Divergence of affinities, serotypes and virulence factor between CTX-M Escherichia coli and non-CTX-M producers

Divergence of affinities, serotypes and virulence factor between CTX-M Escherichia coli and... Abstract This study was undertaken to discern the differences of the multi-locus sequence typing (MLST), O serogroups, and virulence factors among 34 CTX-M-1 Escherichia coli, 49 CTX-M-9 strains and 23 non-CTX-M isolates from chickens in Henan province, China. The MLST scheme yielded 34 sequence types, in which ST155 and ST359 were frequent (17% and 15%, respectively) and associated with zoonotic disease. The irp-2 (20% versus 2%, P = 0.0001), traT (85.3% versus 56.5%, P = 0.019), and sfaS (70.6% versus 0, P = 0.021) were significantly more prevalent in CTX-M-1 E. coli than in non-CTX-M producers. Also, CTX-M-9 isolates carried more irp-2 (17% versus 2%, P = 0.023), iroN (71.4% versus 39.1%, P = 0.019), and iss (79.6% versus 39.1%, P = 0.002) genes. In conclusion, although the 106 isolates encompassed a great genetic diversity, the CTX-M isolates harbored more virulence factor genes than non-CTX-M producers. INTRODUCTION Escherichia coli is a common microorganism responsible for human and animal infections. The treatment of E. coli infections is now threatened by the ever-growing antimicrobial resistance worldwide, especially the emergence of extended-spectrum β-lactamases (ESBLs). Since 1990, when CTX-M was discovered for the first time in an E. coli strain isolated from an ear exudate of a newborn (Bauernfeind et al., 1990), CTX-M-producing E. coli has constituted an emerging public-health concern. Unlike other ESBLs, CTX-M family can be divided into six groups (CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-8, CTX-M-25, KLUC) and others with an intergroup amino acid identity of ≤90% (D’Andrea et al., 2013). As previously described, the CTX-M-1 and CTX-M-9 were the common groups in China (Liu et al., 2015). The dissemination of resistance is associated with genetic mobile elements, such as plasmids, transposons and integrons, that may also carry virulence determinants simultaneously (Da Silva and Mendonca, 2012; Calhau et al., 2015). So, many researchers focus their eyes on the association of antimicrobial resistance with bacterial virulence determinants (Da Silva and Mendonca, 2012; Kumar et al., 2016), but the correction is complex, considering the diversity of antimicrobial resistance genes, virulence factors, and bacterial species. Although few epidemiologic data about the association between virulence traits and CTX-M-producing E. coli are available, these data were apparent conflicting. Lee et al. compared the occurrence of nine virulence factors with the blaCTX-M among pathogenic E. coli isolated from human blood and urine. They discovered that serum resistance-associated outer membrane protein (traT) was significantly more common in the blaCTX-M-1 group strains, while aerobactin receptor (iutA) was more common in the blaCTX-M-9 group isolates (Lee et al., 2010). Pitout et al. also thought that the virulence factor (VFs) were more prevalent in CTX-M producers than in non-producers (Pitout et al., 2005). In contrast, Lavignea et al. evaluated the virulence potential of CTX-M-producing E. coli collected from urinary tract infections and found CTX-M producers had fewer virulence factors and low virulence in vivo (Lavignea et al., 2006). Qin et al. also proved that CTX-M-producing uropathogenic E. coli isolates showed a lower prevalence of fimbrial genes (fimH and sfaD/focG), compared with non-ESBL-producing ones (Qin et al., 2013). The aims of the present study were: (1) to discern the differences of O serogroups among E. coli isolates from chicken (34 CTX-M-1 isolates, 49 CTX-M-9 producers, and 23 non-CTX-M ones) by micro-agglutination; and (2) to use multi-locus sequence typing (MLST) to characterize the genetic diversities of isolates; and (3) to investigate the divergence of 33 virulence genes between CTX-M isolates and non-producers using multiplex PCRs. MATERIALS AND METHODS Bacterial Isolates A total of 106 non-replicate E. coli, including 34 blaCTX-M-1 group, 49 blaCTX-M-9 group, and 23 non-CTX-M producers, were collected from 80 chicken farms in the period of 2014∼2015, which are scattered in 18 prefecture level cities in Henan province in China. The number of farms sampled in each city was not more than 5. Meanwhile, no more than four samples were collected from the same chicken farm. All samples were aseptically obtained from liver swabs as soon as sick chickens died and seeded onto MacConkey agar immediately. According to the Clinical and Laboratory Standards Institute broth micro-diffusion method (CLSI, 2015), the antimicrobial susceptibility of isolates were assessed and E. coli ATCC 25,922 was used as the control strain. Among cefotaxime-resistance isolates, the blaCTX-M genes were investigated by PCR using blaCTX-M subtyping primers and conditions previously reported (Eckert et al., 2006), The PCR products were directly sequenced and analyzed online using BLAST (www.ncbi.nlm.nih.gov/BLAST/). The isolates were considered as the non-CTX-M strains which showed high sensitivity to ceftiofur and cefotaxime with the MICs ≤4μg/mL (Figure 1). Figure 1. View largeDownload slide The relationships between sequence types (STs), CTX-M types, O serogroups, and major virulence related genes of isolates. *new STs. Figure 1. View largeDownload slide The relationships between sequence types (STs), CTX-M types, O serogroups, and major virulence related genes of isolates. *new STs. Multi-Locus Sequence Typings (MLST) MLST was performed, according to the method of the E. coli MLST database website. Seven house-keeping genes: adk, fumC, gyrB, icd, mdh, purA, and recA were targeted for PCR amplification and sequencing. The sequences were submitted to the MLST database for E. coli and the respective sequence types were determined (http:// mlst.ucc.ie/mlst/dbs/Ecoli). The eBURST (http://eburst.mlst.net/) algorithm was used to determine the clonal complexes (CCs) of sequence types, where STs sharing at least 5 of the 7 alleles. The Serotyping Detections All isolates were typified by micro-agglutination in 96-well plates using commercial O antisera (China Veterinary Drug Inspection Institute), according to the manufacturer's instructions. The O antisera included serogroups O1, O2, O5, O8, O14, O26, O36, O50, O54, O78, O88, O111, and O119, which were the main serotypes in chicken E. coli in Henan, China (Xu et al., 2003). Virulence Factor (VF) Typings The 106 E. coli were screened for 33 virulence-related genes by multiplex and single PCR assays, as previously described (Zhao et al., 2009), including 12 adhesin genes (fimH, bmaE, focG, iha, papA, papC, papGI, papGII, papGIII, sfaS, gafD, and afa), 8 iron transport related genes (feoB, fyuA, ireA, iroN, irp-2, iucC, iutA, and sitA), 7 protectin genes (cvaC, iss, kpsMT(k1), kpsMTII, kpsMTIII, rfc, and traT), 3 cytotoxin genes (cdtB, hlyD, and cnf-1), ompT (outer membrane protein gene), ibeA (invasion gene), and malX (high pathogenicity island). The testing was done in duplicate using independently prepared boiled lysates of each isolate. The reference strains from our tested collection, such as E. coli G2 (positive for fimH and feoB), E. coli G3 (positive for iroN, irp-2, iucC, iutA, sitA, iss, cvaC, and traT), E. coli G4 (positive for sfaS), E. coli G8 (positive for papGII, fyuA, and ireA), E. coli G10 (positive for ompT), E. coli G11 (positive for papC), and E. coli G78 (positive for kpsMT(k1), kpsMTII, and ibeA), were used as positive controls after confirmation of PCR products by DNA sequencing. Statistical Analysis The comparison of the virulence-related genes and the serotyping rates among blaCTX-M-1, blaCTX-M-9 and non-CTX-M producing chicken E. coli were documented in Statistical Packages of Social Sciences (SPSS) software for Windows, version 20.0 (IBM Corp., Armonk, NY). RESULTS AND DISCUSSION Genetic Diversity of Strains The 106 E. coli isolates were assigned to 34 distinct sequence types (ST). Novel STs reported in this work include ST4127–4129, ST4143, ST4716–4719, and ST4752–4753 (Figure 1). The most frequent STs were ST155 (n = 18, 17%), followed by ST359 (16, 15%), ST2505 (8, 7.5%), ST48 (5, 4.7%), and ST90 (5, 4.7%). The five common STs accounted for 49.6% (52/106) of the isolates, demonstrating the diversity of lineages, as reported previously (Xu et al., 2016). Among these 52 isolates, 20 isolates (38.5%, 20/52) produced blaCTX-M-1 group enzyme, 24 isolates (46.2%, 24/52) harbored blaCTX-M-9 group, and 8 isolates (15.3%, 8/52) belonged to non-CTX-M producers. In the present work, ST155 and ST359 were shared among CTX-M-1 group E. coli, CTX-M-9 group isolates and non-CTX-M producers, which were proved sharing between avian pathogenic E. coli, uropathogenic E. coli, and human extra-intestinal pathogenic E. coli (ExPEC) (Hussein et al., 2013; Dissanayake et al., 2014; Maluta et al., 2014). Thus, suggesting these STs seem to be important zoonotic-associated STs and should be scrutinized for zoonotic potential. The eBURST analysis revealed that 4 main clonal complexes, CC155 (ST155, ST616 and ST4719), CC90 (ST90, ST410, ST2505, and ST4128), CC156 (ST224, ST156, ST602, ST1706, ST4127, and ST4752), and CC10 (ST10, ST48, ST4717), encompassing 16 STs represented 59 (55.7%) E. coli isolates and the remaining 47 isolates included in the other 18 STs appearing as singletons (Figure 1). The Correlation between Serotypes and CTX-M Clusters O antigens determine O serotypes of the strains, which are composed the outermost part of the lipopolysaccharide layer in E. coli and play a major role in host-pathogen interactions (Debroy et al., 2016). For O serotypes, only 55 out of the 106 E. coli (51.89%) were typeable, suggesting that a large proportion of isolates is caused by other O serotypes which had not been prevalent serotypes in chicken E. coli in Henan (Xu et al., 2003). The 55 typeable isolates belonged to 13 serogroups. The common serotype was O8 (10 isolates), followed by O14 (9 isolates). The large variety of serotyping properties in E. coli also makes it difficult to determine which serotype is truly common in Henan province. Table 1. The virulence factors of E. Coli isolates from chickens (%).   Virulence genes  CTX-M-9 (n = 49)  CTX-M-1 (n = 34)  non-CTX-M (n = 23)  Totals (n = 106)  Adhesion related genes  fimH  43(87.8)  30(88.2)  21(91.3)  94(88.7)    sfaS  0  7(20.6) *#  0  7(6.6)    papGII  5(10.2)  2(5.9)  1(4.3)  8(7.5)    papC  2(4.1)  2(5.9)  2(8.7)  6(5.7)  Iron-related genes  feoB  49(100.0)  34(100.0)  23(100.0)  106(100)    sitA  36(73.5)  25(73.5)  13(56.5)  74(69.8)    irp-2  17(34.7)*  20(58.8)**  2(8.7)  39(36.8)    iucC  17(34.7)  20(58.8)  9(39.1)  46(43.4)    iroN  35(71.4)*#  15(44.1)  9(39.1)  59(55.7)    iutA  13(26.5)  15(44.1)  9(39.1)  37(34.9)    fyuA  8(16.3)  4(11.8)  2(8.7)  14(13.2)    ireA  8(16.3)  2(5.9)  1(4.3)  11(10.4)  Protectins genes  traT  35(71.4)  29(85.3)*  13(56.5)  77(72.6)    iss  39(79.6)**#  16(47.1)  9(39.1)  64(60.4)    cvaC  2(4.1)  9(26.5)#  4(17.4)  15(14.2)    kpsMTII  2(4.1)  1(2.9)  0  3(2.8)    kpsMT(KI)  0  1(2.9)  0  1(0.9)  Outer membrane protein gene  ompT  2(4.1)  1(3.0)  4(17.4)  7(6.6)  Invasin gene  ibeA  0  1(2.9)  0  1(0.9)    Virulence genes  CTX-M-9 (n = 49)  CTX-M-1 (n = 34)  non-CTX-M (n = 23)  Totals (n = 106)  Adhesion related genes  fimH  43(87.8)  30(88.2)  21(91.3)  94(88.7)    sfaS  0  7(20.6) *#  0  7(6.6)    papGII  5(10.2)  2(5.9)  1(4.3)  8(7.5)    papC  2(4.1)  2(5.9)  2(8.7)  6(5.7)  Iron-related genes  feoB  49(100.0)  34(100.0)  23(100.0)  106(100)    sitA  36(73.5)  25(73.5)  13(56.5)  74(69.8)    irp-2  17(34.7)*  20(58.8)**  2(8.7)  39(36.8)    iucC  17(34.7)  20(58.8)  9(39.1)  46(43.4)    iroN  35(71.4)*#  15(44.1)  9(39.1)  59(55.7)    iutA  13(26.5)  15(44.1)  9(39.1)  37(34.9)    fyuA  8(16.3)  4(11.8)  2(8.7)  14(13.2)    ireA  8(16.3)  2(5.9)  1(4.3)  11(10.4)  Protectins genes  traT  35(71.4)  29(85.3)*  13(56.5)  77(72.6)    iss  39(79.6)**#  16(47.1)  9(39.1)  64(60.4)    cvaC  2(4.1)  9(26.5)#  4(17.4)  15(14.2)    kpsMTII  2(4.1)  1(2.9)  0  3(2.8)    kpsMT(KI)  0  1(2.9)  0  1(0.9)  Outer membrane protein gene  ompT  2(4.1)  1(3.0)  4(17.4)  7(6.6)  Invasin gene  ibeA  0  1(2.9)  0  1(0.9)  *significant difference between CTX-M isolates and non-CTX-M ones. **extremely significant difference between CTX-M isolates and non-CTX-M ones. #significant difference between CTX-M-1 isolates and CTX-M-9 ones. View Large In the study, the presence of the O antigens in CTX-M-1 group isolates were significantly higher (82.4%, 28/34) than in CTX-M-9 group (34.7%, 17/49, P = 0.038), which indicated that a certain relation probably existed between the serotypes and CTX-M clusters. In 2011, Emamghorashi et al. ever reported that there was a significant correlation between the presence of O antigens and sensitivity to nalidixic acid and gentamicin (P < 0.05) (Emamghorashi et al., 2011). However, to our knowledge, such correlation has not been described before in CTX-M clusters. On the other hand, there seemed to be a strong correlation between types of O antigen and CTX-M clusters. In 27 typeable CTX-M-1 isolates, about 29.63% (8/27) of isolates were O8 serotype, O26 (11.1%, 3/27) was only found in CTX-M-1 isolates. But in 17 typeable CTX-M-9 isolates, O36 serotype (29.41%, 5/17) was common, and O111 (23.5%, 4/17) was identified only in CTX-M-9 ones. The O26 and O111 belong to non-O157 serogroups, which are increasingly recognized as important foodborne pathogens worldwide (Wang et al., 2013). Also, O26 is the second most prevalent E. coli serotype identified in cases of foodborne illness throughout the world (Lajhar et al., 2017). In the 20th century, some reports documented the plasmid-mediated lipopolysaccharide O antigen expression in Enterobacteriaceae and proved some plasmids were responsible for serotype conversions (Riley et al., 1987; Keenleyside and Whitfield, 1995). In addition, molecular epidemiological studies revealed that plasmids associated with the different blaCTX-M clusters were not all the same. The blaCTX-M-1 group is dominantly harbored by IncF, IncN, IncA/C, and IncI1, while IncF, IncHI2, IncK, IncI1, IncN, IncP, and IncL/M are closely related to the blaCTX-M-9 group (Zhao and Hu, 2013). The Relationship of VF Genes and CTX-M Types Figure 1 also lists the distribution of common VF genes among E. coli producing blaCTX-M-1 cluster, blaCTX-M-9 cluster, and the non-CTX-M producers. There was no difference in aggregate virulence factor scores between CTX-M-producers and non-CTX-M ones (6.5 versus 5.3, P = 0.083), as reported previously (Pitout et al., 2005; Lee et al., 2010). The most prevalent VF genes was feoB (100%), followed by fimH (88.7%, 94/106). The fimH gene, encoding the adhesin FimH as the tip fimbria for type 1 fimbriae, is an important virulence factor for pathogenic E. coli (Krekeler et al., 2013). However, there was no statistically significant difference between them. The CTX-M-producing E. coli harbored significantly more irp-2, sfaS, traT, iroN, and iss genes than non-CTX-M producers, which may enable the former to trigger extra-intestinal infection more readily than the latter (Table 1). The irp-2 (20% versus 2%, P = 0.0001), traT (85.3% versus 56.5%, P = 0.019), and sfaS (70.6% versus 0, P = 0.021) genes were more prevalent in blaCTX-M-1 cluster E. coli than in non-CTX-M producers. At the same time, blaCTX-M-9 cluster isolates carried more irp-2 (17% versus 2%, P = 0.023), iroN (71.4% versus 39.1%, P = 0.019), and iss (79.6% versus 39.1%, P = 0.002) genes than non-CTX-M producers. The irp-2 and iroN virulence factors, belonging to iron acquisition systems, have been directly associated with human urinary tract infection, by allowing bacterial growth even under iron-limiting conditions which are found during the infection process. Paixão et al. also reported that possession of more than one iron transport system seems to play an important role on avian pathogenic E. coli survival (Paixão et al., 2016). The increased serum survival (iss) virulence gene is a distinguishing trait of avian ExPEC, which may enable ExPEC to enter the infection sites, for example upper respiratory tract, urinary tract, et al. (Lee et al., 2010; Maluta et al., 2014). The traT gene also is a serum protection gene. In 2016, Cha et al. found that traT gene was more prevalent in ST131 than in non-ST131 (P = 0.01) (Cha et al., 2016). S fimbriae are similar to type 1 or P fimbriae of E. coli. The sfaS, encoding the adhesin S-fimbrial, were reported to be most often found among ExPEC isolates (Johnson et al., 2008; Antão et al., 2009). In CTX-M-producing E. coli, the statistical analysis showed significant differences in cvaC, sfaS, iroN, and iss genes between the blaCTX-M-1 group and the blaCTX-M-9 group (Table 1). Significantly greater frequencies of cvaC (26.5% versus 4.1%, P = 0.021) and sfaS (20.6% versus 0, P = 0.031) genes were observed in CTX-M-1 cluster E. coli than those in CTX-M-9 cluster isolates. On the contrary, 71.4% of CTX-M-9 isolates carried iroN genes, whereas only 44.1% of CTX-M-1 strains did (P = 0.039), moreover, about 79.6% of CTX-M-9 isolates carried iss gene, whereas 47.1% of CTX-M-1 strains did (P = 0.007). But further studies are required to characterize the association between the VF genes and E. coli pathogenicity. The Relationship between STs and VF distributions It seemed that there was no apparent relationship between STs and VF distributions, but the non-O157 isolates (3of O26 and 4 of O111) conferred more VF genes than the others in Figure 1. All the non-O157 isolates harbored at least 9 VF genes, while the latter was only 13.1%. Meanwhile, the statistical analysis showed significant differences in iucC (100% versus 38.4%, P = 0.005), iutA (85.9% versus 38.4%, P = 0.012), and irp-2 (85.9% versus 32.3%, P = 0.013) genes between the non-O157 E. coli and the others. 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Divergence of affinities, serotypes and virulence factor between CTX-M Escherichia coli and non-CTX-M producers

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© 2017 Poultry Science Association Inc.
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Abstract

Abstract This study was undertaken to discern the differences of the multi-locus sequence typing (MLST), O serogroups, and virulence factors among 34 CTX-M-1 Escherichia coli, 49 CTX-M-9 strains and 23 non-CTX-M isolates from chickens in Henan province, China. The MLST scheme yielded 34 sequence types, in which ST155 and ST359 were frequent (17% and 15%, respectively) and associated with zoonotic disease. The irp-2 (20% versus 2%, P = 0.0001), traT (85.3% versus 56.5%, P = 0.019), and sfaS (70.6% versus 0, P = 0.021) were significantly more prevalent in CTX-M-1 E. coli than in non-CTX-M producers. Also, CTX-M-9 isolates carried more irp-2 (17% versus 2%, P = 0.023), iroN (71.4% versus 39.1%, P = 0.019), and iss (79.6% versus 39.1%, P = 0.002) genes. In conclusion, although the 106 isolates encompassed a great genetic diversity, the CTX-M isolates harbored more virulence factor genes than non-CTX-M producers. INTRODUCTION Escherichia coli is a common microorganism responsible for human and animal infections. The treatment of E. coli infections is now threatened by the ever-growing antimicrobial resistance worldwide, especially the emergence of extended-spectrum β-lactamases (ESBLs). Since 1990, when CTX-M was discovered for the first time in an E. coli strain isolated from an ear exudate of a newborn (Bauernfeind et al., 1990), CTX-M-producing E. coli has constituted an emerging public-health concern. Unlike other ESBLs, CTX-M family can be divided into six groups (CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-8, CTX-M-25, KLUC) and others with an intergroup amino acid identity of ≤90% (D’Andrea et al., 2013). As previously described, the CTX-M-1 and CTX-M-9 were the common groups in China (Liu et al., 2015). The dissemination of resistance is associated with genetic mobile elements, such as plasmids, transposons and integrons, that may also carry virulence determinants simultaneously (Da Silva and Mendonca, 2012; Calhau et al., 2015). So, many researchers focus their eyes on the association of antimicrobial resistance with bacterial virulence determinants (Da Silva and Mendonca, 2012; Kumar et al., 2016), but the correction is complex, considering the diversity of antimicrobial resistance genes, virulence factors, and bacterial species. Although few epidemiologic data about the association between virulence traits and CTX-M-producing E. coli are available, these data were apparent conflicting. Lee et al. compared the occurrence of nine virulence factors with the blaCTX-M among pathogenic E. coli isolated from human blood and urine. They discovered that serum resistance-associated outer membrane protein (traT) was significantly more common in the blaCTX-M-1 group strains, while aerobactin receptor (iutA) was more common in the blaCTX-M-9 group isolates (Lee et al., 2010). Pitout et al. also thought that the virulence factor (VFs) were more prevalent in CTX-M producers than in non-producers (Pitout et al., 2005). In contrast, Lavignea et al. evaluated the virulence potential of CTX-M-producing E. coli collected from urinary tract infections and found CTX-M producers had fewer virulence factors and low virulence in vivo (Lavignea et al., 2006). Qin et al. also proved that CTX-M-producing uropathogenic E. coli isolates showed a lower prevalence of fimbrial genes (fimH and sfaD/focG), compared with non-ESBL-producing ones (Qin et al., 2013). The aims of the present study were: (1) to discern the differences of O serogroups among E. coli isolates from chicken (34 CTX-M-1 isolates, 49 CTX-M-9 producers, and 23 non-CTX-M ones) by micro-agglutination; and (2) to use multi-locus sequence typing (MLST) to characterize the genetic diversities of isolates; and (3) to investigate the divergence of 33 virulence genes between CTX-M isolates and non-producers using multiplex PCRs. MATERIALS AND METHODS Bacterial Isolates A total of 106 non-replicate E. coli, including 34 blaCTX-M-1 group, 49 blaCTX-M-9 group, and 23 non-CTX-M producers, were collected from 80 chicken farms in the period of 2014∼2015, which are scattered in 18 prefecture level cities in Henan province in China. The number of farms sampled in each city was not more than 5. Meanwhile, no more than four samples were collected from the same chicken farm. All samples were aseptically obtained from liver swabs as soon as sick chickens died and seeded onto MacConkey agar immediately. According to the Clinical and Laboratory Standards Institute broth micro-diffusion method (CLSI, 2015), the antimicrobial susceptibility of isolates were assessed and E. coli ATCC 25,922 was used as the control strain. Among cefotaxime-resistance isolates, the blaCTX-M genes were investigated by PCR using blaCTX-M subtyping primers and conditions previously reported (Eckert et al., 2006), The PCR products were directly sequenced and analyzed online using BLAST (www.ncbi.nlm.nih.gov/BLAST/). The isolates were considered as the non-CTX-M strains which showed high sensitivity to ceftiofur and cefotaxime with the MICs ≤4μg/mL (Figure 1). Figure 1. View largeDownload slide The relationships between sequence types (STs), CTX-M types, O serogroups, and major virulence related genes of isolates. *new STs. Figure 1. View largeDownload slide The relationships between sequence types (STs), CTX-M types, O serogroups, and major virulence related genes of isolates. *new STs. Multi-Locus Sequence Typings (MLST) MLST was performed, according to the method of the E. coli MLST database website. Seven house-keeping genes: adk, fumC, gyrB, icd, mdh, purA, and recA were targeted for PCR amplification and sequencing. The sequences were submitted to the MLST database for E. coli and the respective sequence types were determined (http:// mlst.ucc.ie/mlst/dbs/Ecoli). The eBURST (http://eburst.mlst.net/) algorithm was used to determine the clonal complexes (CCs) of sequence types, where STs sharing at least 5 of the 7 alleles. The Serotyping Detections All isolates were typified by micro-agglutination in 96-well plates using commercial O antisera (China Veterinary Drug Inspection Institute), according to the manufacturer's instructions. The O antisera included serogroups O1, O2, O5, O8, O14, O26, O36, O50, O54, O78, O88, O111, and O119, which were the main serotypes in chicken E. coli in Henan, China (Xu et al., 2003). Virulence Factor (VF) Typings The 106 E. coli were screened for 33 virulence-related genes by multiplex and single PCR assays, as previously described (Zhao et al., 2009), including 12 adhesin genes (fimH, bmaE, focG, iha, papA, papC, papGI, papGII, papGIII, sfaS, gafD, and afa), 8 iron transport related genes (feoB, fyuA, ireA, iroN, irp-2, iucC, iutA, and sitA), 7 protectin genes (cvaC, iss, kpsMT(k1), kpsMTII, kpsMTIII, rfc, and traT), 3 cytotoxin genes (cdtB, hlyD, and cnf-1), ompT (outer membrane protein gene), ibeA (invasion gene), and malX (high pathogenicity island). The testing was done in duplicate using independently prepared boiled lysates of each isolate. The reference strains from our tested collection, such as E. coli G2 (positive for fimH and feoB), E. coli G3 (positive for iroN, irp-2, iucC, iutA, sitA, iss, cvaC, and traT), E. coli G4 (positive for sfaS), E. coli G8 (positive for papGII, fyuA, and ireA), E. coli G10 (positive for ompT), E. coli G11 (positive for papC), and E. coli G78 (positive for kpsMT(k1), kpsMTII, and ibeA), were used as positive controls after confirmation of PCR products by DNA sequencing. Statistical Analysis The comparison of the virulence-related genes and the serotyping rates among blaCTX-M-1, blaCTX-M-9 and non-CTX-M producing chicken E. coli were documented in Statistical Packages of Social Sciences (SPSS) software for Windows, version 20.0 (IBM Corp., Armonk, NY). RESULTS AND DISCUSSION Genetic Diversity of Strains The 106 E. coli isolates were assigned to 34 distinct sequence types (ST). Novel STs reported in this work include ST4127–4129, ST4143, ST4716–4719, and ST4752–4753 (Figure 1). The most frequent STs were ST155 (n = 18, 17%), followed by ST359 (16, 15%), ST2505 (8, 7.5%), ST48 (5, 4.7%), and ST90 (5, 4.7%). The five common STs accounted for 49.6% (52/106) of the isolates, demonstrating the diversity of lineages, as reported previously (Xu et al., 2016). Among these 52 isolates, 20 isolates (38.5%, 20/52) produced blaCTX-M-1 group enzyme, 24 isolates (46.2%, 24/52) harbored blaCTX-M-9 group, and 8 isolates (15.3%, 8/52) belonged to non-CTX-M producers. In the present work, ST155 and ST359 were shared among CTX-M-1 group E. coli, CTX-M-9 group isolates and non-CTX-M producers, which were proved sharing between avian pathogenic E. coli, uropathogenic E. coli, and human extra-intestinal pathogenic E. coli (ExPEC) (Hussein et al., 2013; Dissanayake et al., 2014; Maluta et al., 2014). Thus, suggesting these STs seem to be important zoonotic-associated STs and should be scrutinized for zoonotic potential. The eBURST analysis revealed that 4 main clonal complexes, CC155 (ST155, ST616 and ST4719), CC90 (ST90, ST410, ST2505, and ST4128), CC156 (ST224, ST156, ST602, ST1706, ST4127, and ST4752), and CC10 (ST10, ST48, ST4717), encompassing 16 STs represented 59 (55.7%) E. coli isolates and the remaining 47 isolates included in the other 18 STs appearing as singletons (Figure 1). The Correlation between Serotypes and CTX-M Clusters O antigens determine O serotypes of the strains, which are composed the outermost part of the lipopolysaccharide layer in E. coli and play a major role in host-pathogen interactions (Debroy et al., 2016). For O serotypes, only 55 out of the 106 E. coli (51.89%) were typeable, suggesting that a large proportion of isolates is caused by other O serotypes which had not been prevalent serotypes in chicken E. coli in Henan (Xu et al., 2003). The 55 typeable isolates belonged to 13 serogroups. The common serotype was O8 (10 isolates), followed by O14 (9 isolates). The large variety of serotyping properties in E. coli also makes it difficult to determine which serotype is truly common in Henan province. Table 1. The virulence factors of E. Coli isolates from chickens (%).   Virulence genes  CTX-M-9 (n = 49)  CTX-M-1 (n = 34)  non-CTX-M (n = 23)  Totals (n = 106)  Adhesion related genes  fimH  43(87.8)  30(88.2)  21(91.3)  94(88.7)    sfaS  0  7(20.6) *#  0  7(6.6)    papGII  5(10.2)  2(5.9)  1(4.3)  8(7.5)    papC  2(4.1)  2(5.9)  2(8.7)  6(5.7)  Iron-related genes  feoB  49(100.0)  34(100.0)  23(100.0)  106(100)    sitA  36(73.5)  25(73.5)  13(56.5)  74(69.8)    irp-2  17(34.7)*  20(58.8)**  2(8.7)  39(36.8)    iucC  17(34.7)  20(58.8)  9(39.1)  46(43.4)    iroN  35(71.4)*#  15(44.1)  9(39.1)  59(55.7)    iutA  13(26.5)  15(44.1)  9(39.1)  37(34.9)    fyuA  8(16.3)  4(11.8)  2(8.7)  14(13.2)    ireA  8(16.3)  2(5.9)  1(4.3)  11(10.4)  Protectins genes  traT  35(71.4)  29(85.3)*  13(56.5)  77(72.6)    iss  39(79.6)**#  16(47.1)  9(39.1)  64(60.4)    cvaC  2(4.1)  9(26.5)#  4(17.4)  15(14.2)    kpsMTII  2(4.1)  1(2.9)  0  3(2.8)    kpsMT(KI)  0  1(2.9)  0  1(0.9)  Outer membrane protein gene  ompT  2(4.1)  1(3.0)  4(17.4)  7(6.6)  Invasin gene  ibeA  0  1(2.9)  0  1(0.9)    Virulence genes  CTX-M-9 (n = 49)  CTX-M-1 (n = 34)  non-CTX-M (n = 23)  Totals (n = 106)  Adhesion related genes  fimH  43(87.8)  30(88.2)  21(91.3)  94(88.7)    sfaS  0  7(20.6) *#  0  7(6.6)    papGII  5(10.2)  2(5.9)  1(4.3)  8(7.5)    papC  2(4.1)  2(5.9)  2(8.7)  6(5.7)  Iron-related genes  feoB  49(100.0)  34(100.0)  23(100.0)  106(100)    sitA  36(73.5)  25(73.5)  13(56.5)  74(69.8)    irp-2  17(34.7)*  20(58.8)**  2(8.7)  39(36.8)    iucC  17(34.7)  20(58.8)  9(39.1)  46(43.4)    iroN  35(71.4)*#  15(44.1)  9(39.1)  59(55.7)    iutA  13(26.5)  15(44.1)  9(39.1)  37(34.9)    fyuA  8(16.3)  4(11.8)  2(8.7)  14(13.2)    ireA  8(16.3)  2(5.9)  1(4.3)  11(10.4)  Protectins genes  traT  35(71.4)  29(85.3)*  13(56.5)  77(72.6)    iss  39(79.6)**#  16(47.1)  9(39.1)  64(60.4)    cvaC  2(4.1)  9(26.5)#  4(17.4)  15(14.2)    kpsMTII  2(4.1)  1(2.9)  0  3(2.8)    kpsMT(KI)  0  1(2.9)  0  1(0.9)  Outer membrane protein gene  ompT  2(4.1)  1(3.0)  4(17.4)  7(6.6)  Invasin gene  ibeA  0  1(2.9)  0  1(0.9)  *significant difference between CTX-M isolates and non-CTX-M ones. **extremely significant difference between CTX-M isolates and non-CTX-M ones. #significant difference between CTX-M-1 isolates and CTX-M-9 ones. View Large In the study, the presence of the O antigens in CTX-M-1 group isolates were significantly higher (82.4%, 28/34) than in CTX-M-9 group (34.7%, 17/49, P = 0.038), which indicated that a certain relation probably existed between the serotypes and CTX-M clusters. In 2011, Emamghorashi et al. ever reported that there was a significant correlation between the presence of O antigens and sensitivity to nalidixic acid and gentamicin (P < 0.05) (Emamghorashi et al., 2011). However, to our knowledge, such correlation has not been described before in CTX-M clusters. On the other hand, there seemed to be a strong correlation between types of O antigen and CTX-M clusters. In 27 typeable CTX-M-1 isolates, about 29.63% (8/27) of isolates were O8 serotype, O26 (11.1%, 3/27) was only found in CTX-M-1 isolates. But in 17 typeable CTX-M-9 isolates, O36 serotype (29.41%, 5/17) was common, and O111 (23.5%, 4/17) was identified only in CTX-M-9 ones. The O26 and O111 belong to non-O157 serogroups, which are increasingly recognized as important foodborne pathogens worldwide (Wang et al., 2013). Also, O26 is the second most prevalent E. coli serotype identified in cases of foodborne illness throughout the world (Lajhar et al., 2017). In the 20th century, some reports documented the plasmid-mediated lipopolysaccharide O antigen expression in Enterobacteriaceae and proved some plasmids were responsible for serotype conversions (Riley et al., 1987; Keenleyside and Whitfield, 1995). In addition, molecular epidemiological studies revealed that plasmids associated with the different blaCTX-M clusters were not all the same. The blaCTX-M-1 group is dominantly harbored by IncF, IncN, IncA/C, and IncI1, while IncF, IncHI2, IncK, IncI1, IncN, IncP, and IncL/M are closely related to the blaCTX-M-9 group (Zhao and Hu, 2013). The Relationship of VF Genes and CTX-M Types Figure 1 also lists the distribution of common VF genes among E. coli producing blaCTX-M-1 cluster, blaCTX-M-9 cluster, and the non-CTX-M producers. There was no difference in aggregate virulence factor scores between CTX-M-producers and non-CTX-M ones (6.5 versus 5.3, P = 0.083), as reported previously (Pitout et al., 2005; Lee et al., 2010). The most prevalent VF genes was feoB (100%), followed by fimH (88.7%, 94/106). The fimH gene, encoding the adhesin FimH as the tip fimbria for type 1 fimbriae, is an important virulence factor for pathogenic E. coli (Krekeler et al., 2013). However, there was no statistically significant difference between them. The CTX-M-producing E. coli harbored significantly more irp-2, sfaS, traT, iroN, and iss genes than non-CTX-M producers, which may enable the former to trigger extra-intestinal infection more readily than the latter (Table 1). The irp-2 (20% versus 2%, P = 0.0001), traT (85.3% versus 56.5%, P = 0.019), and sfaS (70.6% versus 0, P = 0.021) genes were more prevalent in blaCTX-M-1 cluster E. coli than in non-CTX-M producers. At the same time, blaCTX-M-9 cluster isolates carried more irp-2 (17% versus 2%, P = 0.023), iroN (71.4% versus 39.1%, P = 0.019), and iss (79.6% versus 39.1%, P = 0.002) genes than non-CTX-M producers. The irp-2 and iroN virulence factors, belonging to iron acquisition systems, have been directly associated with human urinary tract infection, by allowing bacterial growth even under iron-limiting conditions which are found during the infection process. Paixão et al. also reported that possession of more than one iron transport system seems to play an important role on avian pathogenic E. coli survival (Paixão et al., 2016). The increased serum survival (iss) virulence gene is a distinguishing trait of avian ExPEC, which may enable ExPEC to enter the infection sites, for example upper respiratory tract, urinary tract, et al. (Lee et al., 2010; Maluta et al., 2014). The traT gene also is a serum protection gene. In 2016, Cha et al. found that traT gene was more prevalent in ST131 than in non-ST131 (P = 0.01) (Cha et al., 2016). S fimbriae are similar to type 1 or P fimbriae of E. coli. The sfaS, encoding the adhesin S-fimbrial, were reported to be most often found among ExPEC isolates (Johnson et al., 2008; Antão et al., 2009). In CTX-M-producing E. coli, the statistical analysis showed significant differences in cvaC, sfaS, iroN, and iss genes between the blaCTX-M-1 group and the blaCTX-M-9 group (Table 1). Significantly greater frequencies of cvaC (26.5% versus 4.1%, P = 0.021) and sfaS (20.6% versus 0, P = 0.031) genes were observed in CTX-M-1 cluster E. coli than those in CTX-M-9 cluster isolates. On the contrary, 71.4% of CTX-M-9 isolates carried iroN genes, whereas only 44.1% of CTX-M-1 strains did (P = 0.039), moreover, about 79.6% of CTX-M-9 isolates carried iss gene, whereas 47.1% of CTX-M-1 strains did (P = 0.007). But further studies are required to characterize the association between the VF genes and E. coli pathogenicity. The Relationship between STs and VF distributions It seemed that there was no apparent relationship between STs and VF distributions, but the non-O157 isolates (3of O26 and 4 of O111) conferred more VF genes than the others in Figure 1. All the non-O157 isolates harbored at least 9 VF genes, while the latter was only 13.1%. Meanwhile, the statistical analysis showed significant differences in iucC (100% versus 38.4%, P = 0.005), iutA (85.9% versus 38.4%, P = 0.012), and irp-2 (85.9% versus 32.3%, P = 0.013) genes between the non-O157 E. coli and the others. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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