Yu-Shan Pan

Molecular characterization of extended-spectrum β-lactamase-producing Escherichia coli isolates from chickens in Henan Province, China

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli has spread rapidly worldwide and poses a serious threat to human and animal health. This study collected 51 non-replicate E. coli isolates from 14 different chicken farms in Henan Province in China from December 2007 to August 2008. The prevalence of ESBL-producing E. coli, molecular characterization of the ESBL-related bla genes, including bla(TEM), bla(SHV) and bla(CTX-M), and the susceptibilities of these bacteria to various antimicrobial agents were determined. Thirty-one of the 51 isolates were positive for an ESBL phenotype and 29 of these isolates carried one or more bla genes. Twenty-two isolates harboured bla(TEM) genes and 15 isolates carried bla(CTX-M) genes (one CTX-M-14, three CTX-M-24 and 11 CTX-M-65). One isolate carried bla(TEM)(-57); the remaining bla(TEM) isolates carried bla(TEM-1) with one silent nucleotide base variation (T18C). We believe that this is the first study to report TEM-57 in E. coli isolates. All isolates harbouring bla(CTX-M-24) and bla(CTX-M-14) and five of the bla(CTX-M-65) isolates also harboured the bla(TEM-1) gene. To our knowledge, this study is the first to describe detection of CTX-M-65-producing E. coli isolated from chickens. None of the isolates contained the bla(SHV) gene. Conjugation experiments demonstrated that bla(CTX-M) and bla(TEM) genes could be transferred to E. coli DH5 alpha. The results indicate that ESBL frequency has reached an alarming level in chicken isolates in China, with TEM-1 and CTX-M-65 enzymes being the two predominant beta-lactamases detected.

Novel arrangement of the blaCTX-M-55 gene in an Escherichia coli isolate coproducing 16S rRNA methylase

A multi‐drug resistant Escherichia coli C21 was isolated from a chicken in China. It was shown to be positive for the presence of the blaTEM‐1, blaCTX‐M‐55 and rmtB genes by PCR. This strain was examined by phylogenetic grouping, conjugation experiments, plasmid analysis, PCR‐based replicon typing and multi‐locus sequence typed (MLST). The genetic environment of blaCTX‐M‐55 was investigated by PCR mapping. The strain belonged to phylogroup A, ST156. The blaCTX‐M‐55 and rmtB genes were found to be present in separate plasmids that belonged to the IncI1 and IncN families, respectively. These antibiotic‐resistant plasmids could be transferred to the recipient strain alone or together. A new arrangement of ISEcp1Δ‐IS1294‐ΔISEcp1‐blaCTX‐M‐55‐ORF477, in which the ISEcp1 element was disrupted by another IS1294 element, was identified initially. Conjugative transfer and IS elements found in this study could lead to the rapid dissemination of blaCTX‐M‐55 and rmtB among strains of Enterobacteriaceae, which could pose a threat to animal husbandry and public health.

A multidrug-resistance region containing blaCTX-M-65, fosA3 and rmtB on conjugative IncFII plasmids in Escherichia coli ST117 isolates from chicken.

The blaCTX-M gene has become the most common gene encoding extendedspectrum b-lactamase (ESBL) in multidrug-resistant Gram-negative bacteria worldwide. To date, 147 subtypes of the CTX-M enzyme have been reported (http://www.lahey.org/Studies/other. asp#table1). Since 2007, blaCTX-M-65 has become one of the predominant blaCTX-M genes in ESBL-producing bacterial isolates from animals in China (Yuan et al., 2009; Deng et al., 2011). The blaCTX-M genes often coexist with genes encoding 16S rRNA methylases such as armA and rmtB, or with fosA3 that confers resistance to fosfomycin (Hou et al., 2012; Lee et al., 2012; Ho et al., 2013). In China, rmtB is the most prevalent 16S rRNA methylase gene among the Enterobacteriaceae. Plasmids in which the four resistance genes blaTEM-1, blaCTX-M-65, fosA3 and rmtB coexist were recently found in Escherichia coli isolates from ducks and dogs in China (Sun et al., 2012; He et al., 2013). Mobile elements mobilize resistance genes. The ISEcp1-blaCTX-M-IS903-iroN structure is a typical transposition unit in the blaCTX-M-9 cluster, to which blaCTX-M-65 belongs (He et al., 2013). The rmtB gene is commonly carried by the Tn3 transposon (Doi et al., 2004) and fosA3 has been found in the IS26-formed composite transposon (Lee et al., 2012). Since chicken was considered as a reservoir for extraintestinal pathogenic E. coli in humans (Bergeron et al., 2012), the detection of blaTEM-1, blaCTX-M-65, rmtB and fosA3 in an isolate of animal origin may represent an emerging threat to public health. In our present study, we characterized a multidrug-resistance region (MRR) of an F33:A-:Bplasmid carrying blaTEM-1, blaCTX-M-65, rmtB and fosA3 that was isolated from an E. coli avian strain of the sequence type 117 (ST117).

Prevalence of tetracycline resistance genes and identification of tet(M) in clinical isolates of Escherichia coli from sick ducks in China

Tetracycline resistance is one of the most frequently encountered resistance properties in bacteria of animal origin. The aim of the present study was to investigate the prevalence and diversity of tetracycline resistance (tet) genes among Escherichia coli clinical isolates from diseased ducks in China and to report the identification and sequencing of the tet(M) gene. The susceptibility of 85 Escherichia coli strains to tetracyclines was determined by broth microdilution, and the presence of tet genes was investigated by multiplex PCR. All of the 85 isolates were fully resistant to both oxytetracycline and tetracycline, and 76.5 % were resistant to doxycycline. Seventy-seven of the isolates (90.6 %) encoded multiple tet genes, with 17.6, 38.8 and 34.1 % encoding two, three and four tet genes, respectively, and only 7.1 % encoded a single tet(A) gene. The MICs of oxytetracycline and tetracycline for all isolates ranged from 16 to ≥128 µg ml(-1) with a MIC90 of >128 µg ml(-1), regardless of the type or number of tet genes encoded. Isolates containing tet(M) commonly had more than one tet gene per strain. The doxycycline resistance rate in the tet(M)-positive isolates was significantly higher than in the tet(M)-negative isolates (P<0.05). A full-length tet(M) gene, including the promoter region, was obtained by PCR in seven of the 41 tet(M)-positive isolates and was sequenced and cloned. The cloned tet(M) gene conferred resistance to tetracyclines in the recombinant Escherichia coli host strain. These results revealed that, in these isolates, the prevalence of multiple tet genes was strikingly high and that tet(M) played a role in doxycycline resistance.

Regulation of the Two-Component Regulator CpxR on Aminoglycosides and β-lactams Resistance in Salmonella enterica serovar Typhimurium

The two-component signal transduction system CpxAR is especially widespread in Gram-negative bacteria. It has been reported that CpxAR contributes to the multidrug resistance (MDR) in Escherichia coli. CpxR is a response regulator in the two-component CpxAR system. The aim of this study was to explore the role of cpxR in the MDR of S. enterica serovar Typhimurium. The minimal inhibitory concentrations (MICs) of various antibiotics commonly used in veterinary medicine for strains JS (a multidrug-susceptible standard strain of S. enterica serovar Typhimurium), JSΔcpxR, JSΔcpxR/pcpxR, JSΔcpxR/pcpxR*, JSΔcpxRΔacrB, JSΔcpxRΔacrB/pcpxR, JSΔcpxRΔacrB/pcpxR*, 9 S. enterica serovar Typhimurium isolates (SH1–9), and SH1–9ΔcpxR were determined by the 2-fold broth microdilution method. The relative mRNA expression levels of ompF, ompC, ompW, ompD, tolC, acrB, acrD, acrF, mdtA, marA, and soxS in strains JS, JSΔcpxR, and JSΔcpxR/pcpxR were detected by real-time PCR. The results showed 2- to 4-fold decreases in the MICs of amikacin (AMK), gentamycin (GEN), apramycin (APR), neomycin (NEO), ceftriaxone (CRO), ceftiofur (CEF), and cefquinome (CEQ) for strain JSΔcpxR, as compared to those for the parental strain JS. Likewise, SH1–9ΔcpxR were found to have 2- to 8-fold reduction in resistance to the above antibiotics, except for NEO, as compared to their parental strains SH1–9. Furthermore, 2- to 4-fold further decreases in the MICs of AMK, GEN, APR, and CEF for strain JSΔcpxRΔacrB were observed, as compared to those for strain JSΔacrB. In addition, CpxR overproduction in strain JSΔcpxR led to significant decreases in the mRNA expression levels of ompF, ompC, ompW, ompD, tolC, acrB, marA, and soxS, and significant increases in those of stm3031 and stm1530. Notably, after all strains were induced simultaneously by GEN to the 15th passage at subinhibitory concentrations, strain JSΔcpxR/pcpxR showed significant increases in mRNA expression levels of the efflux pump acrD and mdtA genes, as compared to strain JSΔcpxR. Our results indicate that the two-component regulator CpxR contributes to resistance of S. enterica serovar Typhimurium to aminoglycosides and β-lactams by influencing the expression level of the MDR-related genes.

Complete Sequence of pEC012, a Multidrug-Resistant IncI1 ST71 Plasmid Carrying blaCTX-M-65, rmtB, fosA3, floR, and oqxAB in an Avian Escherichia coli ST117 Strain

A 139,622-bp IncI1 ST71 conjugative plasmid pEC012 from an avian Escherichia coli D-ST117 strain was sequenced, which carried five IS26-bracketed resistance modules: IS26-fosA3-orf1-orf2-Δorf3-IS26, IS26-fip-ΔISEcp1-blaCTX-M-65-IS903D-iroN-IS26, IS26-ΔtnpR-blaTEM-1-rmtB-IS26, IS26-oqxAB-IS26, and IS26-floR-aac(3)-IV-IS26. The backbone of pEC012 was similar to that of several other IncI1 ST71 plasmids: pV408, pM105, and pC271, but these plasmids had different arrangements of multidrug resistance region. In addition, the novel ISEc57 element was identified, which is in the IS21 family. The stepwise emergence of multi-resistance regions demonstrated the accumulation of different resistance determinants through homologous recombination. To the best of our knowledge, this is the first study to identify a multidrug-resistant IncI1 ST71 plasmid carrying blaCTX-M-65, rmtB, fosA3, floR, and oqxAB in an avian E. coli ST117 strain.

Characterization of an rmtB-carrying IncI1 ST136 plasmid in avian Escherichia coli isolates from chickens.

The rmtB gene, one of the 16S rRNA methylase genes whose products confer high-level resistance to aminoglycosides, is most prevalent among Enterobacteriaceae strains. In this study, eight non-duplicate rmtB-carrying avian Escherichia coli strains from a farm in China were isolated and characterized, and further examined by phylogenetic grouping, conjugation experiments and PCR-based replicon typing. In addition, the genetic environment of rmtB was investigated by cloning and sequencing. Six rmtB-carrying E. coli were identified as phylogroup A, sequence type (ST) 156 (A-ST156), with two assigned to D-ST117; however, all of them carried the same IncI1 ST136 plasmid. The genetic environment of the rmtB gene in these eight plasmids was the same, as shown by PCR mapping. A multidrug-resistant region carrying blaTEM-1, rmtB, a class 1 integron cassette array (intI1-dfrA12-orfF-aadA2-qacEΔ1-sul1) and aacC2 was characterized on the conjugative IncI1 ST136 plasmid. Co-location of the rmtB gene with a class 1 integron cassette array and aacC2 on the conjugative plasmid will facilitate its maintenance and dissemination.

Prevalence, resistance pattern, and molecular characterization of Staphylococcus aureus isolates from healthy animals and sick populations in Henan Province, China

BackgroundStaphylococcus aureus is one of the most prevalent pathogens and a causative agent of a variety of infections in humans and animals. A total of 640 samples were collected from healthy animals and patients from 2013 to 2014 in Henan Province, China, to investigate the prevalence and perform molecular characterization of S. aureus. Antimicrobial resistance and virulence genes were determined and pulsed-field gel electrophoresis (PFGE) and staphylococcal cassette chromosome mec (SCCmec) typing were performed.ResultsOverall, 22.3% (n = 143) of the samples were positive for S. aureus. The prevalence of methicillin-resistant S. aureus (MRSA) was 5.59%. Capsular polysaccharide locus type 5 (Cap5; 56.64%) was the dominant serotype. S. aureus strains showed high resistance to penicillin (96.50%), ciprofloxacin (52.45%), amikacin (67.83%), erythromycin (96.50%), lincomycin (97.20%), and tetracycline (68.53%) and 109 (76.2%) isolates harbored six or more tested resistance genes. The most predominant resistance genes were aphA (52.45%), ermC (53.15%), and tetM (52.45%). Eighty-seven (60.8%) isolates harbored six or more tested virulence genes. The most predominant enterotoxin genes were sed (20.28%), sej (20.98%), sep (14.69%), and set (37.76%). The prevalence of lukED gene was (57.34%), and a small number of isolates carried pvl (5.59%) and TSST-1 (2.80%). A total of 130 (82.52%) isolates could be typed by PFGE with SmaI digestion. PFGE demonstrated that 45 different patterns (P) that were grouped into 17 pulsotypes and 28 separate pulsotypes using a 90% cut-off value. A total of 118 (82.52%) isolates were successfully typed by spa, and 26 spa types were identified, t15075 (14.00%) and t189 (12.59%) were the most common types. SCCmec types were detected from eight MRSA isolates, with the most prevalent type being SCCmec IVa. MRSA-SCCmec Iva-t437 was observed in human isolates.ConclusionThis study revealed a high prevalence of S. aureus in healthy animals and patients from Henan Province, China. Resistant S. aureus exhibited varying degrees of multidrug resistance. The presence of antibiotic resistance and virulence genes may facilitate the spread of S. aureus strains and pose a potential threat to public health, highlighting the need for vigilant monitoring of these isolates at the human–animal interface.

The complete mitochondrial genome of Archangel pigeon

Abstract The Archangel pigeon mitochondrial DNA has 17,235 bp and its structural organization is conserved compared to those of other birds. In this study, we report the basic characteristics of the Archangel mitochondrial genome, including structural organization and base composition of the rRNAs, tRNAs and protein-coding genes, as well as characteristics of tRNAs. These features are applicable for the study of phylogenetic relationships in pigeons.

{μ-N-[(Diphenyl­phosphino)meth­yl]pyridin-2-amine-κ2N1:P}bis­{[2-(2,2′-bipyridin-6-yl)phenyl-κ3N,N′,C1]platinum(II)} bis­(perchlorate)

The title compound, [Pt2(C16H11N2)2(C18H17N2P)](ClO4)2, contains two PtII atoms, bridged by an N-[(diphenylphosphino)methyl]pyridin-2-amine (dppmp) ligand. One Pt atom is coordinated by one P atom from the dppmp ligand, and one C atom and two N atoms from a 6-phenyl-2,2′-bipyridine (pbpy) ligand in a square-planar geometry. The other Pt atom is coordinated by one N atom from the dppmp ligand, and one C atom and two N atoms from another pbpy ligand in a square-planar geometry. There are intramolecular π–π interactions between the pbpy ligands, with a centroid–centroid distance of 3.62 (1) Å between two pyridyl rings. The oxygen atoms of both perchlorate anions are disordered, each over two different positions [occupanicies 0.49 (3)/0.51 (3) and 0.48 (2)/0.52 (2)].