Anders Morten Hay Sørensen

Plasmid-Encoded Multidrug Efflux Pump Conferring Resistance to Olaquindox in Escherichia coli

ABSTRACT We report here the first gene-encoded resistance mechanism to the swine growth enhancer olaquindox. The genetic elements involved in resistance to olaquindox were subcloned and sequenced from a conjugative plasmid isolated from Escherichia coli. The subcloned fragment contained two open reading frames, oqxA and oqxB, that are homologous to several resistance-nodulation-cell-division family efflux systems from different species. The putative protein sequences were aligned to both experimentally verified and putative efflux pumps. We show that oqxA and oqxB are expressed in E. coli. Plasmids containing the oqxAB genes yielded high (>128 μg/ml) resistance to olaquindox in E. coli, whereas strains containing the control plasmid showed low resistance to the drug (8 μg/ml). The oqxAB-encoded pump also conferred high (>64 μg/ml) resistance to chloramphenicol. We demonstrate that the subcloned fragment conferred H+-dependent ethidium efflux abilities to E. coli strain N43. In addition, we show that the efflux system is dependent on the host TolC outer membrane protein when expressed in E. coli.

Measurements of the specific methanogenic activity of anaerobic digestor biomass

A specific methanogenic activity test was tested for its use as a simple procedure suitable for measurement of the activity of the various physiological groups of microorganisms involved in the terminal processes of methanogenesis from complex organic matter. Activity was estimated by supplying sufficient substrate (acetate, propionate, butyrate, H2 or none) to saturate the catabolic systems of the various physiological groups, whereafter the specific methane production rate was determined. Activity was defined as the substrate-dependent methane production rate per unit mass of volatile solids biomass, i.e. the rate with a saturating concentration of substrate present when the background methane production rate had been diluted to an insignificant level. When the digestor was perturbed, the concentration of unused substrate in the biomass obscured the effect of added substrates on the test batches. It was generally found that if the background level of substrates could not be sufficiently lowered by dilution, substrate specific activity tests, as commonly described in the literature, were useless.

Inducible Gene Expression by Nonculturable Bacteria in Milk after Pasteurization

ABSTRACT The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5°C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.

Kinetics of lactate, acetate and propionate in unadapted and lactate-adapted thermophilic, anaerobic sewage sludge : the influence of sludge adaptation for start-up of thermophilic UASB-reactors

SummaryA thermophilic anaerobic sludge digestor was adapted to lactate metabolism. The adapted sludge showed an improved capacity for lactate degradation when tested by a batch activity test, compared to the performance of unadapted sludge. Acetate was the major intermediate produced during the degradation. When adapted sludge was used as the inoculum for a lactate-fed, upflow anaerobic sludge blanket (UASB) reactor, the chemical oxygen demand reduction rate was higher than with unadapted sludge. After 39 days, however, the difference vanished due to an extensive wash-out of sludge from the reactor inoculated with adapted sludge.

Conjugative Plasmid Conferring Resistance to Olaquindox

ABSTRACT A conjugative plasmid, pOLA52, conferring resistance to the antibiotic growth promoter olaquindox has been isolated from Escherichia coli from swine manure. It also confers resistance to ampicillin and chloramphenicol and has a high frequency of transfer between strains of E. coli. Plasmid-borne olaquindox resistance has not been demonstrated before.

Antimicrobial Drug Resistance of Salmonella Isolates from Meat and Humans, Denmark

We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue.

Inactivation of pathogens on pork by steam-ultrasound treatment

The objective of the study was to evaluate a new pathogen inactivation concept that combines application of pressurized steam simultaneously with high-power ultrasound through a series of nozzles. On skin and meat surfaces of pork jowl samples, counts of total viable bacteria were reduced by 1.1 log CFU/cm(2) after treatment for 1 s and by 3.3 log CFU/cm(2) after treatment for 4 s. The mean reduction of 1.7 to 3.3 log CFU/cm(2) on the skin surface was significantly higher than the reduction of 1.1 to 2.5 log CFU/cm(2) on the meat surface. The inactivation of Salmonella Typhimurium, Salmonella Derby, Salmonella Infantis, Yersinia enterocolitica, and a nonpathogenic Escherichia coli was studied on inoculated samples that were treated for 0.5 to 2.0 s. With one exception, no significant differences in reduction were observed among the bacterial types. After treatment for 0.5 s, the 0.9-to 1.5-log reductions of E. coli were significantly higher than the 0.4- to 1.1-log reductions for Salmonella and Y. enterocolitica. Overall, reductions increased by increasing treatment time; reductions were 0.4 to 1.5 log CFU/cm(2) after treatment for 0.5 s and 2.0 to 3.6 log CFU/cm(2) after treatment for 2 s. Reductions on the skin (1 to 3.6 log CFU/cm(2)) were significantly higher than reductions on the meat surface (1 to 2.5 log CFU/cm(2)). The reduced effect on the meat surface may be explained by greater protection of bacteria in deep structures at the muscle surface. No significant difference in reduction was observed between samples inoculated with 10(4) CFU/cm(2) and those inoculated with 10(7) CFU/cm(2), and cold storage of samples for 24 h at 5°C after steam-ultrasound treatment did not lead to changes in recovery of bacteria.

Prediction of Salmonella carcass contamination by a comparative quantitative analysis of E. coli and Salmonella during pig slaughter.

Faecal contamination of carcasses in the slaughterhouse is generally considered to be the source of Salmonella on pork. In this study the hygiene indicator Escherichia coli is used to quantify faecal contamination of carcasses and it is hypothesized that it can be used to predict the quantitative carcass contamination with Salmonella, when the distribution of Salmonella concentrations in faeces is known. Paired pig sample data (faecal samples and carcass swabs) were obtained from five slaughterhouses and analysed for prevalence and concentrations of E. coli and Salmonella. A simple model was developed to describe the faecal contamination of carcasses using the E. coli data. The E. coli results suggested different hygiene performances in different slaughterhouses, and showed that a model assuming that carcasses are predominantly contaminated by their own faeces was not appropriate. Observed Salmonella prevalences were low (on average 1.9% on carcasses) and between slaughterhouses the prevalences ranked differently than the hygiene performance based on the E. coli data suggested. Also, the Salmonella concentrations predicted using E. coli as a faecal indicator were lower than the observed Salmonella concentrations. It is concluded that the faecal carriage of Salmonella together with the faecal contamination of carcasses, as predicted from E. coli data in the animal faeces and hygiene performance of the slaughterhouse, is not sufficient to explain carcass contamination with Salmonella. Our extensive data set showed that other factors than the observed faecal carriage of Salmonella by the individual animals brought to slaughter, play a more important role in the Salmonella carcass contamination of pork.