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Prevalence and characterisation of CTX-M β-lactamases amongst Escherichia coli isolates from healthy food animals in China
International Journal of Antimicrobial Agents 39 (2012) 305–310
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Prevalence and characterisation of CTX-M -lactamases amongst Escherichia coli isolates from healthy food animals in China Hongqing Zheng a,1 , Zhenling Zeng a,1 , Sheng Chen b , Yahong Liu a , Qiongfen Yao a , Yuting Deng a , Xiaojie Chen a , Luchao Lv a , Chao Zhuo c , Zhangliu Chen a , Jian-Hua Liu a,∗ a
College of Veterinary Medicine, National Reference Laboratory of Veterinary Drug Residues, South China Agricultural University, Guangzhou 510642, China Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region c State Key Laboratory of Respiratory Diseases (Guangzhou Medical College), Clinical Microbiology, Guangzhou 510120, China b
a r t i c l e
i n f o
Article history: Received 6 June 2011 Accepted 6 December 2011 Keywords: Extended-spectrum -lactamases Food animals Plasmid CTX-M Escherichia coli
a b s t r a c t The impact of extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae of food animal origins on human health has caught considerable attention worldwide. Intestinal Escherichia coli obtained from healthy food animals (pigs, cattle and poultry) in China were tested for the presence of ESBL genes. CTX-M-producing isolates were further characterised by pulsed-field gel electrophoresis (PFGE), phylogenetic grouping, genetic environment analysis, conjugation and plasmid replicon typing. A total of 127 of the 896 E. coli isolates showed reduced susceptibility to cefotaxime (minimal inhibitory concentration ≥ 2 g/mL). blaCTX-M genes were detected in 111 of the 127 isolates. The most common CTX-M types were CTX-M-14 (n = 40), CTX-M-55 (n = 29) and CTX-M-65 (n = 22), followed by CTX-M-27, -15, -98, -24, -3, -102 and -104. CMY-2 was detected in two isolates. High clonal diversity was found amongst CTXM-producing isolates. Insertion sequence ISEcp1 was observed 42 bp upstream of the start codon of all CTX-M-9 group genes, whereas the spacer region between the right inverted repeats and CTX-M-1 group genes varied from 45 bp to 127 bp. Most blaCTX-M genes were transferable by conjugation. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 of the 70 transconjugants carrying blaCTX-M , respectively. This study demonstrates that commensal E. coli from healthy food animals can be important reservoirs of blaCTX-M genes and may contribute to the dissemination and transfer of these -lactamase genes throughout China. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Extended-spectrum cephalosporins are commonly used as the most appropriate medications to treat infections caused by multidrug-resistant Gram-negative bacteria and are considered as critically important antimicrobial agents by the World Health Organization (WHO) [1]. However, reports of extended-spectrum -lactamases (ESBLs) that can hydrolyse the third- and fourth-generation cephalosporins have become increasingly frequent, and the most common ESBLs that have emerged recently are CTX-M types [2,3]. There are currently more than 110 different CTX-M-type ESBLs recognised (http://www.lahey.org/studies/other.asp#table1). blaCTX-M genes are frequently located on highly mobile genetic elements such as plasmids and transposons, which facilitate their transfer amongst
∗ Corresponding author. Tel.: +86 20 8528 3824; fax: +86 20 8528 3824. E-mail address: [email protected] (J.-H. Liu). 1 These two authors contributed equally to this paper.
different hosts around the world [3]. Of particular concern is the community dissemination of blaCTX-M -carrying Escherichia coli [2]. ESBL-producing E. coli isolates have been increasingly detected in food animals in different countries since 2002 [4]. Food animals colonised with ESBL-producing E. coli have been considered as potential sources of resistant E. coli causing infection in the community and have caught considerable attention worldwide [4]. ESBL-producers were seldom detected in animal isolates in China before 2005 [5–7]. However, the occurrence of ESBL-producing E. coli has dramatically increased in China recently, with CTX-M -lactamases being the most common type [7–9]. There have been few comprehensive studies of ESBLs in China, particularly of the mechanism conferring the rapid distribution of blaCTX-M in food animals. Commensal E. coli isolates in food animals are believed to be the reservoir of ESBL genes [4]. In this study, we investigated the distribution of ESBLs amongst commensal E. coli isolates from common healthy food animals in China, including pigs, cattle, chickens, ducks, geese, partridges and pigeons. Plasmids that carried CTXM were also studied to elucidate the mechanism of transfer and dissemination of -lactamases.
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2. Materials and methods 2.1. Source of Escherichia coli isolates Cloacal samples from poultry (chickens, ducks, geese, partridges and pigeons) and rectal swabs from pigs and cattle were collected from 47 food animal farms located in different geographic areas of China from April 2007 to August 2009 (Table 1). Animals on each farm were randomly selected for sampling based on their age and stage of production. The number of animal samples collected from each farm was based on the size of the farm (ca. 0.2–1% of the total number of animals on each farm). All samples were seeded on MacConkey agar plates and were incubated at 37 ◦ C for 24 h. One suspected colony with typical E. coli morphology and size was selected from all agar plates of each sample and was identified using classical biochemical methods. Species identification was confirmed using an API 20E system (bioMérieux, Marcy l’Étoile, France). 2.2. Antimicrobial susceptibility testing Minimal inhibitory concentrations (MICs) of ampicillin, ceftriaxone, cefotaxime, ceftazidime, cefoxitin, gentamicin, amikacin, chloramphenicol, tetracycline, ciprofloxacin and trimethoprim/sulfamethoxazole (SXT) were determined by the agar dilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. The disk diffusion method was used to determine susceptibility to imipenem and meropenem. Escherichia coli ATCC 25922 was used as the control strain. Isolates were classified as either susceptible or resistant according to the interpretative standards recommended by the CLSI [10,11]. ESBL-producing isolates were screened by double-disk synergy test using both cefotaxime and ceftazidime in the presence or absence of clavulanic acid as recommended by the CLSI. 2.3. Polymerase chain reaction (PCR) amplification and sequencing Genes encoding TEM, CTX-M, SHV, CMY-2 and DHA-1 enzymes as well as the genetic environment of blaCTX-M genes were analysed by PCR amplification as described previously [6,12]. Purified PCR products were directly sequenced from both ends or were cloned in pMD18-T vectors and then sequenced. The DNA sequences and deduced amino acid sequences were compared with genes in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) or the -lactamase classification system (http://www.lahey.org/studies/) to confirm the subtypes of -lactamase genes. 2.4. Strain typing Pulsed-field gel electrophoresis (PFGE) analysis of Xbal-digested genomic DNA was performed to determine the genetic relatedness of CTX-M-producing E. coli isolates using a CHEF-MAPPER System (Bio-Rad Laboratories, Hercules, CA). PFGE patterns were interpreted according to well-established criteria [13]. Isolates that had PFGE patterns with no more than six different bands were considered clonally related. Phylogenetic groups of E. coli isolates carrying blaCTX-M were assigned by a multiplex PCR-based method developed as described previously [14]. Isolates belonging to phylogenetic group B2 were screened with a previously established PCR-based method to identify the O25b-ST131 clone [15]. 2.5. Conjugation experiments and plasmid analysis Transferability of blaCTX-M genes was determined by conjugation experiments using streptomycin-resistant E. coli C600 as the recipient strain as previously described [12]. Ninety-three isolates with
different PFGE patterns were selected for conjugation. Transconjugants were selected on MacConkey agar plates supplemented with cefotaxime (2 g/mL) and streptomycin (2000 g/mL). The presence of blaCTX-M was confirmed by PCR. Incompatibility (Inc) groups were assigned by PCR-based replicon typing of transconjugants [16]. To better clarify IncF plasmids, replicon sequence typing of IncF plasmids was performed according to a previously described procedure [17] and alleles were assigned by submitting the amplicon sequence to the plasmid multilocus sequence typing (MLST) database (http://www.pubmlst.org/plasmid). Plasmids carrying blaCTX-M were extracted from transconjugants using a rapid alkaline lysis method. Plasmids of transconjugants were digested with the endonuclease EcoRI (TaKaRa Biotechnology, Dalian, China) to analyse the restriction fragment length polymorphism (RFLP) profile and to estimate the size of plasmids. 2.6. Nucleotide sequence accession numbers The GenBank accession nos. of the sequence studied herein were HM748991, HM755448, HQ398215, HQ833652 and JF713460. 3. Results 3.1. Antimicrobial susceptibility A total of 896 commensal E. coli collected from 326 pigs, 316 chickens, 88 cattle, 58 ducks, 22 geese, 61 pigeons and 25 partridges were included in this study. Amongst them, 127 isolates (14.2%) (41 from pigs, 44 from chickens, 5 from cattle and 37 from other poultry species) showed reduced susceptibility to cefotaxime (MIC ≥ 2 g/mL) (Table 1). Of the 127 isolates, all were multidrug-resistant and showed resistance to more than two non--lactam antimicrobial agents, including ciprofloxacin (70.9%), gentamicin (71.7%), amikacin (21.3%), chloramphenicol (77.2%) and SXT (94.5%). Some of them were also resistant to other cephalosporins, including ceftazidime (40.9%) (MIC ≥ 16 g/mL), ceftriaxone (97.6%) (MIC ≥ 4 g/mL) and cefoxitin (15.0%) (MIC ≥ 32 g/mL). All of the isolates were susceptible to imipenem and meropenem. ESBL production was detected by the screening method in 117 of the 127 isolates, representing 13.1% of the total 896 commensal E. coli isolates. 3.2. ˇ-Lactamase gene detection Acquired -lactamase genes were detected in most E. coli isolates with a cefotaxime MIC ≥ 2 g/mL. CTX-M-type genes were found to be dominant in these isolates and 111 isolates carried one or two CTX-M genes, representing 12.4% of the total 896 foodanimal-associated E. coli isolates. Fifty-nine of the 127 E. coli carried blaTEM-1 . Two isolates from pigs carried the blaCMY-2 gene. blaSHV and blaDHA-1 genes were not found in any of these isolates. The most predominant CTX-M-encoding gene was blaCTX-M-14 (n = 40), followed by blaCTX-M-55 (n = 29), blaCTX-M-65 (n = 22), blaCTX-M-27 (n = 14), blaCTX-M-15 (n = 5), blaCTX-M-24 (n = 3) and blaCTX-M-3 (n = 2) (Table 2). Three novel variants of CTX-M enzymes (CTX-M-98, CTX-M-102 and CTX-M-104) were identified in three (one from pig and two from ducks), one (from chicken) and one (from chicken) E. coli isolates, respectively. The enzymes CTX-M98 and CTX-M-102 differed from CTX-M-14 by two amino acids (A80V and D242G in the case of CTX-M-98 and A208G and D242G in the case of CTX-M-102), whereas CTX-M-104 differed from CTXM-14 by only one amino acid (N275S). These new blaCTX-M-14 -like genes have been assigned accession nos. in GenBank and Lahey Clinic as follows: HM755448 (CTX-M-98), HQ398215 (CTX-M-102) and HQ833652 (CTX-M-104). In addition, a C348T silent variant of
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Table 1 Origins of Escherichia coli isolates. Farm
Province
Animal species
No. of samples from each farm
No. of E. coli isolates from each farm
No. of E. coli isolates with cefotaxime MIC ≥ 2 g/mL
F1/F2/F3/F4 F5/F6/F7 F8/F9/F10 F11/F12 F13 F14 F15/F16 F17/F18 F19 F20/F21 F22/F23/F24 F25/F26/F27 F28/F29/F30/F31 F32/F33 F34 F35/F36/F37 F38 F39 F40/F41 F42/F43 F44/F45/F46/F47
Guangdong Guangdong Guangdong Guangdong Guangdong Guangdong Guangxi Hainan Sichuan Gansu Gansu Heilongjiang Heilongjiang Hebei Hebei Shandong Henan Henan Shanxi Hunan Jiangxi
Chicken Pig Duck Goose Partridge Pigeon Chicken Chicken Pig Chicken Pig Pig Cow Pig Pigeon Chicken Chicken Pigeon Pigeon Chicken Pig
30/30/30/30 20/20/20 26/26/24 16/16 30 20 30/30 20/22 15 16/18 16/14/16 26/26/26 28/26/30/30 30/26 14 26/24/26 10 28 20/14 22/26 35/35/35/35
27/27/25/26 17/16/14 19/21/18 12/10 25 13 23/27 13/16 12 10/12 13/12/12 20/17/18 20/21/23/24 25/23 10 23/20/22 7 19 11/8 17/21 33/30/31/33
8 5 14 1 12 4 4 3 2 2 1 8 5 2 1 24 3 5 0 0 23
MIC, minimal inhibitory concentration.
blaCTX-M-55 was found in one isolate from chicken and designated here as blaCTX-M-55b . The distribution of CTX-M gene types in different animal species was varied. blaCTX-M-14 and blaCTX-M-55 were found in all animal species tested, blaCTX-M-27 was mainly found in duck isolates and blaCTX-M-65 was found in all animal isolates except for those from calf. blaCTX-M-14 was widely distributed in almost all provinces. 3.3. Phylogenetic groups and clonal analysis Phylogenetic group analysis showed that group A (58; 52.3%) was dominant amongst the isolates that produced CTX-M enzymes, followed by group B1 (26; 23.4%) and group D (21; 18.9%). Only six isolates (four with blaCTX-M-14 , one with blaCTX-M-27 and one with blaCTX-M-3 ) belonged to extraintestinal virulent group B2, and none of them belonged to clone O25b-ST131. PFGE was successfully performed on 99 CTX-M-producers; no PFGE fragment pattern was obtained from the other 12 isolates. Significant clonal diversity was shown in isolates that carried CTX-M -lactamases (Table 2). Within the same farm, clonality was sporadically observed. There were no common clones between the
farms. However, for the three CTX-M-98-producing E. coli isolates, two strains from different farms (one from a duck farm located in South China, the other from a pig farm located in Central China) showed indistinguishable PFGE patterns. 3.4. Genetic environment of blaCTX-M Insertion sequence ISEcp1-like elements were present in the upstream region of blaCTX-M genes in 108 (90.0%) of the 120 blaCTX-M genes. Insertion of ISEcp1 was observed at 42 bp upstream of the start codon of all CTX-M-9 group genes. However, the spacer region between the right inverted repeats and CTX-M-1 group genes was varied: 127 bp upstream of blaCTX-M-3 , 48 bp or 127 bp upstream of blaCTX-M-15 , and 45, 48 or 127 bp upstream of blaCTX-M-55 (Table 3). To our knowledge, this is the first time a 127-bp intergenic region upstream of the blaCTX-M-55 gene has been identified. Interestingly, even in isolates from the same farm, different intergenic regions were found between CTX-M-55 genes and ISEcp1, suggesting that blaCTX-M-55 genes probably emerged from Kluyvera spp. through multiple mobilisation events or from mutation of other CTX-M1-group genes such as blaCTX-M-3 and blaCTX-M-15 . An ISCR1 element
Table 2 Distribution of CTX-M subgroups and alleles amongst Escherichia coli isolates from different animal sources, and clonal relationship of CTX-M-producing E. coli isolates. CTX-M type
CTX-M-9 group CTX-M-14 CTX-M-24 CTX-M-27 CTX-M-65 CTX-M-98 CTX-M-102 CTX-M-104 CTX-M-1 group CTX-M-3 CTX-M-15 CTX-M-55 Any CTX-M
No. of PFGE subtypesa
No. of isolates (%) Pig (n = 326)
Calf (n = 88)
Chicken (n = 316)
Duck and goose (n = 80)
Partridge and pigeon (n = 86)
Total (n = 896)
27 12
4 4
23 11 3 1 6
14 3
16 10
8 1 2
1 5
4
4
84 40 3 14 22 3 1 1 36 2 5 29 111 (12.4)
4 10 1
8 3 5 34 (10.4)
2
2 5 (5.7)
1 1 18 2 1 15 39 (12.3)
1 3 15 (18.8)
PFGE, pulsed-field gel electrophoresis; N/T, non-typeable. a PFGE patterns were interpreted according to the criteria of Tenover et al. [13]. b Number of non-typeable isolates is indicated in parentheses.
4 18 (20.9)
67, N/T (8)b 30, N/T (4) 3 12, N/T (1) 18, N/T (3) 2 1 1 27, N/T (4) 2 4, N/T (1) 21, N/T (3) 85, N/T (12)
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Table 3 Analysis of genetic structure upstream and downstream of the blaCTX-M gene. Type of blaCTX-M
No. of isolates
No. of isolates ISCR1
ISEcp1
Size of intergenic spacer region between ISEcp1 and blaCTX-M (bp)
No. of isolates orf477
IS903
CTX-M-3
2
0
2
127
2
N/A
CTX-M-15
5
0 0
3 2
127 48
3 2
N/A N/A
CTX-M-55a
28
0
12 10 6
45 48 127
10 10 6
N/A
CTX-M-55b CTX-M-14a CTX-M-14b CTX-M-24 CTX-M-27 CTX-M-65 CTX-M-98 CTX-M-102 CTX-M-104
1 39 1 3 14 22 3 1 1
0 0 1 0 0 0 0 0 0
1 30 0 3 13 21 3 1 1
48 42 N/A 42 42 42 42 42 42
1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A 25 0 3 12 19 2 1 1
N/A, not applicable.
was found upstream of the blaCTX-M-14b gene (n = 1). ISEcp1-like and ISCR1 were not found immediately upstream of any other blaCTX-M genes (n = 12). orf477 and IS903 were detected downstream of 34 CTX-M-1 and 63 CTX-M-9 group genes, respectively. The sequence of blaCTX-M-55 with the 127-bp intergenic region and the genetic environment of blaCTX-M-14b were deposited in GenBank (accession nos. HM748991 and JF713460).
3.5. Transferability of blaCTX-M genes and plasmid replicon typing blaCTX-M genes from 70 of 93 isolates were transferred to the recipient by conjugation at frequencies of 10−7 to 10−3 per donor cell. In addition to cefotaxime resistance, the plasmids in some transconjugants encoded co-resistance to non--lactamase antibiotics such as gentamicin (38.6%), amikacin (27.1%), tetracycline (12.9%), chloramphenicol (12.9%), nalidixic acid (7.1%) and SXT (47.1%). Plasmid replicon typing of transconjugants revealed significant plasmid diversity. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 transconjugants, respectively (Table 4). Two plasmid replicons (IncFII in combination with IncFIB or IncI1) were simultaneously present in 11 transconjugants. The replicon types could not be determined in 19 transconjugants. Within FII replicons, F2, F18, F24, F31, F33 and F35 alleles were identified in 12, 1, 2, 3, 5 and 1 transconjugants, respectively. Within FIB replicons, B1 and B6 were identified in 6 and 2 transconjugants, respectively. In addition, the new alleles F38 and F41 were also identified in two transconjugants. Twenty-nine transconjugants were selected for plasmid restriction enzyme digestion analysis according to CTX-M type (13 CTX-M-14, 5 CTX-M-55, 4 CTX-M-65, 4 CTX-M-27, 2 CTX-M-15 and 1 CTX-M-98) and plasmid replicon type (11 IncI, 16 IncFII and 2 IncN). The sizes of the plasmids varied from 40 kb to 100 kb. Most of the plasmids showed diverse digestion patterns. However, in most cases plasmids of the same replicon type carrying CTX-M genes that belonged to the same group shared some bands of the same size (data not shown). Two IncN plasmids (one carrying blaCTX-M-27 and the other carrying blaCTX-M-65 ) had the same plasmid restriction patterns. Two F31:A-:B1 plasmids carrying blaCTX-M-15 showed very similar patterns. Eight F2:A-:B- plasmids carrying diverse CTXM-9 group genes had the same or very similar plasmid restriction patterns. The same was true for four IncI1 plasmids carrying the blaCTX-M-14 gene.
4. Discussion In this study, 896 E. coli isolates originating from food animals were collected from 12 different provinces covering all the seven geographic regions in mainland China during 2007–2009; 14.2% of them were ESBL-producers and 12.4% carried CTX-M -lactamases. Of the 270 E. coli isolates from Guangdong province, 16.3% carried CTX-M -lactamases (data not shown), which was much higher than E. coli isolates collected between 2003 and 2005 in our previous work (2.4%) [6]. The increasing incidence of ESBLs amongst E. coli isolates from food animals may be due to the increasing use of third-generation cephalosporins in food animals in recent years, since fluoroquinolones have become inactive against most E. coli strains in China [18]. Leverstein-van Hall et al. [19] recently reported that Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains, suggesting transmission of ESBL genes from poultry to humans through the food chain. Very recently, a surprisingly high number (50.5%) of faecal samples carrying CTX-M-producing E. coli has been reported in the healthy Chinese population [20]. Although we cannot determine whether the CTX-M-type -lactamase-producing E. coli found in the healthy Chinese have acquired these genes from food animals via the food chain, the high prevalence of CTX-M-type ESBLs amongst commensal E. coli isolates in food animals strongly suggests the significant role of commensal E. coli as ESBL gene reservoirs, which imposes additional risk to patients and even the general community. Therefore, tracking and monitoring the spread of Enterobacteriaceae that produce CTX-M-type ESBLs in foods conducted on the basis of community settings in China are urgently needed for the benefit of public health. Ten types of CTX-M ESBLs were detected in the present study, indicating a high diversity of CTX-M-encoding genes in E. coli isolates from food animals in China. The most common CTX-M enzyme (CTX-M-14) was detected in all animal species from different regions and was also the most frequent type amongst humans and pets in China [12,20–23]. In addition, CTX-M-65, CTX-M-24, CTX-M-27 and CTX-M-15 were also detected in some isolates, which was similar to what has been reported from human isolates in China [20–23]. The epidemiological distribution of CTX-M types appears to be comparable between animal and human bacteria in China. However, the distribution of ESBL types found in the present study is different from food animals of other countries such as France, The Netherlands, Portugal and England where CTX-M-1 or TEM-52 is the dominant type [19,24–26]. A novel variant of the
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Table 4 Replicon sequence typing of plasmids in 70 transconjugants carrying blaCTX-M . blaCTX-M gene type in transconjugant CTX-M-3 (n = 1) CTX-M-15 (n = 2) CTX-M-55 (n = 16) CTX-M-14 (n = 23) CTX-M-24 (n = 1) CTX-M-27 (n = 10) CTX-M-65 (n = 14) CTX-M-98 (n = 2) CTX-M-102 (n = 1) Total (n = 70)
Replicon type (n) N
FII
FII, FIB
FII, I1
I1
A/C
Unknown 1
2 1
2 7
3
2 2
5 2 1
2
5
17
7
CTX-M enzyme, CTX-M-98, was detected in E. coli isolates both from ducks and pigs in this study and was also recently reported to be present in isolates from calf [27] and geese (GenBank accession no. HQ637575), indicating that blaCTX-M-98 has been widely dispersed in different animal species in China. CTX-M-55 was the second common CTX-M enzyme in the present study, as also shown in other studies [7,9]. It was also found in E. coli isolates from pets in mainland China and from animals in Hong Kong [12,27]. However, it was only sporadically detected in other countries except for Thailand and was not common in human isolates in China [20,28,29]. As >80% of the food animals consumed in Hong Kong are imported from mainland China, the prevalence and distribution of CTX-M types in E. coli of food animal origin in Hong Kong may reflect those in mainland China [27]. Thus, CTX-M55 has also widely disseminated in animals in China. CTX-M-15, which is a most common enzyme in many countries [2,3], was only detected in five isolates in this study, similar to the prevalence in isolates from pets [12]. However, it was more common in human isolates in mainland China [20,23]. The different distribution of CTX-M-55 and CTX-M-15 between human and animal isolates may be due to diverse antimicrobial selective pressure. Dissemination of specific clonal groups or clones has been critical for the dramatic increase in the prevalence of blaCTX-M genes, especially blaCTX-M-15 , in many countries [3,30]. However, in this study, high clonal diversity of bacterial hosts was observed, indicating the importance of mobile elements in the dissemination of blaCTX-M genes in China. Plasmids that carried blaCTX-M were also characterised by plasmid replicon typing and fingerprinting analysis. Similar to previous studies [23,31], most CTX-M genes were found to be linked to IncFII or IncI1 plasmids. The most common IncFII type plasmid was F2:A-:B-, which had also been found to be associated with blaCTX-M genes in Enterobacteriaceae isolates from other countries [17,32]. In particular, we observed that plasmids carrying blaCTX-M-14 from epidemiologically unrelated strains (from different farms and geographic regions) had surprisingly similar plasmid restriction patterns (data not shown), indicating the presence of epidemic plasmids in China. blaCTX-M-14 has widely disseminated in China both in human and animal isolates for nearly a decade. However, only very limited information is available on the types of plasmids carrying this gene [22,23]. More studies are needed to determine the linkage of plasmids in humans and animals and to address the factors that contribute to the successful dissemination of some CTX-M types, such as CTX-M-14 and CTXM-55 in China. To our knowledge, this is the first report on PFGE, plasmid analysis and the genetic environment of CTX-M-producing E. coli isolates collected from a variety of animal farms located in different geographic regions of mainland China. The limitation of this study was that more than one-half of the 896 isolates were collected from three provinces (Guangdong, Jiangxi and Heilongjiang), whereas fewer isolates were collected from the other nine provinces. Thus,
2 2
4
3 7 1 2 3 1 17
8 4
1
1 4
1
1 19
the data might not completely represent the actual situation of the occurrence and characterisation of ESBL-producers in food animals. None the less, this study reveals that CTX-M enzymes have been widely distributed in E. coli isolates from different food animal species in mainland China. In conclusion, cephalosporin resistance amongst a high clonal diversity of E. coli from food animals was mainly mediated by blaCTX-M genes harboured by diverse or similar IncFII or IncI1 plasmids. The increasing prevalence of commensal ESBL-producing E. coli in healthy food animals imposes a substantial threat to public health, as the ESBL genes will eventually transfer to human and animal pathogens. Based on these observations, the third- and fourth-generation cephalosporins should be used more prudently in food animals. Acknowledgements The authors are sincerely grateful to Zhiyong Zong for valuable comments on the manuscript. They also thank Alessandra Carattoli and Laura Villa for assigning IncF plasmids, and Dehe Ye, Tao Lei, Wei Tian and Jianxia Hou for sampling and antimicrobial susceptibility testing. Funding: This work was supported by grants from the National Natural Science Foundation of China (Nos. U1031004 and 30972218). Competing interests: None declared. Ethical approval: Not required. References [1] Collignon P, Powers JH, Chiller TM, Aidara-Kane A, Aarestrup FM. World Health Organization ranking of antimicrobials according to their importance in human medicine: a critical step for developing risk management strategies for the use of antimicrobials in food production animals. Clin Infect Dis 2009;49: 132–41. [2] Pitout JD, Laupland KB, Extended-spectrum. -lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008;8:159–66. [3] Rossolini GM, D’Andrea MM, Mugnaioli C. The spread of CTX-M-type extendedspectrum -lactamases. Clin Microbiol Infect 2008;14(Suppl. 1):33–41 [Erratum in: Clin Microbiol Infect 2008;14(Suppl. 5):21–4]. [4] Smet A, Martel A, Persoons D, Dewulf J, Heyndrickx M, Herman L, et al. Broad-spectrum -lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health. FEMS Microbiol Rev 2010;34:295–316. [5] Yang H, Chen S, White DG, Zhao S, McDermott P, Walker R, et al. Characterization of multiple-antimicrobial-resistant Escherichia coli isolates from diseased chickens and swine in China. J Clin Microbiol 2004;42:3483–9. [6] Liu JH, Wei SY, Ma JY, Zeng ZL, Lü DH, Yang GX, et al. Detection and characterization of CTX-M and CMY-2 -lactamases among Escherichia coli isolates from farm animals in Guangdong Province of China. Int J Antimicrob Agents 2007;29:576–81. [7] Li L, Jiang ZG, Xia LN, Shen JZ, Dai L, Wang Y, et al. Characterization of antimicrobial resistance and molecular determinants of -lactamase in Escherichia coli isolated from chickens in China during 1970–2007. Vet Microbiol 2010;144:505–10.
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[8] Yuan L, Liu JH, Hu GZ, Pan YS, Liu ZM, Mo J, et al. Molecular characterization of extended-spectrum -lactamase-producing Escherichia coli isolates from chickens in Henan Province, China. J Med Microbiol 2009;58:1449–53. [9] Li J, Ma Y, Hu C, Jin S, Zhang Q, Ding H, et al. Dissemination of cefotaximeM-producing Escherichia coli isolates in poultry farms, but not swine farms, in China. Foodborne Pathog Dis 2010;7:1387–92. [10] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests for bacteria isolated from animals; approved standard. 3rd ed. Document M31-A3. Wayne, PA: CLSI; 2008. [11] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twentieth informational supplement. Document M100-S20. Wayne, PA: CLSI; 2010. [12] Sun Y, Zeng Z, Chen S, Ma J, He L, Liu Y, et al. High prevalence of blaCTX-M extended-spectrum -lactamase genes in Escherichia coli isolates from pets and emergence of CTX-M-64 in China. Clin Microbiol Infect 2010;16:1475–81. [13] Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsedfield gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–9. [14] Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000;66:4555–8. [15] Clermont O, Dhanji H, Upton M, Gibreel T, Fox A, Boyd D, et al. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15producing strains. J Antimicrob Chemother 2009;64:274–7. [16] Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005;63:219–28. [17] Villa L, García-Fernández A, Fortini D, Carattoli A. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J Antimicrob Chemother 2010;65:2518–29. [18] Lei T, Tian W, He L, Huang XH, Sun YX, Deng YT, et al. Antimicrobial resistance in Escherichia coli isolates from food animals, animal food products and companion animals in China. Vet Microbiol 2010;146:85–9. [19] Leverstein-van Hall MA, Dierikx CM, Cohen Stuart J, Voets GM, van den Munckhof MP, van Essen-Zandbergen A, et al. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin Microbiol Infect 2011;17:873–80. [20] Li B, Sun JY, Liu QZ, Han LZ, Huang XH, Ni YX. High prevalence of CTX-M lactamases in faecal Escherichia coli strains from healthy humans in Fuzhou, China. Scand J Infect Dis 2011;43:170–4.
[21] Liu W, Chen L, Li H, Duan H, Zhang Y, Liang X, et al. Novel CTX-M -lactamase genotype distribution and spread into multiple species of Enterobacteriaceae in Changsha, Southern China. J Antimicrob Chemother 2009;63: 895–900. [22] Zong Z, Yu R. blaCTX-M -carrying Escherichia coli of the O25b ST131 clonal group have emerged in China. Diagn Microbiol Infect Dis 2011;69:228–31. [23] Cao X, Cavaco LM, Lv Y, Li Y, Zheng B, Wang P, et al. Molecular characterization and antimicrobial susceptibility testing of Escherichia coli isolates from patients with urinary tract infections in 20 Chinese hospitals. J Clin Microbiol 2011;49:2496–501. [24] Madec JY, Lazizzera C, Châtre P, Meunier D, Martin S, Lepage G, et al. Prevalence of fecal carriage of acquired expanded-spectrum cephalosporin resistance in Enterobacteriaceae strains from cattle in France. J Clin Microbiol 2008;46:1566–7. [25] Costa D, Vinué L, Poeta P, Coelho AC, Matos M, Sáenz Y, et al. Prevalence of extended-spectrum -lactamase-producing Escherichia coli isolates in faecal samples of broilers. Vet Microbiol 2009;138:339–44. [26] Randall LP, Clouting C, Horton RA, Coldham NG, Wu G, Clifton-Hadley FA, et al. Prevalence of Escherichia coli carrying extended-spectrum -lactamases (CTXM and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009. J Antimicrob Chemother 2011;66:86–95. [27] Ho PL, Chow KH, Lai EL, Lo WU, Yeung MK, Chan J, et al. Extensive dissemination of CTX-M-producing Escherichia coli with multidrug resistance to ‘critically important’ antibiotics among food animals in Hong Kong, 2008–10. J Antimicrob Chemother 2011;66:765–8. [28] Kiratisin P, Apisarnthanarak A, Saifon P, Laesripa C, Kitphati R, Mundy LM, et al. The emergence of a novel ceftazidime-resistant CTX-M extended-spectrum -lactamase, CTX-M-55, in both community-onset and hospital-acquired infections in Thailand. Diagn Microbiol Infect Dis 2007;58:349–55. [29] Whichard JM. CTX-M-producing non-Typhi Salmonella spp. isolated from humans, United States. Emerg Infect Dis 2011;17:97–9. [30] Peirano G, Pitout JD. Molecular epidemiology of Escherichia coli producing CTX-M -lactamases: the worldwide emergence of clone ST131 O25:H4. Int J Antimicrob Agents 2010;35:316–21. [31] Carattoli A. Resistance plasmid families in Enterobacteriaceae. Antimicrob Agents Chemother 2009;53:2227–38. [32] Kim J, Bae IK, Jeong SH, Chang CL, Lee CH, Lee K. Characterization of IncF plasmids carrying the blaCTX-M-14 gene in clinical isolates of Escherichia coli from Korea. J Antimicrob Chemother 2011;66:1263–8.
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Prevalence and characterisation of CTX-M -lactamases amongst Escherichia coli isolates from healthy food animals in China Hongqing Zheng a,1 , Zhenling Zeng a,1 , Sheng Chen b , Yahong Liu a , Qiongfen Yao a , Yuting Deng a , Xiaojie Chen a , Luchao Lv a , Chao Zhuo c , Zhangliu Chen a , Jian-Hua Liu a,∗ a
College of Veterinary Medicine, National Reference Laboratory of Veterinary Drug Residues, South China Agricultural University, Guangzhou 510642, China Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region c State Key Laboratory of Respiratory Diseases (Guangzhou Medical College), Clinical Microbiology, Guangzhou 510120, China b
a r t i c l e
i n f o
Article history: Received 6 June 2011 Accepted 6 December 2011 Keywords: Extended-spectrum -lactamases Food animals Plasmid CTX-M Escherichia coli
a b s t r a c t The impact of extended-spectrum -lactamase (ESBL)-producing Enterobacteriaceae of food animal origins on human health has caught considerable attention worldwide. Intestinal Escherichia coli obtained from healthy food animals (pigs, cattle and poultry) in China were tested for the presence of ESBL genes. CTX-M-producing isolates were further characterised by pulsed-field gel electrophoresis (PFGE), phylogenetic grouping, genetic environment analysis, conjugation and plasmid replicon typing. A total of 127 of the 896 E. coli isolates showed reduced susceptibility to cefotaxime (minimal inhibitory concentration ≥ 2 g/mL). blaCTX-M genes were detected in 111 of the 127 isolates. The most common CTX-M types were CTX-M-14 (n = 40), CTX-M-55 (n = 29) and CTX-M-65 (n = 22), followed by CTX-M-27, -15, -98, -24, -3, -102 and -104. CMY-2 was detected in two isolates. High clonal diversity was found amongst CTXM-producing isolates. Insertion sequence ISEcp1 was observed 42 bp upstream of the start codon of all CTX-M-9 group genes, whereas the spacer region between the right inverted repeats and CTX-M-1 group genes varied from 45 bp to 127 bp. Most blaCTX-M genes were transferable by conjugation. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 of the 70 transconjugants carrying blaCTX-M , respectively. This study demonstrates that commensal E. coli from healthy food animals can be important reservoirs of blaCTX-M genes and may contribute to the dissemination and transfer of these -lactamase genes throughout China. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Extended-spectrum cephalosporins are commonly used as the most appropriate medications to treat infections caused by multidrug-resistant Gram-negative bacteria and are considered as critically important antimicrobial agents by the World Health Organization (WHO) [1]. However, reports of extended-spectrum -lactamases (ESBLs) that can hydrolyse the third- and fourth-generation cephalosporins have become increasingly frequent, and the most common ESBLs that have emerged recently are CTX-M types [2,3]. There are currently more than 110 different CTX-M-type ESBLs recognised (http://www.lahey.org/studies/other.asp#table1). blaCTX-M genes are frequently located on highly mobile genetic elements such as plasmids and transposons, which facilitate their transfer amongst
∗ Corresponding author. Tel.: +86 20 8528 3824; fax: +86 20 8528 3824. E-mail address: [email protected] (J.-H. Liu). 1 These two authors contributed equally to this paper.
different hosts around the world [3]. Of particular concern is the community dissemination of blaCTX-M -carrying Escherichia coli [2]. ESBL-producing E. coli isolates have been increasingly detected in food animals in different countries since 2002 [4]. Food animals colonised with ESBL-producing E. coli have been considered as potential sources of resistant E. coli causing infection in the community and have caught considerable attention worldwide [4]. ESBL-producers were seldom detected in animal isolates in China before 2005 [5–7]. However, the occurrence of ESBL-producing E. coli has dramatically increased in China recently, with CTX-M -lactamases being the most common type [7–9]. There have been few comprehensive studies of ESBLs in China, particularly of the mechanism conferring the rapid distribution of blaCTX-M in food animals. Commensal E. coli isolates in food animals are believed to be the reservoir of ESBL genes [4]. In this study, we investigated the distribution of ESBLs amongst commensal E. coli isolates from common healthy food animals in China, including pigs, cattle, chickens, ducks, geese, partridges and pigeons. Plasmids that carried CTXM were also studied to elucidate the mechanism of transfer and dissemination of -lactamases.
0924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2011.12.001
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2. Materials and methods 2.1. Source of Escherichia coli isolates Cloacal samples from poultry (chickens, ducks, geese, partridges and pigeons) and rectal swabs from pigs and cattle were collected from 47 food animal farms located in different geographic areas of China from April 2007 to August 2009 (Table 1). Animals on each farm were randomly selected for sampling based on their age and stage of production. The number of animal samples collected from each farm was based on the size of the farm (ca. 0.2–1% of the total number of animals on each farm). All samples were seeded on MacConkey agar plates and were incubated at 37 ◦ C for 24 h. One suspected colony with typical E. coli morphology and size was selected from all agar plates of each sample and was identified using classical biochemical methods. Species identification was confirmed using an API 20E system (bioMérieux, Marcy l’Étoile, France). 2.2. Antimicrobial susceptibility testing Minimal inhibitory concentrations (MICs) of ampicillin, ceftriaxone, cefotaxime, ceftazidime, cefoxitin, gentamicin, amikacin, chloramphenicol, tetracycline, ciprofloxacin and trimethoprim/sulfamethoxazole (SXT) were determined by the agar dilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. The disk diffusion method was used to determine susceptibility to imipenem and meropenem. Escherichia coli ATCC 25922 was used as the control strain. Isolates were classified as either susceptible or resistant according to the interpretative standards recommended by the CLSI [10,11]. ESBL-producing isolates were screened by double-disk synergy test using both cefotaxime and ceftazidime in the presence or absence of clavulanic acid as recommended by the CLSI. 2.3. Polymerase chain reaction (PCR) amplification and sequencing Genes encoding TEM, CTX-M, SHV, CMY-2 and DHA-1 enzymes as well as the genetic environment of blaCTX-M genes were analysed by PCR amplification as described previously [6,12]. Purified PCR products were directly sequenced from both ends or were cloned in pMD18-T vectors and then sequenced. The DNA sequences and deduced amino acid sequences were compared with genes in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) or the -lactamase classification system (http://www.lahey.org/studies/) to confirm the subtypes of -lactamase genes. 2.4. Strain typing Pulsed-field gel electrophoresis (PFGE) analysis of Xbal-digested genomic DNA was performed to determine the genetic relatedness of CTX-M-producing E. coli isolates using a CHEF-MAPPER System (Bio-Rad Laboratories, Hercules, CA). PFGE patterns were interpreted according to well-established criteria [13]. Isolates that had PFGE patterns with no more than six different bands were considered clonally related. Phylogenetic groups of E. coli isolates carrying blaCTX-M were assigned by a multiplex PCR-based method developed as described previously [14]. Isolates belonging to phylogenetic group B2 were screened with a previously established PCR-based method to identify the O25b-ST131 clone [15]. 2.5. Conjugation experiments and plasmid analysis Transferability of blaCTX-M genes was determined by conjugation experiments using streptomycin-resistant E. coli C600 as the recipient strain as previously described [12]. Ninety-three isolates with
different PFGE patterns were selected for conjugation. Transconjugants were selected on MacConkey agar plates supplemented with cefotaxime (2 g/mL) and streptomycin (2000 g/mL). The presence of blaCTX-M was confirmed by PCR. Incompatibility (Inc) groups were assigned by PCR-based replicon typing of transconjugants [16]. To better clarify IncF plasmids, replicon sequence typing of IncF plasmids was performed according to a previously described procedure [17] and alleles were assigned by submitting the amplicon sequence to the plasmid multilocus sequence typing (MLST) database (http://www.pubmlst.org/plasmid). Plasmids carrying blaCTX-M were extracted from transconjugants using a rapid alkaline lysis method. Plasmids of transconjugants were digested with the endonuclease EcoRI (TaKaRa Biotechnology, Dalian, China) to analyse the restriction fragment length polymorphism (RFLP) profile and to estimate the size of plasmids. 2.6. Nucleotide sequence accession numbers The GenBank accession nos. of the sequence studied herein were HM748991, HM755448, HQ398215, HQ833652 and JF713460. 3. Results 3.1. Antimicrobial susceptibility A total of 896 commensal E. coli collected from 326 pigs, 316 chickens, 88 cattle, 58 ducks, 22 geese, 61 pigeons and 25 partridges were included in this study. Amongst them, 127 isolates (14.2%) (41 from pigs, 44 from chickens, 5 from cattle and 37 from other poultry species) showed reduced susceptibility to cefotaxime (MIC ≥ 2 g/mL) (Table 1). Of the 127 isolates, all were multidrug-resistant and showed resistance to more than two non--lactam antimicrobial agents, including ciprofloxacin (70.9%), gentamicin (71.7%), amikacin (21.3%), chloramphenicol (77.2%) and SXT (94.5%). Some of them were also resistant to other cephalosporins, including ceftazidime (40.9%) (MIC ≥ 16 g/mL), ceftriaxone (97.6%) (MIC ≥ 4 g/mL) and cefoxitin (15.0%) (MIC ≥ 32 g/mL). All of the isolates were susceptible to imipenem and meropenem. ESBL production was detected by the screening method in 117 of the 127 isolates, representing 13.1% of the total 896 commensal E. coli isolates. 3.2. ˇ-Lactamase gene detection Acquired -lactamase genes were detected in most E. coli isolates with a cefotaxime MIC ≥ 2 g/mL. CTX-M-type genes were found to be dominant in these isolates and 111 isolates carried one or two CTX-M genes, representing 12.4% of the total 896 foodanimal-associated E. coli isolates. Fifty-nine of the 127 E. coli carried blaTEM-1 . Two isolates from pigs carried the blaCMY-2 gene. blaSHV and blaDHA-1 genes were not found in any of these isolates. The most predominant CTX-M-encoding gene was blaCTX-M-14 (n = 40), followed by blaCTX-M-55 (n = 29), blaCTX-M-65 (n = 22), blaCTX-M-27 (n = 14), blaCTX-M-15 (n = 5), blaCTX-M-24 (n = 3) and blaCTX-M-3 (n = 2) (Table 2). Three novel variants of CTX-M enzymes (CTX-M-98, CTX-M-102 and CTX-M-104) were identified in three (one from pig and two from ducks), one (from chicken) and one (from chicken) E. coli isolates, respectively. The enzymes CTX-M98 and CTX-M-102 differed from CTX-M-14 by two amino acids (A80V and D242G in the case of CTX-M-98 and A208G and D242G in the case of CTX-M-102), whereas CTX-M-104 differed from CTXM-14 by only one amino acid (N275S). These new blaCTX-M-14 -like genes have been assigned accession nos. in GenBank and Lahey Clinic as follows: HM755448 (CTX-M-98), HQ398215 (CTX-M-102) and HQ833652 (CTX-M-104). In addition, a C348T silent variant of
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Table 1 Origins of Escherichia coli isolates. Farm
Province
Animal species
No. of samples from each farm
No. of E. coli isolates from each farm
No. of E. coli isolates with cefotaxime MIC ≥ 2 g/mL
F1/F2/F3/F4 F5/F6/F7 F8/F9/F10 F11/F12 F13 F14 F15/F16 F17/F18 F19 F20/F21 F22/F23/F24 F25/F26/F27 F28/F29/F30/F31 F32/F33 F34 F35/F36/F37 F38 F39 F40/F41 F42/F43 F44/F45/F46/F47
Guangdong Guangdong Guangdong Guangdong Guangdong Guangdong Guangxi Hainan Sichuan Gansu Gansu Heilongjiang Heilongjiang Hebei Hebei Shandong Henan Henan Shanxi Hunan Jiangxi
Chicken Pig Duck Goose Partridge Pigeon Chicken Chicken Pig Chicken Pig Pig Cow Pig Pigeon Chicken Chicken Pigeon Pigeon Chicken Pig
30/30/30/30 20/20/20 26/26/24 16/16 30 20 30/30 20/22 15 16/18 16/14/16 26/26/26 28/26/30/30 30/26 14 26/24/26 10 28 20/14 22/26 35/35/35/35
27/27/25/26 17/16/14 19/21/18 12/10 25 13 23/27 13/16 12 10/12 13/12/12 20/17/18 20/21/23/24 25/23 10 23/20/22 7 19 11/8 17/21 33/30/31/33
8 5 14 1 12 4 4 3 2 2 1 8 5 2 1 24 3 5 0 0 23
MIC, minimal inhibitory concentration.
blaCTX-M-55 was found in one isolate from chicken and designated here as blaCTX-M-55b . The distribution of CTX-M gene types in different animal species was varied. blaCTX-M-14 and blaCTX-M-55 were found in all animal species tested, blaCTX-M-27 was mainly found in duck isolates and blaCTX-M-65 was found in all animal isolates except for those from calf. blaCTX-M-14 was widely distributed in almost all provinces. 3.3. Phylogenetic groups and clonal analysis Phylogenetic group analysis showed that group A (58; 52.3%) was dominant amongst the isolates that produced CTX-M enzymes, followed by group B1 (26; 23.4%) and group D (21; 18.9%). Only six isolates (four with blaCTX-M-14 , one with blaCTX-M-27 and one with blaCTX-M-3 ) belonged to extraintestinal virulent group B2, and none of them belonged to clone O25b-ST131. PFGE was successfully performed on 99 CTX-M-producers; no PFGE fragment pattern was obtained from the other 12 isolates. Significant clonal diversity was shown in isolates that carried CTX-M -lactamases (Table 2). Within the same farm, clonality was sporadically observed. There were no common clones between the
farms. However, for the three CTX-M-98-producing E. coli isolates, two strains from different farms (one from a duck farm located in South China, the other from a pig farm located in Central China) showed indistinguishable PFGE patterns. 3.4. Genetic environment of blaCTX-M Insertion sequence ISEcp1-like elements were present in the upstream region of blaCTX-M genes in 108 (90.0%) of the 120 blaCTX-M genes. Insertion of ISEcp1 was observed at 42 bp upstream of the start codon of all CTX-M-9 group genes. However, the spacer region between the right inverted repeats and CTX-M-1 group genes was varied: 127 bp upstream of blaCTX-M-3 , 48 bp or 127 bp upstream of blaCTX-M-15 , and 45, 48 or 127 bp upstream of blaCTX-M-55 (Table 3). To our knowledge, this is the first time a 127-bp intergenic region upstream of the blaCTX-M-55 gene has been identified. Interestingly, even in isolates from the same farm, different intergenic regions were found between CTX-M-55 genes and ISEcp1, suggesting that blaCTX-M-55 genes probably emerged from Kluyvera spp. through multiple mobilisation events or from mutation of other CTX-M1-group genes such as blaCTX-M-3 and blaCTX-M-15 . An ISCR1 element
Table 2 Distribution of CTX-M subgroups and alleles amongst Escherichia coli isolates from different animal sources, and clonal relationship of CTX-M-producing E. coli isolates. CTX-M type
CTX-M-9 group CTX-M-14 CTX-M-24 CTX-M-27 CTX-M-65 CTX-M-98 CTX-M-102 CTX-M-104 CTX-M-1 group CTX-M-3 CTX-M-15 CTX-M-55 Any CTX-M
No. of PFGE subtypesa
No. of isolates (%) Pig (n = 326)
Calf (n = 88)
Chicken (n = 316)
Duck and goose (n = 80)
Partridge and pigeon (n = 86)
Total (n = 896)
27 12
4 4
23 11 3 1 6
14 3
16 10
8 1 2
1 5
4
4
84 40 3 14 22 3 1 1 36 2 5 29 111 (12.4)
4 10 1
8 3 5 34 (10.4)
2
2 5 (5.7)
1 1 18 2 1 15 39 (12.3)
1 3 15 (18.8)
PFGE, pulsed-field gel electrophoresis; N/T, non-typeable. a PFGE patterns were interpreted according to the criteria of Tenover et al. [13]. b Number of non-typeable isolates is indicated in parentheses.
4 18 (20.9)
67, N/T (8)b 30, N/T (4) 3 12, N/T (1) 18, N/T (3) 2 1 1 27, N/T (4) 2 4, N/T (1) 21, N/T (3) 85, N/T (12)
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Table 3 Analysis of genetic structure upstream and downstream of the blaCTX-M gene. Type of blaCTX-M
No. of isolates
No. of isolates ISCR1
ISEcp1
Size of intergenic spacer region between ISEcp1 and blaCTX-M (bp)
No. of isolates orf477
IS903
CTX-M-3
2
0
2
127
2
N/A
CTX-M-15
5
0 0
3 2
127 48
3 2
N/A N/A
CTX-M-55a
28
0
12 10 6
45 48 127
10 10 6
N/A
CTX-M-55b CTX-M-14a CTX-M-14b CTX-M-24 CTX-M-27 CTX-M-65 CTX-M-98 CTX-M-102 CTX-M-104
1 39 1 3 14 22 3 1 1
0 0 1 0 0 0 0 0 0
1 30 0 3 13 21 3 1 1
48 42 N/A 42 42 42 42 42 42
1 N/A N/A N/A N/A N/A N/A N/A N/A
N/A 25 0 3 12 19 2 1 1
N/A, not applicable.
was found upstream of the blaCTX-M-14b gene (n = 1). ISEcp1-like and ISCR1 were not found immediately upstream of any other blaCTX-M genes (n = 12). orf477 and IS903 were detected downstream of 34 CTX-M-1 and 63 CTX-M-9 group genes, respectively. The sequence of blaCTX-M-55 with the 127-bp intergenic region and the genetic environment of blaCTX-M-14b were deposited in GenBank (accession nos. HM748991 and JF713460).
3.5. Transferability of blaCTX-M genes and plasmid replicon typing blaCTX-M genes from 70 of 93 isolates were transferred to the recipient by conjugation at frequencies of 10−7 to 10−3 per donor cell. In addition to cefotaxime resistance, the plasmids in some transconjugants encoded co-resistance to non--lactamase antibiotics such as gentamicin (38.6%), amikacin (27.1%), tetracycline (12.9%), chloramphenicol (12.9%), nalidixic acid (7.1%) and SXT (47.1%). Plasmid replicon typing of transconjugants revealed significant plasmid diversity. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 transconjugants, respectively (Table 4). Two plasmid replicons (IncFII in combination with IncFIB or IncI1) were simultaneously present in 11 transconjugants. The replicon types could not be determined in 19 transconjugants. Within FII replicons, F2, F18, F24, F31, F33 and F35 alleles were identified in 12, 1, 2, 3, 5 and 1 transconjugants, respectively. Within FIB replicons, B1 and B6 were identified in 6 and 2 transconjugants, respectively. In addition, the new alleles F38 and F41 were also identified in two transconjugants. Twenty-nine transconjugants were selected for plasmid restriction enzyme digestion analysis according to CTX-M type (13 CTX-M-14, 5 CTX-M-55, 4 CTX-M-65, 4 CTX-M-27, 2 CTX-M-15 and 1 CTX-M-98) and plasmid replicon type (11 IncI, 16 IncFII and 2 IncN). The sizes of the plasmids varied from 40 kb to 100 kb. Most of the plasmids showed diverse digestion patterns. However, in most cases plasmids of the same replicon type carrying CTX-M genes that belonged to the same group shared some bands of the same size (data not shown). Two IncN plasmids (one carrying blaCTX-M-27 and the other carrying blaCTX-M-65 ) had the same plasmid restriction patterns. Two F31:A-:B1 plasmids carrying blaCTX-M-15 showed very similar patterns. Eight F2:A-:B- plasmids carrying diverse CTXM-9 group genes had the same or very similar plasmid restriction patterns. The same was true for four IncI1 plasmids carrying the blaCTX-M-14 gene.
4. Discussion In this study, 896 E. coli isolates originating from food animals were collected from 12 different provinces covering all the seven geographic regions in mainland China during 2007–2009; 14.2% of them were ESBL-producers and 12.4% carried CTX-M -lactamases. Of the 270 E. coli isolates from Guangdong province, 16.3% carried CTX-M -lactamases (data not shown), which was much higher than E. coli isolates collected between 2003 and 2005 in our previous work (2.4%) [6]. The increasing incidence of ESBLs amongst E. coli isolates from food animals may be due to the increasing use of third-generation cephalosporins in food animals in recent years, since fluoroquinolones have become inactive against most E. coli strains in China [18]. Leverstein-van Hall et al. [19] recently reported that Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains, suggesting transmission of ESBL genes from poultry to humans through the food chain. Very recently, a surprisingly high number (50.5%) of faecal samples carrying CTX-M-producing E. coli has been reported in the healthy Chinese population [20]. Although we cannot determine whether the CTX-M-type -lactamase-producing E. coli found in the healthy Chinese have acquired these genes from food animals via the food chain, the high prevalence of CTX-M-type ESBLs amongst commensal E. coli isolates in food animals strongly suggests the significant role of commensal E. coli as ESBL gene reservoirs, which imposes additional risk to patients and even the general community. Therefore, tracking and monitoring the spread of Enterobacteriaceae that produce CTX-M-type ESBLs in foods conducted on the basis of community settings in China are urgently needed for the benefit of public health. Ten types of CTX-M ESBLs were detected in the present study, indicating a high diversity of CTX-M-encoding genes in E. coli isolates from food animals in China. The most common CTX-M enzyme (CTX-M-14) was detected in all animal species from different regions and was also the most frequent type amongst humans and pets in China [12,20–23]. In addition, CTX-M-65, CTX-M-24, CTX-M-27 and CTX-M-15 were also detected in some isolates, which was similar to what has been reported from human isolates in China [20–23]. The epidemiological distribution of CTX-M types appears to be comparable between animal and human bacteria in China. However, the distribution of ESBL types found in the present study is different from food animals of other countries such as France, The Netherlands, Portugal and England where CTX-M-1 or TEM-52 is the dominant type [19,24–26]. A novel variant of the
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Table 4 Replicon sequence typing of plasmids in 70 transconjugants carrying blaCTX-M . blaCTX-M gene type in transconjugant CTX-M-3 (n = 1) CTX-M-15 (n = 2) CTX-M-55 (n = 16) CTX-M-14 (n = 23) CTX-M-24 (n = 1) CTX-M-27 (n = 10) CTX-M-65 (n = 14) CTX-M-98 (n = 2) CTX-M-102 (n = 1) Total (n = 70)
Replicon type (n) N
FII
FII, FIB
FII, I1
I1
A/C
Unknown 1
2 1
2 7
3
2 2
5 2 1
2
5
17
7
CTX-M enzyme, CTX-M-98, was detected in E. coli isolates both from ducks and pigs in this study and was also recently reported to be present in isolates from calf [27] and geese (GenBank accession no. HQ637575), indicating that blaCTX-M-98 has been widely dispersed in different animal species in China. CTX-M-55 was the second common CTX-M enzyme in the present study, as also shown in other studies [7,9]. It was also found in E. coli isolates from pets in mainland China and from animals in Hong Kong [12,27]. However, it was only sporadically detected in other countries except for Thailand and was not common in human isolates in China [20,28,29]. As >80% of the food animals consumed in Hong Kong are imported from mainland China, the prevalence and distribution of CTX-M types in E. coli of food animal origin in Hong Kong may reflect those in mainland China [27]. Thus, CTX-M55 has also widely disseminated in animals in China. CTX-M-15, which is a most common enzyme in many countries [2,3], was only detected in five isolates in this study, similar to the prevalence in isolates from pets [12]. However, it was more common in human isolates in mainland China [20,23]. The different distribution of CTX-M-55 and CTX-M-15 between human and animal isolates may be due to diverse antimicrobial selective pressure. Dissemination of specific clonal groups or clones has been critical for the dramatic increase in the prevalence of blaCTX-M genes, especially blaCTX-M-15 , in many countries [3,30]. However, in this study, high clonal diversity of bacterial hosts was observed, indicating the importance of mobile elements in the dissemination of blaCTX-M genes in China. Plasmids that carried blaCTX-M were also characterised by plasmid replicon typing and fingerprinting analysis. Similar to previous studies [23,31], most CTX-M genes were found to be linked to IncFII or IncI1 plasmids. The most common IncFII type plasmid was F2:A-:B-, which had also been found to be associated with blaCTX-M genes in Enterobacteriaceae isolates from other countries [17,32]. In particular, we observed that plasmids carrying blaCTX-M-14 from epidemiologically unrelated strains (from different farms and geographic regions) had surprisingly similar plasmid restriction patterns (data not shown), indicating the presence of epidemic plasmids in China. blaCTX-M-14 has widely disseminated in China both in human and animal isolates for nearly a decade. However, only very limited information is available on the types of plasmids carrying this gene [22,23]. More studies are needed to determine the linkage of plasmids in humans and animals and to address the factors that contribute to the successful dissemination of some CTX-M types, such as CTX-M-14 and CTXM-55 in China. To our knowledge, this is the first report on PFGE, plasmid analysis and the genetic environment of CTX-M-producing E. coli isolates collected from a variety of animal farms located in different geographic regions of mainland China. The limitation of this study was that more than one-half of the 896 isolates were collected from three provinces (Guangdong, Jiangxi and Heilongjiang), whereas fewer isolates were collected from the other nine provinces. Thus,
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the data might not completely represent the actual situation of the occurrence and characterisation of ESBL-producers in food animals. None the less, this study reveals that CTX-M enzymes have been widely distributed in E. coli isolates from different food animal species in mainland China. In conclusion, cephalosporin resistance amongst a high clonal diversity of E. coli from food animals was mainly mediated by blaCTX-M genes harboured by diverse or similar IncFII or IncI1 plasmids. The increasing prevalence of commensal ESBL-producing E. coli in healthy food animals imposes a substantial threat to public health, as the ESBL genes will eventually transfer to human and animal pathogens. Based on these observations, the third- and fourth-generation cephalosporins should be used more prudently in food animals. Acknowledgements The authors are sincerely grateful to Zhiyong Zong for valuable comments on the manuscript. They also thank Alessandra Carattoli and Laura Villa for assigning IncF plasmids, and Dehe Ye, Tao Lei, Wei Tian and Jianxia Hou for sampling and antimicrobial susceptibility testing. Funding: This work was supported by grants from the National Natural Science Foundation of China (Nos. U1031004 and 30972218). Competing interests: None declared. Ethical approval: Not required. References [1] Collignon P, Powers JH, Chiller TM, Aidara-Kane A, Aarestrup FM. World Health Organization ranking of antimicrobials according to their importance in human medicine: a critical step for developing risk management strategies for the use of antimicrobials in food production animals. Clin Infect Dis 2009;49: 132–41. [2] Pitout JD, Laupland KB, Extended-spectrum. -lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008;8:159–66. [3] Rossolini GM, D’Andrea MM, Mugnaioli C. The spread of CTX-M-type extendedspectrum -lactamases. Clin Microbiol Infect 2008;14(Suppl. 1):33–41 [Erratum in: Clin Microbiol Infect 2008;14(Suppl. 5):21–4]. [4] Smet A, Martel A, Persoons D, Dewulf J, Heyndrickx M, Herman L, et al. Broad-spectrum -lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health. FEMS Microbiol Rev 2010;34:295–316. [5] Yang H, Chen S, White DG, Zhao S, McDermott P, Walker R, et al. Characterization of multiple-antimicrobial-resistant Escherichia coli isolates from diseased chickens and swine in China. J Clin Microbiol 2004;42:3483–9. [6] Liu JH, Wei SY, Ma JY, Zeng ZL, Lü DH, Yang GX, et al. Detection and characterization of CTX-M and CMY-2 -lactamases among Escherichia coli isolates from farm animals in Guangdong Province of China. Int J Antimicrob Agents 2007;29:576–81. [7] Li L, Jiang ZG, Xia LN, Shen JZ, Dai L, Wang Y, et al. Characterization of antimicrobial resistance and molecular determinants of -lactamase in Escherichia coli isolated from chickens in China during 1970–2007. Vet Microbiol 2010;144:505–10.
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