Board of Directors Meeting Toronto Central Local Health Integration Network Wednesday, February 25, 2015, 4:00 – 7:00 p.m. Boardroom, Toronto Central LHIN 425 Bloor Street East, Suite 201, Toronto, Ontario Directors Present Staff Present Angela Ferrante (Chair) Camille Orridge, Chief Executive Officer Vania Sakelaris, Senior Director, Health System
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Www2.hartwick.eduImpact of Agricultural and Waste Water Treatment Facility Runoff on the Incidence
of Antibiotic Resistant Bacteria in Streams
Due to increased usage of antibiotic drugs over the past few decades, researchers are finding increasing proportions of bacteria in the environment that are resistant to antibiotics.
Areas that are especially affected include streams that receive runoff from farms utilizingantibiotic drugs in their animal feed and from waste water treatment facilities. The goal ofthis study was to determine if these types of pollution are causing an increase in populationsof antibiotic resistant bacteria in streams. Water was collected from three points along astream receiving runoff from agricultural areas and from points above, at, and below theoutflow pipe of a waste water treatment facility. Water was also collected from a locationgeographically removed from these pollution sources. Bacteria filtered from the watersamples were plated on media selective for the growth of coliforms or media selective forthe growth of Acinetobacter. Colonies picked from these plates were grown on mediacontaining ampicillin, chloramphenicol, norfloxacin, streptomycin, or tetracycline, or noantibiotic. Susceptibility or resistance to antibiotics was determined by comparing thepercentage of colonies that grew on media with and without antibiotic. The number ofcoliform bacteria resistant to ampicillin was significantly higher at the waste water treatmentfacility outflow pipe than upstream of the outflow. Greater numbers of coliforms andAcinetobacter resistant to chloramphenicol, streptomycin, and tetracycline were also foundat and below the outflow compared to upstream. Agricultural runoff seems to contribute toan increase in the number of coliform bacteria resistant to ampicillin, streptomycin, andtetracycline, and to the number of Acinetobacter resistant to tetracycline. These resultsappear to indicate that the use of antibiotics in both agriculture and in humans is increasingthe incidence of antibiotic resistant bacteria in lotic environments.
Antibiotics are a vital part of our medical care system and save countless lives every day from infections that were untreatable before the introduction of these drugs.
Unfortunately, antibiotics also select for mutant bacteria that are resistant to concentrationsof previously effective drugs. Antibiotics have become so overused in clinical settings andas prophylactics and growth stimulators in farm animals that many bacteria have developedresistances to such an extent that new antibiotics and higher concentrations of older drugs,which approach levels toxic to the human system, are necessary to treat infections (Houndtand Ochman, 2000). Recent studies suggest a connection between antibiotics used inagriculture and antibiotic-resistant infections in humans (Chee-Sanford et al, 2001). Thedevelopment of antibiotic resistant bacterial strains can lead to serious human healthproblems, both from life-threatening infections and from toxic levels of drugs.
A wide range of antibiotic resistant bacteria have been isolated from polluted environmental samples including Acinetobacter spp. (Guardabassi et al, 1998, 1999 and2000) and members of the family Enterobacteriaceae (Freney et al, 1988, Parveen et al,1997, Mazel et al, 2000, and Goñi-Urriza et al, 2000). Acinetobacter are gram-negative bacteria that occur in many different environments, including soil, water, sewage, foodstuffs and on human skin (Guardabassi et al, 1999). They are typically resistant to _-lactamand aminoglycosidic antimicrobics and are possibly a reservoir of resistance genes inhospital environments, as well as being an increasingly common source of nosocomialinfections (Jawad et al, 1994). Acinetobacter were chosen for examination in this study as agauge of antibiotic resistance in both polluted and unpolluted water samples because of theirubiquity and ability to acquire resistance genes, as well as their clinical importance(Guardabassi et al, 2000).
The Enterobacteriaceae encompasses a number of genera of bacteria isolated from humans and animals including Klebsiella, Enterobacter, Serratia, Yersinia, and Escherichia.
Also included are the coliforms, which are gram-negative, lactose-fermenting bacteria thatlive in the intestines of humans and warm-blooded animals. Like Acinetobacter spp.,members of the Enterobacteriaceae are common causes of nosocomial infections and someof these bacteria can pass resistance genes across species through plasmid exchange either inthe gastrointestinal tract of their human hosts or in sewage systems (Parveen et al, 1997).
The Enterobacteriaceae were chosen for this study because they are commonly found inpolluted aquatic environments (Parveen et al, 1997) and for their importance as a cause ofnosocomial infections (Freney et al, 1988).
The introduction of antibiotics into the environment causes a selective pressure which results in an increase in the proportion of bacteria that are resistant to antibiotics(Houndt and Ochman, 2000). Increased resistance to a wide variety of antibiotics has beenfound in bacteria located in streams receiving runoff from agricultural areas (Chee-Sanfordet al, 2001) and higher frequencies of resistant bacteria have been found in streams receivingwaste water from hospitals (Guardabassi et al, 1998) and sewage from cities (Ogan andNwiika, 1993, Goñi-Urriza et al, 2000, and Parveen et al, 1997). Previous studies haveshown that aquatic isolates of Acinetobacter collected at or downstream from the dischargepoint of waste water treatment facilities are resistant to chloramphenicol and oxytetracycline(Guardabassi et al, 1998 and 1999). A study of fecal coliforms collected from similarlypolluted areas found bacteria resistant to ampicillin, chloramphenicol, tetracycline, andpenicillin (Ogan and Nwiika, 1993) and another study found higher rates of multipleantibiotic resistant (MAR) bacteria in waters polluted with sewage treatment facility effluent(Parveen et al, 1997). This study aimed to investigate whether agricultural runoff and wastewater treatment facility effluent are causing an increase in the proportions of antibioticresistant bacteria in the Oneonta area. Three locations were studied, including anagricultural area of Charlotte Creek, the Susquehanna River above and below the wastewater treatment facility, and Shelley Brook. These streams are at the headwaters of theChesapeake Bay watershed and could be important contributors to reservoirs of antibioticresistant bacteria in this watershed.
MATERIALS AND METHODS Study Sites Shelley Brook represents an area that should not have any introduced pollution. It is located on the upper-tract of Hartwick College's Pine Lake Environmental Campus in WestDavenport, NY and due to this remote location it is not near any residences and has no otherstreams flowing into it. The only potential impact is from foot traffic from a hiking trailwhich crosses it. Charlotte Creek is impacted by non-point sources of pollution.
Agricultural runoff is entering the stream, but it is difficult to tell exactly where this input occurs, and how much effluent enters the water. Conversely, the Susquehanna River is
impacted by a point source of pollution located at the outflow pipe from the waste water
treatment facility. The outflow pipe is a specific point in the river where pollution enters the
water. The exact volume of effluent and what is contained in it can be measured, and the
impact downstream of this point can be compared to the conditions above the point source
Water samples were collected in 1 liter Nalgene bottles from each of seven sites.
One sample was collected from Shelley Brook where it crosses the Jaycee Henderson trail
on the Pine Lake Environmental Campus. Three samples were collected from Charlotte
Creek along Charlotte Creek Road in Davenport, NY, each 50 meters apart. The final three
samples were taken from the Susquehanna River in Oneonta, NY, one from 50 meters above
the outflow pipe of the waste water treatment facility, one from the outflow pipe, and one
from 50 meters below the outflow pipe. Samples were refrigerated over night and processed
the following day.
Bacteria were collected from the water samples by filtering through 0.45 µm pore filters. For the Shelley Brook samples 10 ml, 5 ml, and 1 ml volumes of the sample werefiltered. For the Charlotte Creek and Susquehanna River water samples 1 ml of undilutedsample and 1 ml each of 10-1 and 10-2 dilutions were filtered. Higher volumes were filteredfor Shelley Brook because it is an unpolluted stream and has fewer bacteria than CharlotteCreek or the Susquehanna River, for which smaller volumes resulted in similar numbers ofcolony forming units. The filters were placed on selective media and the plates wereincubated over night at 37˚C for coliforms and 25˚C for Acinetobacter. MacConkey agarwas used for the selection of coliforms, and Leed's Acinetobacter media (Jawad et al, 1994)was used for the selection of Acinetobacter.
Isolated colonies picked from the selection plates with a sterile toothpick were each transferred to one well of a 96-well plate containing 150 µl of Luria-Bertani broth. Only 48
wells of each 96 well plate were inoculated. The plates were incubated over night at 37˚C.
The following day cultures were prepared for freezing by addition of a drop of sterile
glycerol to each well. Each plate was taped shut, placed into a Ziploc storage bag, and
stored at -70˚C.
Frozen stock cultures were used as the inoculum for antibiotic resistance testing. A sterile 48-prong replica-plater was dipped into the frozen stock cultures and then into thewells of a plate, each containing 150 µl of Luria-Bertani broth. The newly inoculated plateswere incubated over night at 37˚C. In order to dilute the cultures to an amount that wouldresult in distinct colonies when plated, 5 µl of freshly grown culture was transferred fromeach well of the plates to 95 µl of sterile water in the corresponding well of a new plate.
The diluted isolates were plated on Mueller-Hinton agar containing one of the fiveantibiotics at one of the three concentrations. The antibiotic plates were arranged in fiverows by antibiotic. In each row was a control plate, three antibiotic plates in order ofincreasing concentration and a second control plate, for a total of twenty-five plates. The48-prong replica-plater was dipped in the wells and stamped on each plate in succession totransfer the diluted inoculum from the microwell plate to the antibiotic and control plates.
The replica-plater was re-inoculated between plates and sterilized between rows. The process was repeated three times for a total of three replicates of each antibiotic set orseventy-five plates. The antibiotic plates were then incubated over night at 37˚C. Theantibiotics used were ampicillin (10, 30, and 50 µg/ml), chloramphenicol (10, 30, and 50µg/ml), norfloxacin (20, 40, and 60 µg/ml), streptomycin (20, 30, and 50 µg/ml), andtetracycline (10, 30, and 50 µg/ml).
The proportion of resistant colonies was calculated as the number of colonies on an antibiotic plate divided by the number of colonies on the control plates. The data wereanalyzed using ANOVA and post-hoc tests in SPSS.
Resistances of Coliform Bacteria
At the Susquehanna River sites there was a significant effect of the waste water treatment facility effluent on the proportions of coliform bacteria that were resistant toampicillin (p < .001), chloramphenicol (p < .001), streptomycin (p < .001), and tetracycline(p < .001) (Table 1). Increases in the proportions of resistant bacteria were found both at theoutflow pipe and downstream of the outflow. The increase in the proportion of resistantbacteria was particularly striking for ampicillin (Figure 1). Upstream of the outflow 62% ofthe coliform bacteria were resistant to ampicillin at the lowest concentration and 23% wereresistant at higher concentrations. At and below the outflow pipe over 95% of the coliformbacteria were resistant to all three concentrations of ampicillin. For chloramphenicol,streptomycin, and tetracycline no coliforms were resistant at the upstream site. At theoutflow and downstream of the effluent input an increase of between 2% and 38% forchloramphenicol, between 4% and 26% for streptomycin, and between 2% and 30% fortetracycline was seen in the proportions of resistant bacteria. No coliforms were resistant tonorfloxacin.
Between 23% and 58% of the coliform bacteria in Shelley Brook were resistant to ampicillin, depending on the concentration. The proportion of resistant coliforms was evenhigher at the Charlotte Creek sites where 26% to 92% were resistant to ampicillin (Figure 2).
The proportion of coliforms resistant to chloramphenicol, streptomycin, and tetracycline waslow across the Shelley Brook and Charlotte Creek sites.
The Susquehanna River site upstream of the waste water treatment facility outflow pipe was chosen for comparison to the Charlotte Creek sites in order to gauge the differencein the proportion of resistant bacteria between a river before a point of effluent impact and astream impacted by non-point sources of pollution. It was found that the two upstreamCharlotte Creek sites had significantly higher proportions of coliforms resistant to ampicillinthan were present above the outflow in the Susquehanna River ( p < .001, Figure 3). Theproportion of resistant coliform bacteria in the Susquehanna River was between 23% and62%, whereas the proportion resistant at the two Charlotte Creek sites was between 51% and92%. The proportion of coliforms resistant to streptomycin was significantly higher at thedownstream site of Charlotte Creek than at the upstream site of the Susquehanna River (p <.001, Figure 4). No coliform bacteria were resistant to streptomycin in the SusquehannaRiver site, but at the Charlotte Creek site between 6% and 12% of the coliforms wereresistant to streptomycin.
Almost 100% of the Acinetobacter in the Susquehanna River were resistant to ampicillin. Between 37% and 65% of the Acinetobacter were resistant to streptomycin, andonly about 2% of the bacteria were resistant to norfloxacin. Significant increases in theproportion of Acinetobacter resistant to chloramphenicol (p < .001, Figure 5) andtetracycline (p = .014) were seen in the outflow and downstream sites of the SusquehannaRiver.
One hundred percent of the Acinetobacter in Shelley Brook were resistant to ampicillin. Between 82% and 96% of the Acinetobacter were resistant to chloramphenicoland between 84% and 96% were resistant to streptomycin. Lower proportions of thesebacteria were resistant to norfloxacin and tetracycline. In Charlotte Creek between 97% and100% of the Acinetobacter were resistant to ampicillin, between 81% and 95% wereresistant to chloramphenicol, and between 47% and 71% were resistant to streptomycin.
Lower proportions of Acinetobacter were resistant to norfloxacin and tetracycline. ShelleyBrook was used as a basis for comparison to Charlotte Creek because it is a more pristinearea that should not be heavily impacted by agricultural pollution. In Charlotte Creek onlyresistance to tetracycline was significantly different from Shelley Brook (p < .001).
When the Charlotte Creek sites were compared to the upstream site of the Susquehanna River in the same manner as was done for the coliforms, significantly higherproportions of Acinetobacter resistant to both chloramphenicol and streptomycin were foundin Charlotte Creek. For chloramphenicol, all three Charlotte Creek sites had higherproportions of resistant Acinetobacter (p <.001, Figure 6) and for streptomycin, the twoCharlotte Creek sites farthest upstream had higher proportions of resistant bacteria (p < .001and p = .006, respectively, Figure 7).
Table 1. Results of a univariate ANOVA used to compare the percentage of resistantcoliform bacteria between the Susquehanna River sites, and between the Shelley Brook andCharlotte Creek sites.
Susquehanna River Site x concentration Site x concentration Site x concentration Site x concentration Shelley Brook and Charlotte Creek
Site x concentration Site x concentration Site x concentration Site x concentration Table 2. Results of a univariate ANOVA used to compare the percentage of resistantAcinetobacter between the Susquehanna River sites, and between the Shelley Brook andCharlotte Creek sites.
Susquehanna River Site x concentration Site x concentration Site x concentration Site x concentration Site x concentration Shelley Brook and Charlotte Creek
Site x concentration P < .001
P < .001
Site x concentration P < .001
P < .001
Site x concentration P < .001
P < .001
P < .001
Site x concentration P < .001
P < .001
Site x concentration
P < .001
Mean Proportion Resistant Figure 1. The proportions of coliform bacteria resistant to ampicillin at the SusquehannaRiver sites.
Mean Proportion Resistant Figure 2. The proportions of coliform bacteria resistant to ampicillin at the Shelley Brook(SB) and Charlotte Creek (CC) sites.
Mean Proportion Resistant Figure 3. The proportions of coliform bacteria resistant to ampicillin in Charlotte Creek(CC) compared to the upstream site of the Susquehanna River (SR). The upstream andmiddle sites of Charlotte Creek were significantly different from the upstream site of theSusquehanna River (p < .001).
Mean Proportion Resistant Figure 4. The proportions of coliform bacteria resistant to streptomycin in Charlotte Creek(CC) compared to the upstream site of the Susquehanna River (SR). The downstream site ofCharlotte Creek was significantly different from the upstream site of the Susquehanna River(p < .001).
Mean Proportion Resistant Figure 5. The proportions of Acinetobacter resistant to chloramphenicol at the SusquehannaRiver (SR) sites.
Mean Proportion Resistant Figure 6. The proportions of Acinetobacter resistant to chloramphenicol in Charlotte Creek(CC) compared to the upstream site of the Susquehanna River (SR). All three sites had asignificantly higher proportion of resistant bacteria than in the Susquehanna River site (p <.001).
Mean Proportion Resistant Figure 7. The proportions of Acinetobacter resistant to streptomycin in Charlotte Creek(CC) compared to the upstream site of the Susquehanna River (SR). The upstream andmiddle sites of Charlotte Creek had a significantly higher proportion of resistant bacteriathan the Susquehanna River site (p < .001 and p = .006, respectively).
The results of this study support the hypothesis that effluent from waste water treatment facilities impacts populations of antibiotic resistant bacteria in the SusquehannaRiver, and additionally that there is some effect of antibiotic use in agriculture or seepagefrom private septic systems on the pools of resistant bacteria in streams.
Significant increases in the proportions of coliform bacteria resistant to ampicillin, chloramphenicol, streptomycin, and tetracycline were found at and below the outflow pipeof a waste water treatment facility. There are two possible mechanisms for these increases.
The effluent may contain antibiotics that are selecting for bacteria in the river that areresistant to antibiotics. Any population of bacteria may contain mutants that are naturallyresistant to antibiotics and exposure to these drugs will kill the normal bacteria in thepopulation while selecting for the resistant mutant strains. The data for streptomycinresistance supports this possibility, as the proportion of resistance is highest at the outflowpipe and then decreases fifty meters downstream, possibly as the selective pressure of theantibiotic becomes more diluted (Chee-Sanford et al, 2001). The clinical use ofstreptomycin is likely the source of this selective pressure in the Susquehanna River. Analternative but not exclusionary hypothesis is that effluent may contain resistant bacteria thatare multiplying and potentially spreading those resistance genes to other bacteria in the riverenvironment (McArthur and Tuckfield, 2000). The water entering the waste water treatmentfacility comes from private homes and A.O. Fox hospital, where the use of antibiotics causes a selective pressure in the human system that will lead to an increase in the proportion ofresistant mutants in the sewage water. The effluent from the waste water treatment facilitymost likely contains some of these resistant mutants.
Interestingly, virtually all of the Acinetobacter isolates were resistant to ampicillin, even at the highest concentrations. Acinetobacter are commonly resistant to _-lactams(Jawad et al, 1994), which most likely explains this phenomenon. Other than resistance toampicillin, only resistance to chloramphenicol and tetracycline showed a significant increaseat the outflow and downstream sites of the Susquehanna River, indicating that the effluentmay be selecting for resistant strains of Acinetobacter in the river, or that clinical isolates ofAcinetobacter with evolved resistance to these drugs are contained in the effluent.
The proportions of resistant coliform bacteria in Shelley Brook were higher than expected and this is most likely due to the production of natural antimicrobics such as small-polypeptide defensins by bacteria competing in the environment (Houndt and Ochman,2000). These interactions may also be occurring at the Charlotte Creek sites, but to a lesserextent because the bottom of the creek has more silt and less decaying organic matter thanShelley Brook. Because Charlotte Creek is impacted by non-point sources of pollution suchas septic tank seepage and agricultural runoff it is much more difficult to assess the impactof this pollution on the numbers of resistant bacteria. The percentage of coliforms resistantto ampicillin was significantly higher than that found in Shelley Brook, and at two of theCharlotte Creek sites it was higher than that found above the outflow point of theSusquehanna River. This suggests that agricultural runoff or additionally private septic tankseepage is impacting the levels of ampicillin resistant coliforms found in these and similarstreams that are influenced by non-point sources of pollution. The levels of resistance inCharlotte Creek to streptomycin were higher than was found at the upstream SusquehannaRiver site and similar to those found at the Susquehanna River site downstream of theoutflow. Streptomycin is often used on apples and pears (Walsh, 292), so it is possible thatthis is a source of pollution in Charlotte Creek.
For the Acinetobacter, virtually 100% of the isolates were resistant to ampicillin.
This was unexpected at the Shelley Brook site where the resistance is theoretically naturallyoccurring, but again this is likely due to the natural _-lactam resistance of Acinetobacter.
Very high levels of resistance to chloramphenicol were also found in Shelley Brook and atall three Charlotte Creek sites, suggesting that there may be competitive microbialinteractions occurring, or that Acinetobacter isolates are naturally resistant tochloramphenicol. Additionally, a very high proportion of Acinetobacter in Shelley Brook,and only a slightly lesser proportion in Charlotte Creek, were resistant to streptomycin. Thisis most likely due to the presence of Streptomyces, the natural source of streptomycin(Demain and Lancini, 1991), in the soil in the streambed and along the shores of ShelleyBrook and Charlotte Creek. The Susquehanna River is a larger and faster-flowing body ofwater, so the bacterial community living in it may be less impacted by the presence ofStreptomyces.
The proportion of Acinetobacter resistant to chloramphenicol at all three of the Charlotte Creek sites was higher than that found above the outflow site of the SusquehannaRiver. This suggests that there is some use of chloramphenicol in the area that drains intoCharlotte Creek that is impacting environmental isolates of Acinetobacter in a waycomparable to how the use of chloramphenicol in medicine is impacting clinical isolates ofAcinetobacter. Additionally, the proportion of Acinetobacter resistant to streptomycin washigher at two of the Charlotte Creek sites than at the upstream site in the Susquehanna River.
This may be due to naturally occurring resistance that can develop in response tointeractions with Streptomyces as was discussed above.
It is very important that we gain a better understanding of how the use of antibiotics in both agriculture and medicine is impacting both environmental and clinical isolates ofbacteria. Antibiotics not only select for resistant strains of bacteria in clinical settings, butthey are also poorly absorbed in both humans and animals, and waste in the form ofagricultural runoff into streams and waste water treatment facility effluent often containsantibiotics that continue to select for resistant strains in the environment. The higher levelsof coliforms resistant to ampicillin, chloramphenicol, and tetracycline found at thedownstream site of the Susquehanna River indicate that the resistant bacteria may not onlybe multiplying, but could also be transferring resistance genes to other bacteria in theenvironment (Kelch and Lee, 1978). This results in reservoirs of resistant bacteria that canpass genes for resistance on to other bacteria ultimately leading to the faster spread ofresistance genes to not only bacteria within the same species, but also to other more diversetypes of bacteria. As genes conferring resistance to common antibiotics spread it becomesincreasingly likely that an individual will become infected with a resistant strain of abacteria that may cause an infection which could be very difficult to treat. Understandinghow our use of antibiotics impacts bacteria in environmental settings may help reduce thespread of these resistance genes. Charlotte Creek flows into the Susquehanna River, andthis river lies at the headwaters of the Chesapeake Bay watershed. Any reservoirs ofresistant bacteria that are contained in this water will only be compounded as more streamsand rivers polluted by agricultural runoff and waste water treatment facility effluentcontribute to the watershed.
Thank you to the Biology Department for funding this project. I would like to thank my advisor, Dr. Mary Allen, for all of her assistance in conducting this research, and RobHunt for ordering all of my supplies. Thank you to Valerie Harwood, University of SouthFlorida Department of Biology, for providing the protocol. Thank you also to Colleen Didasfor helping me collect my samples, and to Dave Miller for continuing this research nextyear.
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William E. Seidelman MDScience and Inhumanity: The Kaiser-Wilhelm/Max Planck Society First Published in: If Not Now an e-journal Volume 2, Winter 2000 Revised February 18, 2001. One hundred years ago this past December a German scientist by the name of Max Karl Ernst Ludwig Planck gave a lecture in Berlin to the German Physical