2012

Antibiotic Resistance in Wastewater Bacteria

Microbiology
In This document I have tried to give an overview of the root cause due to which we face antibiotic resistance in wastewater bacteria & how does it affect us. Moreover some measures to eliminate root causes are also discussed.

Haider Ali & Shujja Haider Institute of Environmental Science & Engineering, National University of Science & Technology 12/2/2012

Contents
1. 2. 3. INTRODUCTION..................................................................................................................................... 1 ANTIBIOTIC RESISTANT BACTERIA ........................................................................................................ 2 WATER AND WASTEWATER CHARACTERISTICS.................................................................................... 4 3.1 3.2 3.3 4. Physical Agents: ............................................................................................................................ 4 Chemical Agents:...........................................................................................................................4 Biological Agents:.......................................................................................................................... 4

INDICATOR ORGANISMS FOR FECAL CONTAMINATION OF WATER..................................................... 5 4.1 Fecal coliforms .................................................................................................................................. 5 4.2 Escherichia coli.................................................................................................................................. 5 4.3 Enterococci........................................................................................................................................ 5

5. 6. 7. 8.

ANTIBIOTIC RESISTANT MICROORGANISMS:........................................................................................6 TARGET ANTIBIOTICS: ........................................................................................................................... 9 SUMMARY AND CONCLUSONS ...........................................................................................................11 REFERENCES ........................................................................................................................................ 12

Antibiotic Resistance In Wastewater Bacteria

1. Introduction

1. INTRODUCTION
To improve the quality of life worldwide antibiotics and pharmaceuticals are commonly used. The release of large amount of pharmaceutical drugs into municipal wastewater results due to incomplete metabolism in humans. The treatment of infections caused by antibiotic resistant organisms is difficult.[1]
[2]

[1]

Antibiotics which are consumed ends up in wastewater in large amount. However, resistance among microorganisms is maintained by antibiotics which may exert selective pressure in wastewater. As compared to surface water, often higher rates and concentrations of antibiotic resistant bacteria and genes encoding antibiotic resistance are commonly detected in wastewater. For the growth of a diverse bacterial community, waste water can also provide favorable conditions, which comprises the basis for the selection and spread of antibiotic resistance. Therefore, in the dissemination and development of antibiotic resistant bacteria wastewater treatment plants plays a vital role. As a nosocomial pathogen Methicillin-resistant Staphylococcus aureus (MRSA) is a large worldwide problem but the knowledge about the occurrence of MRSA is limited.[2] Effective treatment with antibiotics of many diseases that once killed people is now available. However, to commonly used antibiotics,some bacteria have become resistant.Bacteria that are not controlled or killed by antibiotics are called antibiotic resistant bacteria. In the presence of an antibiotic they are able to survive and even multiply. To at least some antibiotics most of the infection-causing bacteria have now become resistant. Multi-resistant organisms (MROs) are bacteria that are resistant to many antibiotics. Serious diseases can be caused by antibiotic resistance and is an important public health problem. Bythe correct use of prescribed antibiotics,minimizing unnecessary prescribing and overprescribing of antibiotics, good hygiene and infection control, antibiotic resistance can be prevented. There are few bacteria which are naturally resistant to few antibiotics. For example on most organisms found in human digestive system (gut), Benzyl penicillin has very little effect.[3]

[3]

Antibiotic Resistance In Wastewater Bacteria

2. Antibiotic Resistant Bacteria

2. ANTIBIOTIC RESISTANT BACTERIA

Antibiotics are cell wall synthesis, protein synthesis and nucleic acid synthesis inhibitors or they can affect the cell membrane integrity by acting on microorganisms. With the increase in demand of antibiotics in this era resistant bacteria are also increases especially in nosocomial environment. It has been reported that there is a global rise in antibiotic resistance. A comparison of strains of E. coli and Salmonella enterica showed that a few decades back they were susceptible to antibiotics while at present time 20 percent of them show resistance to at least one of the antibiotics. In U.S, 46 percent of Streptococcuspneumonia isolates are penicillin resistant, and 30 percent of nosocomial infections are methicillin-resistant Staphylococcus aureus. An investigation showed that E. coli strains in a wastewater treatment plant were resistant to 16 of 24 tested anti-biotics. When the influent to the wastewater treatment plant is from a hospital source the number of antibiotic-resistant strains is increased.From 60 percent of raw wastewater and 36 percent of wastewater effluents Vancomycin-resistant enterococci (VRE) were isolated and it was found that the minimum inhibitory concentrations (MIC) of VRE from hospital wastewaters were much higher than those from residential wastewaters. A large number of antibiotic-resistant bacteria were present in the intestinal tract of patients receiving antibiotic therapy. Bacteria are excreted in large numbers in feces ending up in community wastewater treatment plant. Under appropriate conditions, the genes coding for antibiotic resistance are often located on plasmids (R factors) can be transferred to other bacteria through conjugation that requires cell-to-cell contact, or through other modes of recombination. As a result of the acquisition of antibiotic resistance if the recipient bacteria are potential pathogens, they may be of public health concern. Microorganisms which are drugresistant produce nosocomial and community-acquired human infections that lead to increased morbidity, mortality, and disease incidence. Based on administration of antibiotics to patients exposed to pathogens of environmental origin resistance to antibiotics, including quinolones (e.g., nalidixic acid, ciprofloxacin), can increase the cost and can in turn complicate the therapy. Patients infected with antibiotic-resistant bacterial strains are likely to require hospitalization, sometimes for long periods if they are infected with antibiotic-resistant bacterial strains. In natural environments as well as in engineered systems such as wastewater treatment plants gene transfer between microorganisms is known to occur. To demonstrate the transfer of R plasmids among bacteria in domestic wastewater, survival chambers have been used by the investigators. Between Salmonellaenteritidisand E.coli the highest transfer frequency (2.7 x 1024) was observed. Under low temperature, low nutrient conditions in drinking water and
[4]

Antibiotic Resistance In Wastewater Bacteria

2. Antibiotic Resistant Bacteria

laboratory conditions, plasmid mobilization from genetically engineered bacteria to environmental strains were also demonstrated. The documentation on the occurrence of multiple-antibiotic resistant (MAR) indicator and pathogenic (e.g.,Salmonella) bacteria in water and wastewater treatment plants has been made. The percentage of multiple-antibiotic resistant coliforms varies between less than 1 to about 5 percent of the total coliforms in untreated wastewater. In wastewater treatment plants, chlorination appears to select for resistance to antibiotics. However, chlorination increased the bacterial resistance to some antibiotics (e.g., ampicillin, tetra-cycline) but not to others (e.g., chloramphenicol, gentamicin) according to others observation (Murray et al., 1984). After water and wastewater treatment the proportion of bacteria carrying R factors seems to increase. The percentage of MAR bacteria rose from 15.8 percent in untreated (river) water to 57.1 percent in treated water, in a water treatment plant in Oregon. Furthermore, multiple-antibiotic resistance is associated with resistance to heavy metals (e.g., Cu2,Pb2,Zn2). In both drinking water(Calomiris et al., 1984) as well as in wastewater (Varmaet al., 1976), this phenomenon was observed. Further study is required for the public health significance of this phenomenon.[4]

Antibiotic Resistance In Wastewater Bacteria

3. Water and wastewater Characteristics

3. WATER AND WASTEWATER CHARACTERISTICS

[5]

Mixture of water and dissolved or suspended solids form waste water. Raw wastewater includes following constituents:

3.1 Physical Agents:

They include total solids consisting of floating, colloidal and settle able particles. Other significant physical characteristics are temperature, conductivity, color, density, turbidity, specific weight and specific gravity.

3.2 Chemical Agents:

The chemical constituents are divided into two main categories:

a. Organic constituents
They comprise of Biochemical oxygen demand (BOD) and Chemical Oxygen Demand (COD) parameters.

b. Inorganic constituents
They include nutrients, and metallic and non- metallic constituents.

3.3 Biological Agents:

They include microorganisms of human and animal origin. Organisms which are present in wastewater include algae, bacteria, viruses, protozoa, and fungi. Among to the above agents, biological agents are more important because they include pathogenic bacteria of human origin. In untreated water bacteria, viruses, protozoa and helminthes are potential infectious agents. [5]

Antibiotic Resistance In Wastewater Bacteria

4. Indicator Organisms for Fecal Contamination of Water

4. INDICATOR ORGANISMS FOR FECAL CONTAMINATION OF WATER

Pathogenic bacteria from human origin cause fecal contamination in water which was realized in early 1880s. For the measures of surface water and wastewater quality fecal indicator organism are used. Occurrence offecal indicative in water indicates the potential presence of pathogenic organisms which are capable of posing a threat topublic health has been noted by The Environmental Protection Agency (EPA). Total coliforms, fecal coliforms, enterococci and Escherichia coli are included in fecal indicator organisms. These organisms are discharged into wastewater treatment plants through human feces which are the natural inhabitants of gastrointestinal tracts in humans and warm-blooded animals. Various diseases including cholera, typhoid, hepatitis, diarrhea and endocarditis are caused by fecalindicators.
[6]

4.1 Fecal coliforms

Gram-negative bacteria that live in the digestive tract of warm-blooded animals and humans are called Fecal Coliforms. From fecal origin, they are the indicators of potentially pathogenic bacteria. Fecal coliforms ultimately reach wastewater treatment plants which are excreted in the feces by humans and animals. Hence, in raw wastewater a huge amount of fecal coliforms are observed.

4.2

Escherichia coli

As indicators of microbiological qualityof water, Escherichia coli (E.coli) are used. They arefound naturally in both human and animal intestines and they are gram-negative bacteria. E. coli helps the bodyto absorb important vitamins from food and plays a vital role in digestion. Among several strains, most of E. colistrains are human friendly but few like E. coli 0157:H7 are pathogenic to humans.E.coli 0157:H7 causes several intestinal and extra intestinal infections such as urinary tract infection, meningitis and diarrhea.

4.3

Enterococci

Gram-positive bacterium commonly present in human intestines is known as Enterococci. For humans for many years Enterococci have been recognized as potentially pathogenic bacteria. As the most prevalent species responsible for clinical infections in humans enterococci species, E. faecalis and E. faecium have been identified. Endocarditis, bacteremia, urinary tract infections and intra-abdominal pelvic and soft tissue are infections which are commonly caused by enterococci. Most of infecting strains are originated in human intestines.
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Antibiotic Resistance In Wastewater Bacteria

5. Antibiotic Resistant Microorganisms

5. ANTIBIOTIC RESISTANT MICROORGANISMS:

The ability of microorganisms to withstand the effects of antibiotics is called Antibiotic resistance. As treatment with antibiotic begins, a patient can developan antibiotic resistant infection by contacting a resistant organism, or by having a resistant microbe emerge in the body because the development and proliferation of antibiotic resistance in bacteria is of public health concern. In 1970, as potential environmental contaminants and a threat to public health, antimicrobial agents were described and non-medical uses of antibiotics were questioned. Occurrence of antibiotic resistant organisms in environmental samples and advocated a global public health concern due to these bacteria have been reported in several studies since that time. Drug inactivation or modification, alteration of the target site, alteration in the metabolic pathway, and reduced drug accumulation are the important mechanisms by which microorganisms exhibit resistance to antibiotics.

5.1

Drug inactivation or modification:

The synthesis and secretion of enzymes by resistant bacteria affect the antimicrobial activity of the antibiotics. For example,antibiotic is inactivated by lactamases which is synthesized by antibiotic resistant bacteria hydrolyze the -lactone ring of penicillin

5.2

Alteration of target site:

By attaching to penicillin binding proteins (PBP), Penicillin acts on bacteria, which are essential components for the synthesis of bacterial cell wall. Bythe overproduction of PBPs or by synthesis of PBPs, which have low affinity to penicillin, bacteria can develop resistance to penicillin.

5.3

Alteration of metabolic pathway:

In order to evade the action of antibiotics bacteria are able to modify their metabolic pathways. For example, synthesis of folic acid is inhibited by sulfonamides, and alternate routes for synthesis of folic acid or derepress its synthesis are developed by sulfanomide resistant bacteria

5.4

Reduced drug accumulation:

Reduction of the uptake of the antibiotic by either altering the permeability of the drug or by enhancing active efflux of the drug is possible by bacteria developing resistance to antibiotics[6]
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Antibiotic Resistance In Wastewater Bacteria

5. Antibiotic Resistant Microorganisms

[7]Previously it was believed that Primary resistance is the resistance in bacteria which is acquired by spontaneous mutation. Researchers started to believe that another mechanism beyond spontaneous mutation was responsible for the acquisition of antibiotic resistance because of the wide spread development of multiple antibiotic resistance in many species of bacteria. Lateral or horizontal gene transfer was the mechanisms responsible for the development of resistance. Three possible mechanisms of Horizontal gene transfer (HGT) are: Transduction Transformation Conjunction

Transduction
When bacteria-specific viruses or bacteriophages transfer DNA between two closely related bacteria, transduction occurs.

Transformation
When parts of DNA are taken up by the bacteria from the external environment, process of transformation occurs.

Conjunction:
Due to death of another bacterium, this DNA present in the external environment.When transfer of small pieces of DNA called plasmids takes place and there is direct cell-cell contact between two bacteria, Conjugation occurs. Presence of antibiotic resistant bacteria in wastewater and surface waters has been shown by recent studies. Multi-resistant antibiotic fecal coliforms and enterococci in influent and effluent wastewater from treatment plants were observed by Gallert et al (2005). In wastewater treatment plants across the world multiple anitibiotic resistant organisms have been observed. In one of the treatment plant effluents in Finland, more than 20 % of fecal coliforms were observed were resistant to ampicillin, chloramphenicol, streptomycin, tetracycline and sulfanomide. Fecal coliforms and E. coliinraw sewage were resistant to ampillicin, gentamycin, kanamycin, neomycin and streptomycin as revealed by other studies across the world.Recent studies show that in leukemia patients,fluoroquinolone resistant E. coliisolateshave been identified. A survival benefit to microorganisms and making the elimination of the infections caused by them difficult is provided by antibiotic resistance. The treatment of infections caused by antibiotic resistant bacteria is hard. Hence, a higher dosage of alternative antibiotics to cure these infections is prescribed by the physicians. High doses have potential to produce more
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Antibiotic Resistance In Wastewater Bacteria

5. Antibiotic Resistant Microorganisms

antibiotic-resistant strains of bacteria and have side effects. Hence, study of antibiotic resistance patterns in wastewater bacteria is needed.[7]

Antibiotic Resistance In Wastewater Bacteria

6. Target Antibiotics

6. TARGET ANTIBIOTICS:
[8]

Target antibiotics are: Ciprofloxacin (CIP), Sulfamethoxazole/trimethoprim (SXT) and Vancomycin (VAN)

Based on past work in KSUs environmental engineering laboratories, these compounds were chosen as target antibiotics.Occurrence of CIP, sulfamethoxazole (SMX) and azithromycin (AZI) in municipal wastewater treatment plants has been reported by (Koch et al. 2005; Close 2007).This made it likely that antibiotic resistant strains of bacteria are also included in the microbial biomass in these plants. In combination with sulfamethoxazole and was added as a target antibiotic in this study, trimethoprim is used. VAN is the only drug that is effective to treat the infections caused by resistant enterococcus bacteria and because enterococci were resistant to many antibiotics Vancomycin was selected. Further detail of these target antibiotics is describedin the following sections.

Ciprofloxacin
Ciprofloxacin belonging to the fluoroquinoline group used to treat infections caused by gramnegative and gram-positive bacteriais a widely prescribed antibacterial agent. Patients suffering from cirrhosis can be treated by CIP and is also used for the treatment of urinary tract infections, skin and bone infections, gastrointestinal infections which are causedby multi-drugresistant organisms, lower respiratory tract infections, febrile neutrophenia, and intraabdominal infections

Sulfamethoxazole/Trimethoprim
Trimethoprim is a synergist of the sulfonamide group while Sulfamethoxazole is asulfanomide group of antibiotic. In combination with other drugs, trimethoprim is used. Patients suffering from Wegeners granulomatosis which is a rare disease that primarily affects the upper respiratory tract, kidneys and lungs, SXT is used for their treatment. Inflammation in varioustissues including blood vessels is characterized in this disease. The human immune deficiency virus (HIV) infection and pneumonia caused by Pneumocystis are also treated by SXT.
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Antibiotic Resistance In Wastewater Bacteria

6. Target Antibiotics

Vancomycin
Aglycopeptide antimicrobial agent which is active against infections caused by mainly grampositive bacteria is known as Vancomycin and used as a last resort antibacterial agent. Antibiotic resistant enterococci can be treated with vancomycin when treatment with other antibiotics has failed. It acts synergistically with aminoglycosides for organisms such as enterococci and inhibits synthesis of a cell wall. Infections caused by susceptible organisms resistant to penicillins (methicillin-resistant staphlococcusaureusand multiresistant staphylococcus epidermidis) and infections like pseudo membranous colitis are treated by VAN.
[8]

ROLE OF AMINOGLYCOSIDES IN WASTE WATER

[9]

Due to the fact that antibiotics are not persistent in the environment so they are scarcely studied. They are more readily degraded in the environment as compared to many other antibiotic classes. Furthermore, under acidic conditions aminoglycosides are generally positively charged which may facilitate the adsorption to negatively charged soil and clay particles, and as a result concentration is decreased. However, the occurrence of aminoglycosides in hospital wastewater in one study showed the concentration from 0.4 to 7.6 g per litre.[
9]

WAYS OF TRANSMISSION OF BACTERIA IN HOSPITALS:

[10]

The most common ways through which bacteria can be passed from one patient to another patient are as follows: Contact of hospital staff with contaminated hands Contact with door handles, call bells and overbred tables with contaminated surfaces. are contaminated.
[10]

Contact with equipment such as stethoscopes and blood pressure cuffs which

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Antibiotic Resistance In Wastewater Bacteria

7. Summary and Conclusions

7. SUMMARY AND CONCLUSONS

For municipal wastewater bacteria,antibiotic resistance patterns were analyzed. Fecal coliforms, Escherichia coli and enterococci were the microorganisms studied In the presence of predetermined concentrations of selected antibiotics, wastewater treatment plant influent,disinfected effluent and secondary clarifier effluent were. Ciprofloxacin, sulfamethoxazole/trimethoprim and vancomycin were the antibiotics included for the study. Antibiotics that were once commonly used to treat bacteria have now developed resistance to some antibiotics. For example, infections caused by Staphylococcus aureus (golden staph) and Neisseria gonorrhoeae (the cause of gonorrhea) were usually controlled by penicillin in the past but today they are almost always resistant to benzyl penicillin. Some bacteria have become resistant to almost all of the easily available antibiotics which are the most serious concern with antibiotic resistance and are able to cause serious disease and this is a major public health problem. Methicillin-resistant Staphylococcus aureus(MRSA), vancomycin resistant Enterococcus(VRE) and multi-drug resistant Mycobacterium tuberculosis (MDR-TB) are the most important examples. Most important ways to prevent antibiotic resistance are: Unnecessary prescribing and overprescribing of antibiotics should be minimized. When antibiotics are prescribed for conditions that do not require them or when people expect doctors to prescribe antibiotics for viral illness (antibiotics do not work against viruses), this occurs. Entire course of the prescribed antibiotic should be completed so it can be fully effective and not breed resistance. Appropriate infection control procedures should be used and good hygiene should be practiced.

An efficient way of achieving our target to reduce the content of antibiotic resistant bacteria in the water effluent at the final point is by passing Ultra-Violet light. Thus the final effluent was free from resistant organisms which were released into the surface water.

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Antibiotic Resistance In Wastewater Bacteria

8. References

8. REFERENCES
1. Aggarwal, R., and Krawczynski, K. (2000). Hepatitis E: An overview and recent advances in clinical and laboratory research. Journal of Gastroenterology and Hepatology, 15(1), 9-20. 2. Aggarwal, R., and Krawczynski, K. (2000). Hepatitis E: An overview and recent advances in clinical and laboratory research. Journal of Gastroenterology and Hepatology, 15(1), 9-20. 3. Briles, D. E., Paton, J C., Swiatlo, E., and Crain,M. J. (2006). Pneumococcal 4. Vaccines. Gram-Positive Pathogens, ed by Fischetti, V. A. ASM press, Washington, DC 5. Ash, R. J., Mauck, B., and Morgan, M. (2002). Antibiotic Resistance of GramNegative Bacteria in Rivers, United States. Emerging Infectious Disease, 8(7), 713-716. 6. Altschul, F. S., Gish, W., Miller, W., Myers, W. E., and Lipman D. J. (1990). 7. Basic local alignment search tool. Journal of molecular biology., 215(3), 403410. 8. Bajracharya, B. L., Baral, M. R., Shakya, S., Tuladhar, P., Paudel, M., and Acharya, B. (2006). Clinical profile and antibiotics response in typhoid fever. Kathmandu University Medical Journal, 4 (13), 25-29. Baquero, F. (1997). 9. Carr, A., Tindall, B., Brew, B. J., Marriott, D. J., Harkness, J. L., Penny, R., and Cooper, D. A. (1992). Low dose trimethoprim-sulfamethoxazole prophylaxis for toxoplasmic encephalitis in patients with AIDS. Annals of Internal Medicine, 117(2) 106-111. 10. Chang, J. C., Ossoff, S. F., Lobe, D. C., Dorfman, M. H., Dumais, C. M., Qualls, R. G., and Johnson, J. D. (1985). UV inactivation of pathogenic and indicator microorganisms. Applied and Environmental Microbiology Journal, 49(6), 13611365.

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