Influence of Antibiotic Selection on Genetic Composition of Escherichia coli Populations from Conventional and Organic Dairy Farms

The widespread agricultural use of antimicrobials has long been considered a crucial influence on the prevalence of resistant genes and bacterial strains. It has been suggested that antibiotic applications in agricultural settings are a driving force for the development of antimicrobial resistance, and epidemiologic evidence supports the view that there is a direct link between resistant human pathogens, retail produce, farm animals, and farm environments. Despite such concerns, little is understood about the population processes underlying the emergence and spread of antibiotic resistance and the reversibility of resistance when antibiotic selective pressure is removed. In this study, hierarchical log-linear modeling was used to assess the association between farm type (conventional versus organic), age of cattle (calf versus cow), bacterial phenotype (resistant versus susceptible), and the genetic composition of Escherichia coli populations (E. coli Reference Collection [ECOR] phylogroup A, B1, B2, or D) among 678 susceptible and resistant strains from a previously published study of 60 matched dairy farms (30 conventional and 30 organic) in Wisconsin. The analysis provides evidence for clonal resistance (ampicillin resistance) and genetic hitchhiking (tetracycline resistance [Tet^r]), estimated the rate of compositional change from conventional farming to organic farming (mean, 8 years; range, 3 to 15 years), and discovered a significant association between low multidrug resistance, organic farms, and strains of the numerically dominant phylogroup B1. These data suggest that organic farming practices not only change the frequency of resistant strains but also impact the overall population genetic composition of the resident E. coli flora. In addition, the results support the hypothesis that the current prevalence of Tet^r loci on dairy farms has little to do with the use of this antibiotic.     

Farm disinfectants select for cyclohexane resistance, a marker of multiple antibiotic resistance, in Escherichia coli

AIMS: The aim of this study was to determine if three classes of farm disinfectants were able to select for ciprofloxacin or cyclohexane tolerant [indicative of a multiple antibiotic resistance (MAR) phenotype] Escherichia coli and if cyclohexane-tolerant E. coli could be isolated from farms. METHODS AND RESULTS: Chicken slurry containing ca 1 : 99 ratio ciprofloxacin resistant : susceptible E. coli (10 different resistant strains examined) was treated for 24 h with each of the disinfectants and examined for survival of resistant : susceptible strains. Ciprofloxacin-sensitive (n=5) and -resistant (n=5) E. coli were grown with sublethal concentrations of the disinfectants and then plated to agar containing ciprofloxacin or overlaid with cyclohexane. Escherichia coli (n=389) isolated from farms were tested for cyclohexane tolerance. Minimum inhibitory concentrations (MIC) were determined against representative isolates and mutants. The disinfectants did not select for the ciprofloxacin-resistant E. coli in poultry slurry but following growth with each of the three disinfectants, higher numbers (P < or = 0.023) of cyclohexane-tolerant E. coli were isolated and these had a MAR phenotype. Of the 389 farm E. coli tested, only one was cyclohexane tolerant. CONCLUSIONS: It is possible that in a farm environment, E. coli could be exposed to similar concentrations of the disinfectants that are selected for MAR type organisms under these laboratory conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study suggest that cyclohexane-resistant E. coli are not common on farms, but in view of the ease of isolating them in the laboratory with farm disinfectants, further investigations on farms are warranted.     

Comparison of prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from organic and conventional dairy herds in Wisconsin

The prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion of Campylobacter isolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P = 0.042), but no difference was observed between organic and conventional farms. When we used Campylobacter spp. as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.     

Comparative analysis of the diversity of aerobic spore-forming bacteria in raw milk from organic and conventional dairy farms

Bacterial contamination of raw milk can originate from different sources: air, milking equipment, feed, soil, faeces and grass. It is hypothesized that differences in feeding and housing strategies of cows may influence the microbial quality of milk. This assumption was investigated through comparison of the aerobic spore-forming flora in milk from organic and conventional dairy farms. Laboratory pasteurized milk samples from five conventional and five organic dairy farms, sampled in late summer/autumn and in winter, were plated on a standard medium and two differential media, one screening for phospholipolytic and the other for proteolytic activity of bacteria. Almost 930 isolates were obtained of which 898 could be screened via fatty acid methyl ester analysis. Representative isolates were further analysed using 16S rRNA gene sequencing and (GTG)(5)-PCR. The majority of aerobic spore-formers in milk belonged to the genus Bacillus and showed at least 97% 16S rRNA gene sequence similarity with type strains of Bacillus licheniformis, Bacillus pumilus, Bacillus circulans, Bacillus subtilis and with type strains of species belonging to the Bacillus cereus group. About 7% of all isolates may belong to possibly new spore-forming taxa. Although the overall diversity of aerobic spore-forming bacteria in milk from organic vs. conventional dairy farms was highly similar, some differences between both were observed: (i) a relatively higher number of thermotolerant organisms in milk from conventional dairy farms compared to organic farms (41.2% vs. 25.9%), and (ii) a relatively higher number of B. cereus group organisms in milk from organic (81.3%) and Ureibacillus thermosphaericus in milk from conventional (85.7%) dairy farms. One of these differences, the higher occurrence of B. cereus group organisms in milk from organic dairy farms, may be linked to differences in housing strategy between the two types of dairy farming. However, no plausible clarification was found for the relatively higher number of thermotolerant organisms and the higher occurrence of U. thermosphaericus in milk from conventional dairy farms. Possibly this is due to differences in feeding strategy but no decisive indications were found to support this assumption.     

Genetic diversity and antibiotic resistance patterns in a campylobacter population isolated from poultry farms in Switzerland

The diversity and genetic interrelation of Campylobacter jejuni and C. coli isolated from Swiss poultry were assessed by three independent typing methods. Samples were derived prior to slaughter from 100 randomly selected flocks (five birds per flock) raised on three different farm types. The observed flock prevalence was 54% in total, with 50% for conventional and 69% for free-range farms. Birds held on farms with a confined roaming area had the lowest prevalence of 37%. Campylobacter isolates were characterized by amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism of flaA PCR fragments (flaA-RFLP), and disk diffusion testing for eight antimicrobial agents that are commonly used in veterinary or human medicine in Switzerland. Analysis of the genotypic results indicates that the Campylobacter population in Swiss poultry is genetically highly diverse. Nevertheless, occasionally, isolates with identical or nearly identical characteristics were isolated from different farms or farm types in different locations. Genetic typing by AFLP and flaA-RFLP was found to be complementary. The majority of isolates (67%) were susceptible to all tested antibiotics; however, single, double, and triple resistances were observed in 7%, 23%, and 2% of the strains, respectively. There was no correlation between genotype and antibiotic resistance. Surprisingly, sulfonamide resistance was frequently found together with streptomycin resistance. Our findings illustrate the results of common genetic exchange in the studied bacterial population.     

Genetic basis of macrolide and lincosamide resistance in Brachyspira (Serpulina) hyodysenteriae

Macrolide antibiotic resistance is widespread among Brachyspira hyodysenteriae (formerly Serpulina hyodysenteriae) isolates. The genetic basis of macrolide and lincosamide resistance in B. hyodysenteriae was elucidated. Resistance to tylosin, erythromycin and clindamycin in B. hyodysenteriae was associated with an A-->T transversion mutation in the nucleotide position homologous with position 2058 of the Escherichia coli 23S rRNA gene. The nucleotide sequences of the peptidyl transferase region of the 23S rDNA from seven macrolide and lincosamide resistant and seven susceptible strains of Brachyspira spp. were determined. None of the susceptible strains were mutated whereas all the resistant strains had a mutation in position 2058. Susceptible strains became resistant in vitro after subculturing on agar containing 4 micrograms ml-1 of tylosin. Sequencing of these strains revealed an A-->G transition mutation in position 2058.     

山东省某地区奶牛源大肠埃希菌的血清型、耐药特性及分子特性

【目的】旨在对从山东省某地区4个健康奶牛养殖场分离到的大肠埃希菌进行优势血清型、耐药特性、Ⅰ类整合子基因盒携带情况以及系统进化群分析。【方法】采集194份来自山东省某地区4个规模化奶牛场奶牛新鲜粪便样品,进行大肠埃希菌分离和鉴定,利用常用大肠埃希菌诊断血清进行血清型鉴定;利用10%的绵羊血平板检测溶血性;利用K-B法检测对14种常规抗菌药物的敏感性;利用聚合酶链式反应(PCR)检测革兰阴性菌常见的6大类24种耐药基因、Ⅰ类整合子基因盒结构并对目的条带测序分析;利用细菌多位点序列分型(Multilocus sequence typing,MLST)技术分析大肠埃希菌的ST型并使用eBURST v3软件分析菌株之间的克隆关系。【结果】从194份新鲜粪便样品中分离到171株大肠埃希菌,其中主要为致病性(19.9%)和侵袭性大肠埃希菌(17.0%),优势血清型分别为O128:K67(12/171)和O143:K7(12/171)。另外,具有溶血性的大肠埃希菌阳性率为9.4%(16/171);药敏试验结果显示多重耐药菌株的比率为22.2%,其中对氨苄西林耐药率最高为33.9%,四环素次之,为24.0%;PCR检测耐药基因和整合子结果显示,59.1%的菌株携带β-内酰胺类耐药基因blaTEM,59.1%的菌株携带氨基糖苷类耐药基因ant(2′),未检测到四环素耐药基因tetA和tetB;Ⅰ类整合子的阳性率为4.1%(7/171),dfrA12-aadA2-sul1为优势基因盒结构(4/171);MLST将大肠埃希菌分为8种ST型,其中,ST155(10/171)和ST58(45/171)形成一个克隆复合物且没有发现新的ST型。【结论】本研究证实,从该地区规模化健康奶牛场新鲜粪便中分离到的大肠埃希菌优势血清型为O128:K67和O143:K7;少部分大肠埃希菌具有溶血性;仅对氨苄西林、四环素等具有较高的耐药率;优势基因盒结构为dfrA12-aadA2-sul1;MLST分型显示不同奶牛场分离出亲缘关系较近的菌株,其分布具有多态性,血清型与ST型之间无相关性。本研究表明源自表观健康的奶牛的大肠埃希菌存在多重耐药现象,具有食品公共卫生安全隐患,该研究对于提升规模化奶牛场奶制品的安全生产与质量评估具有一定的理论指导意义。     

Comparison of Prevalence and Antimicrobial Susceptibilities of Campylobacter spp. Isolates from Organic and Conventional Dairy Herds in Wisconsin

The prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion of Campylobacter isolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P = 0.042), but no difference was observed between organic and conventional farms. When we used Campylobacter spp. as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.     

Dairy Farm Age and Resistance to Antimicrobial Agents in <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated from Dairy Topsoil

Antimicrobial agent usage is common in animal agriculture for therapeutic and prophylactic purposes. Selective pressure exerted by these antimicrobials on soil bacteria could result in the selection of strains that are resistant due to chromosomal- or plasmid-derived genetic components. Multiple antimicrobial resistances in Escherichia coli and the direct relationship between antimicrobial agent use over time has been extensively studied, yet the relationship between the age of an animal agriculture environment such as a dairy farm and antibiotic resistance remains unclear. Therefore, we tested the hypothesis that antimicrobial-resistance profiles of E. coli isolated from dairy farm topsoil correlate with dairy farm age. E. coli isolated from eleven dairy farms of varying ages within Roosevelt County, NM were used for MIC determinations to chloramphenicol, nalidixic acid, penicillin, tetracycline, ampicillin, amoxicillin/clavulanic acid, gentamicin, trimethoprim/sulfamethoxazole, cefotaxime, and ciprofloxacin. The minimum inhibitory concentration values of four antibiotics ranged 0.75 to >256 μg/ml, 1 to >256 μg/ml, 12 to >256 μg/ml, and 0.75 to >256 μg/ml for chloramphenicol, nalidixic acid, penicillin, and tetracycline, respectively. The study did not show a direct relationship between antibiotic resistance and the age of dairy farms.     

Reduced Medication in Organic Farming with Emphasis on Organic Dairy Production

Medication is an important focus area in organic animal husbandry. The combination of goals relating to improved animal welfare and reduced use of chemicals in general creates a common wish to reduce medication. Based on data from current Danish research projects in organic dairy farming, one specific organic medication pattern or policy cannot be described. The disease treatment pattern is influenced by many factors, e.g. the interaction with colleagues, veterinarians and agricultural advisors. No significant difference could be found with regard to incidence of mastitis treatments or somatic cell counts in 27 organic and 57 conventional herds. A marked tendency to shorter treatment periods in relation to mastitis treatments was described for organic farms in comparison with conventional farms (1.9 days versus 3.2 days (5 organic and 7 conventional herds)). In a study of development of health advisory service in organic herds, the dialogue between farmer, veterinarian and agricultural cattle advisor changed the treatment pattern markedly during a period of 6 months. Among important future challenges for veterinarians in organic farming is pointed at the constructive, open, and critical interaction with the single organic farmer as well as the organic animal husbandry system in general.     

Similarity of tetracycline resistance genes isolated from fish farm bacteria to those from clinical isolates

Tetracycline-resistant (Tet(r)) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet(r) gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet(r) genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet(r) strains transferred Tet(r) genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet(r) strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.     

Effect of conventional and organic production practices on the prevalence and antimicrobial resistance of Campylobacter spp. in poultry

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.     

Resistance to quinolones in Campylobacter jejuni and Campylobacter coli from Danish broilers at farm level

AIMS: To investigate the prevalence of quinolone resistance among Campylobacter jejuni and Camp. coli isolates from Danish poultry at the farm level, as well as for the whole country. METHODS AND RESULTS: Data and isolates were collected from a national surveillance of Campylobacter in poultry. Quinolone resistance was investigated by determination of minimum inhibitory concentration (MIC) to nalidixic acid and enrofloxacin. Among Camp. jejuni and Camp. coli combined, 7.5% were resistant to nalidixic acid. Quinolone resistance varied considerably from farm to farm, with 0% on some farms and almost 100% on others, but the resistance was evenly distributed geographically. With respect to isolates from farms where resistance was detected, quinolone resistance was higher among Camp. coli (28.7%) than among Camp. jejuni (11.3%). PFGE typing of quinolone-resistant and quinolone-susceptible isolates from four farms indicated that certain resistant isolates belonged to specific clones that were able to persist on the farms during several rotations, even in the absence of selective pressure. Some clones were present and repeatedly isolated in both a quinolone-susceptible and quinolone-resistant variant. CONCLUSIONS: Overall, quinolone resistance among Campylobacter isolates from Danish broilers was 7.5% in 1998 and 1999; it was higher among Camp. coli than Camp. jejuni. Genetic diversity among resistant isolates was lower than among susceptible isolates, and certain clones existed in both a resistant and a susceptible variant. Some resistant clones appeared to persist on the farms and were repeatedly isolated from poultry flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is important for the understanding of persistence and dynamics of Campylobacter in broiler houses. It also highlights the extent, farm-to-farm variation and persistence of quinolone-resistant Campylobacter in broiler houses.     

Role of input self-sufficiency in the economic and environmental sustainability of specialised dairy farms

Increasing input self-sufficiency is often viewed as a target to improve sustainability of dairy farms. However, few studies have specifically analysed input self-sufficiency, by including several technical inputs and without only focussing on animal feeding, in order to explore its impact on farm sustainability. To address this gap, our work has three objectives as follows: (1) identifying the structural characteristics required by specialised dairy farms located in the grassland area to be self-sufficient; (2) analysing the relationships between input self-sufficiency, environmental and economic sustainability; and (3) studying how the farms react to a decrease in milk price according to their self-sufficiency degree. Based on farm accounting databases, we categorised 335 Walloon specialised conventional dairy farms into four classes according to their level of input self-sufficiency. To this end, we used as proxy the indicator of economic autonomy – that is, the ratio between costs of inputs related to animal production, crop production and energy use and the total gross product. Classes were then compared using multiple comparison tests and canonical discriminant analysis. A total of 30 organic farms – among which 63% had a high level of economic autonomy – were considered separately and compared with the most autonomous class. We showed that a high degree of economic autonomy is associated, in conventional farms, with a high proportion of permanent grassland in the agricultural area. The most autonomous farms used less input – especially animal feeding – for a same output level, and therefore combined good environmental and economic performances. Our results also underlined that, in a situation of decrease in milk price, the least autonomous farms had more latitude to decrease their input-related costs without decreasing milk production. Their incomes per work unit were, therefore, less impacted by falling prices, but remained lower than those of more autonomous farms. In such a situation, organic farms kept stable incomes, because of a slighter decrease in organic milk price. Our results pave the way to study the role of increasing input self-sufficiency in the transition of dairy farming systems towards sustainability. Further research is required to study a wide range of systems and agro-ecological contexts, as well as to consider the evolution of farm sustainability in the long term.     

Effect of Conventional and Organic Production Practices on the Prevalence and Antimicrobial Resistance of Campylobacter spp. in Poultry

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.     

New sequence types and multidrug resistance among pathogenic Escherichia coli isolates from coastal marine sediments

The spread of antibiotic-resistant microorganisms is widely recognized, but data about their sources, presence, and significance in marine environments are still limited. We examined 109 Escherichia coli strains from coastal marine sediments carrying virulence genes for antibiotic susceptibility, specific resistance genes, prevalence of class 1 and 2 integrons, and sequence type. Antibiotic resistance was found in 35% of strains, and multiple resistances were found in 14%; the resistances detected most frequently were against tetracycline (28%), ampicillin (16.5%), trimethoprim-sulfamethoxazole (13%), and streptomycin (7%). The highest prevalence of resistant strains was in phylogenetic group A, whereas phylogroup B2 exhibited a significantly lower frequency than all the other groups. Sixty percent of multiresistant strains harbored class 1 or 2 integrase genes, and about 50% carried resistance genes (particularly dfrA and aadA) linked to a class 1 integron. Multilocus sequence typing of 14 selected strains identified eight different types characteristic of extraintestinal pathogens and three new allelic combinations. Our data suggest that coastal marine sediment may be a suitable environment for the survival of pathogenic and antimicrobial-resistant E. coli strains capable of contributing to resistance spread via integrons among benthic bacteria, and they highlight a role for these strains in the emergence of new virulent genotypes.     

Genotypic-phenotypic discrepancies between antibiotic resistance characteristics of Escherichia coli isolates from calves in management settings with high and low antibiotic use

We hypothesized that bacterial populations growing in the absence of antibiotics will accumulate more resistance gene mutations than bacterial populations growing in the presence of antibiotics. If this is so, the prevalence of dysfunctional resistance genes (resistance pseudogenes) could provide a measure of the level of antibiotic exposure present in a given environment. As a proof-of-concept test, we assayed field strains of Escherichia coli for their resistance genotypes using a resistance gene microarray and further characterized isolates that had resistance phenotype-genotype discrepancies. We found a small but significant association between the prevalence of isolates with resistance pseudogenes and the lower antibiotic use environment of a beef cow-calf operation versus a higher antibiotic use dairy calf ranch (Fisher's exact test, P = 0.044). Other significant findings include a very strong association between the dairy calf ranch isolates and phenotypes unexplained by well-known resistance genes (Fisher's exact test, P < 0.0001). Two novel resistance genes were discovered in E. coli isolates from the dairy calf ranch, one associated with resistance to aminoglycosides and one associated with resistance to trimethoprim. In addition, isolates resistant to expanded-spectrum cephalosporins but negative for bla(CMY-2) had mutations in the promoter regions of the chromosomal E. coli ampC gene consistent with reported overexpression of native AmpC beta-lactamase. Similar mutations in hospital E. coli isolates have been reported worldwide. Prevalence or rates of E. coli ampC promoter mutations may be used as a marker for high expanded-spectrum cephalosporin use environments.     

Role of calf-adapted Escherichia coli in maintenance of antimicrobial drug resistance in dairy calves

The prevalence of antimicrobial drug-resistant bacteria is typically highest in younger animals, and prevalence is not necessarily related to recent use of antimicrobial drugs. In dairy cattle, we hypothesize that antimicrobial drug-resistant, neonate-adapted bacteria are responsible for the observed high frequencies of resistant Escherichia coli in calves. To explore this issue, we examined the age distribution of antimicrobial drug-resistant E. coli from Holstein cattle at a local dairy and conducted an experiment to determine if low doses of oxytetracycline affected the prevalence of antimicrobial drug-resistant E. coli. Isolates resistant to tetracycline (>4 microg/ml) were more prevalent in <3-month-old calves (79%) compared with lactating cows (14%). In an experimental trial where calves received diets supplemented with or without oxytetracycline, the prevalence of tetracycline-resistant E. coli was slightly higher for the latter group (P = 0.039), indicating that drug use was not required to maintain a high prevalence of resistant E. coli. The most common resistance pattern among calf E. coli isolates included resistance to streptomycin (>12 microg/ml), sulfadiazine (>512 microg/ml), and tetracycline (>4 microg/ml) (SSuT), and this resistance pattern was most prevalent during the period when calves were on milk diets. To determine if prevalence was a function of differential fitness, we orally inoculated animals with nalidixic acid-resistant strains of SSuT E. coli and susceptible E. coli. Shedding of SSuT E. coli was significantly greater than that of susceptible strains in neonatal calves (P < 0.001), whereas there was no difference in older animals (P = 0.5). These data support the hypothesis that active selection for traits linked to the SSuT phenotype are responsible for maintaining drug-resistant E. coli in this population of dairy calves.     

Variation in the faecal shedding of Salmonella and E. coli O157:H7 in lactating dairy cattle and examination of Salmonella genotypes using pulsed-field gel electrophoresis

AIMS: To examine the variability in faecal shedding of Salmonella and Escherichia coli O157:H7 in healthy lactating dairy cattle and to evaluate the genetic relatedness of Salmonella isolates. METHODS: Faecal samples were obtained from lactating Holstein dairy cattle on four commercial farms in the southwestern US. All farms were within an 8-km radius and were sampled in August 2001, January 2002 and August 2002 (60 cows per farm per sampling; n = 720 total samples). Samples were cultured for E. coli O157:H7 and Salmonella and a portion of the recovered Salmonella isolates were examined for genetic relatedness using pulsed-field gel electrophoresis (PFGE). RESULTS: Faecal shedding of E. coli O157:H7 and Salmonella varied considerably between farms and at the different sampling times. Large fluctuations in the percentage of positive animals were observed from summer to summer for both of these pathogens. Similarly, Salmonella serotype and serotype prevalence varied from farm to farm and within farm from one sampling time to another. Multiple Salmonella genotypes were detected for a number of serotypes and identical genotypes were found on different farms with one genotype of Salmonella Senftenberg identified on three of the four farms. Significance and Impact of the STUDY: This study demonstrated the wide variability in pathogen shedding within and among dairy farms all located in a small geographical region and highlights the complexity of pathogen control at the farm level.     

Genetic Diversity and Antibiotic Resistance Patterns in a Campylobacter Population Isolated from Poultry Farms in Switzerland

The diversity and genetic interrelation of Campylobacter jejuni and C. coli isolated from Swiss poultry were assessed by three independent typing methods. Samples were derived prior to slaughter from 100 randomly selected flocks (five birds per flock) raised on three different farm types. The observed flock prevalence was 54% in total, with 50% for conventional and 69% for free-range farms. Birds held on farms with a confined roaming area had the lowest prevalence of 37%. Campylobacter isolates were characterized by amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism of flaA PCR fragments (flaA-RFLP), and disk diffusion testing for eight antimicrobial agents that are commonly used in veterinary or human medicine in Switzerland. Analysis of the genotypic results indicates that the Campylobacter population in Swiss poultry is genetically highly diverse. Nevertheless, occasionally, isolates with identical or nearly identical characteristics were isolated from different farms or farm types in different locations. Genetic typing by AFLP and flaA-RFLP was found to be complementary. The majority of isolates (67%) were susceptible to all tested antibiotics; however, single, double, and triple resistances were observed in 7%, 23%, and 2% of the strains, respectively. There was no correlation between genotype and antibiotic resistance. Surprisingly, sulfonamide resistance was frequently found together with streptomycin resistance. Our findings illustrate the results of common genetic exchange in the studied bacterial population.