Lien T. Nguyen

Distribution of Tetracycline and Streptomycin Resistance Genes and Class 1 Integrons in Enterobacteriaceae Isolated from Dairy and Nondairy Farm Soils

The prevalence of selected tetracycline and streptomycin resistance genes and class 1 integrons in Enterobacteriaceae (n = 80) isolated from dairy farm soil and nondairy soils was evaluated. Among 56 bacteria isolated from dairy farm soils, 36 (64.3%) were resistant to tetracycline, and 17 (30.4%) were resistant to streptomycin. Lower frequencies of tetracycline (9 of 24 or 37.5%) and streptomycin (1 of 24 or 4.2%) resistance were observed in bacteria isolated from nondairy soils. Bacteria (n = 56) isolated from dairy farm soil had a higher frequency of tetracycline resistance genes including tetM (28.6%), tetA (21.4%), tetW (8.9%), tetB (5.4%), tetS (5.4%), tetG (3.6%), and tetO (1.8%). Among 24 bacteria isolated from nondairy soils, four isolates carried tetM, tetO, tetS, and tetW in different combinations; whereas tetA, tetB, and tetG were not detected. Similarly, a higher prevalence of streptomycin resistance genes including strA (12.5%), strB (12.5%), ant(3″) (12.5), aph(6)-1c (12.5%), aph(3″) (10.8%), and addA (5.4%) was detected in bacteria isolated from dairy farm soils than in nondairy soils. None of the nondairy soil isolates carried aadA gene. Other tetracycline (tetC, tetD, tetE, tetK, tetL, tetQ, and tetT) and streptomycin (aph(6)-1c and ant(6)) resistance genes were not detected in both dairy and nondairy soil isolates. A higher distribution of multiple resistance genes was observed in bacteria isolated from dairy farm soil than in nondairy soil. Among 36 tetracycline- and 17 streptomycin-resistant isolates from dairy farm soils, 11 (30.6%) and 9 (52.9%) isolates carried multiple resistance genes encoding resistance to tetracycline and streptomycin, respectively, which was higher than in bacteria isolated from nondairy soils. One strain each of Citrobacter freundii and C. youngae isolated from dairy farm soils carried class 1 integrons with different inserted gene cassettes. Results of this small study suggest that the presence of multiple resistance genes and class 1 integrons in Enterobacteriaceae in dairy farm soil may act as a reservoir of antimicrobial resistance genes and could play a role in the dissemination of these antimicrobial resistance genes to other commensal and indigenous microbial communities in soil. However, additional longer-term studies conducted in more locations are needed to validate this hypothesis.

Detection of Sorbitol-Negative and Sorbitol-Positive Shiga Toxin-Producing Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, and Salmonella spp. in Dairy Farm Environmental Samples

Six visits were conducted to four dairy farms to collect swab, liquid, and solid dairy farm environmental samples (165 to 180/farm; 15 sample types). The objective of the study was to determine on-farm sources of Campylobacter jejuni, Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC), which might serve as reservoirs for transmission of pathogens. Samples were analyzed using mostly U.S. Food and Drug Administrations Bacteriological Analytical Manual protocols; however, Salmonella spp., L. monocytogenes and STEC were co-enriched in universal pre-enrichment broth. Campylobacter jejuni were enriched in Bolton broth containing Bolton broth supplement. Pathogens were isolated on agar media, typed biochemically, and confirmed using multiplex polymerase chain reaction protocols. Campylobacter jejuni, Salmonella spp., L. monocytogenes, Sorbitol-negative (SN)-STEC O157:H7, and sorbitol-positive (SP)-STEC, respectively, were isolated from 5.06%, 3.76%, 6.51%, 0.72%, and 17.3% of samples evaluated. Whereas other pathogens were isolated from all four farms, SN-STEC O157:H7 were isolated from only two farms. Diverse serotypes of SP-STEC including O157:H7, O26:H11, O111, and O103 were isolated. None of the five pathogen groups studied were isolated from bulk tank milk (BTM). Most pathogens (44.2%) were isolated directly from fecal samples. Bovine fecal samples, lagoon water, bedding, bird droppings, and rat intestinal contents constituted areas of major concern on dairy farms. Although in-line milk filters from two farms tested positive for Salmonella or L. monocytogenes, none of the pathogens were detected in the corresponding BTM samples. Good manure management practices, including control of feral animals, are critical in assuring dairy farm hygiene. Identification of on-farm pathogen reservoirs could aid with implementation of farm-specific pathogen reduction programs.

Evaluation of universal pre-enrichment broth for isolation of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples.

Use of universal pre-enrichment broth (UPB) as a primary enrichment medium for detection of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples was evaluated. There were no differences in bacterial growth between UPB and selective primary enrichment broths for each pathogen inoculated individually or in combination at 10(1) and 10(2) colony forming units/mL. In addition, no differences were observed when UPB and selective primary enrichment broths were compared for detection efficiency of pathogens in artificially contaminated raw milk and fecal samples. Listeria enrichment broth (LEB) was compared with UPB to support growth of L. monocytogenes from naturally contaminated environmental samples. Listeria monocytogenes was isolated from seven of 30 samples enriched in UPB and six of 30 samples enriched in LEB. Dairy farm environmental samples were examined for recovery of the three pathogens using UPB. Subsequent isolation was achieved using selective secondary enrichment of each pathogen. Listeria monocytogenes, Salmonella spp., and E. coli O157:H7 were isolated in 13.4% (30 of 224), 8.9% (20 of 224), and 2.2% (five of 224) of samples, respectively. Isolation rates of the three pathogens were somewhat higher than in previous reports. Overall, UPB supported growth of test pathogens to detectable levels within 24 h. Our results demonstrate that UPB has potential for routine use in isolation of foodborne pathogens from diverse environmental samples.

Phenotypic and Genetic Markers for Serotype-Specific Detection of Shiga Toxin-Producing Escherichia coli O26 Strains from North America

Phenotypic and genetic markers of Shiga toxin-producing Escherichia coli (STEC) O26 from North America were used to develop serotype-specific protocols for detection of this pathogen. Carbohydrate fermentation profiles and prevalence of gene sequences associated with STEC O26 (n = 20) were examined. Non-STEC O26 (n = 17), E. coli O157 (n = 20), E. coli O111 (n = 22), and generic E. coli (n = 21) were used as comparison strains. Effects of supplements: cefixime-tellurite, 4-methylumbelliferyl-beta-D-glucuronide (MUG) and chromogenic additives (5-bromo4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-GlcA) and o-nitrophenyl-beta-D-galactopyranoside (ONPG), added to isolation agar media were examined. Tests for presence of gene sequences encoding beta intimin (eae beta), Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicCh7), enterohemolysin (ehlyA), O26 somatic antigen (wzx), and high pathogenicity island genes (irp2 and fyuA) were conducted using multiplex polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) of XbaI restriction endonuclease genomic DNA digests was used to establish clonality among E. coli O26 strains. Of the 26 carbohydrates tested, only rhamnose had diagnostic value. Rhamnose non-fermenters included STEC O26 (100%), non-STEC O26 (40%), generic E. coli (29%), E. coli O111 (23%), and E. coli O157 (0%). Rhamnose non-fermenting colonies growing on Rhamnose-McConkey agar supplemented with X-GlcA, X-Gal, or ONPG, respectively, were blue, white, or faint yellow, whereas rhamnose-fermenters were red. Blue colonies from X-GlcA-containing media were the most well-defined and easiest to pick for further tests. All STEC O26 were MUG-fluorescent, while STEC O157 (n = 18) were non-fluorescent. E. coli O111 and generic E. coli strains were either MUG-positive or-negative. Serotype-specific detection of STEC O26 was achieved by selecting cefixime-tellurite-resistant, MUG-fluorescent, rhamnose-nonfermenting colonies, which carried stx1, eae beta, irp2, and wzx gene sequences. STEC O26 prevalence in dairy farm environmental samples determined using the developed isolation and genetic detection protocols was 4%. PFGE indicated the presence of one major cluster of E. coli O26 with 72-100% DNA fragment-length digest similarity among test strains. The serotype-specific detection methods described herein have potential for routine application in STEC O26 diagnosis.

Novel Single-Tube Agar-Based Test System for Motility Enhancement and Immunocapture of Escherichia coli O157:H7 by H7 Flagellar Antigen-Specific Antibodies

ABSTRACT This paper describes a novel single-tube agar-based technique for motility enhancement and immunoimmobilization of Escherichia coli O157:H7. Motility indole ornithine medium and agar (0.4%, wt/vol) media containing either nutrient broth, tryptone broth, or tryptic soy broth (TSBA) were evaluated for their abilities to enhance bacterial motility. Twenty-six E. coli strains, including 19 O157:H7 strains, 1 O157:H− strain, and 6 generic E. coli strains, were evaluated. Test bacteria were stab inoculated in the center of the agar column, and tubes were incubated at 37°C for 18 to 96 h. Nineteen to 24 of the 26 test strains (73.1 to 92.3%) were motile in the different media. TSBA medium performed best and was employed in subsequent studies of motility enhancement and H7 flagellar immunocapture. H7 flagellar antiserum (30 and 60 μl) mixed with TSBA was placed as a band (1 ml) in the middle of an agar column separating the top (3-ml) and bottom (3-ml) agar layers. The top agar layer was inoculated with the test bacterial strains. The tubes were incubated at 37°C for 12 to 18 h and for 18 to 96 h. The specificity and sensitivity of the H7 flagellar immunocapture tests were 75 and 100%, respectively. The procedure described is simple and sensitive and could be adapted easily for routine use in laboratories that do not have sophisticated equipment and resources for confirming the presence of H7 flagellar antigens. Accurate and rapid identification of H7 flagellar antigen is critical for the complete characterization of E. coli O157:H7, owing to the immense clinical, public health, and economic significance of this food-borne pathogen.