Competitive effect of commensal faecal bacteria from growing swine fed chlortetracycline-supplemented feed on ß-haemolytic Escherichia coli strains with multiple antimicrobial resistance plasmids.

AIMS: To determine the differences in competitive fitness among Escherichia coli strains with different plasmid profiles when grown in suspension with commensal faecal bacteria from growing swine fed chlortetracycline-supplemented or unsupplemented diets. METHODS AND RESULTS: Five multiple drug-resistant (MDR) E. coli strains that possessed 0, 2, 6 or 8 plasmids were inoculated into anoxic faecal cultures from swine fed an unsupplemented (control) or chlortetracycline (50 g ton(-1))-supplemented (experimental) diet. On days 21 of chlortetracycline supplementation, faecal growth competition studies were performed. MDR E. coli were enumerated at 0, 6 and 24 h. The plasmid-free strain was below culturable limits in both the control and experimental cultures by 24 h. For each plasmid-bearing strain, there was no statistically significant difference in population CFU ml(-1) (P < 0.05) between the control and experimental cultures. CONCLUSIONS: There was no significant effect on the faecal microflora, owing to the inclusion of chlortetracycline, in the swine diets, that affected the growth of E. coli in the competition studies employed. Furthermore, these results suggest that the cost of maintaining plasmids in these E. coli strains had little influence on survivability. SIGNIFICANCE AND IMPACT OF STUDY: Mutations that led to antimicrobial resistance may have a greater impact on survivability than multiple plasmid carriage.

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*.

AIMS: A Lactobacillus buchneri strain NRRL B-30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B-30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B-30929 for fermenting mixed substrates from biomass hydrolysates. METHODS AND RESULTS: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B-30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B-30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1.98-2.35 g l(-1) ethanol from corn stover hydrolysates and 2.92-3.01 g l(-1) ethanol from wheat straw hydrolysates when supplemented with either 0.25x MRS plus 1% corn steep liquor or 0.5x MRS. CONCLUSIONS: The L. buchneri NRRL B-30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are valuable for future research in engineering L. buchneri NRRL B-30929 for fermentative production of ethanol and chemicals from biomass.

Prevalence of class 1 integrons and antimicrobial resistance gene cassettes among enteric bacteria found in multisite group-level cohorts of humans and swine.

The prevalence of antimicrobial resistance genotypic characteristics (class 1 integrons and antimicrobial resistance gene cassettes) among commensal Escherichia coli isolated from humans and swine in a semiclosed, integrated farrow-to-fork population was evaluated in a cross-sectional study. The objective of this study was to establish baseline antimicrobial resistance patterns of enteric bacteria from animals and humans within the study population; specifically, genotypic traits both unique and common to commensal E. coli derived from the different sources were evaluated. There were significant differences between host species; swine isolates were more likely to harbor integrons (odds ratio = 2.33, P = 0.0487). No significant differences were found for facility location, facility type, human housing cohort, or time of day (P > 0.05). There were significant differences (P = 0.006) among swine production groups (fecal samples from boars, dry sows, finishers, growers, intake boars, lactating sows, the lagoon, nursery piglets, influent, and piglets); the grower group was less likely than the nursery group to harbor a class 1 integron (nursery as referent: odds ratio = 0.22, P = 0.04). Among all isolates with an integron present, human isolates were more likely to harbor an antimicrobial resistance gene cassette (odds ratio = 6.36, P = 0.003). When isolates that possessed gene cassettes coding for resistance to specific antimicrobials were compared, no significant differences between host species (P > 0.05) were observed.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase of Haloferax volcanii: role of histidine 398 and attenuation of activity by introduction of negative charge at position 404.

Mutant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases of the halophilic archaeon Haloferax volcanii were constructed to test the proposed mechanism that phosphorylation downregulates the activity of higher eukarya HMG-CoA reductases via charge-charge interaction with the active site histidine. To first verify the sequence-based inference that His 398 is the catalytic histidine of the H. volcanii enzyme, enzyme H398Q was constructed, purified, and assayed for catalysis of three reactions: [1] reductive deacylation of HMG-CoA, [2] reduction of mevaldehyde, and [3] oxidative acylation of mevaldehyde. Enzyme H398Q had low activity for catalysis of reaction [1] or [3], but readily catalyzed mevaldehyde reduction. By analogy to hamster HMG-CoA reductase, we conclude that His 398 is the active site histidine. Mutant forms of the 403-residue H. volcanii enzyme were constructed to model phosphorylation and infer whether attenuated activity involved interaction with His 398. Chimeric H. volcanii-hamster enzymes constructed in an effort to create an active, phosphorylatable chimeric enzyme were inactive or not phosphorylated. We therefore added Asp at position 404 to mimic the introduction of negative charge that would accompany phosphorylation. Enzyme 404D/H398Q was inactive for reaction [1] or [3], but catalyzed reaction [2] at 35% the wild-type rate. These observations are consistent with the model that attenuation of catalytic activity results from an ionic interaction between the imidazolium cation of His 398 and the carboxylate anion of Asp 404.

Archael phosphoproteins. Identification of a hexosephosphate mutase and the alpha-subunit of succinyl-CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus.

When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [gamma-32P]ATP, several radiolabeled polypeptides were observed following SDS-PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35-kDa one, whose protein-phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35- and 50-kDa phosphoproteins were purified and their amino-terminal sequences determined. The former polypeptide's amino-terminal sequence closely matched a conserved portion of the alpha-subunit of succinyl-CoA synthetase, which forms an acid-labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50-kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro-Gly-Thr-Asp/Ser-Gly-Val/Leu-Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6-phosphate to donate its phosphoryl group to the protein.

Persistence of a vancomycin-resistant Enterococcus faecium in an anaerobic continuous-flow culture of porcine microflora in the presence of subtherapeutic concentrations of vancomycin.

Recombined porcine continuous-flow culture (RPCF) maintained in a continuous-flow fermentation system is effective in protecting neonatal and weaned pigs against infection by enteropathogens. In the current study, we demonstrate the effect of RPCF on vancomycin-resistant enterococci (VRE) in the presence and absence of subtherapeutic levels of vancomycin. Also examined was the ability of VRE to transfer vancomycin resistance to endogenous Enterococcus faecalis 137.1. When RPCF was challenged with VRE, the rate of VRE clearance was dependent on the method of challenge. In the control experiment, RPCF was challenged with 7.0 log10/CFU/ml VRE. Clearance of VRE from the culture was observed within 7 days at a rate of 1.44 log10/day. RPCF containing 0.001 microg/ml vancomycin cleared VRE at a slightly lower rate of 0.94 log10/day. RPCF containing 0.01 microg/ml or 0.1 microg/ml vancomycin reduced the level of VRE from 7.0 log10/CFU/ml to 2.0 log10/CFU/ml within 9 days, but failed to clear the VRE after 24 days. During the period of decline, the VRE clearance rate for the 0.01 microg/ml and 0.1 microg/ml vancomycin-treated cultures was 0.52 log10/day, and 0.53 log10/day, respectively. E. faecalis 137.1 endogenous to RPCF did not acquire the vancomycin resistance genes throughout the experiment as evidenced by direct selection, ribotyping, and pulsed-field gel electrophoresis.

Antimicrobial resistance and serotype prevalence of Salmonella isolated from dairy cattle in the southwestern United States.

Mature dairy cattle were sampled over a 2-year period (2001-2002) on six farms in New Mexico and Texas. Fecal samples (n = 1560) were collected via rectal palpation and cultured for Salmonella, and one isolate from each positive sample was serotyped. Three isolates of each serotype, with the exception of Salmonella Newport (n = 12), were examined for susceptibility to 17 antimicrobial agents. Twenty-two different serotypes were identified from a total of 393 Salmonella isolates. Montevideo was the predominant serotype (27%) followed by Mbandaka (15%), Senftenberg (11.4%), Newport (6.4%), Anatum (4.8%), and Give (4.8%). Salmonella Typhimurium and Dublin, two frequently reported serotypes, accounted for only 1% of the observed serotypes in this study. Sixty-four percent of the serotypes were susceptible to all 17 antimicrobials, 14% were resistant to a single agent, and 22% were multiresistant (2-11 types of resistance). All isolates tested were susceptible to amikacin, apramycin, imipenem, ceftriaxone, nalidixic acid, and ciprofloxacin. The most frequent types of resistance were to sulfamethoxazole, tetracycline, streptomycin, kanamycin, chloramphenicol, and ampicillin (ranging from 8.9 to 22.4%). Serotypes demonstrating multiple resistance included Dublin and Give (resistant to three or more antibiotics), Typhimurium (resistant to five antibiotics), and Newport (four and two isolates resistant to six and nine antibiotics, respectively). Class 1 integrons were present in only two Salmonella Dublin isolates and one Salmonella Newport isolate. The most prevalent resistance patterns observed in this study were toward antimicrobial agents commonly used in cattle, while all Salmonella isolates were susceptible to ceftriaxone and ciprofloxacin, antibiotics used in human medicine.

Chicken mim-1 protein, P33, is a heterophil chemotactic factor present in Salmonella enteritidis immune lymphokine.

Lymphokine (ILK) secreted from concanavalin A-stimulated T cells from Salmonella Enteritidis-immune chickens is an undefined mixture of proteins that confers protection against Salmonella infectivity when administered to day-old chicks. It has previously been shown that polyclonal antibodies raised against human granulocyte colony-stimulating factor (GCSF) can neutralize the heterophil activation that is responsible for ILK's protective effect. Western blot analysis of ILK probed with anti-GCSF antibodies detects a prominent protein of mass 33 kDa. We have sequenced the first 20 amino acids of this protein and found it to be identical to residues 24 to 43 of P33, a 326-amino acid protein of unknown function encoded by the chicken mim-1 gene. The primary structure of P33 consists of two 140-residue imperfect repeats that are each homologous to a mammalian neutrophil chemotactic factor termed leukocyte cell-derived chemotaxin 2 (LECT2). We have expressed mim-1 in Escherichia coli and demonstrated in vitro that recombinant P33 is chemotactic for heterophils, the avian equivalent of mammalian neutrophils. We have also constructed a derivative of P33 that consists of residues 33 to 165 (P33[33-165]), the first repeat sequence of P33 that is homologous to LECT2. P33(33-165) is chemotactic for heterophils both in vitro and in vivo, inducing an influx of heterophils into the peritoneum in a response similar to that observed with ILK. These results suggest that P33 functions as a chemotactic factor in chickens and that it plays an active role in ILK-mediated protection against Salmonella infection.

Escherichia coli O157:H7 populations in sheep can be reduced by chlorate supplementation.

Ruminant animals are a natural reservoir of the foodborne pathogen Escherichia coli O157:H7. Some foodborne pathogens (e.g., E. coli) are equipped with a nitrate reductase that cometabolically reduces chlorate. The intracellular reduction of chlorate to chlorite kills nitrate reductase-positive bacteria; however, species that do not reduce nitrate are not affected by chlorate. Therefore, it has been suggested that ruminants be supplemented with chlorate prior to shipment for slaughter in order to reduce foodborne illnesses in human consumers. Sheep (n = 14) were fed a high-grain ration and were experimentally infected with E. coli O157:H7. These sheep were given an experimental product (XCP) containing the equivalent of either 2.5 mM NaNO3 and 100 mM NaCl (control sheep; n = 7) or 2.5 mM NaNO3 and 100 mM NaClO3 (chlorate [XCP]-treated sheep; n = 7). Control and XCP-treated sheep were treated for 24 h; XCP treatment reduced the population of inoculated E. coli O157:H7 (P < 0.05) from 10(2), 10(5), and 10(5) CFU/g in the rumen, cecum, and rectum, respectively, to < 10(1) CFU/g in all three sections of the gastrointestinal tract. The number of sheep testing positive for E. coli O157:H7 was significantly reduced by XCP treatment. In a similar fashion, total E. coli and coliforms were also reduced (P < 0.05) in all three compartments of the intestinal tract. Intestinal pH, total volatile fatty acid production, and the acetate/propionate ratio were unaffected by XCP treatment. On the basis of these results, it appears that chlorate treatment can be an effective method for the reduction of E. coli O157:H7 populations in ruminant animals immediately prior to slaughter.

Effect of feeding the ionophores monensin and laidlomycin propionate and the antimicrobial bambermycin to sheep experimentally infected with E. coli O157:H7 and Salmonella typhimurium.

Escherichia coli O157:H7 and Salmonella are widely recognized as important agents of foodborne disease with worldwide distribution. The use of ionophores in feeding growing ruminants is widespread in the United States and has attracted recent interest due to the apparent temporal relationship between initial ionophore use and the increase in human E. coli O157:H7 cases. Two experiments were conducted to evaluate the effects of short-term feeding of ionophores on fecal shedding, intestinal concentrations, and antimicrobial susceptibility of E. coli O157:H7 and S. typhimurium in growing lambs. Sixteen lambs were used in each experiment, four lambs per treatment group: monensin, laidlomycin propionate, bambermycin, and a control treatment. Lambs were fed a grain and hay (50:50) diet with their respective ionophore for 12 d before experimental inoculation with E. coli O157:H7 or S. typhimurium. Animals were maintained on their respective diets an additional 12 d, and fecal shedding of inoculated pathogens was monitored daily. Lambs were killed and tissues and contents were sampled from the rumen, cecum, and rectum. No differences (P > 0.05) in fecal shedding of Salmonella or E. coli O157:H7 were observed due to treatment. Occurrence of Salmonella or E. coli in luminal contents and tissue samples from the rumen, cecum, and rectum did not differ (P > 0.05) among treatments. Feeding monensin decreased (P < 0.05) the incidence of scours in sheep infected with Salmonella compared with the other treatments. No differences in antimicrobial susceptibility were found in any of Salmonella or E. coli O157:H7 isolates. Results from these studies indicate that short-term ionophore feeding had very limited effects on E. coli and Salmonella shedding or on antimicrobial susceptibility in experimentally infected lambs.

Competitive exclusion of a glycopeptide-resistant Enterococcus faecium in the presence of vancomycin but not equivalent concentrations of tylosin or gentamicin.

The effect of subtherapeutic concentrations of antibiotics (10.0 and 40.0 microg/mL of vancomycin, gentamicin, and tylosin) on the efficacy of a mixed anaerobe culture of chicken microflora (CCF) was studied in a continuous-flow fermentation system. Efficacy of CCF posttreatment was assessed by challenge with glycopeptide-resistant Enterococcus faecium (GRE) at 6.0 log10 cfu/mL. Bacterial enumeration of endogenous CCF isolates, volatile fatty acid (VFA) analysis, and challenge with GRE indicated that CCF efficacy was affected by all antibiotic treatments. Although CCF treated with 10.0 microg/mL of vancomycin eliminated GRE13 at a rate of 0.61 log10 cfu/ mL per day, it was unable to eliminate E. coli, a gram-negative challenge organism. All other antibiotic treatments allowed GRE persistence at approximately 2.0 to 6.5 log10 cfu/mL. All antibiotic-treated cultures had decreased concentrations of acetic and propionic acids. Our data suggest that low concentrations of antimicrobials may adversely affect the microbial ecology of gut microflora with respect to its ability to exclude exogenous bacteria. Moreover, gentamicin had an adverse effect on the inhibitory stringency of CCF even though it showed little anti-anaerobic activity against CCF strict anaerobes in pure culture. Verification of the results in live animals will be necessary to determine if antimicrobial treatment could compromise the effectiveness of normal microflora to serve as a natural host defense against infection.

Effect of lactic acid administration in the drinking water during preslaughter feed withdrawal on Salmonella and Campylobacter contamination of broilers.

The crop is a known source of Salmonella and Campylobacter contamination. We evaluated the use of selected organic acids (0.5% acetic, lactic, or formic) in drinking water during a simulated 8-h pretransport feed withdrawal (FW). Salmonella typhimurium was recovered from 53/100 control crops and from 45/100 of crops from acetic acid-treated broilers. However, treatment with lactic acid (31/100) or formic acid (28/76) caused significant (P < 0.05) reduction in incidence. Reductions of recovered incidence were also associated with reduced numbers of S. typhimurium recovered (e.g., control, log 1.45 cfu/crop; lactic acid, 0.79 cfu/crop). In an additional commercial farm study, broilers were provided 0.44% lactic acid during a 10-h FW (4 h on the farm and 6 h transport) and pre-FW crop, post-FW crop, and pre-chill carcass wash samples were collected for Campylobacter and Salmonella detection. Crop contamination with Salmonella was significantly reduced by lactic acid treatment (6/175) as compared with controls (29/175). Importantly, Salmonella isolation incidence in prechill carcass rinses was significantly reduced by 52.4% with the use of lactic acid (26/175 vs. 55/176). Crop contamination with Campylobacter was significantly reduced by lactic acid treatment (62.3%) as compared with the controls (85.1%). Lactic acid also reduced the incidence of Campylobacter found on pre-chill carcass rinses by 14.7% compared with the controls. These studies suggest that incorporation of lactic acid in the drinking water during pretransport FW may reduce Salmonella and Campylobacter contamination of crops and broiler carcasses at processing.

Effect of drinking-water administration of experimental chlorate ion preparations on Salmonella enterica serovar Typhimurium colonization in weaned and finished pigs.

Foodborne disease caused by Salmonella is of public health and economic significance. In order to assess the practical effectiveness of a new intervention strategy, experimental chlorate preparations (ECP) were administered via the drinking water to weaned and finished pigs that had been orally challenged the previous day with 10(9)-10(10) colony-forming units of Salmonella serovar Typhimurium. After 24 or 36 h ad libitum access to 0X, 1X or 2X ECP treatment (where X is the concentration estimated to deliver a minimal daily effective dose), the pigs were euthanized and gut contents and lymph tissue collected at necropsy were cultured for the challenge Salmonella. Drinking water administration of ECP effectively reduced (p < 0.05) caecal Salmonella concentrations and, with the weaned pigs, tended (p < or = 0.10) to reduce rectal Salmonella concentrations. No negative effects of ECP treatment on water intake and animal wellbeing were observed and only marginal effects on gut fermentation characteristics occurred. The bactericidal effect of administering ECP in drinking water was relatively rapid, with reductions in caecal Salmonella concentrations occurring within 24 h. These results suggest that ECP administered to pigs just days before slaughter may reduce gut concentrations of Salmonella; however, the impacts of such reductions on slaughter hygiene have yet to be determined.

Evaluation of feeding distiller's grains, containing virginiamycin, on antimicrobial susceptibilities in fecal isolates of Enterococcus and Escherichia coli and prevalence of resistance genes in cattle.

Dried distiller's grains (DG) produced from ethanol fermentations dosed with 0 (control), 2, or 20 mg/kg virginiamycin-based product or spiked with virginiamycin (VM) postfermentation were fed to cattle and effects on antimicrobial susceptibility, and prevalence of antimicrobial resistance genes in commensal bacteria was examined. Biological activity assays of DG (from each fermentation) indicated a concentration of 0, 0.7, and 8.9 mg/kg VM, respectively. Twenty-four crossbred beef steers were fed 1 of 4 diets (containing 8% of each of the different batches of DG) and a fourth using 8% of the control DG (0 mg/kg VM) + 0.025 g/kg V-Max50 (positive control) for 7 wk. Fecal samples were collected weekly throughout the experimental period and cultured for Escherichia coli and Enterococcus, and isolates were examined for antimicrobial susceptibility, antimicrobial resistance genes (vatE, ermB, and msrC in Enterococcus), and integrons (E. coli). No treatment differences (P > 0.05) were observed in antimicrobial susceptibility of the E. coli isolates. Enterococcus isolates were resistant to more antimicrobials; however, this was influenced by the species of Enterococcus and not treatment (P > 0.10). The prevalence of ermB was greater (P < 0.05) in the control isolates after 4 and 6 wk while at wk 7, prevalence was greater (P < 0.01) in the 0.7 and 8.9 mg/kg VM treatments. Taken together, the minor treatment differences observed for the presence of ermB coupled with the lack of effect on antimicrobial susceptibility patterns suggest that feeding DG containing VM residues should have minimal if any impact on prevalence of antimicrobial resistance.

Relationships among performance, residual feed intake, and temperament assessed in growing beef heifers and subsequently as 3-year-old, lactating beef cows.

Seventy-four beef heifers were used to evaluate relationships among performance, residual feed intake (RFI), and temperament measured as growing heifers (Phase 1) and subsequently as 3-yr-old lactating beef cows (Phase 2) in the same cohort. In both phases, females were housed in a covered facility and fed similar forage-based diets, and individual feed intakes, BW, BCS, chute scores (CS), exit velocities (EV), and pen scores (PS) were collected throughout the 70-d feeding trials. In Phase 2, cows were milked on trial d 14 (lactation d 28 ± 3.5) and trial d 70 (lactation d 84 ± 3.5) to determine energy-corrected milk (ECM) production. Ultrasonic backfat thickness (BF), and ribeye area (REA) were evaluated on d 0 and 70 of the trial in Phase 2. Heifers were ranked by RFI and placed into Low (<0.5 SD mean RFI; n = 27), Medium (within ± 0.5 SD; n = 23), and High (>0.5 SD mean RFI; n = 24) RFI groups. Body weight, BCS, and ADG were similar among all RFI groups; however, daily DMI differed for all groups (P < 0.01) and was greater (10.76 ± 0.24 kg/d) for High, intermediate (9.88 ± 0.25 kg/d) for Medium, and less (8.52 ± 0.23 kg/d) for Low RFI heifers. When cow performance was analyzed based on RFI rank as heifers, BW, BCS, ADG, RFI, d 14 and d 70 ECM, BF, and REA were similar among RFI groups; however, cows that were most efficient as heifers (Low) had decreased (P < 0.05) daily DMI values (10.30 ± 0.41 kg/d) compared with cows that ranked Medium (11.60 ± 0.44 kg/d) or High (11.50 ± 0.43 kg/d) as heifers. The Pearson rank correlation between Phase 1 and 2 RFI was r = 0.13 (P = 0.30), and Pearson rank correlations showed no relationship (P > 0.1) between RFI and temperament. Phase 1 CS was negatively associated with ADG in Phase 1 (r = -0.28; P = 0.02) and 2 (r = -0.32; P = 0.01), and positively associated with d 14 (r = 0.24; P = 0.04) and 70 (r = 0.25; P = 0.03) ECM. Phase 2 CS was negatively associated with Phase 2 ADG (r = -0.29; P = 0.01) and positively associated with d 14 (r = 0.46; P = 0.001) and 70 (r = 0.33; P = 0.004) ECM. Phase 2 PS also tended to be negatively associated with DMI in Phase 1 (r = -0.20; P = 0.096) and 2 (r = -0.20; P = 0.08). In this study, heifers that were most feed efficient subsequently consumed less feed as lactating cows and maintained similar performance. Feed efficiency was not associated with differences in temperament; however, more excitable females had poorer BW gains and tended to have reduced feed intakes but produced more ECM.

Effects of temporary calf removal before fixed-time artificial insemination on pregnancy rates and subsequent calf performance in suckled beef cows.

Two experiments were conducted to determine the effect of calf removal (CR) on pregnancy rate (PR) and calf performance in suckled beef cows. Cows in both experiments were synchronized with the 7-d CO-Synch + CIDR protocol [i.e., 100-µg injection of GnRH at controlled internal drug release (CIDR) device insertion (d -7) with 25-mg injection of PGF2α at CIDR removal (d 0), followed by injection of GnRH and timed AI (TAI) on d 3]. Cows were blocked by location (6 locations), stratified by days postpartum (DPP) and parity, and assigned to 1 of 2 treatments in Exp. 1: 1) control (Control; n = 156); 2) calves were separated from their dams between d 0 and 3 (CR72; n = 168); and 1 of 4 treatments in Exp. 2: 1) Control (n = 103); 2) CR72 (n = 104); 3) calves were separated from their dams between d 0 and 2 (CR48A; n = 95); and 4) similar to CR48A but CR between d 1 and 3 (CR48B; n = 53). Transrectal ultrasonography of ovarian structures was performed on d 0, 1, 2, 3, 4, and 10 (in a subset of cows) to determine pregnancy status on d 33. Blood samples were collected on d -14, -7, 0, 3, and 10 (in a subset of cows) to determine concentrations of progesterone (P4) and estradiol (E2). Calves were blocked by age as young (25 to 59 d), medium (60 to 79 d), and old (≥80 d), and were weighed on d 0, 3, 33, and 63. Overall PR did not differ among treatments and averaged 50%. Follicle growth rate from d 0 to 3 tended (P = 0.06) to be greater for CR72 (0.42 ± 0.15 mm/d) compared with Control (0.02 ± 0.15 mm/d). Young (-3.9 ± 0.3%) and old (-3.1 ± 0.4%) calves lost a greater (P < 0.001) percent of BW (PBW) during CR than medium-age (-1.6 ± 0.3%) calves exposed to CR72. In Exp. 2, PR were similar among all 3 locations (49%; P = 0.15). Young (-4.8 ± 0.6%) and medium (-3.0 ± 0.5%) calves lost greater (P < 0.01) percent body weight (PBW) during CR than old (-1.4 ± 0.6%) calves within the CR72 treatment. Calves exposed to CR48 (-2.2 ± 0.6%, -1.1 ± 0.6%, and -2.4 ± 0.6% PBW change for young, medium, and old, respectively) lost more BW than calves in the Control group (-3.7 ± 0.4%, -1.7 ± 0.5%, and -2.1 ± 0.5% PBW change for young, medium, and old, respectively). Subsequent calf weights on d 33 and 63 were greater (P < 0.05) in Controls than cows exposed to CR48 or CR72 treatments. We conclude that CR stimulated follicle growth but failed to enhance PR to TAI. However, CR had a negative impact on subsequent calf performance, which differed, depending on the duration and age of the calf when exposed to CR.

Macrolide inactivation gene cluster mphA-mrx-mphR adjacent to a class 1 integron in Aeromonas hydrophila isolated from a diarrhoeic pig in Oklahoma.

OBJECTIVES: To characterize a multidrug-resistant Aeromonas hydrophila isolate (CVM861) that possesses a high-level macrolide inactivation gene cluster (mphA-mrx-mphR), previously only reported in Escherichia coli. METHODS: PCR fragment length mapping, gene sequencing and Southern blotting were used to map the mphA-mrx-mphR gene cluster and flanking elements in CVM861. Conjugation experiments were done to determine whether the multidrug resistance genetic element was mobile. RESULTS: The mphA-mrx-mphR gene cluster mapped downstream of a class 1 integron and upstream of an aph(3') gene, and was present on a Tn21-like element. The gene order determined by sequencing was intI1-dhfrXII-orfF-aadA2-qacDeltaE-sul1-orf5Delta178-tnpA-mphR-mrx-mphA. Horizontal transmission of high-level macrolide resistance from CVM861 to E. coli 47011 was inconsistent; however, a composite plasmid possessing the mphA gene cluster was transferred at a conjugation frequency of 2.02 x 10(-5) per recipient. CONCLUSIONS: An mphA-mrx-mphR gene cluster was present downstream of the In2 integron located on a Tn21-like transposon in an A. hydrophila isolate. Whether this recombination event resulted in the truncation of the orf5 sequence is unknown. The presence of other resistance genes downstream of the mphA-mrx-mphR gene cluster suggests that multiple recombination events have occurred on this genetic element. This is the first known report of the mphA-mrx-mphR gene cluster carried by A. hydrophila and the first known isolation of this cluster in the United States.

Patterns of antimicrobial resistance among commensal Escherichia coli isolated from integrated multi-site housing and worker cohorts of humans and swine.

We examined antimicrobial resistance (AR) phenotypes among commensal Escherichia coli isolated from fecal matter of humans and swine housed in a semi-closed and uniquely integrated multi-site farrow-to-plate operation. Aggregate cohorts of humans consisted of (1) "control" groups of consumers, (2) groups of swine workers, and (3) groups of slaughter-plant workers. Analyses of cross-sectional AR data from 472 human and 376 swine isolates are presented. AR phenotypes among these isolates were compared by (1) host species, (2) facility location, (3) facility type, (4) housing (human) or production (swine) cohort, and (5) sample collection period and time of day. There were significant (p < 0.05) differences in isolates among host-species with swine uniformly at greater risk for (1) AR to four specific antimicrobials (kanamycin, streptomycin, sulfamethoxazole, tetracycline), and (2) multiple resistance phenotypes (p < 0.0001). Facility type and unit location were more often associated with AR differences among swine isolates than among human isolates. Swine production group was significantly associated with AR prevalence (p < 0.05) for nine antimicrobials; in general, purchased boars, suckling piglets, weaned piglets, and lactating sows were at higher risk of AR. There was no significant (p > 0.05) association of human occupational/consumer cohort with AR prevalence. Several unique AR phenotypes were detected in each of the human- and swine-intake groups. These data establish baseline characteristics for an on-going 3-year longitudinal study designed to further characterize AR phenotype and genotype in this population. Host-, facility-, and cohort-specific data demonstrate that sufficient prevalence differences exist to permit the future quantification of AR transmission, should it occur. Based on these cross-sectional data, occupational exposure to either swine-rearing or swine-slaughter facilities does not appear to be associated with the prevalence of phenotypic resistance among the commensal fecal E. coli isolated from this integrated system.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli.

Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases. We report here the characterization of an HMG-CoA reductase from the third domain, the archaea. HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H. volcanii. Subsequently, a portion of the H. volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7. While no HMG-CoA reductase activity was detectable following expression in E. coli, activity could be recovered after extracts were exposed to 3 M KCl. Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P. mevalonii HMG-CoA reductase. Activity was optimal at pH 7.3. Kms were 66 microM (NADPH) and 60 microM [(S)-HMG-CoA]. (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA. H. volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde [optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM [(R,S)-mevaldehyde]] and the oxidative acylation of mevaldehyde [optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM [(R,S)-mevaldehyde]]. These properties are comparable to those of hamster and P. mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism.