Fluoroquinolone Antibiotics in a Hospital Sewage ...

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was based on the solid phase extraction method by Hirsch et al. (18). High performance .... Submitted July 5, 2004; accepted August 17, 2004. Scand J Infect Dis ...
Scand J Infect Dis 36: 752 /755, 2004

Fluoroquinolone Antibiotics in a Hospital Sewage Line; Occurrence, Distribution and Impact on Bacterial Resistance ˚ KE JARNHEIMER1, JAKOB OTTOSON5, RICHARD LINDBERG4, PER-A ¨ M5, MAGNUS JOHANSSON4, MATS TYSKLIND4, THOR-AXEL STENSTRO 2 ¨ RN OLSEN1,3,6 MARI-MALL WINNER and BJO From the 1Department of Infectious Diseases, 2Hospital Pharmacy, Kalmar County Hospital, SE-381 95 Kalmar, Departments of 3Infectious Diseases and 4Environmental Chemistry, Umea˚ University, SE-901 87 Umea˚, 5Department of Bacteriology, SMI, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, and 6Department of Biology and Environmental Science, University of Kalmar, SE-391 82 Kalmar, Sweden 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. B. Olsen, Department of Infectious Diseases, Umea˚ University, SE-901 87 Umea˚, Sweden (Tel. 133006, e-mail. [email protected])

INTRODUCTION Antibiotics are unique in their ability to select for genetic changes in bacteria. Evidence is growing that extensive horizontal transfer of antibiotic resistance genes is occurring in nature between clinical and non-clinical bacteria, between animal and human intestinal bacteria, and between intestinal and soil bacteria (1 /3). The increasing level of bacterial resistance to antibiotics is a major threat to effective treatment of infectious diseases among humans and animals (4 /6). Even without causing major outbreaks, the transmission of isolates with certain antibiotic resistance or virulence traits may be of great importance (2). There is a growing interest in the occurrence, transportation, transformation and effects of antibiotics in the environment (7 /11). After administration, antibiotics are excreted mainly into wastewater. Some are excreted completely unchanged, whereas others are more or less modified by chelate binding, degradation or inactivation by metabolic systems inside or outside the host (12). Hospital sewage lines may have an extraordinary and unique ecosystem, with all the conditions needed for selection and spread of antibiotic resistance among bacteria (13, 14). Most of the free floating bacteria and sediments in wastewater lines have a short residence time, but in small recesses and blind loops sediments can be trapped and exposed to antibiotics for longer periods. In addition, the damp surfaces are tapered with bacterial biofilms. There may be a risk for further selection of antibiotic resistance and horizontal transfer of resistance genes in both sediments and biofilms. Resistance genes can then disperse further during # 2004 Taylor & Francis. ISSN 0036-5548

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sewage treatment practices and finally end up in the environment. Fluoroquinolones (FQ) are a group of several related synthetic compounds with the same mechanisms of bacterial resistance. Consumption has increased worldwide in recent decades as they are often the only reliable oral antibiotic agent against many diseases caused by Gramnegative bacteria. FQ are highly potent and stable drugs with low biodegradability (15) and may be 1 of the most important groups of drugs in the environmental impact on bacterial resistance. On the other hand, FQ absorb firmly to sediment, probably lowering the antimicrobial effect (12). There are few data on the levels of antibiotics in hospital wastewater. Ciprofloxacin was detected in hospital wastewater at concentrations of 3 /87 mg/l and shown to be the main source of genotoxicity in Swiss and German hospital wastewater (15 /17). The mean predicted environmental concentrations of ciprofloxacin and ofloxacin in German hospital effluent were calculated to 0.2 /13.5 mg/l and 0.5 /35.3 mg/l, respectively (11). Here we present an approach for the study of concentrations and distribution of various antibiotics in different matrices in a hospital sewage line with potential effects on antibiotic resistance development. MATERIALS AND METHODS The study was conducted in the main sewage pipeline of a newly established section of Kalmar County Hospital, Kalmar, Sweden. The monitoring equipment allowed sampling from several microhabitats, including biofilm, wastewater and sediment, at selected intervals. The sewage line was connected to a building which opened in September 2000, containing 127 beds (72 general and urology DOI: 10.1080/00365540410021027

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surgery, 33 gynaecology, 22 paediatric). Water supply data for the entire unit were continuously monitored and constituted the major part of the wastewater flow. The amounts of antibiotics used in each ward were monitored by the hospital pharmacy. From those data we calculated the predicted mean concentrations including metabolites of FQ in wastewater. The sampling equipment was installed in a 4-m deep manhole (Fig. 1). Sewage water was pumped from a sump placed laterally to the main furrow in the manhole. This sump was connected directly to the water in the sewage furrow, with its base slightly lower than the bottom of the furrow to ensure the continuous presence of sewage water. In the sump, a submersible sewage pump, ABS Piraya s12-2D (ABS, Wexford, Ireland), was installed and connected to a timer that allowed the pump to start every 10 min and run for 10 s. The pump transported a partial flow of sewage water from the sewer to the test tank. The pump was equipped with a cutting head that disintegrated larger solid contents in the sewage water, preventing clogging of the sampling equipment and the sewage pipe. The water was then transported to a stainless steel open tank, where it was gradually exchanged giving a mean value of the levels of antibiotics in the sewage line. In the tank, solid content sedimented into 100 rows of sampling soda glass centrifuge tubes with round base 100 /34 mm (HECHT, Merck Eurolab, Darmstadt, Germany) and polypropylene tubes of the same size (Merck Eurolab, Darmstadt, Germany) (Fig. 2). Biofilm was sampled on 50 glass plates (100 /100 /6 mm) (Fig. 2). Samples of fluid were also taken direct from the tank to analyse the content of antibiotics in the sewage water. A climate control system was installed to maintain the temperature at around 208C in the building. The sampled sediments represented the part of the sludge with long residence time in the wastewater lines with possibilities of long exposure and accumulation of antibiotics. After installation of the system, sediment samples were obtained for bacterial analyses on 7 occasions with 8- to 12-weeks intervals, all in a study period of 60 weeks. The method for determination of antibiotics in the water phase was based on the solid phase extraction method by Hirsch et al. (18). High performance liquid chromatography (HPLC) was used,

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Fig. 2. The arrangement of sedimentation tubes and biofilm capturing glasses in the stainless test tanks. coupled with tandem mass spectrometry detection. For the solid phase, shaking, ultrasonication and pressurized liquid extraction were used (19). Using these methods, the concentrations of a number of antibiotics in different matrices were analysed (20). Bacteria in the sediments were extracted as previously described by La˚ngmark et al. (21) and analysed in 2 different ways to isolate antibiotic resistant bacteria: i) by growing on selective media and testing for resistance with the paper disc method and ii) direct culturing on selective media containing specific antibiotic

Fig. 1. The arrangement of the pump in the ‘sampling house’ and in the main furrow.

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concentrations. Both methods were in accordance to the Swedish Reference Group for Antibiotics (www.srga.org).

Table II. Ciprofloxacin resistant Enterobacteriacae spp. and faecal enterococci isolated from the sediment

RESULTS

Date yy/mm/dd

To our knowledge, this is the first description of a method that allows continuous long-term sampling in a hospital sewage line. Using this technique, wastewater could be monitored easily without the need for frequent ‘grab sampling’. The system allowed us to identify and measure various antibiotics in different matrices using HPLC, and to calculate the distribution coefficients under relevant conditions. Chemical analysis of the liquid phase resulted in the quantification of ciprofloxacin (0.5 mg/l) and ofloxacin (0.4 mg/l) after 3 months. Five other antibiotics were identified in the same sample, namely; netilmicin, imipenem, meropenem, amoxicillin and norfloxacin. The detection limits for all antibiotics were between 10 and 100 ng/l using a sample volume of 1000 ml. We made a preliminary experimental appraisal of distribution coefficients (Kd) between sludge and water for 6 antibiotics. The following Kd values were determined: amoxicillin (68.2), cloxacillin (52.3), cefadroxil (33.3), norfloxacin (88.4), metronidazole (94.5), sulfamethoxazole (8.6) and clindamycin (3.9). Theoretical Kd values were calculated as previously described (2). The experimental results were generally higher in all antibiotics, except clindamycin, than the calculated Kd values. After 2 months we analysed sediments that had been stored in the box. We found high concentrations of FQ bound to sediment; ciprofloxacin 151.4 ug/g and ofloxacin 31.6 ug/g. Table I shows the calculated mean concentrations of ciprofloxacin and ofloxacin in the liquid phase if no regard to degradation and binding to sediments is taken into account. On several occasions we sampled sediment for isolation and testing of ciprofloxacin resistance in Enterobacteriacae spp. and faecal Enterococcus spp. By both screening methods we measured low frequencies of resistance. The prevalence of ciprofloxacin resistant strains by the paper disc method is presented in Table II. DISCUSSION Continual interference with the wastewater flow carries a very high risk for the sewage line to become blocked. Even Table I. Annual amount of water consumption and quantity of FQ used. Predicted environmental concentration (PEC) and quantity of FQ detected in the liquid phase after 3 months PEC

Found FQ Quantity Water concentration of FQ used consumption

Ciprofloxacin 15 mg/l 0.5 mg/l Ofloxacin 5 mg/l 0.4 mg/l

1314 g 458 g

86,099 m3

00/12/13 01/02/15 01/04/17 01/06/07 01/08/30 01/10/24 02/01/24 Total

No. of Enterobacteriacae spp. tested/resistant

No. of enterococci tested/resistant

44/0 43/2 58/0 16/0 46/0 29/0 37/1

12/2 24/0 24/2 20/0 24/9 30/8 24/0

273/3

160/21

minimal obstacles to the flow in the main sewage furrow lead to quick accumulation of particles and plugging. The design of our system gave a reliable method for longitudinal collection of samples from different matrices without any obstacles. Although our results show lower concentrations of antibiotics in the liquid phase than previously reported (18), they represent the mean concentrations serving as time-integrated samples. Taking the distribution coefficients into account, our calculated mean concentrations of ciprofloxacin and ofloxacin in the water phase were consistent with our experimental findings. The Kd value for norfloxacin was used to calculate the concentration of ciprofloxacin in the sludge. The expected concentration would be more than 40 ug/g. We found even higher concentrations, but in the same order of magnitude as ciprofloxacin (151.4 ug/g) in sediment exposed to antibiotics for 2 months. This implies that determination of the concentration of antibiotics should be undertaken in the relevant compartment or matrix. Because of the variable use of antibiotics and large fluctuations in wastewater flow, antibiotic concentration is expected to differ to a great extent, depending on time of sampling. Solid phase and sediment can therefore serve as timeintegrated samples and will better mirror the amplitudes of antibiotic concentrations in different matrices. The selection of resistant bacteria is driven by the exposure of viable bacteria to active antibiotic substances. There is an obvious risk that resistance development increases if the sediment is constantly exposed to antibiotics. By slowing down the transit time for the sediment through the wastewater line, we believe that the possibility to detect a potential resistance development in the microbial community increases. This increase of residence time serves as a simulation of sludge trapped in recesses and pipe joints. However, despite the high values of FQ found in the sediment far exceeding the MIC50 of susceptible pathogenic bacteria, we found no evidence of resistance development against FQ in the studied indicator bacteria. One plausible explanation could be that FQ firmly bound to sediment has a reduced antibacterial effect (12). Furthermore, bacteria associated with biofilm are, compared to bacteria in a

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planktonic phase, less susceptible to antibiotics also in high concentrations (22). It is also noteworthy that bacteria in damp environments form biofilms even around small particles in different matrices as sediment or liquid phase. The methods used for detection of antibiotic resistance are probably not sensitive enough to rule out the development of bacterial resistance in all matrices investigated from the hospital wastewater line. We also believe that it is more accurate to determine the longitudinal development and amounts of certain resistance genes within the total bacterial community than certain indicator bacteria phenotypic properties. Ciprofloxacin has recently been identified, among other environmental factors and antibiotics, as an unspecific promoter of spread of antibiotic resistance genes in bacterial populations by activating the SOS response (23). As a consequence, search for increasing occurrence of resistance directed to antibiotics other than FQ in the sediment could lead to greater accuracy. The impact of antibiotics in the environment is extremely complex. Although we have some knowledge of the expected antibiotic concentrations in wastewater, little is known about the distribution of these antibiotics and their activity against bacteria in different matrices. To achieve comparable results, allowing risk assessment, there is a need for standardization of sampling, analysis and interpretation. ACKNOWLEDGEMENTS We thank Daniel Glatz, Bert Helmersson and Sten-Uno Frisk for expert technical assistance, and also Dr Paul D Haemig for valuable comments on the manuscript. This work was supported by The Swedish Federation of County Councils, the Swedish Strategic Programmme for the Rational Use of Antimicrobial Agents and Surveillance of Resistance (STRAMA), and the Centre for Environmental Research.

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Submitted July 5, 2004; accepted August 17, 2004