Guobao Tian

Detection of CTX-M-15, CTX-M-22, and SHV-2 extended-spectrum β-lactamases (ESBLs) in Escherichia coli fecal-sample isolates from pig farms in China.

The aim of the present study was to investigate the antibiotic resistance profiles and the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates from two production swine operations in Sichuan Province, China, between August 2002 and February 2007. The prevalence of ESBL-producing E. coli increased dramatically from 2.2% to 10.7% during this period. This increase appeared mostly related to dissemination of CTX-M-type ESBLs among E. coli isolates. Of 212 E. coli isolates studied, 14 harbored ESBL genes. Among them, 13 harbored bla(CTX-M-15/22) and one harbored bla(SHV-2). To our knowledge, this is the first study to identify bla(CTX-M-22) from production animals. One isolate in 2002 harbored bla(SHV-2), indicating that ESBL genes have been present in farm animals in China since at least 2002. Molecular characterization and pulsed-field gel electrophoresis of the ESBL-producing isolates suggested that different mechanisms may be involved in the dissemination of the CTX-M genes and revealed that additional resistance determinants for non-beta-lactam antibiotics were carried by plasmids encoding certain ESBL genes. Results of this study provide an example of how ESBL genes, particularly those of CTX-M lineages, are rapidly spreading among E. coli isolates from commercial pig farms in Sichuan province of China.

Complete genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain H120

The strain H120 of infectious bronchitis virus (IBV) is one of the earliest and representative attenuated live Infectious Bronchitis vaccine strains. To investigate the genomic feature of H120 and further understand its role in the epidemiology of IBV, complete genome of H120 was sequenced and compared with sequences of other IBV strains by phylogenetic and recombination analysis. The complete genome of H120 is 27631 nucleotides in length and has a similar structure with that of Beaudette strain. We found that strain ZJ971 is probably a virulence revertant of H120. Nine amino acids changes and a three-nucleotide deletion were identified in ZJ971. Besides, potential recombination events associated with H120 were found in five IBV strains including H52, KQ6, SAIBK, Ark DPI 11, and Ark DPI 101. This study suggested that H120 might have contributed to the emergence of new IBV variants through both virulence reversion and recombination.

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and beta-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp DNA Stool Mini Kit.

Detection of clinically important β-lactamases in commensal Escherichia coli of human and swine origin in western China.

Data correlating β-lactamases found in commensal Escherichia coli of human and animal origin are limited. In this study, 447 commensal E. coli isolates from the faeces of humans and swine (280 human isolates from four hospitals and 167 swine isolates from seven farms) were collected between September 2006 and January 2009 in western China. For extended-spectrum β-lactamase (ESBL)-producing and other cephalosporin-resistant isolates, the relevant β-lactamase genes (bla(TEM), bla(SHV), bla(CTX-M-1/2/9) group, bla(CMY-2) and bla(KPC)) were detected by PCR analysis. Of the 447 isolates tested, 120 (26.8 %) were confirmed as producing ESBL. Among these, 70 and 40 human isolates carried a member of the bla(CTX-M-1 )group (13 bla(CTX-M-3), 21 bla(CTX-M-15), four bla(CTX-M-22), eight bla(CTX-M-28), four bla(CTX-M-36), 15 bla(CTX-M-55) and five bla(CTX-M-69)) or bla(SHV) (14 bla(SHV-2), seven bla(SHV-5), ten bla(SHV-12), five bla(SHV-57) and four bla(SHV-97)),respectively, whilst six and four swine isolates carried a member of the bla(CTX-M-1 )group (one bla(CTX-M-15) and five bla(CTX-M-22)) or bla(SHV) (three bla(SHV-2) and one bla(SHV-12)), respectively. Furthermore, 59 human and swine isolates and seven human isolates carried bla(CMY-2) and bla(KPC), respectively. These findings indicate that the bla(CTX-M-1) group, including the novel variant bla(CTX-M-69), and bla(SHV) are the predominant ESBL genes in both humans and swine in western China, and bla(CMY-2) is also common in both groups. The carriage rates of broad-spectrum β-lactamases among commensal E. coli was much lower in swine than in humans, suggesting that β-lactamase genes have not established themselves in animal ecosystems in western China.

Phenotypic and genotypic characterization of β-lactam resistance in Klebsiella pneumoniae isolated from swine

Little is known about the antimicrobial resistance mechanisms in Klebsiella pneumoniae from swine in China. Thus, this paper aims to demonstrate the β-lactam resistance phenotypes and genotypes of K. pneumoniae isolates from swine in southwestern China, detect possible new β-lactamase variants, and determine whether or not the variants differ in their antibiotic resistance. Isolates from 58 unrelated diseased swine were collected from 61 pig farms in southwestern China from 2007 to 2009. Among the 58 isolates, 75.8-100% were resistant to β-lactam, 62.0-68.97% to fluoroquinolone, 44.8-46.55% to aminoglycoside, and 8.62-17.24% to β-lactam inhibitors. PCR amplification and DNA sequencing showed that bla(TEM-1) was detected in 100% (n=58) of the isolates, bla(SHV) in 82.76% (n=48), bla(CTX-M) in 39.66% (n=23), and bla(OKP) in 17.24% (n=10). The bla(SHV) types included bla(SHV-1), bla(SHV-11), bla(SHV-12), and bla(SHV-27). None of the isolates harbored bla(KPC), bla(LEN), or bla(GES) gene. Four novel variants (bla(OKP-A-13), bla(OKP-A-14), bla(OKP-A-15), and bla(OKP-A-16)) were identified among the 10 OKP β-lactamase-producing K. pneumoniae isolates resistant to ampicillin, amoxicillin, oxacillin, cefalexin, and cefadroxil. Plasmid analysis and PCR amplification indicated that bla(TEM-1) genes were detected in the total plasmid. Molecular typing by pulsed-field gel electrophoresis revealed the presence of 10 distinct pulsotypes of OKP producer isolates. Plasmid DNA digested with XbaI yielded two to six bands of ca. 0.15-30 kb. Transformants of the 10 OKP producer isolates showed no differences in their antibiotic susceptibility, except for the pulsotype B transformant, which carried bla(CTX-M). In China, β-lactam resistance appeared to be common among K. pneumoniae isolates from swine, suggesting that K. pneumoniae may be a reservoir for the dissemination of β-lactam resistance among Chinese pig farms.

Expression, purification and characterization of a phyA(m)-phyCs fusion phytase.

The phyAm gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichia pastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyAm-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 °C and an optimal pH at 5.5∼6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 °C to 95 °C for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoHf), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyAm.