WGS data from the Illumina and MinION platforms. RESULTS: We identified 10 mcr-3-positive isolates, and all were MDR, mostly resistant to colistin, cefotaxime, ciprofloxacin, doxycycline and florfenicol. mcr-3 was co-present with blaCTX-M-55-qnrS1 on hybrid ST3-IncC-FII conjugatable plasmids (n = 6) and an ST3-IncC non-conjugatable plasmid (n = 1) and embedded into a pCHL5009T-like IncFII plasmid on the Salmonella chromosome (n = 3). Four distinctive genetic contexts surrounded mcr-3 and all but one were closely related to each other and to the corresponding region of IncFII plasmid pCHL5009T. IS15DI was most likely the vehicle for integration of mcr-3-carrying IncFII plasmids into ST3-IncC plasmids and the chromosome and for shaping the MDR regions. In addition, a phylogenetic tree based on the core genome revealed a unique Salmonella lineage (≤665 SNPs) that contained these 10 mcr-3-positive isolates and another 38 (33 from patients) mcr-3-positive Salmonella from five countries. In particular, most of the 51 mcr-3-positive isolates belonged to ST34 and harboured diverse antibiotic resistance genes (ARGs), including mcr-3-blaCTX-M-55-qnrS1, and possessed similar ARG profiles. CONCLUSIONS: Our findings revealed global clonal spread of MDR ST34 Salmonella from clinical isolates co-harbouring mcr-3 with blaCTX-M-55 and qnrS1 and a flexibility of mcr-3 co-transmittance with other ARGs mediated by mobile genetic elements.

No evidence of a role for mitochondrial complex I in Helicobacter pylori pathogenesis.

BACKGROUND: Complex I is the first enzyme complex in the mitochondrial respiratory chain, responsible for generating a large fraction of energy during oxidative phosphorylation. Recently, it has been identified that complex I deficiency can result in increased inflammation due to the generation of reactive oxygen species by innate immune cells. As a reduction in complex I activity has been demonstrated in human stomachs with atrophic gastritis, we investigated whether complex I deficiency could influence Helicobacter pylori pathogenesis. MATERIALS AND METHODS: Ndufs6gt/gt mice have a partial complex I deficiency. Complex I activity was quantified in the stomachs and immune cells of Ndufs6gt/gt mice by spectrophotometric assays. Ndufs6gt/gt mice were infected with H. pylori and bacterial colonization assessed by colony-forming assay, gastritis assessed histologically, and H. pylori -specific humoral response quantified by ELISA. RESULTS: The immune cells and stomachs of Ndufs6gt/gt mice were found to have significantly decreased complex I activity, validating the model for assessing the effects of complex I deficiency in H. pylori infection. However, there was no observable effect of complex I deficiency on either H. pylori colonization, the resulting gastritis, or the humoral response. CONCLUSIONS: Although complex I activity is described to suppress innate immune responses and is decreased during atrophic gastritis in humans, our data suggest it does not affect H. pylori pathogenesis.

Deficiency in mitochondrial complex I activity due to Ndufs6 gene trap insertion induces renal disease.

AIMS: Defects in the activity of enzyme complexes of the mitochondrial respiratory chain are thought to be responsible for several disorders, including renal impairment. Gene mutations that result in complex I deficiency are the most common oxidative phosphorylation disorders in humans. To determine whether an abnormality in mitochondrial complex I per se is associated with development of renal disease, mice with a knockdown of the complex I gene, Ndufs6 were studied. RESULTS: Ndufs6 mice had a partial renal cortical complex I deficiency; Ndufs6gt/gt, 32% activity and Ndufs6gt/+, 83% activity compared with wild-type mice. Both Ndufs6gt/+ and Ndufs6gt/gt mice exhibited hallmarks of renal disease, including albuminuria, urinary excretion of kidney injury molecule-1 (Kim-1), renal fibrosis, and changes in glomerular volume, with decreased capacity to generate mitochondrial ATP and superoxide from substrates oxidized via complex I. However, more advanced renal defects in Ndufs6gt/gt mice were observed in the context of a disruption in the inner mitochondrial electrochemical potential, 3-nitrotyrosine-modified mitochondrial proteins, increased urinary excretion of 15-isoprostane F2t, and up-regulation of antioxidant defence. Juvenile Ndufs6gt/gt mice also exhibited signs of early renal impairment with increased urinary Kim-1 excretion and elevated circulating cystatin C. INNOVATION: We have identified renal impairment in a mouse model of partial complex I deficiency, suggesting that even modest deficits in mitochondrial respiratory chain function may act as risk factors for chronic kidney disease. CONCLUSION: These studies identify for the first time that complex I deficiency as the result of interruption of Ndufs6 is an independent cause of renal impairment.

[Surveillance and analysis on the pathogenic features of Salmonella in Guangdong province in 2010].

OBJECTIVE: In order to better understand the nature of Salmonella infection in diarrheal patients in Guangdong province, the study analyzed the serum types, antibiotic resistance and molecular determinants of the isolated Salmonella strains. METHODS: In year 2010, 8405 diarrhea patients from 16 surveillant hospital in Guangzhou, Zhongshan, Dongguan, Zhuhai, Maoming, Yangjiang and Jiangmen cities in Guangdong province, were recruited in the study. A total of 8405 fecal specimen were collected and subjected to Salmonella isolation and culture. The isolated Salmonella strains were further analyzed via serotyping, antimicrobial susceptibility testing, and PFGE. The χ(2) test was applied to compare the differences between the isolated Salmonella strains in different seasons and districts. BioNumerics software was used to analyze the PFGE results in order to determine the correlation between different Salmonella strains. RESULTS: The positive rate of the surveillant Salmonella in Guangdong province was 3.58% (301/8405) in 2010; with the gender ratio at 1.34:1 (166/124). Salmonella infection was found in all age groups, and most in infants, accounting for 57.48% (173/301). The isolated rates of Salmonella were separately 3.48% (61/1751), 4.97% (134/2695), 3.08% (73/2370) and 2.08% (33/1589) in the four seasons; and the difference was statistically significant (χ(2) = 27.29, P < 0.01). The isolated rates of Salmonella in different regions were as follows: Zhuhai 15.43% (25/162), Maoming 7.53% (18/239), Dongguan 6.51% (39/599), Yangjiang 3.64% (14/385), Zhongshan 3.03% (70/2309), Guangzhou 2.90% (126/4349) and Jiangmen 2.49% (9/362). The difference between regions was statistically significant (χ(2) = 100.75, P < 0.01). Except one strain of the isolated Salmonella cannot be serotyped, the other 300 strains were divided into 42 serotypes, of which Salmonella typhimurium and Salmonella enteritidis were dominant, account for 45.18% (136/301) and 10.96% (33/301) respectively. Although over 85% of Salmonella were sensitive to cephalosporin, ACSSuT resistance patterns (defined as resistance to at least ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline) reached 34.88% (105/301), the highest resistant rate was found in serotype Salmonella typhimurium, as high as 65.44% (89/136). 136 strains of Salmonella typhimurium were divided into 51 PFGE types, showed great genetic diversity. 33 strains of Salmonella enteritidis were divided into 18 PFGE types. The strains with same PFGE pattern may have different drug-resistant patterns, and vice versa. CONCLUSION: Salmonella typhimurium and Salmonella enteritidis were the dominant serotypes causing infectious diarrhea in Guangdong province. Cephalosporin was the primary choice in clinical medicine. However, Salmonella typhimurium was resistant to drug most seriously in Guangdong province. There was no significant correlation between Salmonella resistance patterns and PFGE type.

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy.

Mitochondrial complex I (CI) deficiency is the most common mitochondrial enzyme defect in humans. Treatment of mitochondrial disorders is currently inadequate, emphasizing the need for experimental models. In humans, mutations in the NDUFS6 gene, encoding a CI subunit, cause severe CI deficiency and neonatal death. In this study, we generated a CI-deficient mouse model by knockdown of the Ndufs6 gene using a gene-trap embryonic stem cell line. Ndufs6(gt/gt) mice have essentially complete knockout of the Ndufs6 subunit in heart, resulting in marked CI deficiency. Small amounts of wild-type Ndufs6 mRNA are present in other tissues, apparently due to tissue-specific mRNA splicing, resulting in milder CI defects. Ndufs6(gt/gt) mice are born healthy, attain normal weight and maturity, and are fertile. However, after 4 mo in males and 8 mo in females, Ndufs6(gt/gt) mice are at increased risk of cardiac failure and death. Before overt heart failure, Ndufs6(gt/gt) hearts show decreased ATP synthesis, accumulation of hydroxyacylcarnitine, but not reactive oxygen species (ROS). Ndufs6(gt/gt) mice develop biventricular enlargement by 1 mo, most pronounced in males, with scattered fibrosis and abnormal mitochondrial but normal myofibrillar ultrastructure. Ndufs6(gt/gt) isolated working heart preparations show markedly reduced left ventricular systolic function, cardiac output, and functional work capacity. This reduced energetic and functional capacity is consistent with a known susceptibility of individuals with mitochondrial cardiomyopathy to metabolic crises precipitated by stresses. This model of CI deficiency will facilitate studies of pathogenesis, modifier genes, and testing of therapeutic approaches.

[Surveillance on Salmonella infection in Guangdong province, 2008 - 2009].

OBJECTIVE: To understand the infection of Salmonella (S.) in patients with diarrhea and outbreaks caused by Salmonella to identify the serotypes, resistance to antibiotics and PFGE types of the strains from the surveillance program in Guangdong province. METHODS: S. strains from patients with diarrhea were detected, and all the positive strains collected in routine and outbreak surveillance programs, were tested by serum agglutination, antibiotic susceptibility and PFGE. RESULTS: 71 S. strains were isolated from 1922 stool samples in 2008, with positive rate as 3.7%. 85 S. strains were isolated from 2110 stool samples in 2009, with positive rate as 4.0%. All the 156 strains were divided into 37 serotypes, with S. serotype typhimurium and enteritidis as the most common serotypes. 10 incidents of food poisoning were detected, of which 4 were caused by enteritidis and 3 by typhimurium. A suspected outbreak by enteritidis was discovered and under epidemiological investigation. The findings indicated that 2 of the 4 patients from this outbreak were infected with identical enteritidis isolates. 80% of the 229 isolates were found susceptible to cephalosporins and quinolone and 59.3% of them were multiresistant to the antibiotics. CONCLUSION: S. enteritidis and S. typhimurium were the most common serotypes that caused infectious diarrhoea and food poisoning in Guangdong province.

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations.

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.

Phenotypic and genotypic characterization Vibrio cholerae O139 of clinical and aquatic isolates in China.

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.

[Etiologic characteristics of Vibrio parahaemolyticus strains causing outbreaks and sporadic cases in Guangdong, 2009].

OBJECTIVE: To study the serotypes, virulence features and molecular characteristics of Vibrio parahaemolyticus isolated in food poisoning cases and surveillance program on diarrhea patients in Guangdong, 2009. METHODS: 95 Vibrio parahaemolyticus strains from food poisoning cases and 15 strains from surveillance program on diarrhea patients were serotyped and detected for tdh (thermostable direct hemolysin, tdh) and trh (tdh-related hemolysin gene, trh) by PCR. 81 sero-variant Vibrio parahaemolyticus strains were selected through PFGE subtyping. RESULTS: There were 15 Vibrio parahaemolyticus strains isolated from surveillance program on diarrhea patients and 95 strains were isolated from 11 Vibrio parahaemolyticus-caused food poisoning cases in 2009. Among these strains, O3:K6 (46.67% and 44.21%) and O4:K8 (33.33% and 28.42%) were the dominant serotypes, but not the 7 food-borne strains. There were 93 (84.54%) tdh(+)trh(-), 13 (11.81%) tdh(-)trh(-), and 3 (3.65%) tdh(+)trh(+) strains. The similarity value was between 57.7% to 100.0% of the 81 strains after PFGE sub-typing method and 36 PFGE subtypes were identified. PFGE001 and PFGE029 appeared to be the dominant subtypes. CONCLUSION: O3:K6 and O4:K8 were the most dominant serotypes in Vibrio parahaemolyticus-caused diarrhea and food poisoning cases in Guangdong and tdh were detected in most of the strains. Dominant PFGE subtypes were causing both sporadic and outbreak cases in different areas in Guangdong province.

[Surveillance program on and the distribution related to the virulence-associated genes of Vibrio cholerae in estuary of Pearl River].

OBJECTIVE: To understand the distribution, molecular characteristics and virulence genes of the O1 and O139 Vibrio cholerae isolates from the Pearl River Estuary water. METHODS: Vibrio cholerae isolates collected from the Pearl River estuary waters from January 2009 to December 2010, were tested by PCR for eight virulence-related genes, including cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and the regulatory protein genes (tcpI, toxR). Genetic relation was assessed by pulsed-field gel electrophoresis (PFGE) and the patterns were clustered by BioNumerics. RESULTS: From 1152 aquatic samples, 69 isolates were identified, including 41 Inaba, 18 Ogawa and 10 O139. All the isolates showed ctxA negative, while the hlyA and toxR genes were positive in all the isolates. 34.15% (14/41) of the Inaba strains were hlyA(+) toxR(+) ompU(+) ace(+) zot(+) tcpI(+), while 66.67% (12/18) belonged to Ogawa strains and 70% (7/10) of the O139 strains were hlyA(+) toxR(+). Through PFGE analysis, the O1 isolates formed three clusters in this study. The patterns of O1 isolates differed widely, with the similarity as 72.8% - 100.0%, while the patterns of O139 isolates having the similarity of 69.9% - 95.5%. CONCLUSION: The non-toxigenic O1 and O139 V. cholerae had a wide distribution in the environment of Pearl River estuary water during the non-epidemic period of cholera. All the aquatic isolates presented diversities on the related virulent genes.

[Comparative study on the phenotypic characteristics and molecular typing of foodborne Vibrio parahaemolyticus in Guangdong province].

OBJECTIVE: To understand the phenotypic characteristics of foodborne Vibrio parahaemolyticus in Guangdong province through carrying out a comprehensive comparison including pulse field gel electrophoresis, ribotyping and serotyping. METHODS: 74 different Vibrio parahaemolyticus strains isolated from seafood and cases due to food poisoning in Guangdong province were under serotyping and susceptibility testing, in addition to the testing of direct heat hemolysin (tdh) and the heat hemolysin-related hemolysin hormone (trh) via PCR. Ribosomal genotyping (ribotyping) with EcoR I restriction enzyme was utilized on 74 different Vibrio parahaemolyticus isolates, whereas pulsed-field gel electrophoresis (PFGE) with the Not I restriction enzyme was used on 74 different Vibrio parahaemolyticus isolates. BioNumerics software was used to compare the isolates from different sources, times and places in order to elicit the correlation between different strains. RESULTS: Although Vibrio parahaemolyticus was 100.00% sensitive to chloramphenicol, it still presented different levels of resistance against 13 other antibiotics. Among the 74 different strains of Vibrio parahaemolyticus under testing, 24.32% showed positive for the tdh virulence gene, whereas 4.05% positive for trh. 74 different Vibrio parahaemolyticus strains were found to belong to 26 serotypes, where the O5:K17 and O2:K28 serotypes were dominant in those isolates that causing seafood-poisoning. The O3:K6 serotype was found to be the dominant of those isolates that causing food-poisoning. Based on ribosomal genotyping, the 74 Vibrio parahaemolyticus isolates were divided into 62 different ribotypes, whereas the 74 strains of Vibrio parahaemolyticus were divided into 67 different PFGE types, thus exhibiting considerable genetic diversities of the strains. CONCLUSION: Majority of the isolates causing food-poisoning carried tdh virulence gene. PFGE was shown to have the highest resolution, followed by ribotyping with serotyping being the lowest, where the combination of the three could improve the resolution.

[Automated ribotyping of Salmonella and Staphylococcus aureus in food poisoning of Guangdong province].

OBJECTIVE: To understand the genetic polymorphism of Salmonella and Staphylococcus aureus in Guangdong province, as well as to explore methods for identifying and tracing the source of these two foodborne pathogens. METHODS: Using the automated ribotyping system, two foodborne pathogens were tested with either EcoR I or Pvu II restriction enzymes. BioNumerics software was then applied for image analysis, database establishment and other corresponding analysis. RESULTS: Digestion of 32 Salmonella isolates with Pvu II yielded 19 different ribotypes, and digestion of 14 Salmonella isolates with EcoR I yielded 2 different ribotypes. Staphylococcus aureus isolates showed greater genetic diversity, whereas EcoR I digestion of 49 different isolates yielded 31 different ribotypes. CONCLUSION: Unique Salmonella and Staphylococcus aureus isolates could be identified through ribotyping. Although Salmonella serotyping and ribotyping were not strongly correlated, the combination of both restriction enzymes could be used to more effectively identify the genetic relationship among different strains as well as the source of food poisoning. Thus, not only could the genetic relationships amongst the different strains be inferred through ribotyping skills, the source of food poisoning and mode of transmission could also be determined under the use of this method.