Information on Bacteria Found

              Methods and Materials

              Results

Excel file of raw data (for class use only)

Colony morphology of Bacteria isolated

Description of triclosan resistant isolates

Antibiotic Resistance Profiles
 

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       Triclosan, a broad-spectrum antibacterial agent has recently become popular for use in many products due to its bacteriostatic activity against both gram-positive and gram-negative bacterium.  It is the most potent and widely used member to a class of compounds called 2-Hydroxyphenyl ethers.   Triclosan is a diphenyl ether derivative, 2,4,4’-Trichloro-2’-hydroxydiphenyl ether.  It is most commonly found in personal care products such as toothpastes, deodorants, body washes, detergents, dishwashing liquids, cosmetics, lotion and hand soaps.  It can also be found as an additive in plastics, polymers, and textiles to give these products antibacterial properties.  Triclosan can be found in places least expected, such as baby toys, carpeting, and even hunting clothes.  This highly popular biocide can easily be incorporated into hundreds of products, and during the past 3 decades its use has soared.

        Scientists first thought that bacteria could not become resistant to triclosan because the agent destroyed the whole population of bacteria, acting more as a “grenade than as a bullet.”  This means that it was though that rather than acting like an antibiotic and targeting a single protein, triclosan would act more like bleach and alcohol, affecting multiple cellular targets.   They could not be more wrong.   Affecting multiple cellular targets simultaneously led to the perception that bacteria would not be able to readily develop resistance to triclosan.  However, it has recently been found that triclosan does indeed specifically target an enzyme involved in fatty acid synthesis, an enoyl-acyl carrier protein reductase.  Since this finding, more research has been conducted exploring the possibility that bacteria could become resistant to triclosan and what the implications this may have on the antibiotics currently used to kill bacteria.

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Chuanchuen, R., K. Beinlich, T.T. Hoang, A. Becher, R.R. Karkhoff-Schweizer, and H.P.
Schweizer.  (2001) Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by mulitdrug efflux pumps:  exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing mexCD-oprJ.  Antimicrobial Agents and Chemotherapy.  45:  428-432.

Meade, M.J., R.L. Waddell, T.M. Callahan.  (2000) Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. Dentrificans inactivate triclosan in liquid and solid substrates.  FEMS Letters Submission.

Parisi-Menoutis, A.I., J. Menoutis.  (2001) Triclosan and its impurities.  Technology Review Series. <http://www.quantexlabs.com/triclosan.htm>

Travis, J.  (2000) Popularity of germ fighter raises concern.  Science News.  157:  1-2.

Bacteria Found

As a result of PCR analysis it was determined that the main bacterium were several strains of pseudomonas and serratia.  The strains that were identified were P. putida, P. aeruginosa, P. alcaligenes, and S. marcescens.

 

Pseudomonas: This is a gram-negative aerobic bacillus.  It is a motile bacterium that is capable of producing water-soluble pigments that spread through media. This pigment is usually blue-green in color.  Pseudomonas aeruginosa is commonly found in soil, water, and plants.  However, it is able to survive in any moist environment.  Most human P. aeruginosa infections are nosocomial and generally acquired in a hospital setting.  These infections include skin, eye, urinary tract, open wounds, and can lead to pneumonia, meningitis, abscesses, and septicemia. It is transmitted through direct contact and can even be acquired from plants and flowers.  P. putida is known for its ability to degrade chemicals like trichloroethylene. Treatment of Pseudomonas infections consists of a combination of two antibiotics: for example an anti-pseudomonal penicillin and an aminoglycoside. These bacteria are clinically important because they are resistant to most antibiotics and they are capable of surviving in conditions that few other organisms can tolerate. They also produce a slime layer that is resistant to phagocytosis.

Serratia:

This is gram negative anerobic bacillus.  It is motile and produces characteristic red pigment at room temperature. Serratia marcescens is considered a harmful human pathogen. It naturally occurs in soil and water as well as the intestines and can be found in catheters.  This bacterium can be transmitted via direct contact.  It can be associated with urinary and respiratory infections, endocarditis, osteomyelitis, septicemia, eye infections, and meningitis. Some treatments include cephalosporins, gentamicia, and amikacin.  However, most strains are resistant to some antibiotics. Serratia can be distinguished from other genera belonging to Enterobacteriaceae by its production of three special enzymes: DNase, lipase, and gelatinase.

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Materials and Methods

Experimental Design:

 For this experiment, four bathrooms from the dormitory, Caflisch Hall, on the campus of Allegheny College, Meadville Pennsylvania, were chosen.  Caflisch Hall is broken up into three floors and each floor is broken up into five sections as shown in figure 1.  The bathrooms connecting sections 1 and 2 and sections 4 and 5 were used on both the first and second floor.  The bathroom connecting sections 1 and 2 on the first floor and the bathroom connecting 4 and 5 on the second floor are female while the other two bathrooms are male.

 

   Each bathroom is similar in the amount that it is used.  Each section has on average 10 people living in it, so each total bathroom has about 20 people, 10 from each side using it.  Since this is a dormitory the bathrooms are used for a variety of purposes including showering, going to the bathroom, washing dishes, shaving, and a number of other purposes that may have introduced a number of chemicals into the environment.  All of these were held constant as people living in the dormitory continued their normal lives during the experiment so that conditions would remain the same.  The normal daily cleaning of the bathroom continued through the experiment.  The cleaning crew used the cleaner, Johnson Wax Professional Triad Germicidal Cleaner and Deodorant, which contains the active ingredients 1.140% octyl decyl dimethyl ammonium chloride, .456% dioctyl dimethyl ammonium chloride, .684% didecyl dimethyl ammonium chloride, and 1.520% alkyl dimethyl benzyl ammonium chloride.  The cleaning was done early in the morning.

 In the bathrooms, the normal soap, a non-antibacterial body soap\shampoo was removed from the bathrooms, and replaced with a small, blue soap dispenser.  In the bathrooms connecting sections 1 and 2 on both the first and second floors, Softsoap Brand Hand Soap, Vitamins: Vitamin E Complex was used which contains vitamins A and E, but has no advertised antibacterial ingredients.  In the bathrooms connecting sections 4 and 5 on both the first and second floor, Softsoap Brand Hand Soap, Antibacterial with Extra Moisturizers was used, which contains the active ingredient triclosan.  The soaps were chosen because of their antibacterial and non-antibacterial powers, but also because of their similar color.  The soaps remained in the bathrooms for 6 weeks.  During this duration a similar amount was used from each dispenser, about 24 ounces or 4 ounces per week per dispenser.

Sampling:

 Through the duration of the experiment, samples were taken from the door handle, the pump on the soap dispenser, and the cold water knob of the sink.  In bathrooms with only two sinks, the sink on the right was used, and in bathrooms with three sinks the middle sink was sampled.  These areas were chosen because they are frequently touched while a person is washing their hands.  These samples were taken from both sides of all of the bathrooms late in the evening around 8:00 or 9:00 PM.  The layout of the bathrooms can be seen in figure 2.  The bathrooms were sampled once before the soaps were added into the bathroom (minus the soap dispenser, since it was not yet present) and then 7 times over the six week period of the experiment.

 

 Sampling was done using sterile distilled water and sterile cotton swabs.  30mL of water was poured over the object being sampled and collected in a sterile 50mL polypropyline bottle (LPS).  The cotton swaps were then used to scrape any excess water into the bottle.  The samples were then marked and refrigerated until they were plated.

Plating:

 Plating was done within 24 hours of sampling.  Vacuum filtration was used with .45?m cellulose nitrate membrane filters (at the beginning the filters were from Gelman and then when we ran out we switched to Whatman due to convenience).  The samples were plated on both tryptic soy agar plates and tryptic soy agar plates with .2% triclosan (KIC chemicals).  After the samples were plated they were placed in a 37oC incubator for 48 hours.

Isolation, Antibiotic Resistance, and Staining:

 Isolated colonies that grew on the TSA plus triclosan plates were grown overnight in a nutrient broth at 37oC at 225 rpm’s.  These broths were then used for a standard disk diffusion assay testing the isolates for resistance to 30% kanomycin, 15% erythromycin, 10% ampicillin, 1.25% sulfamethoxazole plus 3.75% trimethoprim, 30% chloramphenical, 5% raifampin, and 30% tetracycline.  These isolates for also stained for identification and examination by standard Gram staining.  Isolates selected for PCR were also tested for resistance to penicillin, ciprofloxacin, ceftazidime, and doxycycline.  PCR was then performed on these multi-drug resistant isolates.

Template Preparation:

 The cell preparation consisted of growing broth cultures of bacteria that were selected for identification overnight at 37oC with shaking.  Then 1.5 ml of bacterial sample were dispensed into each microfuge tube and centrifuged at 6000 rpm for four minutes to pellet cells.  The supernatant was discarded and the cells were then resuspended in 0.5 ml of 1M NaCl by vortexing.  The cells were pelleted again by centrifugation and the supernatant was carefully discarded.  Finally the cell pellets were resuspended in 100 ul of sterile distilled water.

 

PCR Procedure:

 The PCR reaction used 4 ul of the cells prepared above, 75 ul of sterile distilled water, 10ul of 10X PCR buffer, 8 ul of dNTP mix, 0.5 ul of rTaq polymerase (Takara), 1 ul of primer 8f, and 1 ul of primer 518r.   Primer 518r has sequence 5’- ATT ACC GCG GCT GCT GG-3’ with a melting temperature of 57.6oC and primer 8f has sequence 5’- AGA GTT TGA TCC TGG CTC AG – 3’ with a melting temperature of 57.3oC.  The cycling parameters were 95oC for 3 minutes and then 30 cycles of each of the following: 95oC for 30 seconds, 55oC for 30 seconds, and 72oC for 2 minutes.  The final cycle was 72oC for 7 minutes.  The PCR products were then run on 1% agarose gel made with TBE.

Cloning and Transformation of PCR Products:

The cloning procedure that was followed was that described by the Promega pGEM-T vector system.  The resulting plasmid transformed into library efficiency chemically competent DH5? (Gibco-BRL).  A microtansformation using 20 ul of cells was employed.  Briefly, 4 ul of DNA from the miniprep was added to the 20 ul of cells with gentle tapping to mix.  The cells were incubated on ice for 30 minutes and then they were heat shocked for 40 seconds in a 42oC water bath.  They were placed on ice immediately for 2 minutes and then

1 hour. All of the cells were then spread on LB with selective agent (triclosan for experimental, ampicillin for controls) using a sterile spreader.  The plates were incubated overnight at 37oC and the next day the colonies on the plate were counted and transferred to fresh LB+triclosan or LB+ampicillin using sterile toothpicks according to grid.

Survey of the Residents using Caflisch Bathrooms

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Results and Discussion

Experimental Conditions:

        Making use of the dormitory restrooms for the experiment were chosen because of the conformity of the conditions.  Regular housekeeping staff clean and use the same routines from week to week with the same products.  This greatly influences the normal microflora in this environment and helps to keep it consistent.  The restrooms also gave the advantage of allowing the testing of multiple variables (ie. doors, sinks, and soap dispensers).  The normal conditions were easily manipulated to produce our experimental conditions of allowing the regular use of antibacterial or non-antibacterial hand soaps during the six week study.  The restrooms were also regularly used due to normal collegiate traffic, which guaranteed the consistent introduction of new microbes to the environment.  Also, taken into consideration were the differences in male versus female restroom habits.  Female college residents tend to have a more antibacterial product-based hygienic routine and male college residents tend to be quite content with regular soaps and other hygiene products.  The methods of sampling by vacuum filtration techniques were chosen for its higher sensitivity compared with regular swabbing of the sample.  The concentration of triclosan used during plating (0.2%) was chosen because it is a relatively selective amount that will disrupt the cellular membrane synthesis and structure, thereby lysing the cell.  The antibiotic disks that were used in the disk diffusion assay were used because of their broad-spectrum mode of action and to help identify similarities and differences between isolates.  PCR was used as a sensitive molecular technique in order to identify isolates as well.
    Gram Reactions:
        Of the forty-six triclosan isolates taken from the experimental period of six weeks, twenty-four were clearly identified as gram-negative, four were mixed samples, and six were clearly identified as gram-positive.  Twelve of the isolates have an undetermined gram reaction at the present.  The gram-negative isolates are consistent with the regular environment as those microbes are probably of the enteric bacteria super-family.  It can be speculated that the gram-positive isolates may be from soil or oral origin.  These can be further identified using biochemical techniques, though not performed in this study to date, to make certain the microbe origin.  By studying colony morphology on sample plates and cell morphology under microscopy, two genera have been identified including several Pseudomonas spp., and one Serratia marcescans both of which are of the enteric super-family.
    Triclosan Resistance:
    According to the data spreadsheet, a general trend can be seen by looking at the relative resistance between female and male restrooms.  Looking at the 1-1, 1-2, and 2-4, 2-5 samples, which are from the female restrooms, it can be observed that triclosan resistance is more prevalent in these areas than in the corresponding male restrooms.  This can be explained by females being more conscientious and using more antibacterial products for everyday use (ie. facial cleansers, Colgate total toothpaste, hand/dish soap, mouthwashes, etc.).  The 2-4 and 2-5 samples, which are of the triclosan experimental group, show more resistance than the non-triclosan control.  This was consistent with the proposed hypothesis that regular triclosan usage would promote more resistance within the restroom environment.  In the male restrooms, however, approximately an equal amount of triclosan resistance was found present in the sample bacteria.  This can be attributed to perhaps a less likelihood of washing hands before leaving the restroom. (See ASM site http://www.washup.org/page03.htm.).  Therefore, it may be less likely to see a change in flora due to different soaps.  There seemed to be a shift in the types of colonies present from week 1 to week 6.  In the first week, it was observed that there were many more white and yellow colonies than in the later periods.   Also, a dramatic shift between TSA and TSA+tric bacterial colonization was found.  Multiple bacterial levels of colonies encompassing white, yellow, and tan morphologies were significantly reduced in number on the TSA+ tric plates.  The first week of data was left in the incubator for 52 hours and the remaining weeks were only left in for 24-48 hours.  It is assumed that the triclosan resistant colonies are more slowly growing and do not exemplify themselves as much in the latter time period.  Knowing this, in additional studies, it would be useful to allow for a longer incubation period.
Antibiotic Resistance:
        By taking the antibiotic resistance assay of the isolates in combination with colony morphology, it can be seen which isolates actually may be identical species. The bacteria were most resistant to Rifampin and Erythromycin, which makes them the least effective on the microflora found in the restrooms tested.  However, the bacteria showed most susceptibility to Chloroamphenicol, Kanamycin, and Tetracycline respectively.   Several similarities exist between the isolates and suggest a close relationship between if not identical genus and species.  Isolates 1,2,38,and 40 seem to be very closely related.  Isolates 1,2, and 40 may be related to the species and 38 could be related to the genera due to color but have the relatively same antibiotic susceptibility.  Isolates 8, 9, 31 are related, 8 & 9 to the species and by speculation 31 to the genera.    Isolates 13 & 14 may be related to the genera, but not species due to slight differences in color.  There may be other similarities but because there are too many gaps in the data collection it is impossible to make a speculation with any certainty.
    PCR:
        Of the three isolates’ PCR products were sent to Penn State University’s Lab for identification, only one came back positive.  The other two were found to not have enough RNA to make a positive match.  The only result that was received was for isolate #15, which was identified asPseudomonas putida.
    Experimental Improvements:
        The specified dates on the data spreadsheet are ambiguous as to when the plates were either taken or plated.  It would be beneficial for further experimentation to label according to week of the experimental data collected.  Also, uniformity in the colony descriptions would be helpful as to determining similarities between isolates.  This may be controlled by assigning different researchers with a specific task (ie. collection, plating, morphology, etc.), instead of splitting up the actual plates and dividing accordingly.  In this respect, one or two individuals would be assigning characteristics, which would be comparative to the rest and would be less indeterminate.  An additional sampling of the paper towel dispensers would be useful because it is the one of the first objects that is touched right after hand washing in the restroom.  Another sampling between separate edifices of female versus male restroom conditions would be useful for describing more of a grand effect of sex differences.  More control over the housekeeping routine, such as giving different cleansers to provide a more consistent flora with triclosan and non-triclosan products being used.  Also, biochemical techniques for identifications of isolates could be useful and less expensive than PCR.  Most of the isolates appear to be of enteric origin and therefore could be identified via Enterotube II usage, or separate tests of the IMViC series, and/or TSI agar testing.
    Systematic Error:
        Gram stains in some areas are undetermined due to ineffective means of procuring bacteria for proper staining methods.  Data given on the spreadsheet is unclear due to the fact that in some areas isolates claim for TSA+Tric having no growth but still have colonies in the same row.  Errors in colony descriptions by having separate researchers evaluating them under individual discernment.  This made it difficult to assign what each actually meant by the descriptions primarily given.  Additional samples should be taken to ensure reliability and validity of results.
 

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