Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from septicemic broilers Yurong Li,1 Ligong Chen, Xianjun Wu, and Shuying Huo College of Veterinary Medicine, Hebei Agricultural University; North China Research Center of Animal Epidemic Pathogen Biology, China Agriculture Ministry, Baoding, 071001
Key words: APEC, septicemic broiler, virulence, resistance gene, plasmid 2015 Poultry Science 00:1–11 http://dx.doi.org/10.3382/ps/pev008
INTRDUCTION
great concern to poultry veterinarians (Hinton et al., 1986). Many drug-resistant strains and drug resistance genes can be transferred and disseminated between animal and human pathogens, which not only increases the difficulty of treating animal diseases but is also a serious threat to human health (Collignon et al., 2005). Many studies have documented the presence of virulence genes and drug resistance genes in APEC strains (Bass et al., 1999; Oh et al., 2011; Ahmed and Shimamoto, 2013). So far these have rarely been reported systematically in north China. Therefore, the objective of this study was to characterize the molecular bases of virulence and antimicrobial resistance in multidrugresistant APEC strains isolated from septicemic broilers in Hebei in north China.
Escherichia coli is a part of the normal microflora in the poultry intestine, but certain strains, such as those designated as avian pathogenic E. coli (APEC), spread into various internal organs and cause the systemic fatal disease colibacillosis, which is characterized by septicemia with multiple organ lesions, typically pericarditis, airsacculitis, perihepatitis, peritonitis, and other extraintestinal lesions (Oh et al., 2011). APEC is considered a major cause of economic losses due to morbidity, mortality, and condemnation of poultry carcasses worldwide (Ewers et al., 2004; Ahmed and Shimamoto, 2013). There are several virulence traits associated the extraintestinal pathogenesis of APEC, and the relevant virulence gene, such as iutA, hlyF, iss, iroN, and ompT, were carried by plasmids that are considered the most signifficantly associated with APEC strains (Johnson et al., 2008; Ahmed and Shimamoto, 2013). At present, antimicrobial therapy acts as an important tool to reduce both the incidence and the mortality associated with avian colibacillosis. However, resistance to existing antimicrobials is widespread and of
MATERIALS AND METHODS Isolation and Identification of Bacteria Avian E. coli isolates were isolated from the liver, pericardial fluid, heart blood, and spleen collected from 132 septicaemic broilers at 7 to 43 d of age that died of colibacillosis or were culled by veterinarians at 63 large-scale farms in Baoding, Cangzhou, Hengshui, and Xingtai in Hebei Province, China. Sampling was performed randomly between February and July 2013.
C 2015 Poultry Science Association Inc. Received July 14, 2014. Accepted October 21, 2014. 1 Corresponding author: [email protected]
1
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
gentamycin, ofloxacin, norfloxacin, and ceftriaxone. The β -lactamase-encoding genes blaTEM , blaCMY-2 , blaOXA-30 , blaCTX-M-15 , and blaSHV-2 ; the aminoglycosidemodifying enzymes (AME) strA, strB, aph(3 )-IIa, aac(3)-IIa, aac(6 )-Ib, and ant(3 )-Ia; and the plasmidmediated quinolone resistance (PMQR) genes qnrA, qnrB, and qnrS, were also identified in 66 (75.9%), 65 (74.7%), and 6 (6.9%) isolates, respectively. All isolates were evaluated in terms of replicon type. The plasmid replicons were identified in 63 (72.4%) isolates, and the FIB, B/O, and K replicons were the most present. To the best of our knowledge, this is the first report of molecular characterization of antimicrobial resistance in APEC strains from China.
ABSTRACT Avian pathogenic Escherichia coli (APEC) causes extensive mortality in poultry flocks, leading to extensive economic losses. To date, little information has been available on the molecular basis of antimicrobial resistance in APEC in Hebei, China. Therefore, the objective of this study was to characterize the virulence and antimicrobial resistance of multidrug-resistant APEC isolated from septicemic broilers at the molecular level. Among 87 nonrepetitive E. coli isolates, 41 (47.1%) carried 3 or more of the APEC virulence genes iroN, ompT, iss, iutA, and hlyF. All 87 APEC isolates showed multidrugresistant phenotypes, particularly against ampicillin, kanamycin, ciprofloxacin, levofloxacin, streptomycin,
2
LI ET AL.
Pathological materials were collected under sterile conditions. Avian E. coli isolates were cross-inoculated in MacConkey medium (Tianhe Micro-organism Reagent Co. Ltd., Hangzhou, China), and single pink colonies were picked and purified on MacConkey medium. Only one positive sample per broiler was included in this study. In total, 87 E. coli isolates from 63 farms were identified via biochemical testing and were then used for testing.
(CRO), 30 μg; cefotaxime (CTX), 30 μg; gatifloxacin (GAT), 5 μg; gentamycin (GEN), 10 μg; kanamycin (KAN), 30 μg; levofloxacin (LOF), 5 μg; neomycin (NM), 30 μg; norfloxacin (NOR), 10 μg; ofloxacin (OFL), 5 μg; sulbactam/ampicillin 1:1 (SAM), 10 μg/10 μg; spectinomycin (SPE), 100 μg; and streptomycin (STR), 10 μg. The discs were purchased from Hangzhou Tianhe Microorganism Reagent Co. Ltd. (Hangzhou, China).
Drug Susceptibility Test Screening of APEC Virulence Genes by Multiplex PCR A pentaplex PCR assay targeting ffive virulence genes of APEC, iutA, hlyF, iss, iroN, and ompT, was carried out as previously described (Johnson et al., 2008; Ahmed and Shimamoto, 2013). Primers used for pentaplex PCR are listed in Table 1.
Table 1. Primers used for PCR and DNA sequencing. Primer
Sequence 5 to 3
Virulence genes iroN-F AATCCGGCAAAGAGACGAACCGCCT iroN-R GTTCGGGCAACCCCTGCTTTGACTTT ompT-F TCATCCCGGAAGCCTCCCTCACTACTATTA ompT-R GCGTTTGCTGCACTGGCTTCTGATAC hlyF-F GGCCACAGTCGTTTAGGGTGCTTACC hlyF-R GGCGGTTTAGGCATTCCGATACTCAG iss-F CAGCAACCCGAACCACTTGATG iss-R AGCATTGCCAGAGCGGCAGAA iutA-F GGCTGGACATCATGGGAACTGG iutA-R CGTCGGGAACGGGTAGAATCG Lactamases TEM-F ATAAAATTCTTGAAGACGAAA TEM-R GACAGTTACCAATGCTTAATC SHV-F TTATCTCCCTGTTAGCCACC SHV-R GATTTGCTGATTTCGCTCGG OXA-F TCAACTTTCAAGATCGCA OXA-R GTGTGTTTAGAATGGTGA CTX-M-F CGCTTTGCGATGTGCAG CTX-M-R ACCGCGATATCGTTGGT CMY-F GACAGCCTCTTTCTCCACA CMY-R TGGAACGAAGGCTACGTA Aminoglycoside-modifying enzymes (AMEs) strA-F ATGTTCATGCCGCCTGTTTTT strA-R CTAGTATGACGTCTGTCGC strB-F ATGTTCATGCCGCCTGTTTTT strB-R CTAGTATGACGTCTGTCGC aph(3 )-IIa-F ATGAGCCATATTCAACGGGAA TCAGAAAAACTCATCGAGCAT aph(3 )-IIa-R aac(3)-IIa-F ACCCTACGAGGAGACTCTGAATG aac(3)-IIa-R CCAAGCATCGGCATCTCATA aac(6 )-Ib-F ATGACCTTGCGATGCTCTATGA CGAATGCCTGGCGTGTTT aac(6 )-Ib-R ant(3 )-Ia-F ATCTGGCTATCTTGCTGACA ant(3 )-Ia-R TTGGTGATCTCGCCTTTC Plasmid-mediated quiniolone qnrA-F ATTTCTCACGCCAGGATTTG qnrA-F GATCGGCAAAGGTTAGGTCA qnrB-F qnrB-R qnrS-F qnrS-R
GATCGTGAAAGCCAGAAAGG ACGATGCCTGGTAGTTGTCC ACGACATTCGTCAACTGCAA TAAATTGGCACCCTGTAGGC
Target
Reference
iroN
Johnson et al., 2008
ompT
Johnson et al., 2008
hlyF
Johnson et al., 2008
iss
Johnson et al., 2008
iutA
Johnson et al., 2008
blaTEM
Ahmed et al., 2007
blaSHV
Ahmed et al., 2007
blaOXA
Ahmed et al., 2007
blaCTX-M
Ahmed et al., 2007
blaCMY
Ahmed et al., 2007
strA
Xie et al., 2014
strB
Xie et al., 2014
aph(3 )-IIa
Xie et al., 2014
aac(3)-IIa
Zhang et al., 2014
aac(6 )-Ib
Zhang et al., 2014
ant(3 )-Ia
Xie et al., 2014
qnrA
Robicsek et al., 2006; Zhang et al., 2014
qnrB qnrS
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Antimicrobial susceptibility tests were performed using the disc diffusion method, according to the standards and interpretive criteria described by the Clinical and Laboratory Standards Institute. The main process has been described previously (Xie et al., 2014). The following antimicrobials were used: amikacin (AMK), 30 μg; ampicillin (AMP), 30 μg; ciprofloxacin (CIP), 5 μg; cefradine (CRD), 30 μg; ceftriaxone
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI
PCR Screening for Antimicrobial Resistance Genes
PCR-based inc/rep Typing Method of Isolates The necessity of tracing plasmids conferring drug resistance prompted us to develop an inc/rep PCR-based typing (PBRT) method. In this method, 18 pairs of primers (Carattoli et al., 2005) were designed to perform 5 multiplex and 3 simplex PCRs, recognizing the FIA, FIB, FIC, HI1, HI2, I1-Ig, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA replicons, which are representative of the major plasmid incompatibility groups circulating among the Enterobacteriaceae. Primers and methodology have been described previously (Carattoli et al., 2005).
Sequencing and Sequence Analysis PCR was used in a previously described method to screen all 87 isolates (Xie et al., 2014). The PCR products were sequenced by Beijing Sangon Co. Ltd. (Beijing, China). The DNA sequences obtained were compared to the information in the GenBank database of the BLAST network of the National Center for Biotechnology Information.
Biostatistics Two-way frequency tables of source population (Baoding, Cangzhou, Hengshui, and Cangzhou) versus trait (plasmid replicon type) were generated to allow comparisons of the frequency of occurrence of the plasmid replicons across populations. Because there were a number of traits being assessed simultaneously in this study, a resampling-based multiplicity adjustment to the Fisher’s exact test results was applied using the MULTTEST procedure in SAS.
RESULTS Isolation and Identiffication of Multidrug-resistant APEC from Septicemic Broiler We screened all 87 isolates from the affected broilers for each of the 5 genes associated APEC virulence traits. The results showed that 41 out of 87 nonrepetitive E.coli isolates from septicemic broilers carried 3 or more APEC virulence genes (Table 2). The virulence genes were iroN (56 isolates, 64.4%), iutA (53 isolates, 60.9%), iss (37 isolates, 42.5%), hlyF (31 isolates, 35.6%), and ompT (5 isolates, 5.7%). Four isolates (4.6%) were positive for 5 genes, 10 isolates (11.5%) for 4 genes, 27 isolates (31.0%) for 3 genes, 14 isolates (16.1%) for 2 genes, and 13 isolates (14.9%) for one gene. In addition, 19 (21.8%) isolates carried none of the detected virulence traits. Of the antimicrobial susceptibility tests, the results showed that all 87 (100%) APEC isolates showed resistance to more than 5 antimicrobial agents (Tables 2 and 3). The most commonly observed resistance phenotypes were against AMP, KAN, CIP, LOF, STR GEN, and OFL; all were higher than 93%. Moreover, the rate of resistance to SPE was the lowest (20 isolates, 23.0%), followed by NM (46 isolates, 56.9%) and AMK (47 isolates, 56.9%).
Detection and Identiffication of β-lactamase-encoding Genes in APEC PCR and DNA sequencing identiffied β -lactamaseencoding genes in 66 (75.9%) APEC isolates (Tables 2 and 4). The β -lactamase-encoding genes include TEM-encoding genes, blaTEM (14 isolates); the CMY-encoding gene, blaCMY-2 (5 isolates); the OXA-encoding gene, blaOXA-30 (22 isolates); the CTXM encoding gene, blaCTX-M-15 (61 isolates); and the SHV-encoding gene, blaSHV-2 (0 isolates).
Detection and Identiffication of Aminoglycoside-modifying Enzymes in APEC AMEs were inferred to be present by demonstration of the corresponding gene. All 6 AME genes included in the multiplex PCR were present either singly or in combination (Tables 2 and 4). The results show that 65 isolates (74.7%) had AME genes, and frequencies of AME genes were 65.5% strB, 47.2% ant(3 )-Ia, 34.5% aac(6 )-Ib, and 9.2% aph(3 )-IIa.
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
The E.coli isolates were tested for TEM, SHV, CTXM, OXA, and CMY β -lactamase-encoding genes by PCR using universal primers for the corresponding gene families, as described previously (Ahmed et al., 2007; Ahmed and Shimamoto, 2013). The aminoglycosidemodifying enzymes (AME) strA, strB, aph(3 )-IIa, aac(3)-IIa, aac(6 )-Ib, and ant(3 )-Ia were used in multiplex PCR as described previously (Zhang et al., 2014). PCR ampliffication was used for screening of plasmidmediated quinolone resistance (PMQR) genes qnrA, qnrB, and qnrS with speciffic primers, as described previously (Xie et al., 2014). All primers are listed in Table 1.
3
Virulence genes
iroN + ompT + iss + iutA + hlyF
iroN + ompT + iss + iutA + hlyF
iroN + ompT + iss + iutA + hlyF
iroN + ompT + iss + iutA + hlyF
iroN + ompT + iss + iutA
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
A13-1
A13-2
A13-3
A13-4
A13-5
A13-6
A13-7
A13-8
A13-9
A13-10
A13-11
A13-12
A13-13
A13-14 A13-15
A13-16
A13-17
A13-18
A13-19
A13-20
A13-21
A13-22
A13-23
A13-24
A13-25
A13-26
AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, GEN, KAN, LOF, NM, OFL, SAM, STR AMP, CRD, CRO, GAT, LOF, NOR, OFL, SAM, SPE, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMK, AMP, CIP, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMP, CIP, GAT, GEN, KAN, LOF, NOR, OFL, SAM, SPE, STR AMP, GEN, KAN, SAM, STR AMP, AMK, CIP, CRD, CRO, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMP, CRD, CRO, CTX, GEN, KAN, LOF, NM, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SPE, STR AMK, AMP, CIP, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, OFL, STR AMK, AMP, CIP, CRO, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR AMK, AMP, CIP, CRO, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CRD, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR
Resistance phenotypea
Hengshui Baoding Baoding
L/M, FrepB FIC I1, FIC, B/O FrepB
blaCTX-M-15, blaOXA-1, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15
ant(3 )-Ia, strB
blaCTX-M-15, blaCMY-2, strB
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, aph(3 )-IIa, strB blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strB —
blaCTX-M-15, blaTEM-1, aac(6 )-Ib, aph(3 )-IIa, strA, strB blaCTX-M-15, aac(6 )-Ib, ant(3 )-Ia, strB
Xingtai Xingtai Cangzhou Baoding Baoding
FIB FIC, B/O FIB —
Baoding
I1, P, B/O
FIB,K
Baoding
FIA, FIB, P, K, B/O
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15, blaOXA-1
Baoding
Baoding Baoding
K, B/O FIB, FIC, B/O
strB —d
FIB
Baoding
—
blaTEM-1, aac(3)-IIa, strB
Baoding
Baoding
I1
B/O
Baoding
FIB, K, B/O
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strB blaTEM-1
Xingtai
Xingtai
FIB, FIC
—
Baoding
I1, P, FIC, A/C, K, B/O
Baoding
Xingtai
—c
blaCTX-M-15, aph(3 )-IIa, strA, strB, qnrS
FIB
Cangzhou
FIA, FIB, K, B/O
blaCTX-M-15, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia blaCTX-M-15
Baoding
FIB
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strA, strB blaCTX-M-15, aac(3)-IIa, ant(3 )-Ia, strB
ant(3 )-Ia, strA, strB
blaCTX-M-15, blaTEM-1, blaCMY-2, aac(3)-IIa, aac(6 )-Ib, strB blaOXA-1, ant(3 )-Ia, strB
Baoding
N, FIB, K, B/O
blaCTX-M-15, blaTEM-1, aac(3)-IIa, ant(3 )-Ia, strB, qnrS blaCTX-M-15, aac(6 )-Ib, ant(3 )-Ia, strB
City Cangzhou
Replicon typingb
Resistance genes identified
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Isolate
Table 2. Bacterial isolates used in this study and their characteristics.
4 LI ET AL.
Virulence genes
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + hlyF
iroN + iss + hlyF
iroN + iss + hlyF
iroN + iss + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iss + iutA + hlyF
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA iroN + iss
Isolate
A13-27
A13-28
A13-29
A13-30
A13-31
A13-32
A13-33
A13-34
A13-35
A13-36
A13-37
A13-38
A13-39
A13-40
A13-41
A13-42
A13-43
A13-44
A13-45
A13-46
A13-47
A13-48
A13-49
A13-50
A13-51 A13-52
AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, SPE, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, STR AMP, CIP, CRD, CRO, CTX, GEN, KAN, LOF, NM, OFL, SAM, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, NM, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CTX, GEN, KAN, LOF, NM, NOR, OFL, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, NM, NOR, OFL, SAM, STR AMP, CIP, CRO, GAT, GEN, KAN, NM, NOR, OFL, STR AMP, CIP, CRO, GAT, GEN, KAN, LOF, NOR, OFL, STR AMP, CIP, CTX, KAN, LOF, NOR, OFL, AMK, AMP, CIP, CRD, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR
Resistance phenotypea
strB aac(6 )-Ib, ant(3 )-Ia, strB
blaCTX-M-15, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, strB, qnrS blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15, blaOXA-1, ant(3 )-Ia
blaCTX-M-15, blaOXA-1, blaTEM-1, aac(3)-IIa, ant(3 )-Ia, strB blaCTX-M-15, blaOXA-1, aac(3)-IIa
blaOXA-1, blaTEM-1, aac(3)-IIa, strB
blaCTX-M-15, blaOXA-1, ant(3 )-Ia, strB, qnrS blaCTX-M-15, blaOXA-1, blaTEM-1, aac(3)-IIa, aac(6 )-Ib, aph(3 )-IIa, strA, strB —
blaCTX-M-15, ant(3 )-Ia, strB
blaCTX-M-15, blaOXA-1, ant(3 )-Ia, strB
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3)-Ia, strB blaCTX-M-15, blaOXA-1, aac(3)-IIa, aac(6 )-Ib, strB blaCTX-M-15, ant(3 )-Ia, strA, strB
—
blaCTX-M-15, blaTEM-1, blaCMY-2, aac(6 )-Ib, ant(3 )-Ia blaCTX-M-15
blaCTX-M-15, aph(3 )-IIa, strB
blaCTX-M-15, aac(6 )-Ib
blaCTX-M-15, ant(3 )-Ia, strB
blaCTX-M-15, blaTEM-1
blaCTX-M-15, aac(6 )-Ib, ant(3 )-Ia, strB
blaCTX-M-15, blaTEM-1, blaCMY-2, aac(3)-IIa, strB blaCTX-M-15, blaOXA-1, aac(6 )-Ib
Resistance genes identified
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Table 2. continued.
Baoding Cangzhou Cangzhou Baoding Baoding Baoding Baoding Xingtai Cangzhou Cangzhou Baoding
FrepB A/C, FrepB T, FrepB — — — — – FIB, P, K, B/O FIB —
Baoding
FIB, FIC, B/O
Baoding
Baoding
—
I1, FIB, P, B/O
Xingtai
I1
Baoding
Cangzhou
—
I1, P, B
Baoding
I1, K, B/O
Baoding
Baoding
I1, B/O
I1, FIB, P, B/O
Baoding
I1, FIB
Cangzhou
Baoding
—
FIB
Baoding
—
Baoding
Cangzhou
N, FIB, A/C, K
FIA, FIB, P, K, B/O
City
Replicon typingb
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI
5
iroN + iss
iroN + hlyF
iutA + hlyF
iroN
iroN
iroN
iutA
iutA
iutA
iutA
iutA
iss
iss
hlyF
hlyF
hlyF
A13-53
A13-54
A13-55
A13-56
A13-57
A13-58
A13-59
A13-60
A13-61
A13-62
A13-63
A13-64
A13-65
A13-66
A13-67
A13-68
A13-77
A13-76
A13-75
A13-74
A13-73
A13-72
A13-71
A13-70
A13-69
Virulence genes
Isolate
AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, SAM AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, SPE, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO,CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SPE, STR AMK, AMP, CIP, CRO, GAT, KAN, LOF, NM, NOR, OFL, SPE, STR AMK, AMP, CIP, CRO, GAT, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR
AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SPE, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMK, AMP, CIP, CRD, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRD, CRO, CTX, GEN, LOF, NM, NOR, OFL, SAM AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, NOR, OFL, SAM, STR AMP, CIP, CRD, GAT, GEN, KAN, LOF, NOR, OFL, SAM CIP, GAT, LOF, NOR, OFL, STR
Resistance phenotypea
blaCTX-M-15
blaCTX-M-15
—
aph(3 )-IIa, strB
blaCTX-M-15, ant(3 )-Ia, strB
ant(3 )-Ia, strB
aac(3)-IIa, ant(3 )-Ia, strB
blaCTX-M-15
blaTEM-1, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15, aac(3)-IIa, ant(3 )-Ia, strB
blaCTX-M-15
blaCTX-M-15, ant(3 )-Ia, strB
blaCTX-M-15, aac(6 )-Ib, strB
—
blaCTX-M-15, blaCMY-2, ant(3 )-Ia, qnrS
blaCTX-M-15, blaOXA-1
blaCTX-M-15, blaOXA-1, aac(3)-IIa, aac(6 )-Ib, strB blaCTX-M-15, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, aph(3 )-IIa, strB blaCTX-M-15, aac(6 )-Ib, ant(3 )-Ia, strB
aac(6 )-Ib
blaCTX-M-15, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15, aac(6 )-Ib, strB
blaCTX-M-15, blaTEM-1, aac(3)-IIa, strB
blaCTX-M-15, aac(3)-IIa, strB
blaCTX-M-15, blaOXA-1
Resistance genes identified
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Table 2. continued. City Cangzhou Baoding Hengshui Xingtai Baoding Baoding Baoding Baoding Xingtai Baoding Baoding Baoding Hengshui Baoding Cangzhou Hengshui Hengshui Hengshui Baoding Baoding Baoding Hengshui Cangzhou Baoding Xingtai
Replicon typingb FIB FIB, P, K, B/O I1, FIB — — I1 FIB — — I1, FrepB — I1, A/C, K, B/O FIC — FIC, K — K — — — – K, B/O A/C — I1, FIC, K, B/O
6 LI ET AL.
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI
7
Hengshui Baoding
Baoding
—
FrepB P, K
— FIC, B/O
I1,FIC, A/C, K, B/O
blaCTX-M-15
ant(3 )-Ia, strB —
aac(3)-IIa, aph(3 )-IIa, strB aac(3)-IIa, ant(3 )-Ia
strB, qnrS A13-87
A13-85 A13-86
A13-83 A13-84
A13-82
AMK, amikacin; AMP, ampicillin; CIP, ciprofloxacin; CRD, cefradine; CRO, ceftriaxone; CTX, cefotaxime; GAT, gatifloxacin; GEN, gentamycin; KAN, kanamycin; LOF, levofloxacin; NM, neomycin; NOR, norfloxacin; OFL, ofloxacin; SAM, sulbactam/ampicillin 1:1; SPE, spectinomycin; STR, streptomycin b Plasmid Incompatibility Inc group. c, d Negative result
Xingtai Hengshui
— blaCTX-M-15, aac(3)-IIa, ant(3 )-Ia, strB A13-81
A13-80
A13-78 A13-79
a
Cangzhou A/C, FrepB blaCTX-M-15, aac(3)-IIa, ant(3 )-Ia, strB
Baoding
Baoding Baoding FIB K — blaCTX-M-15, blaTEM-1, aac(6 )-Ib, strB
AMK, CIP, GAT, GEN, KAN, LOF, NOR, SPE, STR AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, STR AMP, CIP, CRO, GAT, KAN, LOF, NM, NOR, OFL, STR AMP, CIP, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, GAT, GEN, KAN, LOF, NOR, OFL, SPE, STR AMP, CRO, GEN, KAN, LOF, STR
Replicon typingb
City Resistance genes identified Resistance phenotypea Virulence genes Isolate
We screened all 87 isolates from the affected flocks for each of the 4 genes associated with PMQR. Of the PMQR genes examined, qnrS (6.9%, 6 isolates) was the most frequently identified gene. In addition, none of the isolates carried the qnrA or qnrB gene (Tables 2 and 4).
Distribution of Replicons on Plasmids The PBRT method consists of 5 different multiplex PCRs recognizing 3 different replicon types and 3 simplex PCRs for F, K, and B/O. Eighteen specific primer pairs were designed on the basis of the multiple comparative analysis of nucleotide sequence on the EMBL Gene Databank, for HI1, HI2, I1-Ig, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FrepB , FII, K, and B/O replicons (Carattoli et al., 2005). Table 5 shows the results obtained from these strains by the PBRT method; the plasmid replicons were identified in 63 (72.4%) isolates. The inc/rep typing detected the presence of FIB (26 isolates), B/O (26 isolates), K (20 isolates), I1-Ig (17 isolates), P (10 isolates), FIC (13 isolates), IncFrepB (8 isolates), A/C (7 isolates), FIA (3 isolates), N (2 isolates), L/M (1 isolates), and T (1 isolate) in isolates of this collection; the presence of IncY, IncX, IncW, IncHI2, FII, and IncHI1 was zero. Twenty-four strains were negative for all the replicons tested. No significant differences in plasmid replicon content were observed between APEC isolates from different places (Baoding, Cangzhou, Hengshui, and Cangzhou) in Hebei Province (Table 5).
DISCUSSION Virulence Genes E. coli is a part of the normal microflora found in poultry intestines. However, certain strains, such as those designated as APEC, can spread into various internal organs and cause the systemic fatal disease colibacillosis (Oh et al., 2011). APEC strains contain many virulence factors that enable their extraintestinal pathogenicity. These include production of adhesions, toxins, protections, siderophores, iron transport systems, and invasions (Dziva and Stevens, 2008; Ahmed and Shimamoto, 2013). The 5 essential virulence genes are iutA, hlyF, iss, iroN, and ompT, which are carried on plasmids and are considered good markers for APEC strains from avian fecal isolates (Johnson et al., 2008). In the current study, the virulence genes iroN, iutA, iss, hlyF, and ompT were detected in 64.4, 60.9, 42.5, 35.6, and 5.7% of isolates, respectively. The results obtained in our study were lower than those previously reported (Johnson et al., 2008; Ahmed and Shimamoto,
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Table 2. continued.
Cangzhou
Detection and Identiffication of Plasmid-mediated Quinolone Resistance Genes
8
LI ET AL. Table 3. Antimicrobial resistance of APEC from broilers in Hebei, China. Antimicrobial class
Kanamycin Streptomycin Gentamycin Amikacin Neomycin Spectinomycin Ceftriaxone Cefotaxime Cefradine Ciprofloxacin Levofloxacin Ofloxacin Norfloxacin Gatifloxacin Ampicillin Sulbactam/ampicillin 1:1
Aminoglycosides
Cephalosporins
Fluoroquinolones
Penicllins β -lactamase inhibitors /cephalosporins
Table 4. Frequency of antimicrobial resistance genes of APEC from broilers in Hebei, China. Resistance genes
Number of positive strains (n = 87)
β -lactamases blaTEM blaCTX-M blaOXA blaCMY blaSHV Total
14 (16.1%) 61 (70.1%) 22 (25.3%) 5 (5.7%) 0 (0.0%) 66 (75.9%)
Aminoglycoside-modifying enzymes (AMEs) strA strB aph(3 )-IIa aac(3)-IIa aac(6 )-Ib ant(3 )-Ia Total
6 (6.9%) 57 (65.5%) 8 (9.2%) 25 (28.7%) 30 (34.5%) 41 (47.2%) 65 (74.7%)
Plasmid-mediated quiniolone qnrA qnrB qnrS Total
0 0 6 6
(0.0%) (0.0%) (6.9%) (6.9%)
2013). In the United States, 85.4% of the APEC strains isolated from lesions of birds clinically diagnosed with colibacillosis were positive for at least one of these 5 genes, where iroN, iutA, iss, hlyF, and ompT were detected in 85.3, 80.7, 80.5, 78.2, and 78.6% of isolates, respectively (Johnson et al., 2008). In Egypt, 80.2% of the APEC strains isolated from septicemic broilers carried 3 or more of the APEC virulence genes iroN (73 isolates, 80.2%), ompT (73 isolates, 80.2%), iss (71 isolates, 80.2%), iutA (71 isolates, 78.0%), and hlyF (65 isolates, 71.4%) (Ahmed and Shimamoto, 2013). However, Delicato et al. (2003) obtained a lower prevalence for iss from APEC isolated from poultry with colibacillosis in Brazil, with an incidence of 38.5%. Therefore, it can be concluded that the prevalence of the virulence genes may vary in different regions.
Number of resistant strains (n = 87) 83 82 81 47 46 20 77 51 50 82 82 81 78 67 85 55
(95.4%) (94.3%) (93.1%) (52.9%) (52.9%) (23.0%) (88.5%) (58.6%) (57.5%) (94.3%) (94.3%) (93.1%) (89.7%) (77.0%) (97.7%) (63.2%)
Resistance and Resistance Genes Resistance to existing antimicrobials is widespread and of great concern (Hinton et al., 1986; Ahmed and Shimamoto, 2013; Zhang et al., 2014). In this study, 100% of the tested APEC isolates showed multidrug resistance phenotypes, and 85.1% were resistant to 11 or more antimicrobial agents. Similar resistance phenotypes have been detected in APEC strains isolated from diseased chickens with colibacillosis in Korea (Kim et al., 2007), the United States (Johnson et al., 2008), the United Kingdom (Randall et al., 2010), Australia (Obeng et al., 2011), and more recently in APEC isolated from intensively farmed and free-range poultry in Egypt (Ahmed and Shimamoto, 2013). Exposing the animals’ bacterial flora to the antibiotics encourages the emergence of resistance across a wide range of antibiotics (Barbosa and Levy, 2000). The cumulative effect on the traditional antimicrobials has been multidrug resistance (Szmolka and Nagy, 2013). Perhaps the most striking finding from this study was the widespread resistance to fluoroquinolones. Our results showed that 94.3% of the tested APEC isolates were resistant to ciprofloxacin and levofloxacin, and more than 77.0% were resistant to ofloxacin, norfloxacin, and gatifloxacin. In somewhat similar findings in China, it has been reported in a recent study of clinical E. coli isolates that greater than 70% of all isolates were resistant to ciprofloxacin (Yang et al., 2004). Consequently, fluoroquinolones have become ineffective in veterinary medicine in China (Yang et al., 2004). Similar findings were also reported for E. coli isolates in Spain, wherein 90% of chicken isolates were resistant to ciprofloxacin (Saenz et al., 2001). In regards to resistance mechanisms, a notable high mutation in the quinolone resistance–determining region of gyrA has been found mainly in China (Yang et al., 2004; Xie et al., 2014), while the plasmids carrying qnr genes appear to be rare (Robicsek et al., 2006; Xie et al., 2014). In this study, the detected rates of qnrA, qnrB, and qnrS
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Antimicrobial agent(s) tested
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI
9
Table 5. Occurrence of plasmid replicons and comparison between different groups of E. coli isolates. Replicon profilea (overall %)
FIB (29.9%) B/O (29.9%) K (23.0%) I1 (19.5%) FIC (14.9%) P (11.5%) FrepB (9.2%) A/C (8.0%) FIA (3.4%) N (2.3%) T (1.1%) L/M (1.1%)
P-valueb
No. (%) with profile Baoding
Cangzhou
Hengshui
Xingtai
15 (28.8%) 19 (36.5%) 10 (19.2%) 14 (26.9%) 7 (13.5%) 12 (23.1%) 4 (7.7%) 3 (5.8%) 2 (3.9%) 0 (0%) 0 (0%) 1 (1.9%)
7 5 5 0 2 4 3 4 1 2 1 0
1 1 3 1 2 1 0 0 0 0 0 0
3 1 2 2 2 1 0 0 0 0 0 0
(46.7%) (33.3%) (33.3%) (0%) (13.3%) (26.7%) (20.0%) (26.7%) (6.7%) (13.3%) (6.7%) (%)
(11.1%) (11.1%) (33.3%) (11.1%) (22.2%) (11.1%) (0%) (0%) (0%) (0%) (0%) (0%)
(27.3%) (9.1%) (18.2%) (18.2%) (18.2%) (9.1%) (0%) (0%) (0%) (0%) (0%) (0%)
NS NS NS NS NS NS NS NS NS NS NS NS
Replicons Y, X, W, HI2, HI1, and FII were detected negative. P-values are from resampling-based multiplicity-adjusted Fisher’s exact tests for the null hypothesis that the percentages for each replicon are equal between different isolates (Baoding, Cangzhou, Hengshui, and Xingtai). NS, not statistically significant. b
were 0.0, 0.0, and 6.9%, respectively, which also verifies this theory. Somewhat similar findings have been reported in a study of clinical E. coli isolates from humans that found positivity rates of qnrS, qnrB, and qnrA at 0, 2, and 2%, respectively (Robicsek et al., 2006). Xie et al. (2014) reported the positivity rates of qnrS, qnrB, and qnrA were 8.11, 0.90, and 0%, respectively. Differing from other types of tested antibiotics, resistance rates for the aminoglycosides show great variance. Resistance rates to kanamycin, streptomycin, and gentamycin are higher than 90%, whereas the resistance rate to spectinomycin is only 23.0%. The highly defined daily dose in recent years may be part of the reason. Kanamycin, streptomycin, and gentamycin are commonly used to clinically treat avian colibacillosis, while spectinomycin was rarely used in clinical veterinary medicine due to its high price. It is known that the inactivation of aminoglycoside drugs occurs mainly via plasmid or chromosome-encoded modifying enzymes (AMEs). The AMEs are a diverse set of proteins consisting of three families, aminoglycoside acetyltransferase (aac), aminoglycoside nucleotide transferase (ant), and aminoglycoside phosphoric transferase (aph) (Shaw et al., 1993; Ramirez and Tolmasky, 2010), which eliminate the synergistic bactericidal effect between the cell wall–active agents (Kondo and Hotta, 1999). In this study, most of the resistant isolates harbored modifying enzyme genes. Moreover, two genes, strB and ant(3 )Ia, were the most frequently observed resistance genes in these isolates. These results were similar to observations in other studies in China (Zhang et al., 2014), as well as studies in other countries (Kim et al., 2007; Green et al., 2011; Goncalves et al., 2013). Penicillin derivatives (β -lactams) are broad-spectrum antibacterial agents widely used in human and veterinary medicine (Hasman et al., 2005; Ahmed and Shimamoto, 2013). The resistance to β -lactams in gram-negative bacteria is primarily mediated by β -lactamases. Many types of β -lactamases have been
described. However TEM-, SHV-, OXA-, CMY-, and CTX-M-type β -lactamases are the most common in gram-negative bacteria (Hasman et al., 2005; Ahmed and Shimamoto, 2013). In this study, all groups of β lactamases were identified, including blaTEM-1 , blaCMY-2 , blaOXA-30 , blaCTX-M-15 and blaSHV-2 , and the detection rate was 75.9%. Similar findings have been reported in the APEC strains isolated from avian colibacillosis in Africa, wherein 94.5% of all the isolates were positive for β -lactamase-encoding genes, and blaTEM-1 , blaCMY-2 , blaOXA-30 , blaCTX-M-15 , and blaSHV-2 were also detected (Ahmed and Shimamoto, 2013). Furthermore, blaTEM was detected in Korea, Spain, Australia, and the United Kingdom (Ahmed and Shimamoto, 2013; Wagner et al., 2014). Also detected in the United Kingdom were blaOXA and blaCTX (Randall et al., 2010). In Australia, blaSHV was identified in the APEC strains isolated from poultry (Obeng et al., 2011). The coexistence of 2 or more multidrug resistance genes and virulence-associated genes was common in the APEC of this study. In a recent study by Wang et al. (2014), these findings were confirmed. Wang et al. (2014) reported that the sequence of an APEC strain harbored more than 10 antibiotic resistance genes on 2 plasmids, which concurrently carry several transferrelated elements, thereby facilitating horizontal transfers. Overall, the results of our study confirm that APEC is a cause for concern, as there are obvious limited therapeutic options available for antimicrobial therapy in poultry production.
Plasmid Replicons The epidemiological importance of tracing the plasmids conferring drug resistance prompted us to develop a PCR method based on the replicons (inc/rep PCR) of the major plasmid incompatibility groups among Enterobacteriaceae (Carattoli et al., 2005). This method
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
a
10
LI ET AL.
ACKNOWLEDGMENTS This work was financially supported by Hebei Science and Technology Planning Project (12226602).
REFERENCES Ahmed, A. M., Y. Motoi, M. Sato, A. Maruyama, H. Watanabe, Y. Fukumoto, and T. Shimamoto. 2007. Zoo animals as reservoirs of gram-negative bacteria harboring integrons and antimicrobial resistance genes. Appl. Environ. Microbiol. 73:6686–6690. Ahmed, A. M., and T. Shimamoto. 2013. Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from septicemic broilers. Int. J. Med. Microbiol. 303:475–483. Barbosa, T. M., and S. B. Levy. 2000. The impact of antibiotic use on resistance development and persistence. Drug Resist. Updat. 3:303–311. Bass, L., C. A. Liebert, M. D. Lee, A. O. Summers, D. G. White, S. G. Thayer, and J. J. Maurer. 1999. Incidence and characterization of integrons, genetic elements mediating multiple-drug resistance, in avian Escherichia coli. Antimicrob. Agents Chemother. 43:2925–2929. Carattoli, A. 2011. Plasmids in gram negatives: Molecular typing of resistance plasmids. Int. J. Med. Microbiol. 301:654–658. Carattoli, A., A. Bertini, L. Villa, V. Falbo, K. L. Hopkins, and E. J. Threlfall. 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63:219–228. Collignon, P., H. C. Wegener, P. Braam, and C. D. Butler. 2005. The routine use of antibiotics to promote animal growth does
little to benefit protein undernutrition in the developing world. Clin. Infect. Dis. 41:1007–1013. Delicato, E. R., B. G. de Brito, L. C. Gaziri, and M. C. Vidotto. 2003. Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis. Vet. Microbiol. 94:97–103. Dziva, F., and M. P. Stevens. 2008. Colibacillosis in poultry: Unravelling the molecular basis of virulence of avian pathogenic Escherichia coli in their natural hosts. Avian Pathol. 37: 355–366. Ewers, C., T. Janssen, S. Kiessling, H. C. Philipp, and L. H. Wieler. 2004. Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet. Microbiol. 104:91–101. Goncalves, A., G. Igrejas, H. Radhouani, S. Correia, R. Pacheco, T. Santos, R. Monteiro, A. Guerra, F. Petrucci-Fonseca, F. Brito, C. Torres, and P. Poeta. 2013. Antimicrobial resistance in faecal enterococci and Escherichia coli isolates recovered from Iberian wolf. Lett. Appl. Microbiol. 56:268–274. Green, K. D., W. Chen, and S. Garneau-Tsodikova. 2011. Effects of altering aminoglycoside structures on bacterial resistance enzyme activities. Antimicrob. Agents Chemother. 55:3207–3213. Hasman, H., D. Mevius, K. Veldman, I. Olesen, and F. M. Aarestrup. 2005. Beta-lactamases among extended-spectrum beta-lactamase (ESBL)–resistant Salmonella from poultry, poultry products and human patients in the Netherlands. J. Antimicrob. Chemother. 56:115–121. Hinton, M., A. Kaukas, S. K. Lim, and A. H. Linton. 1986. Preliminary observations on the influence of antibiotics on the ecology of Escherichia coli and the enterococci in the faecal flora of healthy young chickens. J. Antimicrob. Chemother. 18:165–173. Hopkins, K. L., E. Liebana, L. Villa, M. Batchelor, E. J. Threlfall, and A. Carattoli. 2006. Replicon typing of plasmids carrying CTX-M or CMY beta-lactamases circulating among Salmonella and Escherichia coli isolates. Antimicrob. Agents Chemother. 50:3203–3206. Johnson, T. J., Y. Wannemuehler, C. Doetkott, S. J. Johnson, S. C. Rosenberger, and L. K. Nolan. 2008. Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J. Clin. Microbiol. 46: 3987–3996. Johnson, T. J., Y. M. Wannemuehler, S. J. Johnson, C. M. Logue, D. G. White, C. Doetkott, and L. K. Nolan. 2007. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates. Appl. Environ. Microbiol. 73:1976–1983. Kim, T. E., Y. W. Jeong, S. H. Cho, S. J. Kim, and H. J. Kwon. 2007. Chronological study of antibiotic resistances and their relevant genes in Korean avian pathogenic Escherichia coli isolates. J. Clin. Microbiol. 45:3309–3315. Kondo, S., and K. Hotta. 1999. Semisynthetic aminoglycoside antibiotics: Development and enzymatic modifications. J. Infect. Chemother. 5:1–9. Obeng, A. S., H. Rickard, O. Ndi, M. Sexton, and M. Barton. 2011. Antibiotic resistance, phylogenetic grouping and virulence potential of Escherichia coli isolated from the faeces of intensively farmed and free range poultry. Vet. Microbiol. 154:305–315. Oh, J. Y., M. S. Kang, J. M. Kim, B. K. An, E. A. Song, J. Y. Kim, E. G. Shin, M. J. Kim, J. H. Kwon, and Y. K. Kwon. 2011. Characterization of Escherichia coli isolates from laying hens with colibacillosis on 2 commercial egg-producing farms in Korea. Poult. Sci. 90:1948–1954. Ramirez, M. S., and M. E. Tolmasky. 2010. Aminoglycoside modifying enzymes. Drug Resist. Updat. 13:151–171. Randall, L. P., C. Clouting, R. A. Horton, N. G. Coldham, G. Wu, F. A. Clifton-Hadley, R. H. Davies, and C. J. Teale. 2010. Prevalence of Escherichia coli carrying extended-spectrum beta-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009. J. Antimicrob. Chemother. 66:86–95. Robicsek, A., J. Strahilevitz, D. F. Sahm, G. A. Jacoby, and D. C. Hooper. 2006. qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. Antimicrob. Agents Chemother. 50:2872–2874. Ruiz del Castillo, B., L. Vinue, E. J. Roman, B. Guerra, A. Carattoli, C. Torres, and L. Martinez-Martinez. 2013. Molecular
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
could be used to monitor the circulation of plasmids within strains from different environments or to follow the horizontal transmissions of antimicrobial resistance genes among the Enterobacteriaceae (Carattoli et al., 2005). Research studies have shown the diffusion of the prevalent plasmids is in association with the specific resistance gene (Hopkins et al., 2006; Ruiz del Castillo et al., 2013; Wagner et al., 2014). Similarly, a recent study has identified IncFIB, IncFrep, and IncI1 as common replicon types (Johnson et al., 2007). IncFII and IncI1 plasmids are two of a number of plasmid types that are particularly successful in their ability to spread multidrug resistance (Carattoli, 2011). In our study, we found that both IncFIB and IncB/O (29.9%), followed by IncK (23.0%) and IncI1 (17, 19.5%), were the most prevalent plasmid replicon types in E. coli. This may be the reason for the high multidrug-resistant APEC in the APEC researched. In the study, the plasmid profiles of APEC from different places in Hebei showed no significant differences. The main reason is that the four regions are geographically adjacent to each other and have significant interregion contact. In summary, our current study characterized the genetic basis of virulence and antimicrobial resistance in the APEC strains isolated from septicemic broilers in China. The various classes of the resistance genes were identified, and inc/rep PCR was reported in APEC strains. It is important to monitor the occurrence of resistance genes in APEC. It should be emphasized that there is a need to ban the nontherapeutic use of antibiotics, discourage their misuse, and maintain constant vigilance by providing appropriate scientific and technological support for the poultry industry in this region.
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI characterization of multiresistant Escherichia coli producing or not extended-spectrum beta-lactamases. BMC. Microbiol. 13:84. Saenz, Y., M. Zarazaga, L. Brinas, M. Lantero, F. Ruiz-Larrea, and C. Torres 2001. Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain. Int. J. Antimicrob. Agents 18:353–358. Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138–163. Szmolka, A., and B. Nagy. 2013. Multidrug resistant commensal Escherichia coli in animals and its impact for public health. Front. Microbiol. 4:258. Wagner, S., D. L. Gally, and S. A. Argyle. 2014. Multidrug-resistant Escherichia coli from canine urinary tract infections tend to have commensal phylotypes, lower prevalence of virulence determinants and ampC-replicons. Vet. Microbiol. 169:171–178.
11
Wang, X., H. Hao, Z. Xu, H. Zheng, C. Liu, L. Wei, R. Zhang, D. Bi, H. Chen, and C. Tan. 2014. Plasmid-mediated multidrug resistance and virulence in an avian pathogenic Escherichia coli strain isolated in China. J. Global Antimicrobiol. Resist. 1:57–58. Xie, R., S. Huo, Y. Li, L. Chen, F. Zhang, and X. Wu. 2014. Molecular epidemiological survey on quinolone resistance genotype and phenotype of Escherichia coli in septicemic broilers in Hebei, China. Poult. Sci. 93:335–339. Yang, H., S. Chen, D. G. White, S. Zhao, P. McDermott, R. Walker, and J. Meng 2004. Characterization of multiple-antimicrobialresistant Escherichia coli isolates from diseased chickens and swine in China. J. Clin. Microbiol. 42:3483–3489. Zhang, F. Y., S. Y. Huo, Y. R. Li, R. Xie, X. J. Wu, L. G. Chen, and Y. H. Gao. 2014. A survey of the frequency of aminoglycoside antibiotic-resistant genotypes and phenotypes in Escherichia coli in broilers with septicaemia in Hebei, China. Br. Poult. Sci. 55:305–310. Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Key words: APEC, septicemic broiler, virulence, resistance gene, plasmid 2015 Poultry Science 00:1–11 http://dx.doi.org/10.3382/ps/pev008
INTRDUCTION
great concern to poultry veterinarians (Hinton et al., 1986). Many drug-resistant strains and drug resistance genes can be transferred and disseminated between animal and human pathogens, which not only increases the difficulty of treating animal diseases but is also a serious threat to human health (Collignon et al., 2005). Many studies have documented the presence of virulence genes and drug resistance genes in APEC strains (Bass et al., 1999; Oh et al., 2011; Ahmed and Shimamoto, 2013). So far these have rarely been reported systematically in north China. Therefore, the objective of this study was to characterize the molecular bases of virulence and antimicrobial resistance in multidrugresistant APEC strains isolated from septicemic broilers in Hebei in north China.
Escherichia coli is a part of the normal microflora in the poultry intestine, but certain strains, such as those designated as avian pathogenic E. coli (APEC), spread into various internal organs and cause the systemic fatal disease colibacillosis, which is characterized by septicemia with multiple organ lesions, typically pericarditis, airsacculitis, perihepatitis, peritonitis, and other extraintestinal lesions (Oh et al., 2011). APEC is considered a major cause of economic losses due to morbidity, mortality, and condemnation of poultry carcasses worldwide (Ewers et al., 2004; Ahmed and Shimamoto, 2013). There are several virulence traits associated the extraintestinal pathogenesis of APEC, and the relevant virulence gene, such as iutA, hlyF, iss, iroN, and ompT, were carried by plasmids that are considered the most signifficantly associated with APEC strains (Johnson et al., 2008; Ahmed and Shimamoto, 2013). At present, antimicrobial therapy acts as an important tool to reduce both the incidence and the mortality associated with avian colibacillosis. However, resistance to existing antimicrobials is widespread and of
MATERIALS AND METHODS Isolation and Identification of Bacteria Avian E. coli isolates were isolated from the liver, pericardial fluid, heart blood, and spleen collected from 132 septicaemic broilers at 7 to 43 d of age that died of colibacillosis or were culled by veterinarians at 63 large-scale farms in Baoding, Cangzhou, Hengshui, and Xingtai in Hebei Province, China. Sampling was performed randomly between February and July 2013.
C 2015 Poultry Science Association Inc. Received July 14, 2014. Accepted October 21, 2014. 1 Corresponding author: [email protected]
1
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
gentamycin, ofloxacin, norfloxacin, and ceftriaxone. The β -lactamase-encoding genes blaTEM , blaCMY-2 , blaOXA-30 , blaCTX-M-15 , and blaSHV-2 ; the aminoglycosidemodifying enzymes (AME) strA, strB, aph(3 )-IIa, aac(3)-IIa, aac(6 )-Ib, and ant(3 )-Ia; and the plasmidmediated quinolone resistance (PMQR) genes qnrA, qnrB, and qnrS, were also identified in 66 (75.9%), 65 (74.7%), and 6 (6.9%) isolates, respectively. All isolates were evaluated in terms of replicon type. The plasmid replicons were identified in 63 (72.4%) isolates, and the FIB, B/O, and K replicons were the most present. To the best of our knowledge, this is the first report of molecular characterization of antimicrobial resistance in APEC strains from China.
ABSTRACT Avian pathogenic Escherichia coli (APEC) causes extensive mortality in poultry flocks, leading to extensive economic losses. To date, little information has been available on the molecular basis of antimicrobial resistance in APEC in Hebei, China. Therefore, the objective of this study was to characterize the virulence and antimicrobial resistance of multidrug-resistant APEC isolated from septicemic broilers at the molecular level. Among 87 nonrepetitive E. coli isolates, 41 (47.1%) carried 3 or more of the APEC virulence genes iroN, ompT, iss, iutA, and hlyF. All 87 APEC isolates showed multidrugresistant phenotypes, particularly against ampicillin, kanamycin, ciprofloxacin, levofloxacin, streptomycin,
2
LI ET AL.
Pathological materials were collected under sterile conditions. Avian E. coli isolates were cross-inoculated in MacConkey medium (Tianhe Micro-organism Reagent Co. Ltd., Hangzhou, China), and single pink colonies were picked and purified on MacConkey medium. Only one positive sample per broiler was included in this study. In total, 87 E. coli isolates from 63 farms were identified via biochemical testing and were then used for testing.
(CRO), 30 μg; cefotaxime (CTX), 30 μg; gatifloxacin (GAT), 5 μg; gentamycin (GEN), 10 μg; kanamycin (KAN), 30 μg; levofloxacin (LOF), 5 μg; neomycin (NM), 30 μg; norfloxacin (NOR), 10 μg; ofloxacin (OFL), 5 μg; sulbactam/ampicillin 1:1 (SAM), 10 μg/10 μg; spectinomycin (SPE), 100 μg; and streptomycin (STR), 10 μg. The discs were purchased from Hangzhou Tianhe Microorganism Reagent Co. Ltd. (Hangzhou, China).
Drug Susceptibility Test Screening of APEC Virulence Genes by Multiplex PCR A pentaplex PCR assay targeting ffive virulence genes of APEC, iutA, hlyF, iss, iroN, and ompT, was carried out as previously described (Johnson et al., 2008; Ahmed and Shimamoto, 2013). Primers used for pentaplex PCR are listed in Table 1.
Table 1. Primers used for PCR and DNA sequencing. Primer
Sequence 5 to 3
Virulence genes iroN-F AATCCGGCAAAGAGACGAACCGCCT iroN-R GTTCGGGCAACCCCTGCTTTGACTTT ompT-F TCATCCCGGAAGCCTCCCTCACTACTATTA ompT-R GCGTTTGCTGCACTGGCTTCTGATAC hlyF-F GGCCACAGTCGTTTAGGGTGCTTACC hlyF-R GGCGGTTTAGGCATTCCGATACTCAG iss-F CAGCAACCCGAACCACTTGATG iss-R AGCATTGCCAGAGCGGCAGAA iutA-F GGCTGGACATCATGGGAACTGG iutA-R CGTCGGGAACGGGTAGAATCG Lactamases TEM-F ATAAAATTCTTGAAGACGAAA TEM-R GACAGTTACCAATGCTTAATC SHV-F TTATCTCCCTGTTAGCCACC SHV-R GATTTGCTGATTTCGCTCGG OXA-F TCAACTTTCAAGATCGCA OXA-R GTGTGTTTAGAATGGTGA CTX-M-F CGCTTTGCGATGTGCAG CTX-M-R ACCGCGATATCGTTGGT CMY-F GACAGCCTCTTTCTCCACA CMY-R TGGAACGAAGGCTACGTA Aminoglycoside-modifying enzymes (AMEs) strA-F ATGTTCATGCCGCCTGTTTTT strA-R CTAGTATGACGTCTGTCGC strB-F ATGTTCATGCCGCCTGTTTTT strB-R CTAGTATGACGTCTGTCGC aph(3 )-IIa-F ATGAGCCATATTCAACGGGAA TCAGAAAAACTCATCGAGCAT aph(3 )-IIa-R aac(3)-IIa-F ACCCTACGAGGAGACTCTGAATG aac(3)-IIa-R CCAAGCATCGGCATCTCATA aac(6 )-Ib-F ATGACCTTGCGATGCTCTATGA CGAATGCCTGGCGTGTTT aac(6 )-Ib-R ant(3 )-Ia-F ATCTGGCTATCTTGCTGACA ant(3 )-Ia-R TTGGTGATCTCGCCTTTC Plasmid-mediated quiniolone qnrA-F ATTTCTCACGCCAGGATTTG qnrA-F GATCGGCAAAGGTTAGGTCA qnrB-F qnrB-R qnrS-F qnrS-R
GATCGTGAAAGCCAGAAAGG ACGATGCCTGGTAGTTGTCC ACGACATTCGTCAACTGCAA TAAATTGGCACCCTGTAGGC
Target
Reference
iroN
Johnson et al., 2008
ompT
Johnson et al., 2008
hlyF
Johnson et al., 2008
iss
Johnson et al., 2008
iutA
Johnson et al., 2008
blaTEM
Ahmed et al., 2007
blaSHV
Ahmed et al., 2007
blaOXA
Ahmed et al., 2007
blaCTX-M
Ahmed et al., 2007
blaCMY
Ahmed et al., 2007
strA
Xie et al., 2014
strB
Xie et al., 2014
aph(3 )-IIa
Xie et al., 2014
aac(3)-IIa
Zhang et al., 2014
aac(6 )-Ib
Zhang et al., 2014
ant(3 )-Ia
Xie et al., 2014
qnrA
Robicsek et al., 2006; Zhang et al., 2014
qnrB qnrS
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Antimicrobial susceptibility tests were performed using the disc diffusion method, according to the standards and interpretive criteria described by the Clinical and Laboratory Standards Institute. The main process has been described previously (Xie et al., 2014). The following antimicrobials were used: amikacin (AMK), 30 μg; ampicillin (AMP), 30 μg; ciprofloxacin (CIP), 5 μg; cefradine (CRD), 30 μg; ceftriaxone
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI
PCR Screening for Antimicrobial Resistance Genes
PCR-based inc/rep Typing Method of Isolates The necessity of tracing plasmids conferring drug resistance prompted us to develop an inc/rep PCR-based typing (PBRT) method. In this method, 18 pairs of primers (Carattoli et al., 2005) were designed to perform 5 multiplex and 3 simplex PCRs, recognizing the FIA, FIB, FIC, HI1, HI2, I1-Ig, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA replicons, which are representative of the major plasmid incompatibility groups circulating among the Enterobacteriaceae. Primers and methodology have been described previously (Carattoli et al., 2005).
Sequencing and Sequence Analysis PCR was used in a previously described method to screen all 87 isolates (Xie et al., 2014). The PCR products were sequenced by Beijing Sangon Co. Ltd. (Beijing, China). The DNA sequences obtained were compared to the information in the GenBank database of the BLAST network of the National Center for Biotechnology Information.
Biostatistics Two-way frequency tables of source population (Baoding, Cangzhou, Hengshui, and Cangzhou) versus trait (plasmid replicon type) were generated to allow comparisons of the frequency of occurrence of the plasmid replicons across populations. Because there were a number of traits being assessed simultaneously in this study, a resampling-based multiplicity adjustment to the Fisher’s exact test results was applied using the MULTTEST procedure in SAS.
RESULTS Isolation and Identiffication of Multidrug-resistant APEC from Septicemic Broiler We screened all 87 isolates from the affected broilers for each of the 5 genes associated APEC virulence traits. The results showed that 41 out of 87 nonrepetitive E.coli isolates from septicemic broilers carried 3 or more APEC virulence genes (Table 2). The virulence genes were iroN (56 isolates, 64.4%), iutA (53 isolates, 60.9%), iss (37 isolates, 42.5%), hlyF (31 isolates, 35.6%), and ompT (5 isolates, 5.7%). Four isolates (4.6%) were positive for 5 genes, 10 isolates (11.5%) for 4 genes, 27 isolates (31.0%) for 3 genes, 14 isolates (16.1%) for 2 genes, and 13 isolates (14.9%) for one gene. In addition, 19 (21.8%) isolates carried none of the detected virulence traits. Of the antimicrobial susceptibility tests, the results showed that all 87 (100%) APEC isolates showed resistance to more than 5 antimicrobial agents (Tables 2 and 3). The most commonly observed resistance phenotypes were against AMP, KAN, CIP, LOF, STR GEN, and OFL; all were higher than 93%. Moreover, the rate of resistance to SPE was the lowest (20 isolates, 23.0%), followed by NM (46 isolates, 56.9%) and AMK (47 isolates, 56.9%).
Detection and Identiffication of β-lactamase-encoding Genes in APEC PCR and DNA sequencing identiffied β -lactamaseencoding genes in 66 (75.9%) APEC isolates (Tables 2 and 4). The β -lactamase-encoding genes include TEM-encoding genes, blaTEM (14 isolates); the CMY-encoding gene, blaCMY-2 (5 isolates); the OXA-encoding gene, blaOXA-30 (22 isolates); the CTXM encoding gene, blaCTX-M-15 (61 isolates); and the SHV-encoding gene, blaSHV-2 (0 isolates).
Detection and Identiffication of Aminoglycoside-modifying Enzymes in APEC AMEs were inferred to be present by demonstration of the corresponding gene. All 6 AME genes included in the multiplex PCR were present either singly or in combination (Tables 2 and 4). The results show that 65 isolates (74.7%) had AME genes, and frequencies of AME genes were 65.5% strB, 47.2% ant(3 )-Ia, 34.5% aac(6 )-Ib, and 9.2% aph(3 )-IIa.
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
The E.coli isolates were tested for TEM, SHV, CTXM, OXA, and CMY β -lactamase-encoding genes by PCR using universal primers for the corresponding gene families, as described previously (Ahmed et al., 2007; Ahmed and Shimamoto, 2013). The aminoglycosidemodifying enzymes (AME) strA, strB, aph(3 )-IIa, aac(3)-IIa, aac(6 )-Ib, and ant(3 )-Ia were used in multiplex PCR as described previously (Zhang et al., 2014). PCR ampliffication was used for screening of plasmidmediated quinolone resistance (PMQR) genes qnrA, qnrB, and qnrS with speciffic primers, as described previously (Xie et al., 2014). All primers are listed in Table 1.
3
Virulence genes
iroN + ompT + iss + iutA + hlyF
iroN + ompT + iss + iutA + hlyF
iroN + ompT + iss + iutA + hlyF
iroN + ompT + iss + iutA + hlyF
iroN + ompT + iss + iutA
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF
iroN + iss + iutA + hlyF iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
A13-1
A13-2
A13-3
A13-4
A13-5
A13-6
A13-7
A13-8
A13-9
A13-10
A13-11
A13-12
A13-13
A13-14 A13-15
A13-16
A13-17
A13-18
A13-19
A13-20
A13-21
A13-22
A13-23
A13-24
A13-25
A13-26
AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, GEN, KAN, LOF, NM, OFL, SAM, STR AMP, CRD, CRO, GAT, LOF, NOR, OFL, SAM, SPE, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMK, AMP, CIP, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMP, CIP, GAT, GEN, KAN, LOF, NOR, OFL, SAM, SPE, STR AMP, GEN, KAN, SAM, STR AMP, AMK, CIP, CRD, CRO, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMP, CRD, CRO, CTX, GEN, KAN, LOF, NM, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SPE, STR AMK, AMP, CIP, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, OFL, STR AMK, AMP, CIP, CRO, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR AMK, AMP, CIP, CRO, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CRD, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR
Resistance phenotypea
Hengshui Baoding Baoding
L/M, FrepB FIC I1, FIC, B/O FrepB
blaCTX-M-15, blaOXA-1, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15
ant(3 )-Ia, strB
blaCTX-M-15, blaCMY-2, strB
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, aph(3 )-IIa, strB blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strB —
blaCTX-M-15, blaTEM-1, aac(6 )-Ib, aph(3 )-IIa, strA, strB blaCTX-M-15, aac(6 )-Ib, ant(3 )-Ia, strB
Xingtai Xingtai Cangzhou Baoding Baoding
FIB FIC, B/O FIB —
Baoding
I1, P, B/O
FIB,K
Baoding
FIA, FIB, P, K, B/O
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15, blaOXA-1
Baoding
Baoding Baoding
K, B/O FIB, FIC, B/O
strB —d
FIB
Baoding
—
blaTEM-1, aac(3)-IIa, strB
Baoding
Baoding
I1
B/O
Baoding
FIB, K, B/O
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strB blaTEM-1
Xingtai
Xingtai
FIB, FIC
—
Baoding
I1, P, FIC, A/C, K, B/O
Baoding
Xingtai
—c
blaCTX-M-15, aph(3 )-IIa, strA, strB, qnrS
FIB
Cangzhou
FIA, FIB, K, B/O
blaCTX-M-15, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia blaCTX-M-15
Baoding
FIB
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strA, strB blaCTX-M-15, aac(3)-IIa, ant(3 )-Ia, strB
ant(3 )-Ia, strA, strB
blaCTX-M-15, blaTEM-1, blaCMY-2, aac(3)-IIa, aac(6 )-Ib, strB blaOXA-1, ant(3 )-Ia, strB
Baoding
N, FIB, K, B/O
blaCTX-M-15, blaTEM-1, aac(3)-IIa, ant(3 )-Ia, strB, qnrS blaCTX-M-15, aac(6 )-Ib, ant(3 )-Ia, strB
City Cangzhou
Replicon typingb
Resistance genes identified
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Isolate
Table 2. Bacterial isolates used in this study and their characteristics.
4 LI ET AL.
Virulence genes
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + iutA
iroN + iss + hlyF
iroN + iss + hlyF
iroN + iss + hlyF
iroN + iss + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iroN + iutA + hlyF
iss + iutA + hlyF
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA
iroN + iutA iroN + iss
Isolate
A13-27
A13-28
A13-29
A13-30
A13-31
A13-32
A13-33
A13-34
A13-35
A13-36
A13-37
A13-38
A13-39
A13-40
A13-41
A13-42
A13-43
A13-44
A13-45
A13-46
A13-47
A13-48
A13-49
A13-50
A13-51 A13-52
AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, SPE, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, STR AMP, CIP, CRD, CRO, CTX, GEN, KAN, LOF, NM, OFL, SAM, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, NM, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CTX, GEN, KAN, LOF, NM, NOR, OFL, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, NM, NOR, OFL, SAM, STR AMP, CIP, CRO, GAT, GEN, KAN, NM, NOR, OFL, STR AMP, CIP, CRO, GAT, GEN, KAN, LOF, NOR, OFL, STR AMP, CIP, CTX, KAN, LOF, NOR, OFL, AMK, AMP, CIP, CRD, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR
Resistance phenotypea
strB aac(6 )-Ib, ant(3 )-Ia, strB
blaCTX-M-15, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, strB, qnrS blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15, blaOXA-1, ant(3 )-Ia
blaCTX-M-15, blaOXA-1, blaTEM-1, aac(3)-IIa, ant(3 )-Ia, strB blaCTX-M-15, blaOXA-1, aac(3)-IIa
blaOXA-1, blaTEM-1, aac(3)-IIa, strB
blaCTX-M-15, blaOXA-1, ant(3 )-Ia, strB, qnrS blaCTX-M-15, blaOXA-1, blaTEM-1, aac(3)-IIa, aac(6 )-Ib, aph(3 )-IIa, strA, strB —
blaCTX-M-15, ant(3 )-Ia, strB
blaCTX-M-15, blaOXA-1, ant(3 )-Ia, strB
blaCTX-M-15, blaOXA-1, aac(6 )-Ib, ant(3)-Ia, strB blaCTX-M-15, blaOXA-1, aac(3)-IIa, aac(6 )-Ib, strB blaCTX-M-15, ant(3 )-Ia, strA, strB
—
blaCTX-M-15, blaTEM-1, blaCMY-2, aac(6 )-Ib, ant(3 )-Ia blaCTX-M-15
blaCTX-M-15, aph(3 )-IIa, strB
blaCTX-M-15, aac(6 )-Ib
blaCTX-M-15, ant(3 )-Ia, strB
blaCTX-M-15, blaTEM-1
blaCTX-M-15, aac(6 )-Ib, ant(3 )-Ia, strB
blaCTX-M-15, blaTEM-1, blaCMY-2, aac(3)-IIa, strB blaCTX-M-15, blaOXA-1, aac(6 )-Ib
Resistance genes identified
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Table 2. continued.
Baoding Cangzhou Cangzhou Baoding Baoding Baoding Baoding Xingtai Cangzhou Cangzhou Baoding
FrepB A/C, FrepB T, FrepB — — — — – FIB, P, K, B/O FIB —
Baoding
FIB, FIC, B/O
Baoding
Baoding
—
I1, FIB, P, B/O
Xingtai
I1
Baoding
Cangzhou
—
I1, P, B
Baoding
I1, K, B/O
Baoding
Baoding
I1, B/O
I1, FIB, P, B/O
Baoding
I1, FIB
Cangzhou
Baoding
—
FIB
Baoding
—
Baoding
Cangzhou
N, FIB, A/C, K
FIA, FIB, P, K, B/O
City
Replicon typingb
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI
5
iroN + iss
iroN + hlyF
iutA + hlyF
iroN
iroN
iroN
iutA
iutA
iutA
iutA
iutA
iss
iss
hlyF
hlyF
hlyF
A13-53
A13-54
A13-55
A13-56
A13-57
A13-58
A13-59
A13-60
A13-61
A13-62
A13-63
A13-64
A13-65
A13-66
A13-67
A13-68
A13-77
A13-76
A13-75
A13-74
A13-73
A13-72
A13-71
A13-70
A13-69
Virulence genes
Isolate
AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, SAM AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, SPE, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO,CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SPE, STR AMK, AMP, CIP, CRO, GAT, KAN, LOF, NM, NOR, OFL, SPE, STR AMK, AMP, CIP, CRO, GAT, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR
AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SPE, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM, STR AMK, AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMK, AMP, CIP, CRD, CRO, CTX, GEN, KAN, LOF, NM, NOR, OFL, SAM, SPE, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRD, CRO, CTX, GEN, LOF, NM, NOR, OFL, SAM AMK, AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SAM AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, NOR, OFL, SAM, STR AMP, CIP, CRD, GAT, GEN, KAN, LOF, NOR, OFL, SAM CIP, GAT, LOF, NOR, OFL, STR
Resistance phenotypea
blaCTX-M-15
blaCTX-M-15
—
aph(3 )-IIa, strB
blaCTX-M-15, ant(3 )-Ia, strB
ant(3 )-Ia, strB
aac(3)-IIa, ant(3 )-Ia, strB
blaCTX-M-15
blaTEM-1, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15, aac(3)-IIa, ant(3 )-Ia, strB
blaCTX-M-15
blaCTX-M-15, ant(3 )-Ia, strB
blaCTX-M-15, aac(6 )-Ib, strB
—
blaCTX-M-15, blaCMY-2, ant(3 )-Ia, qnrS
blaCTX-M-15, blaOXA-1
blaCTX-M-15, blaOXA-1, aac(3)-IIa, aac(6 )-Ib, strB blaCTX-M-15, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, aph(3 )-IIa, strB blaCTX-M-15, aac(6 )-Ib, ant(3 )-Ia, strB
aac(6 )-Ib
blaCTX-M-15, aac(3)-IIa, aac(6 )-Ib, ant(3 )-Ia, strB blaCTX-M-15, aac(6 )-Ib, strB
blaCTX-M-15, blaTEM-1, aac(3)-IIa, strB
blaCTX-M-15, aac(3)-IIa, strB
blaCTX-M-15, blaOXA-1
Resistance genes identified
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Table 2. continued. City Cangzhou Baoding Hengshui Xingtai Baoding Baoding Baoding Baoding Xingtai Baoding Baoding Baoding Hengshui Baoding Cangzhou Hengshui Hengshui Hengshui Baoding Baoding Baoding Hengshui Cangzhou Baoding Xingtai
Replicon typingb FIB FIB, P, K, B/O I1, FIB — — I1 FIB — — I1, FrepB — I1, A/C, K, B/O FIC — FIC, K — K — — — – K, B/O A/C — I1, FIC, K, B/O
6 LI ET AL.
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI
7
Hengshui Baoding
Baoding
—
FrepB P, K
— FIC, B/O
I1,FIC, A/C, K, B/O
blaCTX-M-15
ant(3 )-Ia, strB —
aac(3)-IIa, aph(3 )-IIa, strB aac(3)-IIa, ant(3 )-Ia
strB, qnrS A13-87
A13-85 A13-86
A13-83 A13-84
A13-82
AMK, amikacin; AMP, ampicillin; CIP, ciprofloxacin; CRD, cefradine; CRO, ceftriaxone; CTX, cefotaxime; GAT, gatifloxacin; GEN, gentamycin; KAN, kanamycin; LOF, levofloxacin; NM, neomycin; NOR, norfloxacin; OFL, ofloxacin; SAM, sulbactam/ampicillin 1:1; SPE, spectinomycin; STR, streptomycin b Plasmid Incompatibility Inc group. c, d Negative result
Xingtai Hengshui
— blaCTX-M-15, aac(3)-IIa, ant(3 )-Ia, strB A13-81
A13-80
A13-78 A13-79
a
Cangzhou A/C, FrepB blaCTX-M-15, aac(3)-IIa, ant(3 )-Ia, strB
Baoding
Baoding Baoding FIB K — blaCTX-M-15, blaTEM-1, aac(6 )-Ib, strB
AMK, CIP, GAT, GEN, KAN, LOF, NOR, SPE, STR AMP, CIP, CRD, CRO, GAT, GEN, KAN, LOF, NM, NOR, OFL, SPE, STR AMP, CIP, CRD, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, GAT, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, CRO, CTX, GAT, GEN, KAN, LOF, STR AMP, CIP, CRO, GAT, KAN, LOF, NM, NOR, OFL, STR AMP, CIP, GEN, KAN, LOF, NOR, OFL, SAM, STR AMP, CIP, GAT, GEN, KAN, LOF, NOR, OFL, SPE, STR AMP, CRO, GEN, KAN, LOF, STR
Replicon typingb
City Resistance genes identified Resistance phenotypea Virulence genes Isolate
We screened all 87 isolates from the affected flocks for each of the 4 genes associated with PMQR. Of the PMQR genes examined, qnrS (6.9%, 6 isolates) was the most frequently identified gene. In addition, none of the isolates carried the qnrA or qnrB gene (Tables 2 and 4).
Distribution of Replicons on Plasmids The PBRT method consists of 5 different multiplex PCRs recognizing 3 different replicon types and 3 simplex PCRs for F, K, and B/O. Eighteen specific primer pairs were designed on the basis of the multiple comparative analysis of nucleotide sequence on the EMBL Gene Databank, for HI1, HI2, I1-Ig, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FrepB , FII, K, and B/O replicons (Carattoli et al., 2005). Table 5 shows the results obtained from these strains by the PBRT method; the plasmid replicons were identified in 63 (72.4%) isolates. The inc/rep typing detected the presence of FIB (26 isolates), B/O (26 isolates), K (20 isolates), I1-Ig (17 isolates), P (10 isolates), FIC (13 isolates), IncFrepB (8 isolates), A/C (7 isolates), FIA (3 isolates), N (2 isolates), L/M (1 isolates), and T (1 isolate) in isolates of this collection; the presence of IncY, IncX, IncW, IncHI2, FII, and IncHI1 was zero. Twenty-four strains were negative for all the replicons tested. No significant differences in plasmid replicon content were observed between APEC isolates from different places (Baoding, Cangzhou, Hengshui, and Cangzhou) in Hebei Province (Table 5).
DISCUSSION Virulence Genes E. coli is a part of the normal microflora found in poultry intestines. However, certain strains, such as those designated as APEC, can spread into various internal organs and cause the systemic fatal disease colibacillosis (Oh et al., 2011). APEC strains contain many virulence factors that enable their extraintestinal pathogenicity. These include production of adhesions, toxins, protections, siderophores, iron transport systems, and invasions (Dziva and Stevens, 2008; Ahmed and Shimamoto, 2013). The 5 essential virulence genes are iutA, hlyF, iss, iroN, and ompT, which are carried on plasmids and are considered good markers for APEC strains from avian fecal isolates (Johnson et al., 2008). In the current study, the virulence genes iroN, iutA, iss, hlyF, and ompT were detected in 64.4, 60.9, 42.5, 35.6, and 5.7% of isolates, respectively. The results obtained in our study were lower than those previously reported (Johnson et al., 2008; Ahmed and Shimamoto,
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Table 2. continued.
Cangzhou
Detection and Identiffication of Plasmid-mediated Quinolone Resistance Genes
8
LI ET AL. Table 3. Antimicrobial resistance of APEC from broilers in Hebei, China. Antimicrobial class
Kanamycin Streptomycin Gentamycin Amikacin Neomycin Spectinomycin Ceftriaxone Cefotaxime Cefradine Ciprofloxacin Levofloxacin Ofloxacin Norfloxacin Gatifloxacin Ampicillin Sulbactam/ampicillin 1:1
Aminoglycosides
Cephalosporins
Fluoroquinolones
Penicllins β -lactamase inhibitors /cephalosporins
Table 4. Frequency of antimicrobial resistance genes of APEC from broilers in Hebei, China. Resistance genes
Number of positive strains (n = 87)
β -lactamases blaTEM blaCTX-M blaOXA blaCMY blaSHV Total
14 (16.1%) 61 (70.1%) 22 (25.3%) 5 (5.7%) 0 (0.0%) 66 (75.9%)
Aminoglycoside-modifying enzymes (AMEs) strA strB aph(3 )-IIa aac(3)-IIa aac(6 )-Ib ant(3 )-Ia Total
6 (6.9%) 57 (65.5%) 8 (9.2%) 25 (28.7%) 30 (34.5%) 41 (47.2%) 65 (74.7%)
Plasmid-mediated quiniolone qnrA qnrB qnrS Total
0 0 6 6
(0.0%) (0.0%) (6.9%) (6.9%)
2013). In the United States, 85.4% of the APEC strains isolated from lesions of birds clinically diagnosed with colibacillosis were positive for at least one of these 5 genes, where iroN, iutA, iss, hlyF, and ompT were detected in 85.3, 80.7, 80.5, 78.2, and 78.6% of isolates, respectively (Johnson et al., 2008). In Egypt, 80.2% of the APEC strains isolated from septicemic broilers carried 3 or more of the APEC virulence genes iroN (73 isolates, 80.2%), ompT (73 isolates, 80.2%), iss (71 isolates, 80.2%), iutA (71 isolates, 78.0%), and hlyF (65 isolates, 71.4%) (Ahmed and Shimamoto, 2013). However, Delicato et al. (2003) obtained a lower prevalence for iss from APEC isolated from poultry with colibacillosis in Brazil, with an incidence of 38.5%. Therefore, it can be concluded that the prevalence of the virulence genes may vary in different regions.
Number of resistant strains (n = 87) 83 82 81 47 46 20 77 51 50 82 82 81 78 67 85 55
(95.4%) (94.3%) (93.1%) (52.9%) (52.9%) (23.0%) (88.5%) (58.6%) (57.5%) (94.3%) (94.3%) (93.1%) (89.7%) (77.0%) (97.7%) (63.2%)
Resistance and Resistance Genes Resistance to existing antimicrobials is widespread and of great concern (Hinton et al., 1986; Ahmed and Shimamoto, 2013; Zhang et al., 2014). In this study, 100% of the tested APEC isolates showed multidrug resistance phenotypes, and 85.1% were resistant to 11 or more antimicrobial agents. Similar resistance phenotypes have been detected in APEC strains isolated from diseased chickens with colibacillosis in Korea (Kim et al., 2007), the United States (Johnson et al., 2008), the United Kingdom (Randall et al., 2010), Australia (Obeng et al., 2011), and more recently in APEC isolated from intensively farmed and free-range poultry in Egypt (Ahmed and Shimamoto, 2013). Exposing the animals’ bacterial flora to the antibiotics encourages the emergence of resistance across a wide range of antibiotics (Barbosa and Levy, 2000). The cumulative effect on the traditional antimicrobials has been multidrug resistance (Szmolka and Nagy, 2013). Perhaps the most striking finding from this study was the widespread resistance to fluoroquinolones. Our results showed that 94.3% of the tested APEC isolates were resistant to ciprofloxacin and levofloxacin, and more than 77.0% were resistant to ofloxacin, norfloxacin, and gatifloxacin. In somewhat similar findings in China, it has been reported in a recent study of clinical E. coli isolates that greater than 70% of all isolates were resistant to ciprofloxacin (Yang et al., 2004). Consequently, fluoroquinolones have become ineffective in veterinary medicine in China (Yang et al., 2004). Similar findings were also reported for E. coli isolates in Spain, wherein 90% of chicken isolates were resistant to ciprofloxacin (Saenz et al., 2001). In regards to resistance mechanisms, a notable high mutation in the quinolone resistance–determining region of gyrA has been found mainly in China (Yang et al., 2004; Xie et al., 2014), while the plasmids carrying qnr genes appear to be rare (Robicsek et al., 2006; Xie et al., 2014). In this study, the detected rates of qnrA, qnrB, and qnrS
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
Antimicrobial agent(s) tested
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI
9
Table 5. Occurrence of plasmid replicons and comparison between different groups of E. coli isolates. Replicon profilea (overall %)
FIB (29.9%) B/O (29.9%) K (23.0%) I1 (19.5%) FIC (14.9%) P (11.5%) FrepB (9.2%) A/C (8.0%) FIA (3.4%) N (2.3%) T (1.1%) L/M (1.1%)
P-valueb
No. (%) with profile Baoding
Cangzhou
Hengshui
Xingtai
15 (28.8%) 19 (36.5%) 10 (19.2%) 14 (26.9%) 7 (13.5%) 12 (23.1%) 4 (7.7%) 3 (5.8%) 2 (3.9%) 0 (0%) 0 (0%) 1 (1.9%)
7 5 5 0 2 4 3 4 1 2 1 0
1 1 3 1 2 1 0 0 0 0 0 0
3 1 2 2 2 1 0 0 0 0 0 0
(46.7%) (33.3%) (33.3%) (0%) (13.3%) (26.7%) (20.0%) (26.7%) (6.7%) (13.3%) (6.7%) (%)
(11.1%) (11.1%) (33.3%) (11.1%) (22.2%) (11.1%) (0%) (0%) (0%) (0%) (0%) (0%)
(27.3%) (9.1%) (18.2%) (18.2%) (18.2%) (9.1%) (0%) (0%) (0%) (0%) (0%) (0%)
NS NS NS NS NS NS NS NS NS NS NS NS
Replicons Y, X, W, HI2, HI1, and FII were detected negative. P-values are from resampling-based multiplicity-adjusted Fisher’s exact tests for the null hypothesis that the percentages for each replicon are equal between different isolates (Baoding, Cangzhou, Hengshui, and Xingtai). NS, not statistically significant. b
were 0.0, 0.0, and 6.9%, respectively, which also verifies this theory. Somewhat similar findings have been reported in a study of clinical E. coli isolates from humans that found positivity rates of qnrS, qnrB, and qnrA at 0, 2, and 2%, respectively (Robicsek et al., 2006). Xie et al. (2014) reported the positivity rates of qnrS, qnrB, and qnrA were 8.11, 0.90, and 0%, respectively. Differing from other types of tested antibiotics, resistance rates for the aminoglycosides show great variance. Resistance rates to kanamycin, streptomycin, and gentamycin are higher than 90%, whereas the resistance rate to spectinomycin is only 23.0%. The highly defined daily dose in recent years may be part of the reason. Kanamycin, streptomycin, and gentamycin are commonly used to clinically treat avian colibacillosis, while spectinomycin was rarely used in clinical veterinary medicine due to its high price. It is known that the inactivation of aminoglycoside drugs occurs mainly via plasmid or chromosome-encoded modifying enzymes (AMEs). The AMEs are a diverse set of proteins consisting of three families, aminoglycoside acetyltransferase (aac), aminoglycoside nucleotide transferase (ant), and aminoglycoside phosphoric transferase (aph) (Shaw et al., 1993; Ramirez and Tolmasky, 2010), which eliminate the synergistic bactericidal effect between the cell wall–active agents (Kondo and Hotta, 1999). In this study, most of the resistant isolates harbored modifying enzyme genes. Moreover, two genes, strB and ant(3 )Ia, were the most frequently observed resistance genes in these isolates. These results were similar to observations in other studies in China (Zhang et al., 2014), as well as studies in other countries (Kim et al., 2007; Green et al., 2011; Goncalves et al., 2013). Penicillin derivatives (β -lactams) are broad-spectrum antibacterial agents widely used in human and veterinary medicine (Hasman et al., 2005; Ahmed and Shimamoto, 2013). The resistance to β -lactams in gram-negative bacteria is primarily mediated by β -lactamases. Many types of β -lactamases have been
described. However TEM-, SHV-, OXA-, CMY-, and CTX-M-type β -lactamases are the most common in gram-negative bacteria (Hasman et al., 2005; Ahmed and Shimamoto, 2013). In this study, all groups of β lactamases were identified, including blaTEM-1 , blaCMY-2 , blaOXA-30 , blaCTX-M-15 and blaSHV-2 , and the detection rate was 75.9%. Similar findings have been reported in the APEC strains isolated from avian colibacillosis in Africa, wherein 94.5% of all the isolates were positive for β -lactamase-encoding genes, and blaTEM-1 , blaCMY-2 , blaOXA-30 , blaCTX-M-15 , and blaSHV-2 were also detected (Ahmed and Shimamoto, 2013). Furthermore, blaTEM was detected in Korea, Spain, Australia, and the United Kingdom (Ahmed and Shimamoto, 2013; Wagner et al., 2014). Also detected in the United Kingdom were blaOXA and blaCTX (Randall et al., 2010). In Australia, blaSHV was identified in the APEC strains isolated from poultry (Obeng et al., 2011). The coexistence of 2 or more multidrug resistance genes and virulence-associated genes was common in the APEC of this study. In a recent study by Wang et al. (2014), these findings were confirmed. Wang et al. (2014) reported that the sequence of an APEC strain harbored more than 10 antibiotic resistance genes on 2 plasmids, which concurrently carry several transferrelated elements, thereby facilitating horizontal transfers. Overall, the results of our study confirm that APEC is a cause for concern, as there are obvious limited therapeutic options available for antimicrobial therapy in poultry production.
Plasmid Replicons The epidemiological importance of tracing the plasmids conferring drug resistance prompted us to develop a PCR method based on the replicons (inc/rep PCR) of the major plasmid incompatibility groups among Enterobacteriaceae (Carattoli et al., 2005). This method
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
a
10
LI ET AL.
ACKNOWLEDGMENTS This work was financially supported by Hebei Science and Technology Planning Project (12226602).
REFERENCES Ahmed, A. M., Y. Motoi, M. Sato, A. Maruyama, H. Watanabe, Y. Fukumoto, and T. Shimamoto. 2007. Zoo animals as reservoirs of gram-negative bacteria harboring integrons and antimicrobial resistance genes. Appl. Environ. Microbiol. 73:6686–6690. Ahmed, A. M., and T. Shimamoto. 2013. Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from septicemic broilers. Int. J. Med. Microbiol. 303:475–483. Barbosa, T. M., and S. B. Levy. 2000. The impact of antibiotic use on resistance development and persistence. Drug Resist. Updat. 3:303–311. Bass, L., C. A. Liebert, M. D. Lee, A. O. Summers, D. G. White, S. G. Thayer, and J. J. Maurer. 1999. Incidence and characterization of integrons, genetic elements mediating multiple-drug resistance, in avian Escherichia coli. Antimicrob. Agents Chemother. 43:2925–2929. Carattoli, A. 2011. Plasmids in gram negatives: Molecular typing of resistance plasmids. Int. J. Med. Microbiol. 301:654–658. Carattoli, A., A. Bertini, L. Villa, V. Falbo, K. L. Hopkins, and E. J. Threlfall. 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63:219–228. Collignon, P., H. C. Wegener, P. Braam, and C. D. Butler. 2005. The routine use of antibiotics to promote animal growth does
little to benefit protein undernutrition in the developing world. Clin. Infect. Dis. 41:1007–1013. Delicato, E. R., B. G. de Brito, L. C. Gaziri, and M. C. Vidotto. 2003. Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis. Vet. Microbiol. 94:97–103. Dziva, F., and M. P. Stevens. 2008. Colibacillosis in poultry: Unravelling the molecular basis of virulence of avian pathogenic Escherichia coli in their natural hosts. Avian Pathol. 37: 355–366. Ewers, C., T. Janssen, S. Kiessling, H. C. Philipp, and L. H. Wieler. 2004. Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet. Microbiol. 104:91–101. Goncalves, A., G. Igrejas, H. Radhouani, S. Correia, R. Pacheco, T. Santos, R. Monteiro, A. Guerra, F. Petrucci-Fonseca, F. Brito, C. Torres, and P. Poeta. 2013. Antimicrobial resistance in faecal enterococci and Escherichia coli isolates recovered from Iberian wolf. Lett. Appl. Microbiol. 56:268–274. Green, K. D., W. Chen, and S. Garneau-Tsodikova. 2011. Effects of altering aminoglycoside structures on bacterial resistance enzyme activities. Antimicrob. Agents Chemother. 55:3207–3213. Hasman, H., D. Mevius, K. Veldman, I. Olesen, and F. M. Aarestrup. 2005. Beta-lactamases among extended-spectrum beta-lactamase (ESBL)–resistant Salmonella from poultry, poultry products and human patients in the Netherlands. J. Antimicrob. Chemother. 56:115–121. Hinton, M., A. Kaukas, S. K. Lim, and A. H. Linton. 1986. Preliminary observations on the influence of antibiotics on the ecology of Escherichia coli and the enterococci in the faecal flora of healthy young chickens. J. Antimicrob. Chemother. 18:165–173. Hopkins, K. L., E. Liebana, L. Villa, M. Batchelor, E. J. Threlfall, and A. Carattoli. 2006. Replicon typing of plasmids carrying CTX-M or CMY beta-lactamases circulating among Salmonella and Escherichia coli isolates. Antimicrob. Agents Chemother. 50:3203–3206. Johnson, T. J., Y. Wannemuehler, C. Doetkott, S. J. Johnson, S. C. Rosenberger, and L. K. Nolan. 2008. Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J. Clin. Microbiol. 46: 3987–3996. Johnson, T. J., Y. M. Wannemuehler, S. J. Johnson, C. M. Logue, D. G. White, C. Doetkott, and L. K. Nolan. 2007. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates. Appl. Environ. Microbiol. 73:1976–1983. Kim, T. E., Y. W. Jeong, S. H. Cho, S. J. Kim, and H. J. Kwon. 2007. Chronological study of antibiotic resistances and their relevant genes in Korean avian pathogenic Escherichia coli isolates. J. Clin. Microbiol. 45:3309–3315. Kondo, S., and K. Hotta. 1999. Semisynthetic aminoglycoside antibiotics: Development and enzymatic modifications. J. Infect. Chemother. 5:1–9. Obeng, A. S., H. Rickard, O. Ndi, M. Sexton, and M. Barton. 2011. Antibiotic resistance, phylogenetic grouping and virulence potential of Escherichia coli isolated from the faeces of intensively farmed and free range poultry. Vet. Microbiol. 154:305–315. Oh, J. Y., M. S. Kang, J. M. Kim, B. K. An, E. A. Song, J. Y. Kim, E. G. Shin, M. J. Kim, J. H. Kwon, and Y. K. Kwon. 2011. Characterization of Escherichia coli isolates from laying hens with colibacillosis on 2 commercial egg-producing farms in Korea. Poult. Sci. 90:1948–1954. Ramirez, M. S., and M. E. Tolmasky. 2010. Aminoglycoside modifying enzymes. Drug Resist. Updat. 13:151–171. Randall, L. P., C. Clouting, R. A. Horton, N. G. Coldham, G. Wu, F. A. Clifton-Hadley, R. H. Davies, and C. J. Teale. 2010. Prevalence of Escherichia coli carrying extended-spectrum beta-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009. J. Antimicrob. Chemother. 66:86–95. Robicsek, A., J. Strahilevitz, D. F. Sahm, G. A. Jacoby, and D. C. Hooper. 2006. qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. Antimicrob. Agents Chemother. 50:2872–2874. Ruiz del Castillo, B., L. Vinue, E. J. Roman, B. Guerra, A. Carattoli, C. Torres, and L. Martinez-Martinez. 2013. Molecular
Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
could be used to monitor the circulation of plasmids within strains from different environments or to follow the horizontal transmissions of antimicrobial resistance genes among the Enterobacteriaceae (Carattoli et al., 2005). Research studies have shown the diffusion of the prevalent plasmids is in association with the specific resistance gene (Hopkins et al., 2006; Ruiz del Castillo et al., 2013; Wagner et al., 2014). Similarly, a recent study has identified IncFIB, IncFrep, and IncI1 as common replicon types (Johnson et al., 2007). IncFII and IncI1 plasmids are two of a number of plasmid types that are particularly successful in their ability to spread multidrug resistance (Carattoli, 2011). In our study, we found that both IncFIB and IncB/O (29.9%), followed by IncK (23.0%) and IncI1 (17, 19.5%), were the most prevalent plasmid replicon types in E. coli. This may be the reason for the high multidrug-resistant APEC in the APEC researched. In the study, the plasmid profiles of APEC from different places in Hebei showed no significant differences. The main reason is that the four regions are geographically adjacent to each other and have significant interregion contact. In summary, our current study characterized the genetic basis of virulence and antimicrobial resistance in the APEC strains isolated from septicemic broilers in China. The various classes of the resistance genes were identified, and inc/rep PCR was reported in APEC strains. It is important to monitor the occurrence of resistance genes in APEC. It should be emphasized that there is a need to ban the nontherapeutic use of antibiotics, discourage their misuse, and maintain constant vigilance by providing appropriate scientific and technological support for the poultry industry in this region.
MOLECULAR CHARACTERIZATION OF AVIAN ESCHERICHIA COLI characterization of multiresistant Escherichia coli producing or not extended-spectrum beta-lactamases. BMC. Microbiol. 13:84. Saenz, Y., M. Zarazaga, L. Brinas, M. Lantero, F. Ruiz-Larrea, and C. Torres 2001. Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain. Int. J. Antimicrob. Agents 18:353–358. Shaw, K. J., P. N. Rather, R. S. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138–163. Szmolka, A., and B. Nagy. 2013. Multidrug resistant commensal Escherichia coli in animals and its impact for public health. Front. Microbiol. 4:258. Wagner, S., D. L. Gally, and S. A. Argyle. 2014. Multidrug-resistant Escherichia coli from canine urinary tract infections tend to have commensal phylotypes, lower prevalence of virulence determinants and ampC-replicons. Vet. Microbiol. 169:171–178.
11
Wang, X., H. Hao, Z. Xu, H. Zheng, C. Liu, L. Wei, R. Zhang, D. Bi, H. Chen, and C. Tan. 2014. Plasmid-mediated multidrug resistance and virulence in an avian pathogenic Escherichia coli strain isolated in China. J. Global Antimicrobiol. Resist. 1:57–58. Xie, R., S. Huo, Y. Li, L. Chen, F. Zhang, and X. Wu. 2014. Molecular epidemiological survey on quinolone resistance genotype and phenotype of Escherichia coli in septicemic broilers in Hebei, China. Poult. Sci. 93:335–339. Yang, H., S. Chen, D. G. White, S. Zhao, P. McDermott, R. Walker, and J. Meng 2004. Characterization of multiple-antimicrobialresistant Escherichia coli isolates from diseased chickens and swine in China. J. Clin. Microbiol. 42:3483–3489. Zhang, F. Y., S. Y. Huo, Y. R. Li, R. Xie, X. J. Wu, L. G. Chen, and Y. H. Gao. 2014. A survey of the frequency of aminoglycoside antibiotic-resistant genotypes and phenotypes in Escherichia coli in broilers with septicaemia in Hebei, China. Br. Poult. Sci. 55:305–310. Downloaded from http://ps.oxfordjournals.org/ at University of California, San Francisco on March 27, 2015
