Whole genome sequence of Streptococcus pluranimalium SP21-2, a porcine strain harbouring optrA and lsa(E) with chromosomal location.

OBJECTIVES: The aim of this study was to characterise the whole genome sequence of multidrug-resistant Streptococcus pluranimalium strain SP21-2 of swine origin in China. METHODS: Illumina Miseq (200X coverage) and Nanopore PromethION platform (100X coverage) were used for genome sequencing. Rapid Annotation using Subsystem Technology (RAST) was used to annotate the genome of SP21-2. The antimicrobial resistance genes (ARGs) were identified using ResFinder-4.1. RESULTS: The assembled circular genome of S. pluranimalium SP21-2 was 1,987,058 bp in length with a GC content of 39.54%, and no plasmid sequence was detected. A total of 2086 coding sequences were predicted by RAST. Oxazolidinone-phenicol resistance gene, optrA, and pleuromutilin-lincosamide-streptogramin A resistance gene, lsa(E), are both located on chromosomes, associated with IS1216 and ISS1S, respectively. In addition, SP21-2 harbours lnu(B) (lincosamide), ant (6)-Ia and aac(6')-aph(2") (aminoglycoside), erm(B) (macrolide), and tet(O) (tetracycline). CONCLUSION: We firstly report the oxazolidinone-phenicol gene, optrA, and pleuromutilin-lincosamide-streptogramin A resistance gene, lsa(E), in S. pluranimalium. In this strain, we firstly identified ISS1S and IS1216 carrying ARGs in S. pluranimalium, which will provide a valuable reference to understanding potential transfer mechanisms of ARGs in S. pluranimalium.

Genome sequence of multidrug-resistant Erysipelothrix rhusiopathiae ZJ carrying several acquired antimicrobial resistance genes.

OBJECTIVES: This study aimed to determine the genetic environment of antimicrobial resistance genes (ARGs) in Erysipelothrix rhusiopathiae strain ZJ isolated from a pig with symptoms of swine erysipelas in China. METHODS: Illumina MiSeq (200× coverage) and PacBio RS II (100× coverage) platforms were used for genome sequencing. ARGs and prophages were identified using ResFinder 3.0 and PHASTER, respectively. A conjugation experiment, induced prophage infection and long-term passage assay were performed to determine the transferability and stability of ARGs in this strain. RESULTS: The assembled circular genome of E. rhusiopathiae ZJ was 1 945 689 bp with a GC content of 36.48%; no plasmid sequence was detected. Eleven acquired ARGs were identified in the genome. A novel integrative and conjugative element (ICE) encoding a multidrug resistance (MDR) gene cluster [aadE-apt-spw-lsa(E)-lnu(B)-aadE-sat4-aphA3] was identified in strain ZJ. A prophage Φ1605 harbouring mef(A)-msr(D) and tet(M) was also found in this strain, which can take a circular form and can be induced by mitomycin C to infect E. rhusiopathiae G4T10 for ARG transfer. CONCLUSION: To our knowledge, this is the first report of a complete genome sequence of E. rhusiopathiae carrying multiple ARGs obtained from a pig farm. This is the first identification of a novel chimeric ICE carrying a MDR gene cluster and a prophage carrying ARGs in E. rhusiopathiae, which will provide a valuable reference to understand the potential transfer mechanism of MDR gene clusters carried by ICEs and prophages in Gram-positive bacteria.

Two novel Salmonella genomic island 1 variants in Proteus mirabilis isolates from swine farms in China.

Four different Salmonella genomic island 1 (SGI1) variants, including two novel variants, were characterized in one Salmonella enterica serovar Rissen sequence type ST1917 isolate and three Proteus mirabilis isolates from swine farms in China. One novel variant was derived from SGI1-B with the backbone gene S021 disrupted by a 12.72-kb IS26 composite transposon containing the dfrA17-aadA5 cassettes and macrolide inactivation gene cluster mphA-mrx-mphR. The other one was an integron-free SGI1 and contained a 183-bp truncated S025 next to IS6100 and S044.

First report of macrolide resistance gene erm(T) harbored by a novel small plasmid from Erysipelothrix rhusiopathiae.

The macrolide resistance gene erm(T) was identified for the first time in a porcine Erysipelothrix rhusiopathiae isolate from swine in China. The novel 3,749-bp small plasmid pER29, which carries erm(T), had a G+C content of 31% and four distinct open reading frames. The presence of pER29 increased by at least 128-fold the MICs of clindamycin and erythromycin for E. rhusiopathiae. The fitness cost of pER29 could be responsible for the low frequency of erm(T) in E. rhusiopathiae.

Molecular characteristics of Salmonella genomic island 1 in Proteus mirabilis isolates from poultry farms in China.

Six out of the 64 studied Proteus mirabilis isolates from 11 poultry farms in China contained Salmonella genomic island 1 (SGI1). PCR mapping showed that the complete nucleotide sequences of SGI1s ranged from 33.2 to 42.5 kb. Three novel variants, SGI1-W, SGI1-X, and SGI1-Y, have been characterized. Resistance genes lnuF, dfrA25, and qnrB2 were identified in SGI1 for the first time.

Dynamics of quinolone resistance in fecal Escherichia coli of finishing pigs after ciprofloxacin administration.

Escherichia coli resistance to quinolones has now become a serious issue in large-scale pig farms of China. It is necessary to study the dynamics of quinolone resistance in fecal Escherichia coli of pigs after antimicrobial administration. Here, we present the hypothesis that the emergence of resistance in pigs requires drug accumulation for 7 days or more. To test this hypothesis, 26 pigs (90 days old, about 30 kg) not fed any antimicrobial after weaning were selected and divided into 2 equal groups: the experimental (EP) group and control (CP) group. Pigs in the EP group were orally treated daily with 5 mg ciprofloxacin/kg of body weight for 30 days, and pigs in the CP group were fed a normal diet. Fresh feces were collected at 16 time points from day 0 to day 61. At each time point, ten E. coli clones were tested for susceptibility to quinolones and mutations of gyrA and parC. The results showed that the minimal inhibitory concentration (MIC) for ciprofloxacin increased 16-fold compared with the initial MIC (0.5 µg/ml) after ciprofloxacin administration for 3 days and decreased 256-fold compared with the initial MIC (0.5 µg/ml) after ciprofloxacin withdrawal for 26 days. GyrA (S83L, D87N/ D87Y) and parC (S80I) substitutions were observed in all quinolone-resistant E. coli (QREC) clones with an MIC ≥8 µg/ml. This study provides scientific theoretical guidance for the rational use of antimicrobials and the control of bacterial resistance.

Comparative dynamic distribution of avian infectious bronchitis virus M41, H120, and SAIBK strains by quantitative real-time RT-PCR in SPF chickens.

Avian infectious bronchitis is an acute, highly contagious disease of chickens. To study the differences of dynamic distribution between nephropathogenic infectious bronchitis virus (IBV) strains such as SAIBK and other strains (the M41 and H120 strains), relative quantitative real-time reverse transcription-polymerase chain reaction was developed by housekeeping gene selection. Glyceraldehyde-3-phosphate dehydrogenase and Ubiquitin were chosen for normalization in this experimental set. Then nine tissues, the trachea, thymus, liver, spleen, lungs, kidney, pancreas, proventriculus, and bursa of Fabricius, were analyzed and compared to determine the tropism of IBV infection. In this research, the kidney and the lung were established of the most sensitive organs in IBV infection. The pancreas and the liver are candidates for antigen detection. The trachea and the spleen can be used as references for histological diagnosis, but they are not suitable for antigen detection; proventriculus might be an important target in IBV infection; the thymus and the bursa of Fabricius were not sensitive organs in IBV infection.

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

Data correlating ß-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 ß-lactamase (ESBL)-producing and other cephalosporin-resistant isolates, the relevant ß-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 ß-lactamases among commensal E. coli was much lower in swine than in humans, suggesting that ß-lactamase genes have not established themselves in animal ecosystems in western China.

Erythromycin resistance and virulence genes in Enterococcus faecalis from swine in China.

This study aims to describe the erythromycin resistance phenotypes and genotypes, and the prevalence of virulence genes of Enterococcus faecalis isolated from swine in China. A total of 117 nonreplicate E. faecalis isolates, obtained from 502 clinical samples taken from different pig farms between 2007 and 2009 were included in the study. Minimum inhibitory concentrations were determined using the broth microdilution method. All of the isolates were screened for the presence of seven virulence genes (ace, asa1, cylA, efaA, esp, gelE, and hyl). In addition, the DNA from rythromycin-resistant isolates were amplified with primers specific for erythromycin resistance erm(A), erm(B), erm(C), mef(A/E), and msr(C) genes. Results show that erythromycin, tylosin, and ciprofloxacin resistance rates in E. faecalis were 66.67% (n=78), 66.67% (n=78), and 64.10% (n=75), respectively. About 69.23% of isolates (n=81) were positive for gelE, 48.72% (n=57) for ace, 15.38% (n=18) for efa, 7.69% (n=9) for asa1, and 6.84% (n=8) for esp. Among the erythromycin-resistant isolates, erm(B) (n=54) was the most prevalent resistance gene, followed by erm(A) (n=37). A significant correlation was found between the presence of the gelE virulence gene and erythromycin resistance (P<0.05). These findings suggest that enterococci from swine should be regarded with caution because they can be reservoirs for antimicrobial resistance and virulence genes.

Phenotypic and genotypic characterization of ß-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 ß-lactam resistance phenotypes and genotypes of K. pneumoniae isolates from swine in southwestern China, detect possible new ß-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 ß-lactam, 62.0-68.97% to fluoroquinolone, 44.8-46.55% to aminoglycoside, and 8.62-17.24% to ß-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 ß-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, ß-lactam resistance appeared to be common among K. pneumoniae isolates from swine, suggesting that K. pneumoniae may be a reservoir for the dissemination of ß-lactam resistance among Chinese pig farms.

Detection of CTX-M-15, CTX-M-22, and SHV-2 extended-spectrum beta-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.