Characterization of a novel microcin that kills enterohemorrhagic Escherichia coli O157:H7 and O26.

A novel phenotype was recently identified in which specific strains of Escherichia coli inhibit competing E. coli strains via a mechanism that was designated "proximity-dependent inhibition" (PDI). PDI-expressing (PDI(+)) E. coli is known to inhibit susceptible (PDI(-)) E. coli strains, including several enterohemorrhagic (EHEC) and enterotoxigenic (ETEC) E. coli strains. In this study, every strain from a genetically diverse panel of E. coli O157:H7 (n = 25) and additional strains of E. coli serovar O26 were susceptible to the PDI phenotype. LIVE/DEAD staining was consistent with inhibition by killing of susceptible cells. Comparative genome analysis identified the genetic component of PDI, which is composed of a plasmid-borne (Incl1) operon encoding a putative microcin and associated genes for transport, immunity, and microcin activation. Transfer of the plasmid to a PDI(-) strain resulted in transfer of the phenotype, and deletion of the genes within the operon resulted in loss of the inhibition phenotype. Deletion of chromosomally encoded tolC also resulted in loss of the inhibitory phenotype, and this confirmed that the putative microcin is most likely secreted via a type I secretion pathway. Deletion of an unrelated plasmid gene did not affect the PDI phenotype. Quantitative reverse transcription (RT)-PCR demonstrated that microcin expression is correlated with logarithmic-phase growth. The ability to inhibit a diversity of E. coli strains indicates that this microcin may influence gut community composition and could be useful for control of important enteric pathogens.

Discrimination between Mycoplasma and Acholeplasma species of bovine origin using digitonin disc diffusion assay, nisin disc diffusion assay, and conventional polymerase chain reaction.

Microbiological culture of milk samples has been used as a standard diagnosis for Mycoplasma mastitis. This technique is effective in isolating mollicutes that are Mycoplasma-like; however, isolates may be misinterpreted as Acholeplasma species, which are indistinguishable from Mycoplasma species by culture. A study to contrast the abilities of 2 culture-based tests, digitonin and nisin disc diffusion assays and a conventional polymerase chain reaction (PCR) technique, to discriminate between Mycoplasma and Acholeplasma was performed using 16S ribosomal RNA gene partial sequencing as the gold standard of comparison. A total of 288 bovine mollicute field isolates (248 from milk and 40 from other organ sites) and 13 reference strains were tested. Results obtained from the digitonin disc diffusion assay when it was performed with all field isolates were 92.7% and 99.0% in agreement with the gold standard using 5 mm and 3 mm of zone of growth inhibition as thresholds, respectively. Considering only milk isolates, agreements between the digitonin disc diffusion assay with the gold standard were 97.2% and 100% using 5 mm and 3 mm of zone of growth inhibition as thresholds, respectively. Culture identification using the nisin disc diffusion assay and the PCR was in a 100% agreement with the gold standard. Comparable results using culture-based nisin and digitonin disc diffusion assays, and PCR, to distinguish Mycoplasma and Acholeplasma species was found, especially for isolates from bovine milk.

Proximity-dependent inhibition in Escherichia coli isolates from cattle.

We describe a novel proximity-dependent inhibition phenotype of Escherichia coli that is expressed when strains are cocultured in defined minimal media. When cocultures of "inhibitor" and "target" strains approached a transition between logarithmic and stationary growth, target strain populations rapidly declined >4 log CFU per ml over a 2-h period. Inhibited strains were not affected by exposure to conditioned media from inhibitor and target strain cocultures or when the inhibitor and target strains were incubated in shared media but physically separated by a 0.4-µm-pore-size membrane. There was no evidence of lytic phage or extracellular bacteriocin involvement, unless the latter was only present at effective concentrations within immediate proximity of the inhibited cells. The inhibitory activity observed in this study was effective against a diversity of E. coli strains, including enterohemorrhagic E. coli serotype O157:H7, enterotoxigenic E. coli expressing F5 (K99) and F4 (K88) fimbriae, multidrug-resistant E. coli, and commensal E. coli. The decline in counts of target strains in coculture averaged 4.8 log CFU/ml (95% confidence interval, 4.0 to 5.5) compared to their monoculture counts. Coculture of two inhibitor strains showed mutual immunity to inhibition. These results suggest that proximity-dependent inhibition can be used by bacteria to gain a numerical advantage when populations are entering stationary phase, thus setting the stage for a competitive advantage when growth conditions improve.

Bibersteinia trehalosi inhibits the growth of Mannheimia haemolytica by a proximity-dependent mechanism.

Mannheimia (Pasteurella) haemolytica is the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS; Ovis canadensis) under experimental conditions. Paradoxically, Bibersteinia (Pasteurella) trehalosi and Pasteurella multocida have been isolated from BHS pneumonic lungs much more frequently than M. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized that B. trehalosi overgrows or otherwise inhibits the growth of M. haemolytica. Growth curves (monoculture) demonstrated that B. trehalosi has a shorter doubling time ( approximately 10 min versus approximately 27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared to M. haemolytica. During coculture M. haemolytica growth was inhibited when B. trehalosi entered stationary phase (6 h) resulting in a final cell density for M. haemolytica that was 6 to 9 logs lower than expected with growth in the absence of B. trehalosi. Coculture supernatant failed to inhibit M. haemolytica growth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition of M. haemolytica when both pathogens were cultured in the same media but separated by a filter (0.4-microm pore size) that limited contact between the two bacterial populations. There was significant growth inhibition of M. haemolytica when the populations were separated by membranes with a pore size of 8 mum that allowed free contact. These observations demonstrate that B. trehalosi can both outgrow and inhibit M. haemolytica growth with the latter related to a proximity- or contact-dependent mechanism.

Antimicrobial resistance in beef and dairy cattle production.

Observational studies of cattle production systems usually find that cattle from conventional dairies harbor a higher prevalence of antimicrobial resistant (AMR) enteric bacteria compared to organic dairies or beef-cow operations; given that dairies usually use more antimicrobials, this result is not unexpected. Experimental studies have usually verified that application of antimicrobials leads to at least a transient expansion of AMR bacterial populations in treated cattle. Nevertheless, on dairy farms the majority of antibiotics are used to treat mastitis and yet AMR remains relatively low in mastitis pathogens. Other studies have shown no correlation between antimicrobial use and prevalence of AMR bacteria including documented cases where the prevalence of AMR bacteria is non-responsive to antimicrobial applications or remains relatively high in the absence of antimicrobial use or any other obvious selective pressures. Thus, there are multi-factorial events and pressures that influence AMR bacterial populations in cattle production systems. We introduce a heuristic model that illustrates how repeated antimicrobial selection pressure can increase the probability of genetic linkage between AMR genes and niche- or growth-specific fitness traits. This linkage allows persistence of AMR bacteria at the herd level because subpopulations of AMR bacteria are able to reside long-term within the host animals even in the absence of antimicrobial selection pressure. This model highlights the need for multiple approaches to manage herd health so that the total amount of antimicrobials is limited in a manner that meets animal welfare and public health needs while reducing costs for producers and consumers over the long-term.

Assessment of phenotypic and genotypic diversity of Escherichia coli shed by healthy lactating dairy cattle.

A study was conducted to assess the diversity among fecal Escherichia coli from healthy lactating cattle. E. coli (n = 100) isolates from 10 healthy lactating dairy cows of a Pennsylvania dairy herd were examined for phenotypic and genotypic characteristics. Results revealed 26, 58, 10, and 6 E. coli isolates belonged to phylogenetic groups A, B1, B2, and D respectively. Overall, 63 serotypes, nine antibiotic resistance profiles, and 65 pulsed-field gel electrophoresis (PFGE) profiles were observed among the 100 isolates. Based on the combination of phenotypic and genotypic characteristics, the 100 E. coli isolates were classified into 76 clonal types. The numbers of different phenotypic and genotypic characteristics of E. coli were observed for each cow at ranges of 2-10, 1- 4, 2-10, and 4-10 for serotypes, antibiograms, PFGE profiles, and clonal types, respectively. The Chao1 estimator was used to estimate diversity among fecal E. coli. It was estimated that a range of 3-55, 1- 4, 2-55, and 8-55 fecal isolates from one cow would be required to include all possible types of E. coli based on serotype, antibiotic resistance profile, PFGE profile, and clonal type respectively. Based on the findings of the study it can be inferred that 1) dairy cattle should be considered as a significant reservoir of genotypically and phenotypically diverse E. coli, and 2) epidemiological investigations that focus on commensal bacteria should take into consideration the diversity within the species being investigated; if not addressed adequately, inappropriate sample size could lead to inaccurate study findings.

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.

Antimicrobial-resistant enteric bacteria from dairy cattle.

A study was conducted to understand the descriptive and molecular epidemiology of antimicrobial-resistant gram-negative enteric bacteria in the feces of healthy lactating dairy cattle. Gram-negative enteric bacteria resistant to ampicillin, florfenicol, spectinomycin, and tetracycline were isolated from the feces of 35, 8, 5, and 42% of 213 lactating cattle on 74, 39, 9, 26, and 82% of 23 farms surveyed, respectively. Antimicrobial-resistant gram-negative bacteria accounted for 5 (florfenicol) to 14% (tetracycline) of total gram-negative enteric microflora. Nine bacterial species were isolated, of which Escherichia coli (87%) was the most predominant species. MICs showing reduced susceptibility to ampicillin, ceftiofur, chloramphenicol, florfenicol, spectinomycin, streptomycin, and tetracycline were observed in E. coli isolates. Isolates exhibited resistance to ampicillin (48%), ceftiofur (11%), chloramphenicol (20%), florfenicol (78%), spectinomycin (18%), and tetracycline (93%). Multidrug resistance (> or =3 to 6 antimicrobials) was seen in 40% of E. coli isolates from healthy lactating cattle. Of 113 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 93% of isolates, while the remaining 7% isolates carried the tet(A) determinant. DNA-DNA hybridization assays revealed that tet determinants were located on the chromosome. Pulsed-field gel electrophoresis revealed that tetracycline-resistant E. coli isolates (n = 99 isolates) belonged to 60 subtypes, which is suggestive of a highly diverse population of tetracycline-resistant organisms. On most occasions, E. coli subtypes, although shared between cows within the herd, were confined mostly to a dairy herd. The findings of this study suggest that commensal enteric E. coli from healthy lactating cattle can be an important reservoir for tetracycline and perhaps other antimicrobial resistance determinants.

Prevalence of enterotoxin and toxic shock syndrome toxin genes in Staphylococcus aureus isolated from milk of cows with mastitis.

Staphylococcus aureus isolated from milk of cows with mastitis were evaluated for the prevalence of 16 enterotoxin genes (sea-see and seg-seq) and toxic shock syndrome toxin gene (tsst-1). Of 78 S. aureus examined, 73 (93.6%) were positive for one or more enterotoxin genes and these were divided into 36 groups by the presence of different enterotoxin genes. Enterotoxin genes including sen (84.6%), sem (71.8%), sei (60.3%) and sed (52.6%) were found frequently, while seg (24.4%), seq (16.7%), seo (12.8%), and seb (1.3%) were found at lower frequencies. Toxic shock syndrome toxin (tsst-1) gene was detected in 20 (25.6%) isolates and was always found in combination with other enterotoxin genes. The majority (88.5%) harbored more than one enterotoxin gene in different combinations. Eight S. aureus isolates (10.3%) were positive for sed, sei, sem, and sen; six (7.7%) possessed sed, seg, sei, sem, sen, and tsst-1; five (6.4%) had sei, sem, and sen; and four (5.1%) had sei, and sen. One isolate was positive for seb along with other SE genes including sed, seh, sem, sen, seq, and tsst-1. None of the isolates carried other enterotoxin genes (sea, sec, see, sej, sek, sel, and sep). PFGE profiles revealed 15 distinct pulsotypes among the 78 S. aureus isolates evaluated. PFGE and enterotoxin gene profiles did not match with each other because a single pulsotype carried different combinations of enterotoxin genes. The majority of S. aureus isolated from milk of mastitic cows carried newly described SE genes sem, sen and sei along with classical SE genes, sed and tsst-1. This is the first report describing the high prevalence of newly described enterotoxin genes, sem and sen in S. aureus from bovine mastitis. The high prevalence of enterotoxin genes and tsst-1 in S. aureus may be important as it is relevant to udder pathogenicity and food hygiene.

Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves.

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.

Evaluation of five selective media for isolation of catalase-negative gram-positive cocci from bulk tank milk.

Five selective media including Edwards modified medium, Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L), Streptococcus selective medium, Streptosel agar, and thallium-crystal violet-toxin-ferric citrate medium were evaluated for the isolation of streptococci and streptococci-like organisms from raw milk. The sensitivity and specificity of these selective media for streptococci and streptococci-like organisms were determined by using American Type Culture Collection reference strains. Under experimental conditions Edwards modified medium with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) showed the highest sensitivity (100%) and specificity (100%) for streptococci and streptococci-like organisms followed by thallium-crystal violettoxin-ferric citrate medium, Edwards modified medium, Streptococcus selective medium, and Streptosel agar. Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) allowed growth of all streptococci and streptococci-like organisms, while inhibiting growth of the staphylococci and gram-negative reference strains. Bulk tank milk samples from 114 dairy herds were spiral plated onto Edwards modified medium with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L). A total of 344 isolates (at least three isolates from each sample) were randomly selected and identified to their species. This medium permitted growth of 328 streptococci and streptococci-like organisms belonging to genera Aerococcus, Enterococcus, Gemella, Lactococcus, Streptococcus, and Vagococcus. When Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) was evaluated using bulk tank milk samples, the sensitivity and specificity of this medium for streptococci and streptococci-like organisms were observed to be 100 and 87.5%, respectively. The positive predictive value for streptococci and streptococci-like organisms was observed to be 99.4%. The results of the study indicate that Edwards modified medium supplemented with colistin sulfate (5 mg/L) and oxolinic acid (2.5 mg/L) can be used as a selective medium for the isolation of streptococci and streptococci-like organisms from bulk tank milk.