Virulent and toxigenic Escherichia coli strains are responsible for a number of diseases in humans and other animals, ranging from diarrhea in children and neonatal livestock to life-threatening extraintestinal infections. In humans, strains of ETEC are responsible for 280 to 400 million cases of diarrhea in children and approximately 380,000 deaths annually.1 In fact, ETEC is the most common cause of traveler's diarrhea, a worldwide public health problem of major proportions. Enteropathogenic E coli express intimin, an outer membrane protein, which is responsible for the intimate attachment of the bacteria onto ileal enterocytes followed by destruction of surface microvilli2 that subsequently leads to malabsorption and osmotic diarrhea.3
Cattle and other domestic ruminants have been considered the major reservoir of STEC.4,5 The STEC group of toxigenic E coli constitutes an important emerging group of zoonotic foodborne pathogens.6–8 The STEC group includes E coli O157:H7 as well as numerous non-O157 serogroups of potential public health importance.6,9,10 Shiga-toxin–producing E coli are responsible for severe diseases in humans and other animals mediated by cytolytic toxins (Stx1 and Stx2) that interfere with protein biosynthesis in target cells.11 Although these toxins may exist independent of each other, concurrent expression of Stx1, Stx2, and intimin has also been reported.3 Acting together, the 3 toxins are often associated with an acute inflammatory response that leads to serious mucosal damage in the small intestine and manifests as a bloody mucoid diarrhea. In addition to causing severe diarrhea in neonatal ruminants,2 STEC are responsible for fulminant diseases in humans, including hemorrhagic colitis and hemolytic uremic syndrome.6,12 Hemolytic uremic syndrome, in particular, is a life-threatening disease characterized by acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia that often progresses into thrombotic thrombocytopenic purpura.10 Concurrent expression of STa with Stx1, Stx2, and intimin has been reported for neonatal ruminants with diarrhea.13,14 Ruminant-specific ETEC strains also frequently coexpress STa or heat-labile enterotoxin13,14 together with colonization factors such as K99 and F41.15
Similar to the situation in other bacteria, many of the drug-resistance mechanisms in E coli are associated with chromosomal and mobile genetic determinants that encode resistance effector molecules. Of importance to public and animal health, drug-resistance-associated genetic elements may also encode virulence factors.16,17 In a study17 conducted in India, investigators detected a significant association between a number of virulence factors and expression of extended-spectrum β-lactamase by extraintestinal E coli. Similar studies16,18 based on E coli isolates from chickens have also revealed concurrent expression of virulence factors and antimicrobial-resistance genetic factors. In another study,19 sequence analysis was used to characterize a hybrid plasmid that coencodes an MDR E coli phenotype and virulence factors. In that study,19 investigators clearly detected a link between the MDR phenotype and multiple virulence factors. Against a background of limited therapeutic options, multivirulent MDR bacteria17–19 pose a serious emerging challenge to animal and public health that needs to be urgently addressed. Fortunately, reports20–22 on virulence factor–based multivalent vaccines provide evidence that immunization may be a practical control strategy to adopt against infections caused by MDR bacterial pathogens. The purpose of the study reported here was to assess samples obtained from diarrheic neonatal calves and determine the prevalence of multivirulent MDR E coli important in animal and public health.
Materials and Methods
Sample—A total of 97 E coli isolates were cultured from 97 intestinal or fecal samples obtained from neonatal (≤ 2 weeks old) calves with diarrhea. The calves were raised at various beef or dairy farms located within North Dakota, western Minnesota, and northern South Dakota. Samples were collected by veterinary pathologists from dead calves during necropsy and by veterinarians or producers from live diarrheic neonatal calves. Samples were submitted to the North Dakota State University Veterinary Diagnostic Laboratory for routine testing. The analysis of samples was conducted at the Veterinary Diagnostic Laboratory and the Department of Veterinary and Microbiological Sciences at North Dakota State University during January to May 2010.
Antimicrobial susceptibility testing—Susceptibility profiles of the E coli isolates were determined for 17 antimicrobial agents via the broth dilution antimicrobial susceptibility assay by use of the MIC susceptibility system.a The 17 antimicrobial agents belonged to 9 classes: penicillins (ampicillin and penicillin), cephalosporins (ceftiofur), aminoglycosides (gentamicin, neomycin, and spectinomycin), tetracyclines (chlortetracycline and oxytetracycline), macrolides (tiamulin, tilimicosin, tulathromycin, and tylosin tartrate base), lincosides (clindamycin), quinolones (danofloxacin), sulfonamides (sulfadimethoxine and sulfamethoxazole-trimethoprim), and amphenicols (florfenicol).
Each of the 97 E coli isolates was streaked on MacConkey agar and incubated at 37°C for 18 to 24 hours. The following day, inocula were prepared by suspending 1 or 2 colonies in 5 mL of sterile double-distilled water; the bacterial suspensions were then standardized to 0.5 McFarland units with the built-in nephelometer in the autoinoculator of the MIC susceptibility system. Ten microliters of each bacterial suspension was obtained with a calibrated disposable inoculating loopb and suspended in cation-adjusted Mueller-Hinton broth with Tris EDTA–sodium chloride buffer. A dosing head was placed on the tube containing the Mueller-Hinton broth, and 50 μL of bacterial suspension was inoculated with the autoinoculator into a 96-well bovine-porcine MIC plate. The MIC plates were incubated at 35°C for 18 to 24 hours, after which they were evaluated via the MIC susceptibility system autoreader. Isolates resistant to ≥ 3 classes of the antimicrobial agents were categorized as MDR E coli.
Multiplex PCR testing—Bacterial samples were selected from a single colony after overnight growth or from a pure culture. Samples were collected with a pipette tip and suspended in 200 μL of nuclease-free water. All bacterial suspensions were boiled for 10 minutes and centrifuged at 16,000 × g for 30 seconds to pellet bacterial debris. Five microliters of the supernatant was used as the DNA template in the multiplex PCR assay.
The PCR reaction and primer pairs used to amplify the virulence genes in the E coli isolates were incorporated into a multiplex PCR reaction as described elsewhere.3 Six virulence factors (Stx1, Stx2, STa, intimin, F41, and K99) were evaluated via multiplex PCR assay with primer pairs (Appendix). The multiplex PCR assay was performed in a 25-μL reaction that contained 1× Taq reaction buffer,c a final concentration of 3mM MgCl2, 0.2mM deoxyribonucleotide triphosphate mix, 2μM of each primer, 1 U of DNA polymerase,d and 5 μL of DNA template. Amplification was conducted in a thermocycler with the following cycling conditions: 95°C for 2 minutes; 35 cycles at 94°C for 30 seconds, 57°C for 90 seconds, and 72°C for 90 seconds; a final incubation at 72°C for 10 minutes; and holding at 4°C. Multiplex PCR products were examined on 3% agarose gels after electrophoresis at a constant voltage of 150 V for 80 minutes; gels then were stained with 0.5 μg of ethidium bromide/mL and developed by UV transillumination. A 100-bp DNA laddere was used to identify amplified products.
Data analysis—Data were entered into a spreadsheet program.f Graphs were created, which provided visual insight into the distribution of proportions among the virulent and avirulent E coli isolates cultured from samples obtained from the diarrheic neonatal calves. To quantify the proportions and assess significant differences of multivirulent MDR E coli, the data were analyzed with a statistical program.g Further statistical analysis to compare the proportions of susceptible and MDR E coli was conducted via a 2-sample test of proportions. The z test statistic (large sample test) was used to provide the P value to reject the null hypothesis. When the proportions were 100%, no test was performed. Results were considered significant at values of P < 0.05.
Results
Results of antimicrobial susceptibility testing—Analysis of the multiplex PCR results for the 6 virulence factors revealed that 23 of 97 (23.7%) E coli isolates were virulent, whereas 74 of 97 (76.3%) were avirulent. Further analysis revealed that 20 of 23 (87.0%) virulent E coli isolates were MDR (ie, resistant to ≥ 3 classes of antimicrobial agents). Because of an extreme pattern of resistance against almost all of the 17 antimicrobial agents, 2 of the 20 virulent MDR E coli isolates could also be defined as extensively drug resistant (Table 1).
Results of a multiplex PCR assay used to determine the expression patterns of 6 virulence factor genes in 23 Escherichia coli isolates cultured from samples obtained from diarrheic neonatal (≤ 2 weeks old) calves.
| Identification No.* | E coli virulence factor gene | Location of farm of origin | |||||
|---|---|---|---|---|---|---|---|
| Stx1 | Intimin | F41 | K99 | STa | Stx2 | ||
| 10–67 | + | + | − | − | − | − | New Salem, ND |
| 10–751 | − | − | + | + | + | − | Page, ND |
| 10–753 | − | − | + | + | − | − | Sebeka, Minn |
| 10–1188 | − | − | − | − | + | − | Cavalier, ND |
| 10–1414 | − | − | + | + | + | − | Kintyre, ND |
| 10–1591 | − | − | + | + | + | − | New Salem, ND |
| 10–1702 | + | − | + | + | + | − | Middle River, Minn |
| 10–1753 | + | + | + | − | − | − | Rutland, ND |
| 10–2021 | − | − | − | + | + | − | Morristown, SD |
| 10–20821 | + | + | − | − | − | − | Fort Ransom, ND |
| 10–2355 | − | − | + | − | − | − | Ashley, ND |
| 10–24142 | − | − | + | + | + | − | Buchanan, ND |
| 10–27033 | + | + | − | − | − | − | Claire City, SD |
| 10–2741 | − | − | + | + | + | − | Jamestown, ND |
| 10–2865 | − | − | + | + | + | − | Litchville, ND |
| 10–29164 | − | − | + | + | + | − | Glen Ullin, ND |
| 10–2917 | − | + | − | − | − | − | Flasher, ND |
| 10–3027 | + | − | − | + | + | − | Halma, Minn |
| 10–3128 | + | − | − | + | − | − | Glenfield, ND |
| 10–3165 | + | + | − | − | − | − | Red Lake Falls, Minn |
| 10–3239 | − | − | + | + | − | − | Enderlin, ND |
| 10–34125 | + | − | + | + | − | − | Minot, ND |
| 10–34216 | − | − | + | + | + | − | Long Prairie, Minn |
Identification numbers with a superscript numeral correspond to the 6 virulent MDR E coli strains included in multiplex PCR gels.
+ = Positive result. − = Negative result.
The 23 virulent E coli isolates had variations in resistance to the 17 antimicrobial agents (Table 2). When the susceptibility data for the virulent E coli were considered on the basis of the various classes of antimicrobial agents, the highest resistance was detected against each of 3 macrolide-lincoside antimicrobial agents (all 23 [100%] virulent isolates were resistant against tiamulin, tilmicosin, and clindamycin). Interpretation of the MIC data was not possible for tulathromycin and tylosin tartrate base, which were the other 2 macrolides included in the antimicrobial susceptibility testing panel. For the tetracyclines, 20 of 23 (87.0%) virulent E coli isolates were resistant against both chlortetracycline and oxytetracycline. For the penicillins, 23 of 23 (100%) virulent isolates were resistant against penicillin, and 17 of 23 (74.0%) were resistant against ampicillin. Notably, variations in resistance were seen for the 2 sulfonamides, of which 17 of 23 (74.0%) virulent isolates were resistant against sulfadimethoxine and 10 of 23 (43.5%) virulent isolates were resistant against trimethoprim-sulfamethoxazole. The least resistance (highest susceptibility) was detected for ceftiofur (a cephalosporin; only 2/23 [8.7%] virulent isolates were resistant), gentamicin (an aminoglycoside; 4/23 [17.4%] virulent isolates were resistant), and spectinomycin (an aminoglycoside; 9/23 [39.1%] virulent isolates were resistant).
Results (number [%]) of antimicrobial susceptibility testing of 23 virulent E coli isolates cultured from samples obtained from diarrheic neonatal calves.
| Antimicrobial | Resistant | Susceptible | Intermediate | No interpretation possible |
|---|---|---|---|---|
| Ampicillin | 17 (74.0) | 6 (26.1) | 0 (0) | 0 (0) |
| Ceftiofur | 2 (8.7) | 21 (91.3) | 0 (0) | 0 (0) |
| Chlortetracycline | 20 (87.0) | 2 (8.7) | 1 (4.4) | 0 (0) |
| Clindamycin | 23 (100) | 0 (0) | 0 (0) | 0 (0) |
| Danofloxacin | 0 (0) | 0 (0) | 0 (0) | 23 (100) |
| Florfenicol | 15 (65.2) | 0 (0) | 8 (34.8) | 0 (0) |
| Gentamicin | 4 (17.4) | 16 (69.6) | 3 (13.0) | 0 (0) |
| Neomycin | 17 (74.0) | 6 (26.1) | 0 (0) | 0 (0) |
| Oxytetracycline | 20 (87.0) | 2 (8.7) | 1 (4.4) | 0 (0) |
| Penicillin | 23 (100) | 0 (0) | 0 (0) | 0 (0) |
| Spectinomycin | 9 (39.1) | 0 (0) | 14 (61.0) | 0 (0) |
| Sulfadimethoxine | 17 (74.0) | 6 (26.1) | 0 (0) | 0 (0) |
| Tiamulin | 23 (100) | 0 (0) | 0 (0) | 0 (0) |
| Tilmicosin | 23 (100) | 0 (0) | 0 (0) | 0 (0) |
| Trimethoprim-sulfamethoxazole | 10 (43.5) | 13 (56.5) | 0 (0) | 0 (0) |
| Tulathromycin | 0 (0) | 0 (0) | 0 (0) | 23 (100) |
| Tylosin tartrate base | 0 (0) | 0 (0) | 0 (0) | 23 (100) |
Analysis of the antimicrobial susceptibility data revealed that 60 of 74 (81.0%) avirulent E coli isolates were also MDR (Table 3). The 74 avirulent isolates also had variation in resistance against the antimicrobial agents. They were most resistant against clindamycin (74/74 [100%]), penicillin (74/74 [100%]), sulfadimethoxine (62/74 [83.8%]), and tiamulin (74/74 [100%]) and had the lowest resistance against gentamicin (8/74 [10.8%]).
Results (number [%]) of antimicrobial susceptibility testing of 74 avirulent E coli isolates cultured from samples obtained from diarrheic neonatal calves.
| Antimicrobial | Resistant | Susceptible | Intermediate | No interpretation possible |
|---|---|---|---|---|
| Ampicillin | 41 (55.4) | 33 (44.6) | 0 (0) | 0 (0) |
| Ceftiofur | 19 (25.7) | 55 (74.3) | 0 (0) | 0 (0) |
| Chlortetracycline | 54 (73.0) | 18 (24.3) | 2 (2.7) | 0 (0) |
| Clindamycin | 74 (100) | 0 (0) | 0 (0) | 0 (0) |
| Danofloxacin | 0 (0) | 0 (0) | 0 (0) | 74 (100) |
| Florfenicol | 43 (58.1) | 2 (2.7) | 29 (39.2) | 0 (0) |
| Gentamicin | 8 (10.8) | 66 (89.2) | 0 (0) | 0 (0) |
| Neomycin | 44 (59.5) | 30 (40.5) | 0 (0) | 0 (0) |
| Oxytetracycline | 55 (74.3) | 19 (25.7) | 0 (0) | 0 (0) |
| Penicillin | 74 (100) | 0 (0) | 0 (0) | 0 (0) |
| Spectinomycin | 26 (35.1) | 7 (9.5) | 41 (55.4) | 0 (0) |
| Sulfadimethoxine | 62 (83.8) | 12 (16.2) | 0 (0) | 0 (0) |
| Tiamulin | 74 (100) | 0 (0) | 0 (0) | 0 (0) |
| Tilmicosin | 74 (100) | 0 (0) | 0 (0) | 0 (0) |
| Trimethoprim-sulfamethoxazole | 27 (36.5) | 47 (63.5) | 0 (0) | 0 (0) |
| Tulathromycin | 0 (0) | 0 (0) | 0 (0) | 74 (100) |
| Tylosin tartrate base | 0 (0) | 0 (0) | 0 (0) | 74 (100) |
Results of multiplex PCR assay—Of the 97 E coli isolates cultured from samples obtained from diarrheic neonatal calves and subsequently tested with a multiplex PCR assay, 23 (23.7%) had positive results for genes that encode ≥ 1 of the 6 virulence factors, whereas 74 (76.7%) had negative results for any of the 6 virulence factor genes and therefore were categorized as avirulent. Of the 23 E coli isolates that were PCR positive for at least 1 of the 6 virulent factors, 20 (87.0%) were confirmed to be MDR, and 3 (13.0%) were of intermediate antimicrobial susceptibility. On the other hand, 60 of 74 (81.1%) avirulent E coli isolates were MDR, as defined by resistance against ≥ 3 classes of antimicrobial agents.
When the 6 virulence factor genes were considered separately, 15 of 23 (65.2%) virulent E coli isolates had positive results for K99, 14 (60.9%) for F41, 12 (52.2%) for STa, 9 (39.1%) for Stx1, 6 (26.1%) for intimin, and 0 (0%) for Stx2. Of the 23 virulent E coli isolates evaluated, 20 (87.0%) expressed ≥ 2 virulence factors, and only 3 (13.0%) had positive results for 1 virulence factor (STa, F41, and intimin, respectively; Table 1). Furthermore, 8 of 23 (34.8%) virulent E coli isolates evaluated with the multiplex PCR assay expressed STa along with the attachment factors K99 and F41, and 1 of 23 (4.4%) had positive results for the virulence factors STa, F41, intimin, and Stx1. The second most frequent coexpression pattern for virulence factor genes was Stx1 and intimin, which constituted 5 (21.7%) of all virulent MDR E coli isolates. Interestingly, another of the isolates had positive results for 4 virulence factor genes (Stx1, F41, K99, and STa). Results were obtained for multiplex PCR analysis of 6 of 23 virulent and 4 of 74 avirulent E coli isolates (Figure 1). Data obtained from testing of the samples for other enteric pathogens (eg, Cryptosporidium spp, rotavirus, and coronavirus) were not included because they were considered beyond the scope of the present study.
Photograph of a 3% agarose gel stained with ethidium bromide depicting results of multiplex PCR analysis of 6 virulent MDR strains (lanes 1 through 6) and 4 avirulent MDR strains (lanes 8 through 11) of Escherichia coli cultured from fecal or intestinal material obtained from diarrheic neonatal (≤ 2 weeks old) calves. Lane 7 is a positive control E coli strain for all 6 virulence genes, and lane 12 is a negative control (no template). Lane M is a 100-bp DNA ladder. Virulence genes are listed in the left column, with arrows indicating the band or bands that correspond to each virulence gene.
Citation: American Journal of Veterinary Research 73, 12; 10.2460/ajvr.73.12.1944
Graphically, the proportions of virulent and avirulent E coli isolates resistant against the various antimicrobial agents were extremely similar, and there appeared to be no difference in the proportions of resistant isolates in both E coli groups. These results were consistent with results of the statistical analysis conducted with the z test in which values of P > 0.05 implied that there was not a significant difference in antimicrobial resistance between the 2 E coli groups (Table 4). Therefore, it can be concluded that no relationship existed between virulence status and resistance against the antimicrobial agents. However, there was variation in resistance among the antimicrobial agent classes, with some classes having higher resistance than others. A comparison of proportions of resistance of antimicrobial agents was conducted to assess whether a difference existed between virulent and avirulent E coli isolates. Resistance against ceftiofur differed, but not significantly (P = 0.084), between virulent and avirulent isolates.
Comparison of the number (%) of isolates with antimicrobial resistance between 23 virulent and 74 avirulent E coli isolates cultured from samples obtained from diarrheic neonatal calves.
| Antimicrobial | Virulent E coli | Avirulent E coli | P value* |
|---|---|---|---|
| Ampicillin | 17 (73.9) | 41 (55.4) | 0.114 |
| Penicillin | 23 (100) | 74 (100) | ND |
| Ceftiofur | 2 (8.7) | 19 (25.7) | 0.084 |
| Florfenicol | 15 (65.2) | 43 (58.1) | 0.727 |
| Chlortetracycline | 20 (87.0) | 54 (73.0) | 0.168 |
| Oxytetracycline | 20 (87.0) | 55 (74.3) | 0.206 |
| Clindamycin | 23 (100) | 74 (100) | ND |
| Danofloxacin | 0 (0) | 0 (0) | ND |
| Gentamicin | 4 (17.4) | 8 (10.8) | 0.402 |
| Neomycin | 17 (73.9) | 44 (59.5) | 0.210 |
| Spectinomycin | 9 (39.1) | 26 (35.1) | 0.728 |
| Tiamulin | 23 (100) | 74 (100) | ND |
| Tilmicosin | 23 (100) | 74 (100) | ND |
| Tulathromycin | 0 (0) | 0 (0) | ND |
| Tylosin tartrate base | 0 (0) | 0 (0) | ND |
| Trimethoprim-sulfamethoxazole | 10 (43.5) | 27 (36.5) | 0.547 |
| Sulfadimethoxine | 17 (73.9) | 62 (83.8) | 0.369 |
Values were considered to differ significantly at P < 0.05.
ND = Not determined.
Discussion
Analysis of the data for the study reported here revealed a comparable and high prevalence of multidrug resistance among both virulent and avirulent E coli isolates cultured from samples obtained from diarrheic neonatal calves (Tables 2–4). The study findings also indicated a high degree of resistance against the 17 antimicrobial agents tested, except for gentamicin and ceftiofur, to which isolates had relatively high susceptibility, and danofloxacin, tulathromycin, and tylosin tartrate base, for which the interpretation of MIC data was not possible. Similar to results reported in another study,23 findings of the present study suggested that intraspecies exchange of drug-resistance genetic determinants within a mammalian bovine host or environment may have played a role in the phenotypic evolution of the MDR E coli isolates investigated in the study reported here. Class 1 integrons are widely reported among gram-negative bacteria and have been detected with similar frequencies in isolates cultured from clinical cases and in pathogens and commensals isolated from livestock.23,24 Class 1 integrons have been detected in MDR E coli isolates cultured from fecal samples obtained from diarrheic calves.h
In the present study, the highest antimicrobial resistance was against drugs that belong to the macrolide-lincoside, penicillin, and tetracycline classes, whereas intermediate susceptibility was detected for sulfamethoxazole-trimethoprim, and the highest susceptibility was recorded for the aminoglycosides (gentamicin and spectinomycin) and a cephalosporin (ceftiofur). However, the other sulfonamide (sulfadimethoxine) had poor susceptibility, which suggested that trimethoprim most likely potentiated the efficacy of the sulfamethoxazole in the sulfamethoxazole-trimethoprim formulation. Although antimicrobial agent–specific resistance mechanisms cannot be ruled out for the E coli isolates in the present study, such broad-spectrum antimicrobial resistance may be attributed to upregulated drug efflux pumps and altered porin gene expression among the E coli strains. Several investigators have reported that upregulated efflux pumps24–28 and altered membrane porins2,7,29,30 play important roles in drug-resistance mechanisms. Recently, our laboratory group detected upregulated transcription of genes that encode TolC, a component of the membrane-based trimeric AcrAB:TolC pump, and a novel porin (YiaT) in MDR E coli isolates cultured from samples obtained from diarrheic calves.31
For the β-lactam antimicrobial agents in the present study, there was high resistance against drugs that belong to the penicillin class, whereas most of the E coli isolates were highly susceptible to ceftiofur, a cephalosporin. Because both penicillins and cephalosporins belong to the β-lactamase group of antimicrobial agents, findings of the present study would appear to suggest that class A β-lactamases (penicillinases) and not class C β-lactamases (cephalosporinases)32 are likely to predominate among the E coli isolates evaluated. Considering our data from another perspective, it is plausible to conclude that broad-spectrum β-lactamases such as tumor epithelial marker-133 are likely to have an extremely low prevalence among the E coli strains; however, this supposition must be corroborated in future studies. Tumor epithelial marker-1 is a plasmidic β-lactamase frequently found among members of the family Enterobacteriaceae and imparts broad-spectrum resistance against penicillins and cephalosporins alike, except the aminothiazol cephalosporins.33 Given the high resistance against the tetracyclines, the E coli isolates of the present study must also be evaluated for molecular markers of tetracycline resistance (eg, tetA and tetM genes34) and tetracycline-specific efflux systems.35
Of the 97 E coli isolates cultured from samples obtained from the diarrheic calves in the present study, 23 (23.7%) were virulent and 74 (76.3%) were avirulent. Even though some of the genetic determinants (eg, Stx1) may potentially cause severe disease in humans, testing of E coli isolates for all possible virulent factors of public health importance was beyond the scope of the present study. Results of the PCR-positive isolates revealed that attachment factors had the highest prevalence, with 65.2% and 60.9% of the virulence factor–PCR-positive isolates having positive results for the K99 and F41 genes, respectively. These data strongly underscore the need for mandatory inclusion of these attachment factors in vaccines designed to protect neonatal calves against E coli infections. Not surprisingly, STa was the most frequently expressed toxin, constituting 52.2% of the PCR-positive isolates, followed by Stx1 (39.1%) and intimin (26.1%). Interestingly, none of the isolates in the present study had positive results for Stx2.
The status of calves (alive, dead, or moribund and subsequently died) and types of farms (dairy or beef) could potentially have been associated with resistant determinants but were not evaluated in the present study. The most interesting aspect of the experimental data indicated that 20 of 23 (87.0%) virulence factor–positive E coli isolates were MDR and also had a multivirulence factor phenotype. This finding is of concern because it underscores the challenges regarding antibacterial treatment in humans and other animals. The most predominant virulence factor coexpression pattern among the MDR E coli isolates was K99-STa-F41, which constituted 34.8% of all the PCR-positive strains, followed by Stx1-intimin (21.7%) and STa-F41-intimin-Stx1 (4.4%). This information is likely to be useful to animal health scientists who are developing vaccines against agents that cause diarrhea in neonatal calves. Although we did not obtain follow-up information on the outcome of antimicrobial treatment in the calves infected with virulent MDR E coli, the expectation is that there was a high case fatality rate. The experimental data warrant consideration of novel approaches that prioritize antibacterial treatments (eg, vaccination and immunotherapy) that are not based on antimicrobial administration. Multivalent vaccines that incorporate toxigenic and E coli attachment factors have been investigated and can be protective,21 which underscores the fact that such immunogens could be adopted to protect immunized humans and other animals against MDR bacterial infections.
In the study reported here, we detected multivirulent MDR E coli in samples obtained from diarrheic neonatal calves. Most of these MDR E coli isolates carry multiple virulence factors of potential importance for animal and public health. The study findings also indicate a comparable proportion of multidrug resistance among virulent and avirulent E coli, which suggests that there may be a continuous exchange of virulence factor genes between the 2 groups. Detection in diarrheic neonatal calves of multivirulent MDR E coli pathogens with virulence factors important in health of humans and domestic animals underscores challenges for the prevention and treatment of infections in humans and other animals.
Because antimicrobial treatment is becoming less effective, we strongly believe that vaccination will provide a better alternative to the management of such infections. Our research group advocates for a paradigm shift based on developing therapeutic tools for the treatment, prevention, and management of infections caused by MDR pathogens. Specifically, we propose a novel approach that targets bacterial membrane–based efflux pumps or porins (or both) in the development of vaccines against MDR pathogens. This approach is supported by results of a study36 in China in which investigators abrogated chloramphenicol resistance with anti-TolC antibodies in drug-resistant E coli. Future studies will be needed to determine the effectiveness of such an approach.
ABBREVIATIONS
| ETEC | Enterotoxigenic Escherichia coli |
| MDR | Multidrug resistant |
| MIC | Minimum inhibitory concentration |
| STEC | Shiga-toxin–producing Escherichia coli |
Sensititre MIC, TREK Diagnostic Systems Inc, Cleveland, Ohio.
Disposable inoculating loop, Becton, Dickinson & Co, Franklin Lakes, NJ.
Green GoTaq reaction buffer, Promega, Madison, Wis.
GoTaq DNA polymerase, Promega, Madison, Wis.
DNA ladder, Promega, Madison, Wis.
Excel, Microsoft Corp, Redmond, Wash.
SAS, version 9.2, SAS Institute Inc, Cary, NC.
Parmar K. Molecular analysis of highly multi-drug resistant Escherichia coli isolated from scouring calves in North Dakota. MS thesis, Department of Veterinary and Microbiological Sciences, College of Agriculture, Food Systems, and Natural Resources, North Dakota State University, 2007.
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