A longitudinal study of antimicrobial resistant faecal bacteria in sediments collected from a hospital wastewater system.

OBJECTIVE: The objective with this study was to determine and follow antimicrobial resistance in faecal bacteria over time in hospital wastewater pipe sediment. A further aim was to determine bacterial growth rates of sensitive, intermediate and resistant intestinal enterococci in different ciprofloxacin concentrations as a measure of bacterial fitness. METHODS: A system enabling the collection of settled particles over time was installed at Kalmar County Hospital. Samples were collected bi-monthly for a 14-month period. Coliform bacteria and enterococci were isolated from the sediment with standard methods and investigated for resistance to ciprofloxacin (CIP), imipenem (IMI), trimetroprim-sulfamethoxazole (TS), ampicillin (AMP) and vancomycin (VAN) by the disc diffusion method. Resistant isolates were further typed with the PhenePlateTM system. Growth assessments were performed with an automated spectrophotometer. RESULTS: The rate of intestinal enterococci resistance was <0.6, 1.3, 1.9 and 13% to VAN, IMI, AMP and CIP respectively. Coliform resistance frequencies were 1.1, 2.2 and 2.2% to CIP, IMI and TS respectively. At two sampling occasions, significantly higher rates of ciprofloxacin resistant enterococci were found and the establishment of a resistant clone in the sewer was indicated by the PhP-analysis. Ciprofloxacin resistant intestinal enterococci had a significantly longer lag-phase time than sensitive isolates, but from 500 µg ml(-1) (half MIC) resistant isolates had a competitive advantage in terms of significantly faster generation time. DISCUSSION: Despite high concentration of antimicrobials in the sediment, resistance frequencies were generally low. This can depend on limited growth possibilities for faecal bacteria. However, the establishment of a resistant clone shows that hospital sewers can serve as a reservoir for antibiotic resistant bacteria.

A comparative study of three methods to evaluate an intervention to improve empirical antibiotic therapy for acute bacterial infections in hospitalized patients.

BACKGROUND: In order to limit the use of broad-spectrum antibiotics, standardized empirical therapy against acute bacterial infections has been advocated. METHODS: Guidelines for acute bacterial infections recommending increased usage of benzylpenicillin and restricted use of fluoroquinolones and cephalosporins have been implemented in Kalmar County, Sweden. We evaluated this strategy by recording therapy in patients with bacteraemia, antibiotic requisition, and point prevalence surveys prior to this intervention and at 6 and 12 months after. RESULTS: Comparing the methods simultaneously, there was good agreement between them and an overall significant change in antibiotic usage. There was a significant shift from cefuroxime to cefotaxime and a borderline significant increase in the use of benzylpenicillin (p = 0.057). Based on the defined daily dose (DDD), a highly significant decrease in total cefotaxime and cefuroxime usage was observed that was not detected when applying the prescribed daily dose (PDD), which is adapted to local treatment practices. No change was found in mortality in Staphylococcus aureus bacteraemia or the incidence of Clostridium difficile infection. CONCLUSIONS: We conclude that the implementation of the new guidelines has resulted in a significant change in antibiotic usage, which could be conveniently monitored by antibiotic requisition if PDD is used in addition to DDD.

Quantification of genes encoding resistance to aminoglycosides, beta-lactams and tetracyclines in wastewater environments by real-time PCR.

In this study real-time PCR assays, based on the LUX-technique, were developed for quantification of genes mediating resistance to aminoglycosides [aac(6 ')-Ie + aph(2 ' ')], beta-lactams (mecA), and tetracyclines (tetA and tetB), for use in wastewater environments. The developed assays were applied on DNA extracted from three wastewater-associated environments: soil from an overland flow area treating landfill leachates, biofilm from a municipal wastewater treatment plant, and sludge from a hospital wastewater pipeline. The highest concentration of all genes was observed in the hospital pipeline and the lowest in the overland flow system. TetA and aac(6 ')-Ie + aph(2 ' ') could be detected in all environments. The tetB gene was detected in the overland flow area and the hospital wastewater pipeline and mecA was detected in the wastewater treatment plant and the hospital pipeline. The developed LUX real-time PCR assays were shown to be fast and reproducible tools for detection and quantification of the four genes encoding antibiotic resistance in wastewater.

Determination of antibiotic substances in hospital sewage water using solid phase extraction and liquid chromatography/mass spectrometry and group analogue internal standards.

A method for determination of antibiotics in hospital sewage water has been developed and validated. Analogue internal standards for fluoroquinolones, sulfonamides, trimethoprim, beta-lactams (penicillins and cephalosporins), nitroimidazoles and tetracyclines were successfully used for calibration and shown to generally improve precision compared to external calibration. Matrix components caused ion suppression/enhancement effects during the MS detection for all analytes studied. Two effects were observed: general suppression and short-term variations in the MS response. In the hospital sewage water large temporal variations in the analyte concentration were observed during the course of the sampling period (seven grab samples in 13 h). Analyte concentrations varied within the following ranges (in microg l(-1)): ciprofloxacin, 3.6-101.0; metronidazole, 0.1-90.2; sulfamethoxazole, 0.4-12.8; ofloxacin, 0.2-7.6; trimethoprim, 0.6-7.6; and doxycycline, 0.6-6.7.

Fluoroquinolone antibiotics in a hospital sewage line; occurrence, distribution and impact on bacterial resistance.

In hospital sewage lines, human faecal bacteria are exposed to antibiotics, posing a risk for selection of antibiotic resistant microorganisms. We constructed a system for continuous sampling in a hospital sewage line that allowed us to study longitudinal establishment of bacteria, concentrations of antibiotics, and selection of bacterial resistance in waste water, sediment and biofilm. The focus in this study was on fluoroquinolones, a widely used group of antibiotics with increasing resistance problems. We found low levels of ciprofloxacin and ofloxacin in waste water but high concentrations in sediment. Despite the high levels of fluoroquinolones bound to sediment, we did not find any development of resistance against fluoroquinolones in Enterobacteriacae spp. and faecal enterococci isolated from sediment.