Abstract: The aim of this study was to investigate the distribution of high pathogenicity island (HPI) in Escherichia coli (E. coli) isolated from cows with clinical and subclinical mastitis in China. Milk samples from 750 cows with clinical and subclinical mastitis in seven provinces were used to screen Gram-negative bacteria by MacConkey agar. 115 E. coli isolates were further identified from the Gram-negative bacteria by microbiological tests. Total DNA extraction from E. coli was used to detect HPI genes by PCR. The PCR results showed that in 115 samples, 29, 22 and 20 were positive for genes irp2, fyuA and intB, respectively. Sequence analysis of randomly selected 10 PCR products showed that homology of genes irp2, fyuA and intB were 97.1, 98.2 and 97.2% identical to the published sequences. In addition, HPI+ isolates were associated with fyuA (75.86%) and asn_tRNA locus (68.96%). We further analyzed the association of the prevalence of HPI and O serotypes and found that HPI occurred in several serotypes including O149, O93, O9 and O7. Therefore, we concluded that HPI was widely distributed in the E. coli isolates from cows with clinical and subclinical mastitis in China. The prevalence of HPI is associated with special serotypes in E.coli from cows with clinical and subclinical mastitis.
INTRODUCTION
High-pathogenicity island (HPI), a large chromosomal DNA fragment of Yersinia pestis, is highly associated with the mouse-lethal phenotype of Yersinia (Bearden et al., 1997; Buchrieser et al., 1998a; Alvarez and Cardineau, 2010; Carniel et al., 1996). HPI carries the gene fyuA, which is specific for the pesticin receptor and the iron repressible protein (irp) loci encoding the siderophore yersiniabactin (Ybt). The functional core of HPI is called irp2-fyuA gene cluster, the major gene element encoding an iron uptake system mediated by Ybt. The Ybt is associated with asparagine-specific tRNA loci and carries an integrase gene, int, often associated with a phage genome (Carniel et al., 1996; Rakin et al., 1999; Gehring et al., 1998; Buchrieser et al., 1998b). Irp2, one of the major structural genes of the core conservative region of HPI, has been used as a marker to detect HPI. FyuA gene coded for the yersiniabactin receptor FyuA and also served as a receptor for pesticin (Lucier et al., 1996; Rakin et al., 1994). Besides pathogenic Yersinia species, HPI has been reported as a part of the genomes of other enterobacteria such as Klebsiella spp., Citrobacter spp. and Escherichia coli (E. coli) (Hacker et al., 1999; Schubert et al., 1998).
Dairy cattle with clinical mastitis caused by Escherichia coli exhibits a wide range of disease severity, from mild, with only local inflammatory changes of the mammary gland, to severe, with significant systemic derangement. Escherichia coli is one of the commonest causes of bovine clinical mastitis (Green et al., 2005) and a major problem in lactating dairy cows (Kobori et al., 2004). It has been reported that the incidence of E. coli mastitis has recently increased in some countries (Green et al., 2005; Guler and Gunduz, 2007). Numerous studies in identifying virulence factors of E. coli isolated from cows with clinical mastitis have been conducted (Barrow and Hill, 1989; Kaipainen et al., 2002). However, the role of HPI in the E. coli caused bovine mastitis is still unclear. The present study has detected the prevalence of HPI using specific HPI markers from 115 E. coli isolated from milk samples, which were collected from 750 cows with clinical and subclinical mastitis in different areas of China.
MATERIALS AND METHODS
Bacterial isolation: Milk samples from 750 cows with clinical and subclinical mastitis in different dairy farms of seven provinces (Beijing, Inner Mongolia, Gansu, Sichuan, Chongqing, Guizhou and Yunnan) in China were collected between 2008 and 2009. Clinical and subclinical mastitis were identified by the Lanzhou Mastitis Test (LMT) and clinical examination (Li et al., 2002). Approximately 5 mL of milk were collected in sterile vials after disinfection with 70% ethanol and removal of the first 3 to 4 streams of milk. Samples were cultured in MacConkeys (MAC) agar. The isolates were identified as E. coli based on colony morphology and color, Gram stain and API-20E Enteric Identification System (Wenz et al., 2006) and stored at -70°C.
Identification of O-serotyping: Pure cultures of bacterial strains (Escherichia coli isolates) were grown on nutrient agar slants and sent to Bacterial products testing center, China Institute of Veterinary Drugs Control, (Beijing, China) for serotyping.
Bacterial pre-culturing and extraction of DNA templates: Each bacterial strain (Escherichia coli isolates) grown in LB agar at 37°C for 24 h was selected single colony and transfered into LB nutrient broth overnight with vigorous agitation at 37°C. The bacterial culture was enriched by centrifugation at 12000 g and DNA samples were extracted using a bacterial genomic DNA extraction kit (from TaKaRa Biotechnology Co., Ltd. (Dalian, P.R.China)). The productions were used directly or stored at -70°C before PCR as DNA templates.
PCR detection of HPI in E. coli: The specific PCR primers were respectively designed according to the published gene sequences of irp2, fyuA and asn_tRNA_intB in Genebank. PCR assays were performed by the Applied Biosystems (2720 Thermal Cycler America). The primers were synthesized by Sangon Biological Engineering Technology and Service Co. Ltd. (Shanghai, P.R. China). All the PCR reagents were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, P.R. China). Irp2, fyuA and asn_tRNA_intB genes were detected using the three pairs of specific primers as following protocols.
The PCR assay was carried out in a total volume of 50 μL of mixture containing 5 μL of 10x PCR buffer (Mg2+), 5 IU of Taq polymerase, 4 μL of dNTP mixture (each 2.5 mmol L-1), 1 μL of HPI primers set (each 50 mmol L-1), 2 μL of DNA template and deionized water to a final volume of 50 μL. The amplified DNA products were separated by electrophoresis on 1% agarose gel, stained with ethidium bromide and detected under ultraviolet light. The expected sizes of PCR products should be 276, 1071 and 1512 bp, respectively. The sequences of the forward (FP) and reverse (RP) primers used for PCR reactions and PCR amplification Tm were listed in Table 1.
DNA sequencing and sequence comparison: Ten PCR products from irp2-positive, fyuA-positive or intB-positive E. coli strains were randomly selected for amplification. The amplified DNA products were purified and connected to the pMD18-T carrying agent, then which were transformed into E. coli JM109 and cultured. Eventually, positive clones were selected to extract plasmid DNAs and identified by PCR. DNA sequencing was performed by Sangon Biological Engineering Technology and Service Co. Ltd. (Shanghai, P.R.China). The nucleotide sequences were analyzed with the DNAStar and BLAST in NCBI.
RESULTS
Survey for the presence of irp2, fyuA and intB genes among E. coli strains: We isolated 115 E. coli strains from a total of 750 mastitc milk samples. After analyzing all three HPI-genes irp2, fyuA and intB in each of 115 E. coli, found that 22 (19.13%) of the 115 E.coli strains harbored both irp2 and fyuA gene at the same time, while 7 (6.09%) of that only harbored genes irp2. Therefore, totally 29 (25.22%) E. coli isolates were HPI-positive strains. In addition, in the 29 HPI-positive E. coli, 20 (68.96%) strains were intB postitive, indicating that most HPI-positive E. coli isolates carried the intB gene and the HPI was linked at asn_tRNA locus (Fig. 1-3).
Sequence analysis of the genes of irp2, fyuA, asn_tRNA_intB in E. coli: After analyzed by DNAStar and MEGA programs, we found that all 10 irp2 gene sequences were 276 bp and the sequence homology was identical to the published irp2 sequences (97.1%). The sequence homology of 5 irp2+fyuA+intB+ strains and 3 irp2+fyuA-intB+ strains and 2 irp2+fyuA¯intB¯ strains exceeded 98.3%. The fyuA gene sequences were 1071 bp and sequence homology was identical to the published fyuA sequences (98.2%). The asn_tRNA_intB gene sequences were 1512 bp and sequence homology was identical to the published asn_tRNA_intB sequences (97.2%). The phylogenetic tree of isolated strains on the genes of asn_tRNA_intB in E. coli was seen (Fig. 4-6).
HPI and O-serotyping in E. coli: For the 115 E. coli strains, 63 isolates were identified as 39 serogroups, 3 isolates were self-clumpinged and 49 isolates could not be serotyped. As we shown (Table 2), O93, O9, O146, O7, O74 serotypes were the most prevalent serogroups.
| Table 1: | Oligonucleotide primers used in the study |
| Fig. 1: | PCR for detection irp2 in partial isolates, M. eDNA Marker DL2000, 1. S433014 strains; 2, 3 and 5; irp2+ strains; 4, 6 and 7; irp2 strains |
| Fig. 2: | PCR for detection fyuA in partial isolates, M. DNA Marker DL2000, 1. S433014 strains, 2, 3, 4, 5 and 6; fyuA+ strains, 7. fyuA strains |
| Fig. 3: | PCR for detection asn_tRNA_intB in partial isolates, M. DNA Marker DL2000, 1. S452621 strains, 2 and 7: asn_tRNA_intB strains; 3, 4, 5 and 6: asn_tRNA_intB strains |
| Fig. 4: | The phylogenetic tree of isolated strains on irp2 gene sequences, 1, 2, 3, 4, 5. seq. isolated strains irp2 gene sequences |
| Fig. 5: | The phylogenetic tree of isolated strains on fyuA gene sequences, 6, 7, 8, 9, 10. seq. isolated strains fyuA gene sequences |
| Fig. 6: | The phylogenetic tree of isolated strains on asn_tRNA_intB gene sequences, 11, 12, 13, 14, 15. seq. isolated strains asn_tRNA_intB gene sequences |
Among the 39 serogroups, 22 HPI-positive strains belongs to 16 different serogroups, 6 HPI-positive strains were non-serotyping and 1 was self-clumpinged. Furthermore, comparing to the detection rates of HPI in the most prevalent serogroups, O93 serogroup was the highest one than O9 (33.3%), O7 (33.3%), O74 (33.3%) and O146 (20%). While among non-prevalent serogroups, O149 serogroups were 100% HPI-positive strains.
| Table 2: | Relation statistic between HPI+ and isolates serotype |
| *Ratio = No. of HPI positive isolates/No. of isolates | |
DICUSSION
HPI was first discovered in pathogenic Yersinia strains and has recently been found in other enterobacteria. Schubert et al. (1998) demonstrated that the fyuA-irp gene cluster is absent in Shigella, S.enterica serotypes Enteritidis and Typhimurium and Shiga toxin-producing E.coli (EHEC). The gene cluster is infrequently detected in E.coli strains of EPEC, ETEC and EIEC pathotypes. Strikingly, the EAEC pathotype, which causes acute and chronic diarrhea in infants (Huppertz et al., 1997; Frederick, 2011; Naidu et al., 2007), frequently harbors a chromosomal fyuA-irp gene cluster (Buchrieser et al., 1998a; Hacker et al., 1999; Schubert et al., 1998). The HPI of Yersinia spp. is responsible for lethality for mice and for biosynthesis and uptake of the siderophore yersiniabactin. Yersiniabactin-mediated iron acquisition is thought to be the main function of the HPI (Gehring et al., 1998) and genes involved in yersiniabactin biosynthesis, transport and regulation (irp1 to irp9, ybtA and fyuA) are clustered on the “core” of the HPI. Although, HPI was the important virulence factor of pathogenicity E. coli isolates from humans, piglets and rabbits (Clermont et al., 2001; Bach et al., 2000; Penteado et al., 2002), there is no study reporting HPI of E. coli isolates from bovine mastitis milk in China. This is critical problem for dairy industry in China now.
The irp2 and fyuA genes are the two major structure of the “core” of the Yersinia HPI. Both of them are extremely conservative and usually exist at the same time. In our study, we found that 22 (19.13%) of the strains harbored both irp2 and fyuA gene at the same time, while 7 (6.09%) of that only harbored the irp2 genes. Totally 29 (25.22%) E. coli isolates were HPI-positive strains. The detection rate is lower than that from humans (32.25-34.9%) (Yong et al., 2002; Changyun and Jianguo, 2000) and avians (44.9%) (Wenjie et al., 2006), but higher than that from pigs (12.7-16.66%) (Xiang et al., 2006; Darong et al., 2006). These results indicate that horizontal gene transfer of HPI occurs between Yersinia strains and E. coli strains and Yersinia HPI was widespread in E. coli isolates from bovine mastitis milk and confered virulence-associated functions. The data of results also offered certain evidences for horizontal gene transfer of Yersinia HPI. The results of our survey indicated that the irp2 and fyuA genes may not be simultaneously expressed in one strain. Because of the fyuA gene was only detected in 75.86% of HPI-positive strains. The absence of fyuA gene, may reduce the iron-uptaken ability and subsequently attenuate the virulence for these HPI-positive strains without fyuA gene. This reduced virulence may result from gene mutation or gene recombination in the course of horizontal gene transfer of HPI among bacterial species for adaptation to a new environment. The functions of the fyuA-irp gene cluster in E. coli strains still remains unclear.
The HPI in Yersinia is located next to the asn tRNA bacterial attachment site. The asnT locus is linked to a gene which is highly homologous with a phage derived integrase determinant termed int that codes for a P4-like integrase. The instability of Yersinia HPI might be due to containing some mobile elements in Yersinia HPI, such as potential insertion sequences and integrase. Partial deletion of int might result in a non-functional integrase and incline to stabilization of the HPI in the chromosome of those isolates (Schubert et al., 1999). In our research, 64% of Yersinia HPI-positive strains from bovine mastitis milk carried the intB gene and inserted the asn-tRNA site. This is in line with some reports that Yersinia HPI from humans and pigs mostly linked to the asn-tRNA site (Bach et al., 2000; Wenjie et al., 2006). As recent report from Germany demonstrated that the HPI of O26 STEC strains and two E. coli strains isolated from blood poisoning avians had same deletion at its left junction, leading to a truncated integrase gene int (Karch et al., 1999). Therefore, we conclude that the HPI of the STEC O26 group represents a new and unique type of HPI with a partially deleted int. The deletion in the int gene may result in a nonfunctional integrase and subsequent fixation of the HPI in the genome of this STEC clonal lineage.
We also demonstrated that O93, O9, O146, O7 serotypes were dominant serotypes in all E. coli isolated from seven provinces in China. However, the Yersinia HPI was expressed differently among these different dominant serotypes and all two isolates of non-dominant serotype O149 are HPI positive. These results indicate that the distribution of Yersinia HPI existed distinction among different serotype strains. HPI might be only associated with special serotypes, not with dominant serotypes.
In summary, the findings in this study demonstrate that Yersinia HPI was widely distributed in the Escherichia coli isolates from cows with clinical and subclinical mastitis in China. The prevalence of HPI is probably associated with special serotypes in E. coli from cows with clinical and subclinical mastitis.