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. 2018 Mar 1;84(6):e02430-17. doi: 10.1128/AEM.02430-17

Molecular Epidemiology of Dairy Cattle-Associated Escherichia coli Carrying blaCTX-M Genes in Washington State

Josephine A Afema a,#, Sara Ahmed a,*,#, Thomas E Besser b, Lisa P Jones a, William M Sischo c, Margaret A Davis a,✉
Editor: Christopher A Elkinsd
PMCID: PMC5835732  PMID: 29305512

ABSTRACT

An increase in the prevalence of commensal Escherichia coli carrying blaCTX-M genes among dairy cattle was observed between 2008 and 2012 in Washington State. To study the molecular epidemiology of this change, we selected 126 blaCTX-M-positive and 126 blaCTX-M-negative isolates for determinations of the multilocus sequence types (MLSTs) and antibiotic resistance phenotypes from E. coli obtained during a previous study. For 99 isolates, we also determined the blaCTX-M alleles using PCR and sequencing and identified the replicon types of blaCTX-M-carrying plasmids. The blaCTX-M-negative E. coli isolates comprised 76 sequence types (STs) compared with 32 STs in blaCTX-M-positive E. coli isolates. The blaCTX-M-positive E. coli isolates formed three MLST clonal complexes, accounting for 83% of these isolates; 52% of blaCTX-M-negative E. coli isolates clustered into 10 clonal complexes, and the remainder were singletons. Overall, blaCTX-M-negative E. coli isolates had more diverse genotypes that were distinct to farms, whereas blaCTX-M-positive E. coli isolates had a clonal population structure and were widely disseminated on farms in both regions included in the study. Plasmid replicon types included IncI1 which predominated, followed by IncFIB and IncFIA/FIB. blaCTX-M-15 was the predominant CTX-M gene allele, followed by blaCTX-M-27 and blaCTX-M-14. There was no significant association between plasmid replicon types and bacterial STs, and neither clonal complexes nor major plasmid groups were associated with two discrete dairy-farming regions of Washington State.

IMPORTANCE Infections caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli occur globally and present treatment challenges because of their resistance to multiple antimicrobial drugs. Cattle are potential reservoirs of ESBL-producing Enterobacteriaceae, and so understanding the causes of successful dissemination of blaCTX-M genes in commensal bacteria will inform future approaches for the prevention of antibiotic-resistant pathogen emergence.

KEYWORDS: Escherichia coli, extended-spectrum β-lactamases, dairy cattle, molecular epidemiology, plasmids, sequence types

INTRODUCTION

Infections caused by Enterobacteriaceae such as Escherichia coli or Klebsiella pneumoniae that are resistant to third-generation cephalosporins are a public health concern because they are challenging to treat, and their prevalence has increased in the United States (1) and worldwide (2) in recent decades. Enterobacteriaceae exhibit resistance to expanded-spectrum cephalosporins mainly by producing extended-spectrum β-lactamases (ESBLs), which are enzymes that hydrolyze β-lactam antibiotics. The ESBL families include TEM, SHV, OXA, CTX-M, and other variants. The CTX-M β-lactamases are of particular interest because they have become the predominant ESBL worldwide (3–5), and more than 120 CTX-M enzymes have been described (6, 7). The success of CTX-M β-lactamase-producing lineages has been attributed to several factors, such as an efficient mobilization and dissemination of CTX-M genes by mobile genetic elements (insertion sequences, transposons, and plasmids), the association of plasmids that harbor CTX-M genes with successful bacterial clones that are widely disseminated, the low fitness cost of CTX-M β-lactamase production, and antibiotic selection pressure (7).

The global problem of E. coli isolates that are resistant to critically important antibiotics requires investigations of how and where antibiotic selection is occurring, where reservoirs of resistant bacterial strains exist, and how these strains disseminate. We recently reported that E. coli isolated from dairy cattle in Washington State in 2008 did not carry CTX-M genes, while isolates collected in 2011 did carry CTX-M genes (8), confirming the recent emergence of these strains in the cattle population in that region. To investigate whether this relatively sudden widespread prevalence of blaCTX-M-carrying E. coli isolates was due to the dissemination of one or a few clones, we applied multilocus sequence typing to determine the genotypes among blaCTX-M-carrying and non-blaCTX-M-carrying E. coli isolates and examined their distributions across farms and regions. To assess the possibility of plasmid horizontal transfer rather than clonal expansion, we also characterized CTX-M genes and plasmids that encode CTX-M enzymes and determined the associations between blaCTX-M alleles, plasmid replicon types, and genotypes. We also compared blaCTX-M alleles, plasmid replicon types, and genotypes from this study with those from other studies, particularly studies of blaCTX-M-carrying E. coli isolates that are known to cause clinical infections in humans.

RESULTS

Comparison of MLST diversity and distribution between blaCTX-M-positive and -negative E. coli isolates.

The sequence type (ST) distribution was less diverse in E. coli isolates carrying blaCTX-M (Simpson's index of diversity [SID] = 0.98; 95% confidence interval [CI], 0.97 to 0.99) than in E. coli isolates lacking blaCTX-M (SID = 0.93; 95% CI, 0.91 to 0.95). We found 76 different STs among the 126 blaCTX-M-negative E. coli isolates and 32 STs among the 126 blaCTX-M-positive E. coli isolates. An eBURST analysis of the blaCTX-M-positive E. coli isolates based on multilocus sequence type (MLST) genotypes identified three clonal complexes (CC10, CC58, and CC88), which accounted for 105 isolates (83.3% of the blaCTX-M-positive E. coli isolates) and 21 singletons (see Table S3 in the supplemental material). CC10 was the predominant clonal complex, which included 6 STs and accounted for 50% of the blaCTX-M-positive isolates, and was found on 16 of 18 farms sampled. Among the 63 blaCTX-M-positive isolates within the CC10 complex, ST744 was most frequent (n = 20), followed by ST48 (n = 16) and ST761 (n = 15). CC58 was the second largest clonal complex, accounting for 9.5% of blaCTX-M-positive E. coli isolates from 5 different farms. The third largest clonal complex (CC88) was also found on 5 separate farms. Overall, 65.1% of blaCTX-M-positive E. coli isolates were associated with these three clonal complexes (Fig. 1; Table 1). As was true for blaCTX-M-positive E. coli isolates, CC10 and CC58 were the most common clonal complexes among the blaCTX-M-negative E. coli isolates but represented smaller proportions of the total (15.1% and 12.7%, respectively). The blaCTX-M-negative E. coli isolates included a significantly higher proportion of singleton STs than the blaCTX-M-positive E. coli isolates (P < 0.05) (Fig. 2; Table 1). When all of the isolates were analyzed together, the blaCTX-M-negative and -positive E. coli isolates were often associated with the same or closely linked STs, with the exception of a few STs; for example, ST44, ST162, and ST361 had 4 to 6 isolates each and were exclusively blaCTX-M positive. The association between ST744, ST761, and ST48 and blaCTX-M carriage is noticeable in this analysis (Fig. 3).

FIG 1.

FIG 1

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Minimum spanning tree analysis of 126 blaCTX-M-positive Escherichia coli isolates based on MLST genotypes. Each circle represents a genotype, and the partitions within a circle are individual isolates. There are three main clonal complexes, and each is surrounded by a gray zone. Thick lines connect genotypes that differ at one locus, and thin lines connect genotypes that differ by two or more loci. Shading indicates the region from which isolates were obtained: pale gray indicates eastern Washington (n = 78) and dark gray indicates northwestern Washington (n = 48).

TABLE 1.

Multilocus sequence types and clonal complexes observed in 126 blaCTX-M-positive and 126 blaCTX-M-negative E. coli isolates from cattle on 18 dairy farms in Washington State

Clonal complexa blaCTX-M-positive E. coli
 blaCTX-M-negative E. coli
ST No. of isolates (%) ST No. of isolates (%)
10 10, 44, 48, 744, 761, 3476 63 (50) 10, 34, 48, 744, 761, 1137, 1415 19 (15.1)
58 58, 155 12 (9.5) 58, 155, 4091, 4092 16 (12.7)
88 23, 88, 90 7 (5.5) 23, 88, 90 5 (3.97)
29 21, 29, 16, 723, 765 11 (8.7)
No founder 14 (11.1)
Singletons 44 (34.9) 61 (48.4)
Total 126 (100) 126 (100)
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a

Clonal complexes were defined using the eBURST algorithm from blaCTX-M-negative E. coli and blaCTX-M-positive E. coli MLST data. The clonal complexes correspond to the predicted founder. “No founder” refers to the inability of the algorithm to predict the founder. The likelihood ratio chi-square P value was ≤0.001 for a 2 × k test for differences in the percentages of each clonal complex within blaCTX-M-negative and blaCTX-M-positive E. coli isolates.

FIG 2.

FIG 2

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Minimum spanning tree analysis of 126 blaCTX-M-negative Escherichia coli isolates based on MLST genotypes. Each circle or node represents a genotype, and the partitions within a node are individual isolates. Each clonal complex is surrounded by a gray zone. Thick lines connect genotypes that differ by one locus, and thin lines connect genotypes that differ by two or more loci. Shading indicates the region from which isolates were obtained: pale gray indicates eastern Washington (n = 78) and dark gray indicates northwestern Washington (n = 48).

FIG 3.

FIG 3

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Minimum spanning tree analysis of 252 Escherichia coli isolates based on MLST genotypes. Each circle represents a sequence type, and the partitions within each circle represent individual isolates. Clonal complexes are surrounded by gray zones. Within each circle, dark gray represents blaCTX-M-positive Escherichia coli isolates (n = 126) and light gray represents blaCTX-M-negative Escherichia coli isolates (n = 126).

Human disease-associated sequence types.

ST69 (n = 4) and ST95 (n = 1) were detected among the blaCTX-M-negative E. coli isolates, and one ST69 isolate was blaCTX-M positive. These sequence types are considered pandemic human disease-associated STs (Table 2; Fig. 1 and 2) (9).

TABLE 2.

Distribution of CTX-M genes and plasmid replicon types among 99 blaCTX-M-positive E. coli isolates from dairy cattle in Washington State

No. of isolates
CCa STb CTX-M group 1
 CTX-M group 9
 Plasmid replicon types
CTX-M-15 CTX-M-55 CTX-M-14 CTX-M-24 CTX-M-27 CTX-M-65 I1 FIB NT FIA/FIB N FIA F FIA/I1 B/0 A/C
CC10 ST761 4 1 8 3 5 1 1 2
ST48 1 1 5 2 2 1 3
ST744 10 2 4 7 3 2 1 2 1
ST10 2 1 1
ST4096 1 1
ST44 5 1 4
ST685 1 1
CC58 ST58 4 2 4 2
ST155 5 4 1
CC88 ST23 1 1
ST88 9 3 2 3 1
ST90 1 1
Singletons or no founders ST162 3 2 1
ST86 2 1 3
ST361 4 1 1 1 1
ST540 2 3 1 1 2 1
ST1163 1 1 1 1
ST154 1 1
ST345 1 1
ST448 1 1
ST219 1 1
ST1629 1 1
ST3018 1 1
ST32 1 1
ST4249 1 1
ST227 1 1
ST3042 1 1
ST163 1
ST362 1 1
ST69 1 1
ST746 1 1
ST38 1
ST349 1 1
Total 50 1 16 1 26 5 31 27 10 9 8 7 4 1 1 1
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a

CC, clonal complex.

b

ST, sequence type.

Plasmid replicon types and CTX-M alleles among blaCTX-M-positive E. coli isolates.

Plasmid replicon typing was performed on 99 blaCTX-M-positive E. coli isolates from which we were able to isolate the blaCTX-M-carrying plasmids. Nine replicon types were identified among 89 isolates; an additional 10 isolates could not be typed using the multiplex PCR method (10, 11). IncI1 plasmids were most frequent (n = 31), followed by IncFIB (n = 27), IncFIA/FIB, IncN, IncFIA, and others (Table 3).

TABLE 3.

Relationship of plasmid replicon types and CTX-M genes among 99 blaCTX-M-positive E. coli isolates from cattle on dairy farms in Washington State

Plasmid replicon type No. of isolates
CTX-M group 1
 CTX-M group 9
 Subtotal
CTX-M-15 CTX-M-55 CTX-M-14 CTX-M-24 CTX-M-27 CTX-M-65
IncI1 24 2 4 1 31
IncFIB 8 7 1 10 1 27
IncFIA/FIB 7 1 1 9
IncFIA 1 1 2 3 7
IncF 2 2 4
IncFIA/I1 1 1
IncA/C 1 1
IncB/O 1 1
IncN 1 4 3 8
NTa 6 2 2 10
Total 50 1 16 1 26 5 99
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a

NT, nontypeable: no PCR product was obtained using the standard set of primers for replicon typing (10).

We detected 6 different blaCTX-M alleles, of which the most frequent was blaCTX-M-15 (n = 50), followed by blaCTX-M-27 (n = 26) and blaCTX-M-14 (n = 16), while blaCTX-M-65, blaCTX-M-24, and blaCTX-M-55 were uncommon (Table 3). Overall, blaCTX-M-15 and blaCTX-M-27 were widely distributed among the different STs. There was an association between MLST clonal complexes and CTX-M alleles (P = 0.0008); the isolates in CC10 tended to carry blaCTX-M-15 or blaCTX-M-27 alleles; those in CC88 mostly carried blaCTX-M-15 alleles (Fig. 4).

FIG 4.

FIG 4

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Distributions of blaCTX-M genes among the different MLST genotypes in blaCTX-M-positive E. coli isolates (n = 99) from dairy cattle in Washington State. NT, nontypeable.

There was an association between replicon types and blaCTX-M alleles (P = 0.007). For instance, IncI1 and IncFIA/FIB predominantly carried blaCTX-M-15 alleles, while IncFIB carried blaCTX-M-27, blaCTX-M-15, and blaCTX-M-14 alleles in almost equal proportions. There was no clear association between clonal complex and plasmid replicon type, including the 10 plasmids that were nontypeable (P = 0.178) (Table 2).

Geographic distribution of MLSTs.

The four major clonal complexes did not differ significantly in the proportions that were found in the two regions of Washington State (Table S3).

Antibiotic resistance among blaCTX-M-positive and blaCTX-M-negative E. coli isolates.

The proportions of blaCTX-M-positive E. coli isolates with resistance to ampicillin, ceftiofur, cephalothin, chloramphenicol, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim-sulfamethoxazole were higher than those of blaCTX-M-negative isolates (Table 4).

TABLE 4.

Percentages of blaCTX-M-positive and blaCTX-M-negative E. coli isolates resistant to a panel of 12 antibiotics

Antibiotic Point estimate (% [95% CI])
CTX-M-positive E. coli CTX-M-negative E. coli
Amikacin 4.0 (1.7–9.0) 4.7 (2.2–10.0)
Amoxicillin-clavulanic acid 15.1 (9.9–22.3) 22.2 (15.8–30.2)
Ampicillin 99.2 (95.6–100)a 38.9 (30.8–47.6)a
Ceftiofur 99.2 (95.6–100)a 13.4 (8.6–20.5)a
Cephalothin 99.2 (95.6–100)a 27.0 (20.0–35.3)a
Chloramphenicol 52.3 (43.7–60.9)a 30.2 (22.8–38.7)a
Gentamicin 7.9 (4.4–14.0) 9.5 (5.5–15.9)
Nalidixic acid 35.7 (27.9–44.4)a 7.9 (4.4–14.0)a
Streptomycin 77.8 (69.8–84.2)a 46.0 (37.6–54.7)a
Sulfisoxazole 73.0 (64.7–80.0)a 45.2 (36.8–53.9)a
Tetracycline 86.5 (79.5–91.4)a 51.6 (43.0–60.1)a
Trimethoprim-sulfamethoxazole 66.7 (58.1–74.3)a 27.8 (20.7–36.2)a
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a

Higher percentage of blaCTX-M-positive isolates are resistant than blaCTX-M-negative isolates.

DISCUSSION

We recently reported an increase in commensal E. coli isolates carrying blaCTX-M resistance genes in dairy cattle, and to assess the possibility that this represented the clonal dissemination of one or a few clones of E. coli, we genotyped blaCTX-M-positive and blaCTX-M-negative commensal E. coli isolates from dairy cattle across Washington State. We found that a large proportion of blaCTX-M-positive E. coli isolates belonged to a few MLST clonal complexes, while blaCTX-M-negative E. coli isolates were significantly more diverse with regard to sequence types. The predominance of a few clones among blaCTX-M-positive E. coli isolates is consistent with other studies that report few genotypes accounting for a large proportion of multidrug-resistant strains (9).

The primary clonal complexes of commensal blaCTX-M-positive E. coli isolates were a subset of those found in blaCTX-M-negative E. coli isolates, suggesting a scenario in which the acquisition of blaCTX-M plasmids by E. coli genotypes that were already fit and established in the dairy animal niche resulted in an increase in their fitness in the presence of antimicrobial selection pressure. The wide distribution of blaCTX-M-positive E. coli clones across a broad geography could be the result of clonal dissemination via movement of cattle, humans, contaminated vehicles, feed, and possibly birds and insects (8, 12, 13), followed by a diversification through normal microevolutionary mechanisms, such as mutation and horizontal gene transfer mediated by plasmids and bacteriophages (14, 15). An expectation of this scenario would be that a single ST of E. coli could be associated with a diversity of plasmid types, which is consistent with our observations. For example, in our data, ST744 isolates (n = 16) were associated with six different plasmid types associated with three different blaCTX-M alleles (Table 2).

We detected small numbers of ST69 and ST95, which, like ST131 and ST393, have been referred to as pandemic clonal lineages of human-associated extraintestinal pathogenic E. coli (ExPEC). The ST69 isolates in this study were primarily blaCTX-M negative, which is consistent with reports that ST69 strains rarely produce ESBL. To our knowledge, these were the only human disease-associated pandemic STs detected in our study (9). It has been suggested that food-producing animals are reservoirs of resistant bacteria that cause clinical infections in humans (16–18); however, compelling quantitative evidence is lacking. For example, a systematic review of 34 original studies found that six studies provided evidence for whole bacterium transmission, and 13 studies provided evidence for the transmission of resistance via mobile genetic elements from food animals to humans; most of these were of poultry or retail meats. Conversely, findings from 17 studies did not support whole bacterium transmission, and two studies did not provide evidence for the transfer of resistance via mobile genetic elements from food animal sources to humans (19).

Among the STs within the ST10 clonal complex, ST48, ST744, and ST761 were the most frequent and had the strongest association with blaCTX-M (Fig. 1 through 3). Although ST761 was not detected in the northwestern region of Washington State, the other two in that complex (ST48 and ST744) were detected in both regions, suggesting that they represent widely disseminated types (Fig. 2). ST744 has been frequently reported from widely dispersed locations around the world, including from food animals in Australia (20), broiler chickens in Algeria (21), bovine mastitis in Germany (22), cattle in France (23), and hospitalized patients in Hong Kong (24), many with ESBL and/or carbapenem resistance characteristics. ST48 has also frequently been reported from diverse sources and locations and is reportedly associated with resistance (25–30).

The IncI1 and IncF plasmid families were the most frequent replicon types detected in this study, which is consistent with reports that IncI1 and IncF were the most frequent replicon types among blaCTX-M-15-carrying plasmids in Enterobacteriaceae from human, livestock, and environmental sources in European studies (31–33). IncFIB has been reported to be the most common replicon type in commensal and pathogenic E. coli isolates originating from avian, human, and poultry meat (34), and it has been associated with virulence (35). Both IncI and IncF plasmid groups are considered to be easily transmissible narrow-host-range plasmids and clustered with each other in an analysis of their gene content in comparison with that of IncN, IncP, and IncW plasmids (36, 37). Unlike IncI1 and IncFIA/FIB that were mainly associated with blaCTX-M-15, IncFIB, the second most common replicon type in this study, was associated with blaCTX-M-15, blaCTX-M-14, and blaCTX-M-27 genes (Table 3).

blaCTX-M-15 was the most frequent blaCTX-M allele in this study, followed by blaCTX-M-27 and blaCTX-M-14 (Table 3). Consistent with our findings, blaCTX-M-15 was the dominant allele among cattle-derived ESBL-producing E. coli isolates in the United Kingdom and Lebanon (38, 39). In contrast with our results, the prevalence of blaCTX-M-15-producing E. coli was reported to be low among ESBL-producing E. coli isolates from nonhuman sources (33, 40). A review of ESBL genes in E. coli isolates from human and nonhuman sources from Europe and Asia found that overall, blaCTX-M-1 was the most frequent allele in cattle/pig-derived isolates (72%), followed by blaCTX-M-14 (14%) and blaCTX-M-15 (8%); however, regional differences in prevalence were observed (40). Other studies indicate that blaCTX-M-14 is prevalent among cattle-derived ESBL-producing E. coli isolates (41, 42), while blaCTX-M-27 is uncommon in ESBL-producing E. coli isolates from cattle/pigs (40). Whereas the real explanation for the observed differences in the distribution of blaCTX-M alleles is unknown, plausible explanations include environmental or geographical factors, differences in β-lactamase activity against specific antimicrobial substrates, regional founder effects, the mobility of plasmids that mediate these genes, and other resistance and virulence determinants (43, 44).

Conclusion.

We observed low MLST diversity among commensal E. coli isolates carrying blaCTX-M in dairy cattle across Washington State and a lack of association between geographic region and MLST clonal complexes, suggesting that clonal bacterial spread played a role in the rapid increase of blaCTX-M prevalence that was observed between 2008 and 2012 (8). However, we did not detect any association between the blaCTX-M plasmid replicon types and the ST or clonal complexes of their host bacteria. In addition, the blaCTX-M-negative E. coli isolates, although more diverse, included the same clonal complexes that were prominent among the blaCTX-M-positive E. coli isolates. Certain E. coli STs are apparently host associated, while blaCTX-M plasmid replicon types are found in multiple vertebrate hosts and have been referred to as “epidemic plasmids,” suggesting an important role of horizontal gene transfer in the epidemiology of blaCTX-M-associated E. coli (45).

To elucidate all of the causal factors driving a population change in resistant bacteria, we would need a complex model of multiple interacting forces, including animal movements, environmental distribution systems such as water flow, local antimicrobial use, and selective pressure, and the fitness of bacteria due to plasmid and other genetic acquisitions. Ideally, such a model could simulate changes in any of these factors to predict changes in commensal as well as clinical bacterial populations that may impact public health.

MATERIALS AND METHODS

Sample collection.

Samples for this study were collected during the summer and fall of 2012 as described in a previous study (8). The 30 dairy farms in that study were chosen based on their willingness to collaborate and their geographic dispersion across the state. For 21 of the 30 farms sampled in that study, susceptible E. coli isolates with no blaCTX-M were available because nonselective as well as selective media were used, but three of those 21 farms had no E. coli isolates carrying blaCTX-M. To be able to compare the genotypes of E. coli isolates with no blaCTX-M to genotypes of E. coli isolates carrying blaCTX-M, we used isolates from the remaining 18 dairy farms. They were located in northwestern (region 1) and eastern (region 2) Washington State. Fecal samples were collected from the rectums of individual calves or pooled from fresh fecal pats from adult lactating cow pens, were placed on ice, and were transported to the laboratory for processing (8).

Bacterial culture, isolation, and confirmation.

Escherichia coli isolates were cultured from fecal samples as previously described (46). Briefly, each sample was directly plated onto unsupplemented MacConkey agar and incubated overnight, and three presumptive E. coli colonies were inoculated in a 96-well plate containing brain heart infusion (BHI) broth and incubated. Buffered glycerol was added to each well, and the plates were stored at −80°C. In addition, to select for ESBL strains, each fecal sample (5 g) was placed in nutrient broth supplemented with cefotaxime (2 μg/ml) (Research Products International Corp., Mount Prospect, IL, USA) and incubated overnight. Then, 100 μl nutrient broth was plated onto MacConkey agar plates supplemented with cefepime (4 μg/ml) (Apotex Corp., Weston, FL, USA). Similarly, three single colonies were picked from each plate, inoculated in 96-well plates, and stored. The isolates were confirmed as E. coli by PCR amplification of the uidA gene (47).

Detection of the blaCTX-M gene and isolate selection.

The presence of blaCTX-M was determined by PCR (48). We grouped preweaned calves into age groups of 7 days each (1 to 7 days, 8 to 14 days, 14 to 21 days, 22 to 28 days, 35 to 42 days, 43 to 49 days, 50 to 56 days, and 57 to 63 days) and adults into one age group. From each group, one to four samples were chosen by generating a random number list using the Rand function in Excel version 3 (Microsoft, Redmond, WA), sorting by random number within the group, and choosing the first isolate in the list. Because we selected similar numbers of blaCTX-M-positive and -negative isolates from each age group, we could not make inferences about the distribution of CTX-M by animal age. From each of the 18 farms, an average of 14 isolates (standard deviation [SD], 5.5), including one to four from adult cows and 1 to 4 from each age group of preweaned calves, were chosen for a final isolate set of 126 blaCTX-M-negative and 126 blaCTX-M-positive isolates (total, 252 isolates). Ninety-six isolates were from farms in northwestern Washington and 156 isolates were from farms in eastern Washington (see Table S1 in the supplemental material). To add to the sample size of isolates for the purpose of studying the distribution of plasmid types and blaCTX-M alleles, an additional 23 isolates from farms not included in the current study, but from which plasmids could be isolated, were used. These additional isolates were obtained from nine different farms in eastern Washington (region 2) during the previous study (8), one to three isolates per farm. They were obtained during the same time period as the 252 isolates that were chosen for the MLST comparison between blaCTX-M-carrying and noncarrying E. coli isolates. Although northwestern Washington (region 1) was not represented in this additional set, the aim was to get as complete a picture as possible of replicon types, and so we used these for that purpose only (8, 49) (see Table S2).

Nucleic acid extraction.

Each isolate was streaked on a blood agar plate, and a single colony was inoculated into 3 ml BHI broth and incubated overnight at 37°C. Genomic DNA from each isolate was extracted using a Qiagen DNeasy blood and tissue kit according to the manufacturer's instructions (Qiagen Sciences, Inc., Germantown, MD) and stored at 4°C.

MLST determination.

All isolates were genotyped according to the Achtman seven-locus MLST scheme. Briefly, internal fragments of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) were amplified using published primers and protocols (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/documents/primersColi_html). The amplified products were sequenced bidirectionally, the raw trace files were read, and the sequences were assembled into single contigs using BioNumerics version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium). Each unique allele was assigned a number, the allelic profile for each isolate was determined, and sequence types (STs) were assigned using BioNumerics (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). The sequences of novel alleles were submitted to the curator for new allele and ST assignment. The genotypic relationships among isolates were determined by generating minimum spanning trees using BioNumerics. Within a clonal complex, each ST has to match with at least one other ST at 6 loci. The eBURST algorithm was used to determine the founder ST of a clonal complex (http://eburst.mlst.net/).

Antibiotic resistance testing.

The 252 E. coli isolates were tested for resistance to 12 antibiotics (amikacin, 30 μg; amoxicillin-clavulanic acid, 20/10 μg; ampicillin, 10 μg; ceftiofur, 30 μg; cephalothin, 30 μg; chloramphenicol, 30 μg; gentamicin, 10 μg; nalidixic acid, 30 μg; streptomycin, 10 μg; sulfisoxazole, 0.25 μg; tetracycline, 30 μg; and trimethoprim-sulfamethoxazole, 1.25/23.75 μg) using the disc diffusion assay according to the Clinical and Laboratory Standards Institute guidelines (50).

CTX-M groups and types.

To determine blaCTX-M groups, we first screened for blaCTX-M group 1 using previously described protocols and primers, followed by PCR to detect blaCTX-M group 9 (51). To determine blaCTX-M types, we performed PCR using sequencing primers as previously described (46, 52). The primers used to determine blaCTX-M groups and types are shown in Table 5. The PCR products were submitted to Functional Biosciences (Madison, WI, USA) for sequencing, and the specific blaCTX-M alleles were identified using the NCBI Basic Local Alignment Search Tool (53).

TABLE 5.

PCR Primers used in this study

Target Primer name Sequence (5′→3′) Reference
blaCTX-M Pan CTX-M forward TTT GCG ATG TGC AGT ACC AGT AA 48
Pan CTX-M reverse CGA TAT CGT TGG TGG TGC CAT A
blaCTX-M (group I) CTXM1-F3 forward GAC GAT GTC ACT GGC TGA GC 59
CTXM1-R2 reverse AGC CGC CGA CGC TAA TAC A
blaCTX-M (group II) TOHO1-2F forward GCG ACC TGG TTA ACT ACA ATC C 59
TOHO1-1R reverse CGG TAG TAT TGC CCT TAA GCC
blaCTX-M (group III) CTXM8.WSAgroupIII.F forward AGA CCT GAT TAA CTA CAA TCC CAT TA 51
CTXM8.WSAgroupIII.R reverse ACT TTC TGC CTT CTG CTC TGG C
blaCTX-M (group IV) CTXM914F forward GCT GGA GAA AAG CAG CGG AG 59
CTXM914R reverse GTA AGC TGA CGC AAC GTC TG
blaCTX-M sequencing primers CTXM-1 upstream forward ATG TTG TTG TTA ATT CGT CTC 46
CTXM-1 upstream reverse CGT TAT CGC TGT ACT GTA G
CTXM-2 downstream forward TTA ACT ATA ATC CGA TTG CG 46
CTXM-2 downstream reverse TTT CTG CCT TAG GTT GAG
blaCTX-M (CTX-M-9 group) M9 upper forward ATG GTG ACA AAG AGA GTG CA 60
M9 lower reverse CCC TTC GGC GAT GAT TCT C
uidA UAL-754 forward AAA ACG GCA AGA AAA AGC AG 47
UAR-900 Reverse ACG CGT GGT TAC AGT CTT GCG
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Plasmid isolation, characterization, and replicon typing.

To isolate and characterize blaCTX-M-associated plasmids, two procedures were used. Conjugation experiments were performed by growing blaCTX-M-positive E. coli and GeneHog recipient cells (Invitrogen) separately in LB broth at 37°C for 6 h. Then, blaCTX-M-positive E. coli and GeneHog recipient cells were plated together on LB medium supplemented with rifampin (100 μg/ml) and cefepime (16 μg/ml) to select for recipient cells carrying the blaCTX-M plasmid. Conjugants were screened by denaturing chromosomal DNA using alkaline sodium dodecyl sulfate, removing protein and cell debris by phenol-chloroform extraction (54), and confirming the presence of blaCTX-M using PCR (48). If conjugation was not successful, plasmid DNA was extracted using a GeneJET plasmid Midiprep kit (Thermo Fisher Scientific, Waltham, MA, USA) or by using a published protocol for larger plasmids (55). The plasmid DNA was then electroporated into competent E. coli DH10B or GeneHog cells (Invitrogen) in 2-mm-path-length cuvettes using a GenePulser (Bio-Rad). Transformants were immediately placed into SOC medium (MP Biomedicals, Solon, OH, USA) (500 ml) and incubated for 1 h at 37°C with agitation (300 rpm). Transformants were then plated on LB agar plates supplemented with cefepime (16 μg/ml). The isolates were confirmed to have a single plasmid by preparing plasmid DNA as above (54), and the blaCTX-M gene was confirmed using PCR (48).

Plasmid replicon typing was performed as described by Johnson and Nolan (10), using a modification of the PCR method described by Carattoli et al. (11). The procedure involves three multiplex PCR panels using previously described primer pairs, targets, control strains, and run conditions, and the approach can detect 18 replicon types.

Statistical analyses.

The proportions of blaCTX-M-carrying and non-blaCTX-M-carrying E. coli isolates that were resistant to the antibiotics tested were compared using R package Hmisc. The associations between genotype and blaCTX-M allele and between plasmid replicon type and blaCTX-M allele were determined using Pearson's chi-square test of independence in R software. The comparisons between proportions were tested for significance using the Pearson's chi-square test in the software WinPepi (56). The Simpson's index of diversity was calculated according to the formula developed by Hunter and Gaston (57), and 95% confidence intervals surrounding the indices of diversity were calculated using the formula of Grundmann et al. (58).

Supplementary Material

Supplemental material
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ACKNOWLEDGMENT

This project was supported by funding from National Integrated Food Safety Initiative (NIFSI; grant no. 2010-51110-21131) from the USDA National Institute of Food and Agriculture.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02430-17.

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