Gentamicin- and Ciprofloxacin-Resistant Enterobacteriaceae in Cattle Farms in Israel: Risk Factors for Carriage and the Effect of Microbiological Methodology on the Measured Prevalence

Abstract

Our objectives were to establish a methodology for surveillance of ciprofloxacin-resistant Enterobacteriaceae and gentamicin-resistant Enterobacteriaceae (CPRE and GNRE, respectively) in cattle and to study the prevalence and risk factors for carriage of these bacteria in a national survey. This was a point prevalence study conducted from July to October 2013 in Israel. Stool samples were collected from 1,226 cows in 123 sections of 40 farms of all production types. The number of CPRE- and GNRE-positive cows was highest in quarantine stations and fattening farms and was lowest in pasture farms (p < 0.01). The number of CPRE- and GNRE-positive cows was lowest in dairy farm sections containing adult cows (>25 months) and highest in calves (<4 months) (p < 0.001). In bivariate analysis, other variables that were significant risk factors for CPRE and GNRE carriage included fewer troughs, crowding, lack of manure cleaning, and recent arrival of new calves. Antimicrobial prophylaxis was given almost exclusively to calves and was associated with a higher prevalence of carriers (p < 0.001). Compared to the use of nonselective media (MacConkey agar alone), the use of selective media (MacConkey agar with 10 μg/ml of ciprofloxacin or 5 μg/ml of gentamicin) increased the sensitivity of screening for CPRE and GNRE by 6.6- and 13.5-fold, respectively. CPRE and GNRE were identified in 609 (49.7%) and 840 (68.5%) samples, respectively. This study provides novel data regarding both the epidemiology of CPRE and GNRE carriage in livestock and the microbiological methodology for their surveillance.

Introduction

Since the advent of the first antimicrobials, antimicrobial-resistant bacteria (AMRB) have evolved in conjunction with the use of antimicrobial agents targeting them.¹

This observation has attracted attention to the possibility of sources of AMRB acquisition other than healthcare, including the food and livestock industries. As a result, many European countries have initiated surveillance programs for AMRB in livestock animals, including cattle.² These programs regularly test for resistance in two indicator bacteria, E. coli and enterococci. Surveillance samples (e.g., feces) are typically inoculated onto media that are not antimicrobial selective (e.g., MacConkey agar), and antimicrobial susceptibility testing (AST) is performed on randomly selected E. coli isolates. This method is fundamentally different from the AMRB surveillance methods for human samples, where various selective media for a specific resistance trait, for example, extended-spectrum β-lactamase (ESBL),³ are used. Hence, the method used in livestock may lead to underestimation of the prevalence of AMRB in the population being studied.

Beside the β-lactams, aminoglycosides and quinolones are probably the most important groups of antimicrobial agents that are used in human medicine as well as veterinary medicine.^4,5 Although there have been several publications, including a recent publication by our group⁶ regarding the prevalence of ESBL-producing Enterobacteriaceae (ESBLPE) in cattle,^6–8 the prevalence of resistance to other major antimicrobial groups, including aminoglycosides and quinolones, has never been studied in cattle. Furthermore, the effect of antimicrobial consumption and other potential risk factors on the prevalence of these resistance traits has never been studied.

The objectives of this work were (1) to determine the prevalence of gentamicin-resistant Enterobacteriaceae and ciprofloxacin-resistant Enterobacteriaceae (GNRE and CPRE, respectively) carriage among cattle; (2) to analyze risk factors for carriage; and (3) to study the effects of different microbiological methods on the measured prevalence of GNRE, CPRE, and ESBLPE in a nationwide survey of cattle farms in Israel.

Materials and Methods

Study design and data collection

This was a point prevalence study conducted from July to October 2013 in cattle farms from the main farming locations in Israel. A comprehensive description of the study design and the data variables can be found in the first part of this study, which described the prevalence and risk factors for ESBLPE carriage in cattle.⁶

Microbiological methods

Stool (∼1 g) was inoculated in brain-heart infusion (BHI) broth and incubated overnight at 36°C. A broth aliquot of 10 μl was subcultured onto two selective media: (1) GNRE-MacConkey agar with 5 μg/ml of gentamicin⁹ and (2) CPRE-MacConkey agar with 10 μg/ml of ciprofloxacin (Hylabs, Rehovot, Israel).¹⁰ In addition, a subset of 128 specimens that included 40 samples that were tested positive to each of the AMR bacteria was chosen to evaluate the difference in the sensitivity of selective versus nonselective surveillance methods (SSM).

In the SSM, an aliquot of 10 μl was subcultured onto the two selective media described above as well as on CHROMagar™ ESBL agar plates for the detection of ESBL-producing Enterobacteriaceae (Hylabs). Suspicious colonies were defined as lactose-fermenting colonies (MacConkey-based agars) or according to the manufacturer's instructions (CHROMagar ESBL). In the non-selective surveillance method (NSSM), an aliquot of 10 μl was subcultured onto MacConkey agar without antimicrobials and a single lactose-fermenting colony was randomly selected, as practiced by several surveillance programs.^4,5

Identification was performed using the ENTEROTEST™ kit with a Citrate test (Hylabs) or the VITEK-2 system (bioMérieux, France) in equivocal cases. ESBL testing was performed by the combined disk method using ceftazidime and cefotaxime disks alone and with clavulanic acid. AST for gentamicin, ciprofloxacin, tetracycline, streptomycin, trimethoprim–sulfamethoxazole, chloramphenicol, cephalothin, amoxicillin–clavulanate, fosfomycin, ertapenem, colistin, and nitrofurantoin was performed on all the isolates detected in the subset of 128 isolates described above by disk diffusion and interpreted according to CLSI criteria.¹¹ Nonsusceptibility to colistin was first screened for by disk diffusion followed by minimal inhibitory concentration (MIC) testing by gradient method (Etest^®; bioMérieux, France) for isolates showing a diameter of less than 10 mm.

Following the validation of the CPRE/GNRE selective media on the subset of 128 samples (see below), CPRE and GNRE were defined based on growth of lactose-fermenting colonies on their respective selective media.

Data analysis

The two outcomes, GNRE carriage and CPRE carriage, were analyzed separately. Outcomes were reported as the mean prevalence of carriers per section. Because the denominator was identical or similar in nearly all sections (Tables 1 –3), the mean prevalence per section was presented as the numerator only (i.e., the mean number of carriers) rather than as a proportion. For continuous covariates, we calculated the mean and standard deviation (SD) and the prevalence ratio, that is, the change in prevalence of carriers per one unit change in the predictor.¹² For categorical covariates, we calculated the number and percentage of sections in each category, the mean number (±SD) of GNRE or QNRE carriers per section in each category (as the measure of prevalence), and the prevalence ratio.

Table 1.

Farm Types and Prevalence of GNRE and CPRE in Israeli Farms

			GNRE			CPRE
Farm type	Farms (N)	Sections (N)	Mean number of carriers per section ± SD	Prevalence ratio	p	Mean number of carriers per section ± SD	Prevalence ratio	p
Dairy cattle	17	66	5.8 ± 3.1	0.578	<0.01	4.2 ± 3.4	0.418	<0.01
Fattening farm	7	16	9.3 ± 1.4	0.970		7.3 ± 2.8	0.770
Pasture farm	6	13	4.9 ± 3.3	0.492		4.2 ± 3.5	0.417
Mixed pasture and feedlot^a	5	12	8.5 ± 1.7	0.845		4.8 ± 3.0	0.481
Quarantine station	2	4	10.8 ± 1.5	Ref.		10.8 ± 1.5	Ref.
Mixed dairy feedlot^b	3	12	8.3 ± 2.3	0.833		4.8 ± 3.3	0.475

Six sections of pasture and feedlot each.

Nine dairy section and three feedlots.

CPRE, ciprofloxacin-resistant Enterobacteriaceae; GNRE, gentamicin-resistant Enterobacteriaceae; SD, standard deviation.

Table 2.

Descriptive Statistics and Bivariate Analysis of Variables Related to GNRE Carriage in Israeli Cattle

Covariate	Mean ± SD of covariate/category	Mean number of GNRE carriers per section ± SD	No. (%) of sections in category	PR	p
Continuous covariates
Cattle cleanliness (%)	35.1 ± 34.5			1.000	0.975
Trough, N	3.1 ± 2.4			0.959	0.041
Categorical covariates
Age (month)	<4 m	9.1 ± 1.8	32 (26.0)	1.784	<0.001
	5–10 m	7.3 ± 3.1	29 (23.6)	1.433
	11–24 m	5.6 ± 3.1	36 (29.3)	1.112
	>25 m	5.1 ± 3.0	26 (21.1)	Ref.
Trough cleanliness	Dirty	6.3 ± 3.2	16 (13.0)	0.790	0.330
	Partially dirty	6.9 ± 3.3	82 (66.7)	0.868
	Clean	6.7 ± 2.7	21 (17.1)	Ref.
	Missing		4 (3.3)
Cooling system	Fan	6.6 ± 3.0	39 (31.7)	0.984	0.085
	Fan + nebulizers	5.5 ± 3.1	18 (14.6)	0.758
	Nothing	7.3 ± 3.2	66 (53.7)	Ref.
Crowdedness (head/m²)	<5 m²	8.4 ± 2.2	24 (19.5)	1.743	0.003
	5–10 m²	6.6 ± 3.4	23 (18.7)	1.291
	11–20 m²	6.4 ± 3.4	40 (32.5)	1.199
	21–30 m²	7.1 ± 2.9	20 (16.3)	1.368
	>30 m²	5.6 ± 3.2	16 (13.0)	Ref.
Manure cleaning method	Tractor	6.1 ± 3.1	38 (30.9)	0.710	<0.001
	Automatic shovel	6.0 ± 3.0	18 (14.6)	0.698
	Slatted floors	3.0 ± 2.0	6 (4.9)	0.378
	Nothing	7.6 ± 2.8	46 (37.4)	Ref.
	Missing		15 (12.2)
No. of cattle per section	<199	7.8 ± 2.8	18 (14.6)	Ref.	0.374
	200–499	7.2 ± 3.3	46 (37.4)	0.939
	500–799	6.2 ± 3.3	43 (35.0)	0.831
	800–999	6.3 ± 2.8	16 (13.0)	0.831
No. of veterinarian visit/week	0	7.4 ± 3.0	36 (29.3)	0.728	0.582
	1	6.6 ± 3.8	17 (13.8)	0.680
	2	6.6 ± 2.9	60 (48.8)	0.651
	3	5.5 ± 4.0	8 (6.5)	0.550
	5	11.5 ± 2.1	2 (1.6)	Ref.
New cattle in farm in preceding month	None	6.1 ± 3.1	91 (74.0)	0.605	<0.001
	From Israel	8.4 ± 2.3	25 (20.3)	0.858
	From overseas	10.4 ± 1.1	7 (5.7)	Ref.
Antimicrobial prophylaxis	No	6.3 ± 3.1	90 (73.2)	0.699	<0.001
	Yes	8.3 ± 2.8	33 (26.8)	Ref.
Vaccination other than mandatory	Yes	7.1 ± 3.0	23 (18.7)	Ref.	0.737
	No	6.8 ± 3.2	100 (81.3)	0.953

PR, prevalence ratio.

Table 3.

Descriptive Statistics and Bivariate Analysis of Variables Related to CPRE Carriage in Israeli Cattle

Covariate	Mean ± SD of covariate/category	Mean number of CPRE carriers per section ± SD	No. (%) of sections in category	PR	p
Numeric covariate
Cattle cleanliness (%)	35.1 ± 34.5			1.004	0.018
Trough, N	3.1 ± 2.4			0.909	<0.001
Categorical covariate
Age (month)	< 4 m	8.4 ± 2.3	32 (26.0)	2.716	<0.001
	5–10 m	4.3 ± 3.4	29 (23.6)	1.428
	11–24 m	3.6 ± 3.0	36 (29.3)	1.105
	>25 m	3.3 ± 2.6	26 (21.1)	Ref.
Trough cleanliness	Dirty	4.1 ± 3.3	16 (13.0)	0.413	<0.001
	Partially dirty	4.7 ± 3.5	82 (66.7)	0.523
	Clean	6.1 ± 3.7	21 (17.1)	Ref.
	Missing		4 (3.3)
Cooling system	Fan	4.8 ± 3.2	39 (31.7)	0.836	<0.001
	Fan + nebulizers	1.8 ± 1.9	18 (14.6)	0.276
	None	5.9 ± 3.5	66 (53.7)	Ref.
Crowdedness (head/m²)	<5 m²	7.3 ± 3.3	24 (19.5)	2.071	<0.001
	5–10 m²	5.1 ± 3.2	23 (18.7)	1.480
	11–20 m²	4.4 ± 3.3	40 (32.5)	1.024
	21–30 m²	3.6 ± 3.6	20 (16.3)	0.848
	>30 m²	4.3 ± 3.5	16 (13.0)	Ref.
Manure cleaning method	Tractor	3.5 ± 3.1	38 (30.9)	0.457	<0.001
	Automatic shovel	3.7 ± 2.9	18 (14.6)	0.460
	Slatted floors	1.8 ± 1.2	6 (4.9)	0.217
	None	6.4 ± 3.4	46 (37.4)	Ref.
	Missing		15 (12.2)
No. of cattle per section	<199	5.6 ± 3.4	18 (14.6)	Ref.	0.002
	200–499	5.7 ± 3.6	46 (37.4)	1.030
	500–799	4.0 ± 3.4	43 (35.0)	0.655
	800–999	4.8 ± 3.7	16 (13.0)	0.875
No. veterinarian visit/week	0	5.5 ± 3.4	36 (29.3)	0.549	0.230
	1	4.9 ± 3.6	17 (13.8)	0.505
	2	4.5 ± 3.3	60 (48.8)	0.441
	3	4.6 ± 4.6	8 (6.5)	0.445
	5	11.5 ± 2.1	2 (1.6)	Ref.
New cattle in farm in preceding month	None	4.4 ± 3.4	91 (74.0)	0.437	<0.001
	From Israel	5.4 ± 3.2	25 (20.3)	0.549
	From overseas	10.4 ± 1.1	7 (5.7)	Ref.
Antimicrobial prophylaxis	No	4.1 ± 3.2	90 (73.2)	0.505	<0.001
	Yes	7.3 ± 3.4	33 (26.8)	Ref.
Vaccination other than mandatory	Yes	5.2 ± 3.4	23 (18.7)	Ref.	0.832
	No	4.9 ± 3.6	100 (81.3)	0.963

Prevalence ratios were estimated using mixed-effects robust Poisson regression, with farm as a random effect and number of cows within the section as an offset term. Multiple imputation of missing data was performed, but bivariate analyses with the imputed data for the relevant covariates did not converge; therefore, all analyses were conducted with available data only. All covariates with p-values ≤0.1 and with no missing values were entered into the multivariable models. Statistical analyses were conducted using SAS^© version 9.2.

Results

Risk factors for GNRE and CPRE carriage

The study included 1,226 cows housed in 123 sections in 40 farms, the majority of which were dairy farms (Table 1). Overall, GNRE was identified in 840 samples (68.5%) and CPRE in 609 (49.7%) samples. GNRE and CPRE carriage was most common in the quarantine stations and fattening farms and least common in pasture farms (p < 0.01) (Table 1). There was no significant association between geographical location and prevalence of GNRE or CPRE carriage (data not shown).

Descriptive statistics and bivariate analysis of section-level risk factors for GNRE and CPRE carriage are presented in Tables 2 and 3, respectively. In bivariate analysis, the following variables were significantly and positively associated with both GNRE and CPRE carriage: young age, greater crowding, lack of manure cleaning, new cattle arriving on the farm in the previous month, and use of antimicrobial prophylaxis. For both outcomes, having more troughs per sections was protective: for each additional trough, the prevalence of GNRE carriage decreased by 4% and the prevalence of CPRE carriage decreased by 9%. For CPRE only, the presence of a cooling system was protective. Curiously, subjective assessments of the cleanliness of cows and of troughs and a high number of heads per section were also significantly inversely associated with CPRE carriage.

The prevalence of GNRE and CPRE carriage was highest in calves, gradually declining in adult cows (Tables 2 and 3). This pattern was most pronounced in dairy farms and was lacking in pasture farms (for CPRE) and fattening farms (for GNRE) (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr). This suggests that GNRE and CPRE carriage is acquired mostly in calves and gradually resolves with maturation. It is likely that the differences in the prevalence of GNRE and CPRE carriage between farms are the result of the differences in farms’ age structure. For instance, quarantine stations included calves only and consequently had the highest prevalence; pasture farms had no calves younger than 4 months and thus had the lowest prevalence.

In bivariate analysis, sections in which antimicrobial prophylaxis was not used had 30% lower prevalence of GNRE carriage and 49% lower prevalence of CPRE carriage. Antimicrobial prophylaxis was administered in 33 sections (27%) in all farm types. Twenty-six of the 33 sections (79%) where antimicrobial prophylaxis was used housed calves aged <4 months. The most common agents were tetracyclines (n = 26 sections, 79%), either as chlortetracycline or doxycycline. Other agents included norofloxacin (n = 4), cephalosporins (cephalexin or ceftiofur, n = 4), anticoccidiosis agents (n = 4), sulfa agents (n = 3), and gentamicin (n = 1). In eight sections, more than one agent was given, most commonly in addition to tetracycline.

In multivariable analysis (data not shown), no variables were statistically significant. There were nearly significant associations between lack of a cooling system and CPRE carriage (p = 0.08) and between the use of antibiotic prophylaxis and GNRE carriage (p = 0.06).

Effect of sampling methods on detection of GNRE, CPRE, and ESBLPE

The effect of the sampling methodology on detection of GNRE, CPRE, and ESBLPE E. coli was compared on a subset of 128 samples. Using the SSM, we identified 40 (31.2%) samples that were positive for each of these resistance types. The specificity of the selective media used for GNRE and CPRE detection was 100%. Using the NSSM, we identified only 1 ESBLPE (0.7%), 3 GNRE (2.3%), and 6 CPRE (4.6%); in 120 of the 128 samples (93.7%), no AMRB was identified. Thus, the use of SSM increased the sensitivity of detecting AMRB between 6.6-fold (CPRE) and 40-fold (ESBLPE) compared with NSSM. Susceptibility testing for other antimicrobials was performed by the NSSM method only, yielding E. coli resistant to tetracycline in 70 of the 128 samples (54.6%), streptomycin in 33 samples (25.7%), trimethoprim-sulfamethoxazole in 32 samples (25%), chloramphenicol in 11 samples (8.5%), cephalothin in 7 samples (5.4%), amoxicillin–clavulanate in 2 samples (1.5%), and fosfomycin in 1 sample (0.7%). Resistance to ertapenem, colistin, and nitrofurantoin was not identified.

Discussion

The presence of AMRB in livestock has been the focus of increased interest because of its possible role in the dissemination of these bacteria to humans.¹³ Epidemiological data concerning the risk factors for AMRB carriage in animals are relatively scarce, probably due to both the anthropocentric nature of the studies and the methodological difficulties in performing such studies. Among gram-negative bacteria, the resistance mechanism that has been by far the main focus of scientific interest is the ESBL.¹⁴ Thus, epidemiological studies in cattle have focused only on ESBL producers,^8,15 despite the fact that third-generation cephalosporins are not commonly used in veterinary medicine,⁴ and despite the importance of other antimicrobial groups (e.g., quinolones) in both veterinary and human medicine. For these reasons, our study is unique in providing novel data regarding the prevalence and risk factors for CPRE and GNRE carriage in cattle specifically and in livestock in general.

Although several studies have examined the molecular mechanisms underlying CPRE and GNRE in livestock,^16–22 data regarding the prevalence of these bacteria are very limited. The prevalence of GNRE and CPRE carriage in our study was high (68.5% and 49.7%, respectively); it is higher than the prevalence of ESBLPE (23.7%) found in our recent study from the same sample.⁶ Two recent reports of E. coli isolates from clinical cultures in cattle with diarrhea in Korea²⁰ and cattle with endometritis in China²³ found that the prevalence of CPRE was less than half of that found in our study. This difference is explained in part by differences in study design: although described as prevalence studies, the Chinese and Korean studies actually examined proportions of resistant isolates among clinical isolates rather than actual prevalence in their population.

The design of our study more closely resembles that of the AMRB surveillance programs that are routinely performed in several European Union countries.² In these programs, a single E. coli isolate is randomly picked from fecal cultures (one per epidemiological unit) collected at the slaughterhouse and the burden of resistance is measured by determining the proportion of resistant isolates among those picked. The proportions of CPRE and GNRE measured in these programs are much lower than in our study, especially with regard to gentamicin, for which the highest resistance burden (reported from Germany) was only 6.4%. The likely reason why the prevalence of AMR in our study was so much higher than that reported by the European surveillance systems is the difference in the microbiological methodology, that is, the use of antimicrobial-selective media (SSM), as done in our study, versus random testing of isolates (NSSM). In our study, we have demonstrated that SSM is 6.6-fold (CPRE) to 40-fold (ESBLPE) more sensitive than NSSM. Our study underlines the importance of using the SSM methodology to produce sensitive and comparable measurements of AMRB prevalence. Although this may be cumbersome and expensive, it should be used for at least one representative agent in each antimicrobial class tested.

An important contribution of this study is the validation of two MacConkey-based selective media for GNRE and CPRE surveillance in cattle. These media were previously used in a few human studies^9,10 but were never used in livestock. Both were found to be 100% specific, omitting the need for confirmatory testing, which makes them very practical for large-scale surveys. To accurately assess the prevalence of other resistance traits (e.g., to trimethoprim–sulfamethoxazole), other antimicrobial-selective media must be developed and validated.

We tested AMRB other than GNRE and CPRE (e.g., tetracycline- and streptomycin-resistant Enterobacteriaceae) by NSSM in a subset of the samples. Here, the resistance proportions were similar to the reports from other countries (e.g., Germany and Belgium).² The proportion of tetracycline resistance was high(54.6%), probably as a result of the common use of this agent for prophylaxis.

In analyzing the risk factors for CPRE and GNRE carriage, we faced an unexpected problem. Unlike our previous study of ESBLPE,⁶ the very high prevalence of these bacteria (especially GNRE) in the population likely compromised our ability to discern the effects of the different factors on prevalence.

As with ESBLPE, the use of antimicrobial prophylaxis, most commonly tetracycline agents, was associated with increased carriage of both CPRE and GNRE in bivariate analysis. In the latter, it was found to be nearly statistically significant in multivariable analysis. As prophylaxis was used almost exclusively in calves, it is difficult to determine the differential role of this factor. However, the persistence of carriage in the older age groups suggests that its role in CPRE and GNRE is limited compared with ESBLPE.⁶

An interesting finding in both CPRE and GNRE was the high prevalence found in farms that received new cattle in the preceding month, especially from overseas. This may indicate that international transfer of cattle might facilitate the spread of certain AMRB, as was also found to be the case in humans.²⁴

Several hygiene and infrastructure-related variables were identified as risk factors for increased prevalence of CPRE and GNRE by the bivariate analyses. These include crowdedness and absence of manure cleaning (both CPRE and GNRE) and lack of cooling system (CPRE). Strangely, clean troughs and high rate of cattle cleanliness were also associated with increased prevalence of CPRE. Since many of these factors (e.g., crowdedness and lack of cooling system) were common in the calves sections (data not shown), it is very difficult to determine in certain their independent role in the evolution and spread of these resistance traits. Notwithstanding, it is plausible to assume that factors such as crowdedness and lack of manure cleaning can certainly augment the dissemination of CPRE and GNRE within a cowshed and thus can be a potential target for intervention.

Our study has a number of limitations. In addition to the difficulty related to the overall high prevalence of carriage (discussed above), the differences between the grazing types made the assessment of the role of certain variables, for example, crowdedness, problematic. We suspect that the inability to identify statistically significant variables in the multivariable analysis is likely related to these two factors.

Despite these limitations, this study provides novel data regarding both the epidemiology of CPRE and GNRE carriage in livestock and the microbiological methodology of their surveillance. This knowledge may be especially important in both interpreting the data generated by existing AMRB surveillance programs and designing future surveillance studies.

Footnotes

Disclosure Statement

No competing financial interests exist.

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