1. 【Introduction】
Since 2000, E. coli and other Enterobacteriaceae species producing CTX-M-type ESBLs (CTX-M) have been commonly isolated from community-acquired extraintestinal infections in humans and their companion animals and from food-producing animals worldwide (Carattoli 2008).
The blaCTX-M-related genes were transferred separately to plasmids including complex class 1 integrons and transposons from chromosomes of different Kluyvera species (i.e., K. ascorbata, K. cryocrescens, K. georgiana); thereby, CTX-M has derived in five CTX-M clusters (CTX-M-1/-2/-8/-9/-25) from base sequence homology (Cantón et al. 2012).
CTX-M confers resistance against penicillins, oxyimino-cephalosporins, and monobactams (Bonnet 2004).

2. Hierarchical function
Spread
chromosome
Fig. Hierarchical complexity of blaCTX-M genes within genetic structure and bacterial clones participation in the mobilization, spread, and maintenance
Spread
Plasmid
Spread
Insertion sequence and CTX-M gene
Class1 Integron
Transposon
Mobilization and expression
Maintenance

3. 【Background】
Plasmidic CMY-2, derived from the AmpC β-lactamase of Citrobacter freundii, and the dominant AmpC β-lactamase among Enterobacteriaceae (Salmonella enterica) in humans and animals worldwide (Tamang et al. 2012)
CMY-type β-lactamases, cephamycinases that confer resistance to cephamycins and oxyimino-cephalosporins
【Aims】
to determine the herd prevalence of Enterobacteriaceae producing CTX-M and CMY among numerous dairy farms in Japan
to evaluate the genetic relatedness among E. coli isolates derived from dairy cattle by serotyping, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE)

4. 【Materials and methods】
A total of 897 faecal samples: each of 897 cattle on a total of 381 dairy farms localized in Nemuro Subprefecture of Hokkaido Prefecture, Japan, between May and Dec. in 2007, and between July and Dec. in 2009
Identification: ID 32 E API system (Sysmex bioMérieux）
A total of 250 isolates → CLSI combination disc ESBL confirmatory tests (CLSI 2008) and a chromogenic oxyimino-cephalosporins hydrolysis test (Cica-β-test I; Kanto Chemical, Tokyo, Japan) to detect ESBLs, plasmidic AmpC β-lactamases and metallo-β-lactamases (MBL)
Screening for MBL: Sodium mercaptoacetic acid (SMA) double-disc synergy test (SMA-test) : using 1 SMA and
The 897 cattle: 550 calves (< 12 months old), 80 heifers　(≥ 12 months old to first parturition) and 267 cows
Culture: directly streak 50 μg faeces onto a BTB lactose agar plate containing 2 μg ml-1 cefotaxime and 8 μg ml-1 　vancomycin → Incubation: 35°C for 40 h (Shiraki et al. 2004)
The cattle affected by diarrhoea or dictyocauliasis
2 CAZ discs (Eiken Chemical, Tokyo, Japan)
Screening for ESBLs
Faecal samples
【Materials and methods】

5. 【Materials and method】
Isolates positive for the ESBL confirmatory tests and/or the Cica-β-test, and the SMA-test-negative
The blaTEM and blaSHV genes： PCR using grpou-specific primers (Kojima) and bidirectional sequencing
For the CTX-M-positive isolates:　PCR and bidirectional sequencing using group-specific PCR primers for blaCTX-M-1 group (Mena et al. 2006), blaCTX-M-2 group and blaCTX-M-9 group (Kojima et al. 2005)
For the AmpC-positive isolates : PCR using type-specific primers (blaCMY-1 and blaCMY-2 genes) (Kojima)
 Serotyped of E. coli (O and H antigens): pathogenic E. coli antisera ‘SEIKEN’ Set 1 for O antigens, Set 2 for H antigens (Denka Seiken, Tokyo, Japan)
Multiplex PCR for the presence of blaCTX-M genes (Xu et al. 2007), and plasmid-mediated AmpC β-lactamase genes (i.e., CMY, ACC, FOX, MOX, DHA, CIT, and EBC groups) (Pérez-Pérez and Hanson 2002)
MLST: protocols and database on the E. coli MLST website (
PCR and sequencing for ESBLs and AmpC
Serotyping and MLST of E. coli　　　　
【Materials and method】

6. 【Materials and methods】
PFGE: the PulseNet standardized laboratory protocol (CDC 2004). 　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 Restriction enzyme: Xba I or Bln I (Roche Applied Science, Mannheim, Germany) 　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 CHEF-DR III electrophoresis systems (Bio-Rad, Hercules, CA, USA)
Disc diffusion: cefoxitin, kanamycin, chloramphenicol and levofloxacin (CLSI 2008 and 2011)
Dendrogram of the combined PFGE patterns of Xba I and BlnI： BioNumerics software version 5·1 (Applied Maths, Austin, TX, USA)
MICs of 23 antimicrobial by broth microdilution： customer-designed, microtiter panel (Opt Panel MP; Kyokuto Pharmaceutical Tokyo, Japan) (CLSI 2008 and 2011)
The breakpoints for veterinary pathogens were used for 12 antimicrobial agents (CLSI 2008).　
The breakpoints for human Enterobacteriaceae isolates were used for 12 antimicrobial agents (CLSI 2011).
E. coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were used as quality-control strains. 　
Antimicrobial susceptibility tests
PFGE of E. coli　　　　
【Materials and methods】

10. Table 4 CTX-M, TEM, SHV and ST of 23 E. coli strains
CTX-M, TEM, and SHV genes n
E. Coli MLST (18 STs)
CTX-M-15 4
394
744
1167
1266
CTX-M-15+TEM-1 3
540
2325
CTX-M-15+CMY-2+TEM-1 1
CTX-M-2 5
10 （n=3）
48（STC10）
2324
CTX-M-2+TEM-1 58
CTX-M-2+SHV-11
2437
CTX-M-2+CMY-2 46
CTX-M-2+CMY-2+SHV-11
44(STC10)
CTX-M-14+TEM-1 10 57 69
CTX-M-14+CMY-2+TEM-1 88
CMY-2
1284
CMY-2+TEM-1
2438

12. 【 Results】
Incidence of isolation and herd prevalence of CTX-M and CMY-2  
28 Enterobacteriaceae strains producing CTX-M-15/2/14 and/or CMY-2 were isolated from 28 (3·1 %) of the 897 fecal samples from 20 dairy farms.
① blaCTX-M-15 : 7 E. coli isolates (25·0 %) 　　　
　 ② blaCTX-M-2: 12 isolates (42·9 %; 7 E. coli; 3 K. pneumoniae,
　　　　1 Enterobacter cloacae, 1 Citrobacter freundii)
　 ③ blaCTX-M-14; 3 E. coli isolates (10·7 %)
　　④ 6 E. coli isolates (21.4%): blaCMY-2 and/or blaCTX-M-2/15/14 　　　
12 of 23 E. coli (52.2%): blaTEM-1 or blaSHV-11
4 (50%) of 8 CTX-M-15–producing E. coli：blaTEM-1 
MLST, serotyping and PFGE of 23 E. coli isolates  
23 E. coli isolates → 18 STs/ 21 PFGE types
No predominant E. coli strain of a particular ST/PFGE type/serotype was shown in one farm and among the farms.　

14. 【 Results】 Antimicrobial susceptibility and antimicrobial use
CTX-M-2/14 producers showed high susceptibility rates to CAZ, cefmetazole (CMZ), moxalactam, carbapenems, gentamicin (GEN), amikacin (AMK) and fluoroquinolones (FQs).
MICs for CAZ CMZ, enrofloxacin and ciprofloxacin were higher for the E. coli isolates producing CTX-M-15 and CMY-2 than for the isolates producing CTX-M-2/14.
Between 2006 and 2010, our veterinary clinics used 2,016 daily doses of ceftiofur and 1,408 daily doses of enrofloxacin (based on the weight of an adult cow) per year for approximately130,000 cows.
The spread of blaCTX-M/CMY-2 may be due to the frequent use of mastitis preparations consisting of 1st-, 2nd-generation cephalosporins and using waste milk mixed with cephalosporins to feed calves.

15. 【Conclusion】 【 Significance and impact of study 】
Three clusters of CTX-M (CTX-M-15, CTX-M-2, CTX-M-14) had spread among Japanese dairy farms.
The incidence of isolation of E. coli and 3 genera of Enterobacteriaceae producing CTX-M-15/2/14 and/or CMY-2 in bovine faeces was 28 (3·1 %) of the 897 fecal samples.
The herd prevalence of these Enterobacteriaceae was 20 (5·2 %) of the 381 dairy farms.
The 23 E. coli isolates showed clonal diversity, as assessed by MLST analysis and PFGE typing.
The 8 E. coli isolates producing CTX-M-15 belonged to 6 STs and most harbored TEM-1 and were resistant to CAZ and FQs.
This is the first report on the prevalence of multidrug-resistant CTX-M-15–producing E. coli among Japanese dairy farms.
【 Significance and impact of study 】

16. 【References】
Bonnet, R. (2004) Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother
48, 1–14.
Canton, R., González-Alba, J.M. and Galán J.C. (2012) CTX-M enzymes: origin and diffusion. Front Microbial 3, article 110.
(1–19)
Carattoli, A. (2008) Animal reservoirs for extended spectrum β-lactamase producers. Clin Microbiol Infect 14 (Suppl 1), 117–
123.
Centers for Disease Control and Prevention (CDC). (2004) One-day (24-28 h) standardized laboratory protocol for molecular
　subtyping of Escherichia coli O157:H7, non-typhoidal Salmonella serotypes, and Shigella sonnei by pulsed field gel
electrophoresis (PFGE). Sections 5.1, 5.2, 5.4. Atlanta, GA: CDC
Clinical and Laboratory Standards Institute (CLSI). (2008) Performance Standards for Antimicrobial Disk and Dilution 　
　Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard, 3rd edn, M31-A3. Wayne, PA: CLSI.
Clinical and Laboratory Standards Institute (CLSI). (2011) Performance Standards for Antimicrobial Susceptibility Testing,
21st Informational Supplement, M100-S21. Wayne, PA: CLSI.
Kojima, A., Ishii, Y., Ishihara, K., Esaki, H., Asai, T., Oda, C., Tamura, Y., Takahashi, T. and Yamaguchi, K. (2005)
　Extended-spectrum-β-lactamase-producing Escherichia coli strains isolated from farm animals from 1999 to 2002: Report
from the Japanese Veterinary Antimicrobial Resistance Monitoring Program. Antimicrob Agents Chemother 49, 3533−3537.
Mena A., Plasencia V., Garcia L., Hidalgo O., Avestarán J.I., Alberti S., Borrell N., Pérez J.L. and Oliver A. (2006)
　Characterization of a large outbreak by CTX-M-1-producing Klebsiella pneumoniae and mechanisms leading to in vivo
carbapenem resistance development. J Clin Microbiol 44, 2831−2837.
Pérez-Pérez F.J. and Hanson N.D. (2002) Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using
　multiplex PCR. J Clin Microbiol 40, 2153−62.
Shiraki, Y., Shibata, N., Doi, Y. and Arakawa, Y. (2004) Escherichia coli producing CTX-M-2 β-lactamase in cattle, Japan.
Emerg Infect Dis 10, 69−75.
Tamang, M.D., Nam, H.-M., Jang, G.-C. Kim, S.-R., Chae, M.H., Jung, S.-C., Byun, J.-W. Park, Y.H. and Lim, S.-K. (2012)
　Molecular characterization of extended-spectrum-β-lactamase-producing and plasmid-mediated AmpC β-lactamase-
producing Escherichia coli isolated from stray dogs in South Korea. Antimicrob Agents Chemother 56, 2705–2712.
Xu, L., Ensor, V., Gossain, S., Nye, K. and Hawkey, P. (2007) Rapid and simple detection of blaCTX-M genes by multiplex PCR
assay. J Med Microbiol 54, 1183–1187.

17. Sample: Accepted-paper
Ohnishi M, Okatani AT, Esaki H, Harada K, Sawada T, Murakami M, Marumo K, Kato Y, Sato R, Shimura K, Hatanaka N, Takahashi T Herd Prevalence of Enterobacteriaceae producing CTX-M-type and CMY-2 β-lactamases among Japanese dairy farms.
J. Appl. Microbiol. 115:xx-xx
Sample: Accepted-paper