The misuse of antimicrobial agents has been identified as one of the major forces resulting in the rapid spread of resistance, but the nature of this relationship is complex. Resistance to antimicrobials is sometimes brought about by a change in the bacterial genome, which can occur by the transfer of antimicrobial resistance genes which may be found in some of the bacteria found in the environment and transferred to those without the resistance genes and also through other horizontal gene transfer elements  . Products that are used in disinfection and sterilization, as well as heavy metals used in industries and households along with antibiotics, creating selective pressure in the environment that lead to mutations in microorganisms  .
Indiscriminate waste dumping enhances the breeding of microorganisms that pose a danger to the human population. Urban wastes contain a wide range of things that may have come from different sources e.g. hospitals, animal sheds and may carry antimicrobial resistance bacteria belonging to the human and animal commensal flora, mainly Enterobacteriaceae  . Waste degradation is enhanced by the presence of soil microorganisms that create a conducive environment for the resistant bacteria e.g. Salmonella species and E. coli to thrive in thus becoming potential human pathogens and may cause severe health hazards  . The presence of rodents in these dumpsites and garbage collection areas enhance the spread of antimicrobial resistant bacteria to other areas. Previous studies have focused on the identification of these disease vectors on dumpsites and have reported cockroach, housefly, black garbage fly, and stable fly to be the most prevalent disease vectors on the dumpsites and garbage collection areas   .
Enterobacteriaceae are one of the major causes of infections and deaths around the world. The prevalence of antimicrobial resistance in this family of bacteria e.g. Escherichia coli, Salmonella and Shigella has raised over the years. One of the major reasons for the increase is the spread of Klebsiella pneumoniae carbapenemase (KPC), a class A serine carbapenemase that was originally isolated from Klebsiella pneumoniae in 1996  .
Resistance in pathogenic organisms poses a distinct clinical challenge. However commensal bacteria may enhance the spread of resistant bacteria as they may act as reservoirs of the resistance genes which may have been acquired through various horizontal gene transfer elements. This, therefore, increases the carriage levels of resistant organisms. The resistant microorganisms from clinical samples may gain their entry into the environment through fecal contamination and various wastes from the hospital that has not been sterilized properly. Escherichia coli and Klebsiella species were used in this study as an indicator species as they are commonly associated with humans and animals disease and have also been used in other studies to gauge the spread of acquired resistance  .
2. Materials and Methods
2.1. Study Area
Dumpsites near schools, residential areas and the municipal general waste dumping sites in Nairobi area were selected for the study and sampled. In order to verify the most accessed area of these dumpsites and garbage collection areas by the street families and other people relying on dumpsites for a living, a qualitative survey of the dumpsites and garbage collection areas was conducted. The purpose of the survey was to determine the most appropriate area to sample for the main study. This was done by visiting the dumpsites and garbage collection areas before the start of the study and surveying the areas and identifying potential barriers to our study.
2.2. Sampling Points
A total of 17 dumpsites (permanent dumping area) and garbage collection areas (temporary dumping area where garbage is dumped awaiting collection) were randomly sampled in different parts of Nairobi area (Figure 1).
Figure 1. Aerial view of sampling sites in Dandora sampling area.
2.3. Sample Collection
Sample collection was randomly carried out in different days in seventeen different points. A total of 126 samples were collected during the sampling period. At each sampling station, the sub-surface soil, mixed solids, leaking water, stagnant water, swabs and food samples were collected from one squire foot area into sterile sampling bottles and appropriately labeled. Six samples were collected from each site. The samples were then transported to the laboratory for analysis.
2.4. Determination of Microbial Load in the Dumpsites and Garbage Collection Areas
One gram (1 g) of each solid samples and 1 mL of the liquid samples and swabs were suspended in 10 mL physiological sterile saline. Serial dilutions of 10 fold, 5 fold, and 1 fold dilutions were prepared from the 10 mL suspension and transferred onto duplicate molten Plate Count Agar (PCA) mixed and allowed to cool at room temperature. This was then incubated at 37˚C for 24 hours. Colonies were determined from duplicate plates and the average counts recorded as mean viable bacteria (colony forming units [CFUs] of the sample. The low and high CFUs were reached by dividing the dumpsites and garbage collection areas into two, those that had CFUs above 5.0 were considered to be high and those below 5.0 considered being low.
2.5. Isolation and Identification of E. coli and Klebsiella Species
A loop full (1 µl) of the mixture incubated in buffered peptone water was then transferred onto MacConkey agar plates and incubated at 37˚C for 24 hours for isolation of E. coli and Klebsiella species. After overnight incubation the plates were then examined for growth and presumptive identification of E. coli and Klebsiella species for lactose-fermenting colonies (pink). Presence of pink non- mucoid colonies for E. coli and pink mucoid for Klebsiella species were further identified by biochemical tests.
2.6. Biochemical Identification of the Isolates
Pure discrete colonies of E. coli and Klebsiella species from primary cultures were identified by gram staining and biochemical tests. Colonies that appeared as gram negative rods were tested for indole test, methyl-red test, Voges- Proskauer test and citrate utilization (IMViC). Presence of E. coli was interpreted as a positive reaction for indole and methyl-red tests and a negative reaction for Voges-Proskauer test and lack of citrate utilization. Additionally presence of Klebsiella species was interpreted as a negative reaction for indole and methyl-red tests and a positive reaction for Voges-Proskauer test and citrate utilization  .
2.7. Antimicrobial Susceptibility Testing
The antimicrobial susceptibility testing was done on 286 isolates on Mueller- Hinton agar plates (Oxoid). The isolates were tested against the following antibiotics; Ampicillin (10 μg), Cefpodoxime (10 μg), Ceftazidime (30 μg), Cefoxitin (30 μg) Cefepime (30 μg), Amoxicillin-Clavulanic acid (10/100μg ratio), Ciprofloxacin (10 μg), Tetracycline (30 μg), Trimethoprim Sulfamethoxazole (30 μg), Gentamicin (10 μg), Chloramphenicol (30 μg), Streptomycin (25 μg), Nalidixic acid (10 μg), and Meropenem (10 μg). The plates were then incubated at 37˚C for 18 - 24 hours. The inoculums for susceptibility testing were compared against the McFarland 0.5 turbidity standards with E.coli ATCC 25,922 strain being used as the control standard for quality assurance of media and the antimicrobial discs. The interpretation of results was according to Clinical and Laboratory Standards Institute guidelines  .
3.1. Contamination Levels of the Dumpsites and Garbage Collection Areas
The lowest CFU from any given sampling point was 1.1 × 1011 that was recorded in Dandora dumpsite. The site with the highest CFU value was Muthurwa estate dumpsite that recorded 8.2 × 1010. Other sites with high CFU counts were Umama garbage collection area (Komarock), Kawangware market dumpsite, Kenyatta staff quarter garbage collection area, Kweria garbage collection area, City market garbage collection area, Central police garbage collection area, Kibera dumpsite, and Kenyatta market dumpsite while Seven of the dumpsites and garbage collection areas (Ayany dumpsite, Dandora dumpsite, Ngara market garbage collection area, Muthurwa market garbage collection area, Masai market dumpsite, Mareba garbage collection area (Kibera), District Commissioner garbage collection area (Kibera) recorded CFUs below 2.0 × 1010 Table 1.
3.2. Antimicrobial Susceptibility Profile of E. coli and Klebsiella Species
Resistance pattern to all antimicrobials for E. coli and Klebsiella was above 5% except for ciprofloxacin (3.3% E.coli, and 2.4% Klebsiella), meropenem (1.7% E.coli and 1.8% Klebsiella) and gentamicin (3.3% E.coli and 0% Klebsiella) Table 2.
3.3. Antimicrobial Resistance in Dumpsites and Garbage Collection Areas with High and Low Colony Forming Units
In general, resistances frequencies were similar for E. coli and Klebsiella obtained from samples with high CFUs to those obtained from samples with low CFUs. In the dumpsites and garbage collection areas that had high CFUs, such as Muthurwa estate dumpsite, Central police garbage collection area, City market garbage collection area, City park market dumpsite Figure 2(a), there were high resistance prevalence’s of above 25% to streptomycin, ampicillin, tetracycline and trimethoprim sulfamethoxazole for isolates belonging to both species. In contrast, there was low resistance to meropenem, gentamicin, and ciprofloxacin (≤5%) in both species. The study also found that 42% of E. coli isolates were resistant to ampicillin compared to 59% of Klebsiella isolates found in this study.
Table 1. Average microbial load of the samples from dumpsites and garbage collection areas and their characteristics.
Key: Seepage-leakage of water into the ground in and around the dumpsite or garbage collection area, Fecal contamination―presences of feces on the dumpsite or garbage collection area, Domestic/Rodents―presence of livestock (e.g. goats) or rodents (e.g. rats) on the dumpsite and garbage collection area, Human activity―presence of humans on or adjacent to the dumpsite and garbage collection area.
Table 2. Antibiotic susceptibility pattern of E. coli and Klebsiella among the antibiotics tested.
Figure 2. (a) antimicrobial resistance in dumpsites and garbage collection areas with high Colony Forming Units, (b) -antimicrobial resistance in dumpsites and garbage collection areas with low Colony Forming Units.
Key: AMP-ampicillin (10 μg), CPD-cefpodoxime (10 μg), CAZ-ceftazidime (30 μg), FOX-cefoxitin (30 μg), FEP-cefepime (30 μg), CIP-ciprofloxacin (10 μg), AMC-amoxicillin clavulanic acid (10 μg), TE-tetracycline (30 μg), MRP-meropenem (10 μg), S-streptomycin (10 μg), NA-nalidixic acid (10 μg), C-chloramphenicol (10 μg), GEN-gentamicin (10 μg), SXT-trimethoprim sulfamethoxazole (30 μg).
Resistance to gentamicin among isolates from sites recording high CFUs was only observed for E. coli (6%) while resistance to meropenem from the same population of isolates, was observed in Klebsiella (2.8%).
There was high resistance (>20%) to streptomycin, ampicillin, tetracycline, trimethoprim sulfamethoxazole and cefpodoxime for both species in the dumpsites and garbage collection areas that had low CFUs such as the Ngara market garbage collection area, Ayany dumpsite, Dandora dumpsite, Muthurwa market garbage collection area Figure 2(b). In such sites, there was a low resistance of E. coli and Klebsiella to meropenem, gentamicin, and ciprofloxacin (<5%). The only E. coli strains found to be resistant to gentamicin were from the sites with low CFUs.
3.4. Antimicrobial Resistance Profiles
The isolates were classified into three resistance profiles (those that were fully susceptible, those resistant to 1 - 3 antimicrobials and those resistant to more than 3 antimicrobials (MDROs) as shown in Table 3. There were no isolates that
Table 3. Distribution of isolates with different resistance profiles across dump sites and garbage collection areas.
Key: The fully susceptible are those that did not show any resistance while resistance is showing resistance to 1-3 antimicrobials and MDROs are those that show resistance to more than 3 antimicrobials.
were fully susceptible in 7 (41%) of the seventeen dumpsites and garbage collection areas sampled. Most (61%) of the isolates were resistant to 1 - 3 antimicrobials. Another 23% of isolates were resistant to more than 3 antimicrobials and were thus multidrug resistant (MDROs). In the Umama garbage collection area (Komarock) 5 (71%) out of the 7 isolates recovered, were MDROs but no MDROs strain was recovered from the Kawangware market dumpsite. The sites with the highest prevalence of MDROs strains included Muthurwa estate dumpsite (66%) and Umama garbage collection area (Komarock) (71%) that both recorded high CFUs as well as shown earlier in Table 1.
Based on the qualitative survey, this study showed that there was evidence of human contact with garbage and dumpsites surveyed. Some of these sites had high CFUs indicating a high possibility of contamination with fecal material. It is, therefore, possible that these interactions pose a serious danger to the public who work and sell their salvaged merchandise from such sites. Such merchandise may include fruits and vegetables. This may result in the spread of pathogens to the unsuspecting public. These results are in agreement with a study done in Nigeria that found that waste scavenging poses a great threat to the public. In addition, such dumpsites allow the growth of many pathogenic bacteria including those that may be MDR  . Human and animal scavengers were invariably at the site at all times.
This study’s results show that the mean colony counts obtained from the dumpsite and garbage collection areas in residential areas and market places close to the dumpsite were relatively high. Among the dumpsites and garbage collection areas that had high CFUs, 70% were found in residential areas and 20% in market areas. In the dumpsites and garbage collection areas that had low CFUs, 57% were in residential areas and 14% in market areas. These high CFUs were probably as a result of the presence of fecal contamination and human activity in the dumpsites and garbage collection areas. The results obtained in this study correlates with that of Odeyemi, 2012 that showed that the mean total bacterial counts obtained from the dumpsite and in the residential area are relatively higher than those obtained at neighbouring streams or samples collected at least 50 m away from dumpsites  . This shows how fast resistant bacteria from the environment can gain entry into the human body through contamination from these dumpsites and garbage collection areas, thus creating high health concerns in the public health sector. It is also worthy of note that the heaviest bacteria burden in this study was found at Muthurwa estate dumpsite and that the least was at Dandora dumpsite. This may not be too surprising since Muthurwa estate dumpsite had fecal contamination and a high number of trespassers thus bringing and taking a high number of the bacteria with them. Dandora dumpsite was the only dumpsite with a structured management system. This is the largest dumpsite in East and Central Africa, where rubbish/garbage from different parts of the city is dumped. This potentially offers a chance of transfer of pathogens from such sites to human residential sites since Dandora dumpsite is closely located to an ever busy residential area.
This present study also determined antibiotic resistance profile of E. coli and Klebsiella from the sampled sites. A high proportion of E. coli and Klebsiella strains were resistant to ampicillin (42%, 59%), streptomycin (46%, 46%) and trimethoprim/sulfamethoxazole (29%, 29%) respectively. While these values may be lower than those reported from clinical studies  These results suggest that resistance to antimicrobials is rising and this may be due to either the intrinsic resistance of many microorganisms to antibiotics or acquired resistance of the organisms enabled by the transfer of resistance of drug resistance plasmids. A high level of resistance has been found with members of the family Enterobacteriaceae which are increasingly becoming MDR. The origin of this resistance can probably be traced to the fecal constituent of the wastes or dump produced by people or animals that have been treated indiscriminately with various antibiotics and also to antibiotics production naturally by soil microorganisms  . The resistance prevalence of the two species was almost similar in all the antimicrobials used in this study. This indicates that the action of the antimicrobial to the two species works in an almost similar way thus the close resistance prevalence.
The highest multidrug resistance was found in Umama garbage collection area (71%), and the lowest multidrug resistance was found in Kenyatta staff quarter garbage collection area (4%). This difference may be due to the difference in diversity of dumpsites and garbage content and possible difference in the amount of human and animal fecal contaminants in different sites. Some sites recorded an MDR prevalence of more than 50% indicating that resistance to antimicrobials is on the rise and thus may lead to an increase in resistance related complications. The presence of high MDR phenotypes among environmental samples is an indication that resistant clinical samples are gaining entry into these sites. The possible factors driving the emergence of MDR phenotype could be the poor use of combined therapy.
Poor waste disposal and recycling practices are still rampant across the dumpsites and garbage collection areas. From the study, there was a high occurrence of Klebsiella and E. coli across the dumpsites and garbage collection areas indicating environmental contamination. Most of the dumpsites and garbage collection areas with high CFUs were found in residential areas indicating that contamination of the dumpsites and garbage collection areas maybe on the rise due to human interference. Isolates from dumpsites and garbage collection areas were resistant to a high proportion of the antimicrobials tested. This shows that clinical bacterial strains are gaining access into the environment thus the high resistance prevalence. The most effective antimicrobial was gentamicin to E. coli and meropenem to both isolates. The most non-effective antimicrobials were ampicillin and streptomycin indicating that the future of the antimicrobials is at risk with the changing bacterial pressure. A high frequency of MDR isolates was recorded in the garbage collection areas than in dumpsites. These findings concluded that human activities during collection of the garbage could play a role in the contamination of the dumpsites with potential clinical isolates which can help spread antimicrobial resistance to the environmental strains.
Grateful acknowledgement is expressed to my able supervisors Ms. Winnie Mutai and Dr. John Kiiru for their continuous efforts towards making this study a reality. Few, if any, projects are written without the support and professional contributions of many people. This work is no exception. I admired my supervisors’ patience, enthusiasm, motivation and immense knowledge they imparted to me during this period. They have guided me in the time of research and writing of this thesis.
GW wrote the proposal and drafted the manuscript. She also did the lab work throughout the study. Ms. WM assisted in the cleanup of the proposal, drafting, and submission of the manuscript. Dr. Kiiru also assisted in the cleanup of the proposal and supervision of the laboratory work. Mr. MM and LO assisted in the laboratory and statistical analysis. All the authors read and approved the manuscript.
GW is a master’s student at the University of Nairobi Medical Microbiology department. Miss. WM is a lecturer at the University of Nairobi Medical microbiology department who has vast experience in proposal presentation, writing, and microbial techniques. Dr. JK guided through the study and he is a senior researcher at the Kenya Medical Research Institute. MM and LO are graduate assistants working with the University of Nairobi who have a wealth of experience in laboratory research and data analysis.