Genetic features of livestock-associated Staphylococcus aureus ST9 isolates from Chinese pigs that carry the lsa(E) gene for quinupristin/dalfopristin resistance.

Whole-genome sequencing (WGS) was used to investigate the genetic features of the recently identified lsa(E) gene in porcine S. aureus ST9 isolates. Three quinupristin/dalfopristin-resistant isolates harboring the lsa(E) gene (two MRSA and one MSSA) were sequenced. Phylogenetic analysis of 184S. aureus genomes showed that ST9 porcine isolates belong to a distinct sequence cluster. Further analysis showed that all isolates were deficient in the recently described type IV restriction-modification system and SCCmec type XII was identified in the two MRSA isolates, which included a rare class C2 mec gene complex. A 24kb ΨSCC fragment was found in the MRSA and MSSA isolates sharing 99% nucleotide sequence homology with the ΨSCCJCSC6690 (O-2) element of a ST9 MRSA isolate from Thailand (accession number AB705453). Comparison of these ST9 isolates with 181 publically available S. aureus genomes identified 24 genes present in all (100%) ST9 isolates, that were absent from the most closely related human isolate. Our analysis suggests that the sequenced quinupristin/dalfopristin-resistant ST9 lineage represent a reservoir of mobile genetic elements associated with resistance and virulence features.

Molecular typing of Chinese Streptococcus pyogenes isolates.

Streptococcus pyogenes causes human infections ranging from mild pharyngitis and impetigo to serious diseases including necrotizing fasciitis and streptococcal toxic shock syndrome. The objective of this study was to compare molecular emm typing and pulsed field gel electrophoresis (PFGE) with multiple-locus variable-number tandem-repeat analysis (MLVA) for genotyping of Chinese S. pyogenes isolates. Molecular emm typing and PFGE were performed using standard protocols. Seven variable number tandem repeat (VNTR) loci reported in a previous study were used to genotype 169 S. pyogenes geographically-diverse isolates from China isolated from a variety of disease syndromes. Multiple-locus variable-number tandem-repeat analysis provided greater discrimination between isolates when compared to emm typing and PFGE. Removal of a single VNTR locus (Spy2) reduced the sensitivity by only 0.7%, which suggests that Spy2 was not informative for the isolates screened. The results presented support the use of MLVA as a powerful epidemiological tool for genotyping S. pyogenes clinical isolates.

Staphylococcus aureus ST398 from slaughter pigs in northeast China.

To describe the prevalence and population structure of Staphylococcus aureus bacteria that colonize pigs at slaughterhouses in northeastern China, nose swabs were collected from pigs in two slaughterhouses in Harbin, Heilongjiang Province, China in 2009. S. aureus isolates were characterized by multilocus sequence typing (MLST), spa typing, SCCmec typing, antimicrobial susceptibility testing and pvl gene detection. A total of 200 S. aureus isolates were collected from 590 pigs (33.9%, 200/590), of which 162 (81%, 162/200) were methicillin-susceptible S. aureus (MSSA) and 38 (19%, 38/200) were methicillin-resistant S. aureus (MRSA). Ninety-nine of the MSSA isolates (99/162, 61.1%) were ST398, which represented the dominant sequence type overall. Eighty-seven isolates were ST9 (87/200, 43.5%), and all MRSA belonged to that sequence type which consisted of the spa types t899 and t2922. Among the MSSA strains, t034, t899 and t4358 were the most dominant spa types (139/162, 85.8%). All MRSA isolates harbored SCCmec type IVb. The pvl gene was only detected in 3 ST7/t2119 MSSA isolates. All MRSA but more importantly also 82.7% (134/162) of the MSSA isolates were resistant to six or more antibiotics. Moreover, a novel resistance determinant-lsa(E) was identified among 22% (44/200) of all isolates. In conclusion, pigs in northeast China are frequently colonized with ST398 MSSA. MRSA with this sequence type, typically associated with pigs in Europe, was not found. High levels of multiple antibiotic resistance among MRSA isolates as well as MSSA isolates are a public health concern.

Molecular subtyping and antimicrobial susceptibilities of Campylobacter coli isolates from diarrheal patients and food-producing animals in China.

The aim of this study was to determine the molecular subtyping and antimicrobial susceptibility characteristics of Campylobacter coli isolates from different sources in China. One hundred thirteen C. coli isolates were subtyped by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and porA and flaA short variable region (SVR) nucleotide sequences. Cluster analysis was performed based on the PFGE and sequence types (ST). Eighty-four PFGE patterns (SmaI) were observed in 113 isolates. Fifty-four STs (28 novel) and three clonal complexes (CC), 86% of which were clustered to CC828, were observed, as well as 52 porA and 37 flaA-SVR sequence alleles. MLST, porA, and flaA-SVR analysis demonstrated that many isolates from diarrheal patients shared identical genotypes with chicken isolates. Minimum inhibitory concentration values of 10 antibiotics were analyzed for 109 isolates isolated in 2011 using the E-test method. The most frequently observed resistance agents were nalidixic acid (100%) and ciprofloxacin (100%), followed by levofloxacin (99%), tetracycline (94%), metronidazole (93%), erythromycin (61%), streptomycin (72%), gentamicin (59%), ampicillin (50%), and chloramphenicol (29%). Multidrug resistance was detected in 108 of 109 C. coli isolates (99%).

Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expressions of TNF-α and IL-1ß.

Our previous study has verified that activation of group Ⅰ metabotropic glutamate receptors (mGluRⅠ) in the red nucleus (RN) facilitate the development of neuropathological pain. Here, we further discussed the functions and possible molecular mechanisms of red nucleus mGluR Ⅱ (mGluR2 and mGluR3) in the development of neuropathological pain induced by spared nerve injury (SNI). Our results showed that mGluR2 and mGluR3 both were constitutively expressed in the RN of normal rats. At 2 weeks post-SNI, the protein expression of mGluR2 rather than mGluR3 was significantly reduced in the RN contralateral to the nerve lesion. Injection of mGluR2/3 agonist LY379268 into the RN contralateral to the nerve injury at 2 weeks post-SNI significantly attenuated SNI-induced neuropathological pain, this effect was reversed by mGluR2/3 antagonist EGLU instead of selective mGluR3 antagonist ß-NAAG. Intrarubral injection of LY379268 did not alter the PWT of contralateral hindpaw in normal rats, while intrarubral injection of EGLU rather than ß-NAAG provoked a significant mechanical allodynia. Further studies indicated that the expressions of nociceptive factors TNF-α and IL-1ß in the RN were enhanced at 2 weeks post-SNI. Intrarubral injection of LY379268 at 2 weeks post-SNI significantly suppressed the overexpressions of TNF-α and IL-1ß, these effects were reversed by EGLU instead of ß-NAAG. Intrarubral injection of LY379268 did not influence the protein expressions of TNF-α and IL-1ß in normal rats, while intrarubral injection of EGLU rather than ß-NAAG significantly boosted the expressions of TNF-α and IL-1ß. These findings suggest that red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expressions of TNF-α and IL-1ß. mGluR Ⅱ may be potential targets for drug development and clinical treatment of neuropathological pain.

Surveillance of macrolide-resistant Mycoplasma pneumoniae in Beijing, China, from 2008 to 2012.

Macrolide resistance rates of Mycoplasma pneumoniae in the Beijing population were as high as 68.9%, 90.0%, 98.4%, 95.4%, and 97.0% in the years 2008 to 2012, respectively. Common macrolide-resistant mobile genetic elements were not detected with any isolate. These macrolide-resistant isolates came from multiple clones rather than the same clone. No massive aggregation of a particular clone was found in a specific period.

Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis typing of Acinetobacter baumannii in China.

A panel of seven variable-number tandem-repeat (VNTR) markers was selected for Acinetobacter baumannii typing analysis (MLVA-7). Compared with pulsed-field gel electrophoresis (PFGE), MLVA-7 provided greater discrimination. We modified the criteria for MLVA complex assignments proposed previously, and a remarkable congruence between MLVA-7- and PFGE-based strain clustering was observed.

Draft genome sequences of two Streptococcus pyogenes strains involved in abnormal sharp raised scarlet fever in China, 2011.

A scarlet fever outbreak caused by Streptococcus pyogenes occurred in China in 2011. To determine the genomic features of the outbreak strains, we deciphered genomes of two strains isolated from the regions with the highest incidence rates. The sequences will provide valuable information for comprehensive study of mechanisms related to this outbreak.

Antibiotic sensitivity of 40 Mycoplasma pneumoniae isolates and molecular analysis of macrolide-resistant isolates from Beijing, China.

MICs of eight antibiotics were detected with 40 Chinese Mycoplasma pneumoniae isolates. Thirty-eight isolates (95%) were macrolide resistant. Each macrolide-resistant isolate harbored an A2063G or A2064G point mutation in the 23S rRNA gene. All 40 isolates (100%) were type I strains, but they might have originated from different clones.

Characterization of Staphylococcus aureus strains associated with food poisoning in Shenzhen, China.

To characterize isolates of Staphylococcus aureus that were associated with staphylococcal food poisoning between 2006 and 2009 in Shenzhen, Southern China, a total of 52 Staphylococcus aureus isolates from 11 outbreaks were analyzed by using multilocus sequence typing (MLST), spa typing, and pulsed-field gel electrophoresis (PFGE). PCR analysis was used to analyze the staphylococcal enterotoxin (SE) genes sea to sei, and antimicrobial susceptibility testing was also performed. ST6 was the most dominant sequence type (ST), constituting 63.5% (34/52) of all of the isolates in 7 outbreaks. The next most common ST was ST943, which constituted 23.1% (12/52) of the isolates that were collected from 3 outbreaks. t701, t091, and t2360 were the most predominant spa types, constituting 67.3% (35/52) of the isolates that were collected from 11 outbreaks. Three PFGE types, (types A, B, and C) were the most frequently observed types, constituting 84.6% (44/52) of all of the isolates. The enterotoxin gene that we detected most frequently was sea (45/52; 86.5%). Four SE gene profiles were observed, including sea (n = 45), sec-seh (n = 3), seb (n = 2), and seg-sei (n = 2). With respect to antibiotic resistance, penicillin resistance was the most common (96.2%; 50/52), followed by resistance to tetracycline (28.8%; 15/52). Approximately 30.8% (16/52) of the isolates were resistant to at least two antibiotics, and 7.7% (4/52) of the isolates were resistant to three or more drugs. The two predominant S. aureus lineages, (i) PFGE types A and B with ST6 and (ii) PFGE type C with ST943, were identified in the outbreaks.

Sequence analysis of the p1 adhesin gene of Mycoplasma pneumoniae in clinical isolates collected in Beijing in 2008 to 2009.

The p1 genes of 60 Mycoplasma pneumoniae clinical isolates were sequenced and compared to previously reported p1 gene sequences. An AGT trinucleotide variable-number tandem repeat was identified that ranged in copy number from 5 to 14 among the isolates. In addition, a novel p1 gene variant named 2c was identified in 6 of the isolates.

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.

High DNA Uptake Capacity of International Clone II Acinetobacter baumannii Detected by a Novel Planktonic Natural Transformation Assay.

Acquisition of novel resistance genes is a key driver of multidrug resistance in the nosocomial pathogen Acinetobacter baumannii. To investigate the DNA uptake ability among clinical A. baumannii strains, a planktonic salt-free transformation assay was developed. A total of 142 clinical A. baumannii isolates with divergent genetic distance were selected, and 86 of them belong to international clonal lineage II (ICL2). Using this new transformation assay, 38% of the clinical A. baumannii isolates were natural competent. Among the multidrug-resistant (MDR) isolates, the transformable isolates all belonging to the ICLs, and showed significant higher transformation frequency compared with sensitive isolates. In addition, some of the ICL2 isolates triggered competence much earlier than the sensitive isolates with similar transformation frequencies. This may give them more opportunities to obtain successful transformation in their natural environment and provides an important clue to explain the severe drug resistance and clinical successfulness of ICL2.

Biofilm may not be Necessary for the Epidemic Spread of Acinetobacter baumannii.

Biofilm is recognized as a contributing factor to the capacity of Acinetobacter baumannii to persist and prosper in medical settings, but it is still unknown whether biofilms contribute to the spread of A. baumannii. In this study, the biofilm formation of 114 clinical A. baumannii isolates and 32 non-baumannii Acinetobacter isolates was investigated using a microtiter plate assay. The clonal relationships among A. baumannii isolates were assessed using pulsed-field gel electrophoresis and multilocus sequence typing, and one major outbreak clone and 5 other epidemic clones were identified. Compared with the epidemic or outbreak A. baumannii isolates, the sporadic isolates had significantly higher biofilm formation, but no significant difference was observed between the sporadic A. baumannii isolates and the non-baumannii Acinetobacter isolates, suggesting that biofilm is not important for the epidemic spread of A. baumannii. Of the multidrug-resistant (MDR) A. baumannii isolates in this study, 95.7% were assigned to international clone 2 (IC2) and showed significantly lower biofilm formations than the other isolates, suggesting that biofilm did not contribute to the high success of IC2. These findings have increased our understanding of the potential relationship between biofilm formation and the epidemic capacity of A. baumannii.

[Phenotype and genotype of antimicrobial resistance on nasal Staphylococcus aureus isolates from healthy people].

OBJECTIVE: To investigate the antimicrobial susceptibility and molecular nature related to the resistance on macrolides from nasal Staphylococcus (S.) aureus isolates among healthy people. METHODS: A total of 100 S. aureus isolates collected from 2009 to 2011 were tested for antimicrobial susceptibility by E-test. Double disc test (D-test) was used to detect the inducible clindamycin resistance. All S. aureus isolates were characterized by spa typing. Macrolides resistance genes were detected and compared with isolates that were collected clinically or from the livestock. RESULTS: High resistance rates on erythromycin or clindamycin was noticed, with 52% and 27%, respectively. Inducible clindamycin resistance was identified in 29 of the 100 (29%) isolates. In total, the 100 isolates were assigned to 35 spa types. The most common spa types were found to be t189, t571, t002, t796, t437, t034 and t701, that accounted for 51.0% of all the isolates. erm (C) (57.7%) and erm (B) (34.6%) were found as the dominant genes in 52 S. aureus isolates from healthy people. On the other hand, erm (A) and erm (C) were identified in 95.0% S. aureus isolates from patients and all the livestock, respectively. CONCLUSION: erm (C) and erm (B) carrying S. aureus strains were circulating in healthy people and these genes were distributed in different S. aureus clones.

Culture-Independent Detection and Genotyping of Mycoplasma pneumoniae in Clinical Specimens from Beijing, China.

A duplex real-time PCR assay was designed for simultaneous detection and genotyping of Mycoplasma pneumoniae (M. pneumoniae). The detection/typing performance of this duplex PCR method, targeting specific genes for M. pneumoniae type 1 (mpn 459) and type 2 (mpna 5864), was compared to that of the previously published MpP1 real-time PCR assay and the genotyping method for the adhesin P1 gene (mpn 141). A total of 1,344 throat swab specimens collected from patients in Beijing, China were tested for M. pneumoniae by bacterial culture, MpP1 real-time PCR assay, and our duplex PCR assay, and positive detection rates of 26.9%, 34.4%, and 33.7%, respectively, were obtained. The duplex PCR method demonstrated high sensitivity and accuracy for detecting and genotyping M. pneumoniae, and significant differences in genotyping ability were observed when compared to the conventional P1 gene-based method. M. pneumoniae type 1 was the predominate genotype from 2008 to 2012 in Beijing, and a shift from type 1 to type 2 began to occur in 2013. To our knowledge, this is the first reported incidence of a type shift phenomenon of M. pneumoniae clinical isolates in China. These genotyping results provide important information for understanding recent changes in epidemiological characteristics of M. pneumoniae in Beijing.

[Cultivation and serial propagation of a new rotavirus causing adult diarrhea in primary human embryo kidney cells].

OBJECTIVE: To investigate the methodology of cultivation of the new rotavirus that causes adult diarrhea. METHODS: 10% suspension of new rotavirus positive stool specimens in DMEM with 100 microgram/ml trypsin was made and centrifuged at the speed of 3 000 rpm for 10 minutes. The supernatant was filtered with 0.45 micrometer-pore-sized membrane filter, and the filtrate was incubated at 37 degrees C for 60 minutes. Primary human embryo kidney (PHEK) cells were prepared and seeded into roller tubes at the concentration of 1 ml odf cell suspension per tube. At 1 hour prior to inoculation, the PHEK cells were rinsed and refed with serum-free DMEM. Immediately before inoculation, the DMEM was decanted, and 200 microliter of prepared filtrate was inoculated into each tube. The tubes were incubated at 37 degrees C for 1 hour. At the end of 1 hour 800 microliter of DMEM containing trypsin (100 microgram per ml) were added to each tube. The tubes were placed in a roller tube apparatus at 37 degrees C for 3 days, removed and frozen-thawed once. 200 microliter of the cell culture fluids (CCF) were used for inoculation of each tube for next virus passage. Detection of new rotavirus from CCF was carried out by PAGE, IF, EM and IEM. RESULTS: New rotavirus was successfully isolated in cultured cells from 1 of 10 specimens. The isolated virus, designated J19, was propagated in PHEK cells for 28 passages. RNA patterns of J19 strain were identical to that of original new rotavirus inoculum and different from that of group A, B, C rotavirus. PHEK cells infected with the J19 strain were detected by IF. Infected cells reacted with convalescent antisera to the new rotavirus, but did not react with convalescent antisera to ADRV and hyperimmune antisera against group A, B rotavirus. Rotavirus particles were detected by EM, IEM in infected CCF of J19 strain. Other viral particles were not observed. The particles were aggregated with convalescent antisera to new rotavirus. CONCLUSION: We were successful in adapting a new rotavirus to serial propagation in PHEK cells. The J19 strain is a kind of new rotavirus different from group A, B, C rotavirus. This is the first report on the cultivation and propagation of the new rotavirus in cultured cells.

Comparative analysis of the virulence characteristics of epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from Chinese children: ST59 MRSA highly expresses core gene-encoded toxin.

This study aims to investigate the prevalence of a novel cell wall-anchored protein gene, sasX, and to obtain information on the genetic basis for the pathogenic potential of the MRSA strains isolated from Chinese children. The molecular and virulence characteristics of the clinical strains were analyzed. Twenty-two sequence types (STs) were obtained, with six epidemic clones ST59, ST239, ST1, ST910, ST88, and ST338 accounting for 35.8, 22, 6.6, 6.6, 5.3, and 4.1% respectively. The expression levels of hla, psmα, and RNAIII were higher in ST59 than in other STs (p < 0.05). The sasX gene was detected in 26 (10.7%) MRSA isolates. ST239-MRSA-SCCmecIII-t037 (61.5%) was the predominant sasX-positive MRSA clone. The expressions of PSMα and RNAIII were higher in sasX-positive ST239 isolates than in sasX-negative ST239 ones (p < 0.01). Notably, the percentage of invasive infection in infections caused by sasX-positive ST239 MRSA was higher than that by sasX-negative ST239 MRSA (p = 0.008). This study indicated that ST59 was the predominant clone in the MRSA isolates obtained from Chinese children and might have stronger pathogenic potential. The prevalence of the sasX gene in the MRSA isolates from children was relatively low. Furthermore, the sasX gene might be related to the expressions of PSMα and RNAIII and infection invasiveness.

Detection of Mycoplasma pneumoniae by colorimetric loop-mediated isothermal amplification.

Mycoplasma pneumoniae (M. pneumoniae) is one of the most important pathogens that cause respiratory tract infection in children and adults. In this study, we describe a rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method to detect M. pneumoniae. The specificity and sensitivity of this assay were detected with 21 common respiratory pathogens and 39 M. pneumoniae DNA. The sensitivity of LAMP was 100% among 39 M. pneumoniae isolates and the specificity was 100% among 9 members of other Mycoplasma and 12 common respiratory pathogens. The lowest detectable limit (LDL) of this assay was 102 copies, which detected by a series of standard M. pneumoniae DNA. To evaluate the clinical applicability of the LAMP assay, a total of 80 clinical samples were examined by conventional PCR, real-time PCR and the LAMP assays, respectively. The positive rates were 15.0%, 32.5% and 26.3%, respectively. This colorimetric LAMP assay demonstrated a high level of sensitivity comparable with that of conventional PCR for the detection of M. pneumoniae. It is a valuable method for simple, cost-effective and rapid detection of M. pneumoniae in the rural areas and basic clinical of China.

Comparative genomics of Helicobacter pylori strains of China associated with different clinical outcome.

In this study, a whole-genome CombiMatrix Custom oligonucleotide tiling microarray with 90,000 probes covering six sequenced Helicobacter pylori (H. pylori) genomes was designed. This microarray was used to compare the genomic profiles of eight unsequenced strains isolated from patients with different gastroduodenal diseases in Heilongjiang province of China. Since significant genomic variation was found among these strains, an additional 76 H. pylori strains associated with different clinical outcomes were isolated from various provinces of China. These strains were tested by polymerase chain reaction to demonstrate this distinction. We identified several highly variable regions in strains associated with gastritis, gastric ulceration, and gastric cancer. These regions are associated with genes involved in the bacterial type I, type II, and type III R-M systems. They were also associated with the virB gene, which lies on the well-studied cag pathogenic island. While previous studies have reported on the diverse genetic characterization of this pathogenic island, in this study, we find that it is conserved in all strains tested by microarray. Moreover, a number of genes involved in the type IV secretion system, which is related to horizontal DNA transfer between H. pylori strains, were identified in the comparative analysis of the strain-specific genes. These findings may provide insight into new biomarkers for the prediction of gastric diseases.