A Conjugative MDR pMG1-Like Plasmid Carrying the lsa(E) Gene of Enterococcus faecium With Potential Transmission to Staphylococcus aureus.

lsa(E) is a pleuromutilin, lincosamide, and streptogramin A (PLSA phenotype) resistance gene that was first described in S. aureus and was thought to have been transferred from Enterococcus sp. This study aimed to elucidate the prevalence of the lsa(E) gene among E. faecium isolates at a tertiary teaching hospital and to evaluate the transferability of the lsa(E) gene from E. faecium to S. aureus in vitro. A total of 96 E. faecium strains isolated from one hospital in Beijing in 2013 were analysed for quinupristin-dalfopristin (QDA) resistance genes, and multilocus sequence typing (MLST) was performed. The transferability of QDA resistance between ten E. faecium strains and four S. aureus strains was determined by filter mating. Genome sequencing of the transconjugant was performed. A total of 46 E. faecium isolates (46/96, 47.92%) tested positive for lsa(E), while two isolates (2/96, 2.08%) tested positive for lsa(A). Thirty-six lsa(E)-positive strains (36/46, 78.3%) belonged to ST78. Among 40 mating tests, lsa(E) was successfully transferred through one conjugation at a frequency of 1.125 × 10-7 transconjugants per donor. The QDA resistance of the transconjugant N7435-R3645 was expressed at a higher level (MIC = 16 mg/L) than that of the parent S. aureus strain (MIC = 0.38 mg/L). Next-generation sequencing (NGS) analysis of the transconjugant N7435-R3645 showed that the complete sequence of the lsa(E)-carrying plasmid pN7435-R3645 had a size of 92,396 bp and a G + C content of 33% (accession no. MT022086). The genetic map of pN7435-R3645 had high nucleotide similarity and shared the main open reading frame (ORF) features with two plasmids: E. faecium pMG1 (AB206333.1) and E. faecium LS170308 (CP025078.1). The rep gene of pN7435-R3645 showed 100% identity with that of pMG1, although it did not belong to the rep1-19 family but instead a unique rep family. Multiple antibiotic resistance genes, including lsa(E), aadE and lnu(B), erm(B), ant6-Ia, and lnu(B), were present on the plasmid. In conclusion, an lsa(E)-carrying plasmid that can be transferred by conjugation from E. faecium to S. aureus in vitro was identified. This multidrug resistance (MDR) pMG1-like plasmid may act as a vector in the dissemination of antimicrobial resistance among species.

Antimicrobial susceptibility of Streptococcus sp. to quinupristin-dalfopristin in China.

This study aimed to determine the in vitro activity of quinupristin-alfopristin against Streptococcus sp. isolated in China. This agent is not yet available for clinical use, but it has been tested against a high proportion of resistant Staphylococcus aureus strains. A total of 156 streptococcal isolates, which were recovered from various geographic areas and diseases, were tested using the Etest (AB Biodisk, Solna, Sweden). Quinupristin-alfopristin showed excellent activity against all of the tested streptococci isolates. These results provide useful data for the clinical use of quinupristin-alfopristin in China.

Molecular epidemiological characteristics of Streptococcus pyogenes strains involved in an outbreak of scarlet fever in China, 2011.

OBJECTIVE: To investigate molecular characterization of streptococcus pyogenes isolates involved in an outbreak of scarlet fever in China in 2011. METHODS: Seventy-four Streptococcal pyogenes involved in an outbreak of scarlet fever were isolated from pediatric patients in the areas with high incidence in China from May to August of 2011. Emm genotyping, pulsed-field gel electrophoresis (PFGE), superantigen (SAg) genes and antimicrobial susceptibility profiling were analyzed for these isolates. RESULTS: A total of 4 different emm types were identified. Emm12 was the most prevalent type which contained four predominating PFGE patterns corresponding to four different virulence and superantigen profiles. Emm12 (79.7%) and emm1 (14.9%) accounted for approximately 94% of all the isolates. The speA gene was all negative in emm12 isolates and positive in emm1 isolates. All strains were resistant to erythromycin, and 89.4% of them were resistant to erythromycin, tracycline, and clindamycin simultaneously. CONCLUSION: Several highly diversified clones with a high macrolide resistance rate comprise a predominant proportion of circulating strains, though no new emm type was found in this outbreak. The data provide a baseline for further surveillance of scarlet fever, which may contribute to the explanation of the outbreak and development of a GAS vaccine in China.

In vitro protein expression profile of Campylobacter jejuni strain NCTC11168 by two-dimensional gel electrophoresis and mass spectrometry.

OBJECTIVE: To investigate the protein expression profiles of the major food-borne pathogen Campylobacter jejuni NCTC11168. METHODS: Membrane and soluble cellular proteins were extracted from the genome-sequenced C. jejuni strain NCTC11168. Protein expression profiles were determined using two-dimensional gel electrophoresis (2-DE). All the detected spots on the 2-DE map were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis. RESULTS: A total of 537 and 333 spots were detected from the whole cell and membrane-associated proteins of C. jejuni NCTC11168 cultured on Columbia agar medium at 42 °C by 2-DE and Coomassie Brilliant Blue staining, respectively. Analyses of whole cell and membrane-associated proteins included 399 and 133 spots, respectively, which included 182 and 53 functional proteins identified by MALDI-TOF/TOF analysis. CONCLUSION: The comprehensive expression protein profiles of C. jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation.

Comparative genomic analysis of gene variations of two Chinese Yersinia pestis isolates from vaccine strain EV76.

OBJECTIVE: To investigate genomic variations of two Chinese Yersinia pestis isolates that were isolated from different plague foci obtained from vaccine strain EV76 from the Yunnan province of China. METHODS: A microarray containing 12 000 probes covering the entire genome of seven Yersinia pestis and two Yersinia pseudotuberculosis strains, was used. PCR assays were performed to confirm microarray results. RESULTS: The gene variations detected included the absence of five genes related to the synthesis of betaine in both EV76 and another sequenced attenuated strain, KIM D27. Several genes related to phage-related membrane proteins were found to be absent in the Antiqua biovar Yunnan strain, 485, which was isolated from a rodent plague foci. CONCLUSION: These findings provide initial insight into the distinct strains isolated from natural foci, within their genomic context, including Yunnan Y. pestis strains. This information will be used therefore to establish subsequent comparisons of these sequences with published complete genomes of other strains.

Assessment of comparative genomic hybridization experiment by an in situ synthesized CombiMatrix microarray with Yersinia pestis vaccine strain EV76 DNA.

OBJECTIVE: The quality of microarray data influences the accuracy of comparative genomic analyses to a large extent. To ensure that the results obtained by using an in situ synthesized microarray are accurate, data quality is to be assessed by evaluating the melting temperature (Tm) of probes, probability of false synthesis rates, and fragmentation of labeled targets. METHODS: DNA from the Yersinia pestis vaccine strain EV76 was used for microarray analyses. Microarray results were confirmed by PCR. Statistical and bioinformatics methods were employed to perform microarray data analyses and evaluation. RESULTS: Correlation coefficients of the three datasets were above 0.95 after two-time stripping and hybridization with a labeled DNA with the size of fragmentation being 200 bp - 2 kb, which showed that the hybridization results were highly reproducible. Correlation coefficients were lower with the values ranging from 0.87 to 0.92 between the datasets generated from hybridization with different sizes of the labeled DNA fragment. For the relationship between Tm and signal intensity, there was a different distribution of Tm in the lowest 300 or 3,000 probes with a range of 70 °C-72 °C and the highest 300 or 3,000 probes with a range of 72 °C-74 °C. CONCLUSION: The results of this study suggest that the initial microarray design may affect the accuracy of final analyses and that the probe Tm and the size of the labeled fragment may be the two factors of the greatest importance.

Time-series gene expression profiles in AGS cells stimulated with Helicobacter pylori.

AIM: To extend the knowledge of the dynamic interaction between Helicobacter pylori (H. pylori) and host mucosa. METHODS: A time-series cDNA microarray was performed in order to detect the temporal gene expression profiles of human gastric epithelial adenocarcinoma cells infected with H. pylori. Six time points were selected to observe the changes in the model. A differential expression profile at each time point was obtained by comparing the microarray signal value with that of 0 h. Real-time polymerase chain reaction was subsequently performed to evaluate the data quality. RESULTS: We found a diversity of gene expression patterns at different time points and identified a group of genes whose expression levels were significantly correlated with several important immune response and tumor related pathways. CONCLUSION: Early infection may trigger some important pathways and may impact the outcome of the infection.

Detection of genetically modified crops by combination of multiplex PCR and low-density DNA microarray.

OBJECTIVE: To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. METHODS: Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1). RESULTS: A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. CONCLUSION: A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

[In-vitro activity of rabeprazole, lansoprazole, and esomeprazole against Helicobacter pylori].

OBJECTIVE: To investigate the antimicrobial activity of Pariet, Tekpron, Nexium, respectively, against Helicobacter pylori (H. pylori) in vitro. METHODS: Antimicrobial effects of these medicines were evaluated through detection of MICs for 3 H. pylori strains isolated from different countries. RESULTS: The MIC(99) contents were 2.25 mg/L, 42.5 mg/L and 360 mg/L, respectively, for the three medicines. The strains under testing exhibited the same susceptibility to each medicine. Nexium did not inhibit the bacteria under the concentration of 3.6 - 36 mg/L with more and bigger H. pylori colonies seen when compared with controls. CONCLUSIONS: The growth inhibitory activity appeared to be different among the three PPI medicines under investigation, with Rabeprazole the most potential agent of the three. Data suggested that the action of growth inhibition in vitro was resting on the characteristic of the given PPI as well as the supplements of the medicine.