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Open Access Journal of Microbiology & Biotechnology Research Article 13 min read

Prevalence of Pathogenic Microorganisms and Determination of Possible Presence of Antimicrobial Residues in Industrial and Medical Wastes

Chakraborty D, Das KK and Munshi SK*
* Corresponding author
ISSN: 2576-7771  10.23880/oajmb-16000209  Received: December 16, 2021  Published: December 29, 2021
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Keywords
Microbiological Quality Antimicrobial Residues Pharmaceutical Waste Medical Waste
Abstract

Waste materials discharged from any industries without any treatment may cause serious environmental problems which may consequently affect public health. Present study design to screen the proliferation of microorganisms in different waste and environmental samples near industrial areas along with the assessment of the presence of antibacterial substances. The existence of the total viable bacteria and fungi was estimated up to 107 cfu/g and 104 cfu/g, respectively, in solid wastes. For liquid wastes, the total viable bacteria were recovered up to 107 cfu/mL and fungi were observed up to 105 cfu/mL. Both types of samples were found to be contaminated with an array of pathogenic bacteria including Klebsiella spp., Staphylococcus spp., and Vibrio spp. Conversely, some medical and pharmaceutical waste samples were found to inhibit the growth of laboratory isolates tested which indicate the presence of antibiotics residues. Finally, the huge microbial load and the possible presence of antibiotic or other antimicrobial residues render the samples a major public health concern.

Introduction

The increase of industrialization in Bangladesh contributes to various kinds of environmental pollution, which may directly or indirectly cause ecological imbalance within the environmental ecosystem as well as seriously damage the biosystem [1]. With the increase of population, the demand for various rubbers, plastic, cloths, papers, and many other products is also increasing day by day. Therefore, to achieve the extensive requirement of a huge population in Bangladesh, much more industries are developing gradually [2]. Different organic and inorganic pollutants and many xenobiotic compounds are introduced to environments from these industrial toxic wastes [3, 4, 5]. Discharge of this untreated or partially treated industrial waste in the aquatic environment may cause a significant change in the behavior of aquatic life. Moreover, when they release onto the soil it can change the physicochemical and biological properties of soil color which is responsible for sexual mutation in fish, amphibians, and birds [6, 7].

Solid and liquid waste can also be generated from domestic, medical, commercial, or agricultural exploits, surface runoff, or storm water [8]. About 75 to 90 percent of total wastes can be classified as domestic wastes, which are usually non- hazardous wastes generated by administrative, food, and cleaning services, among others. The remaining 10 to 25 percent are considered hazardous wastes mainly healthcare- related wastes which are referred to as hospital or medical wastes [9]. Medical wastes consist of considerable amounts of chemicals and microbial agents including microbiological cultures, infectious blood samples, human body parts, etc. [10]. Healthcare and clinical settings are considered to be the reservoirs for large numbers of pathogenic microorganisms [11]. Along with their undesirable environmental effect, these wastes can similarly cause serious health problems to humans [12]. These wastes may establish the perfect site for the exchange of resistance genes between medical and environmental pathogens [13, 14]. Furthermore, the pathogens in wastewater are exposed to a wide range of biocides that could act as a selective pressure for the evolution of resistance [15]. However, antibiotic residues from hospital waste that may enter into the environment may gradually accumulate in the environmental segment and are responsible for increasing antibiotic resistance [13]. The presence of antibiotic-resistant bacteria in the waste or environmental samples is one of the biggest health concerns as they can transmit resistant genes through horizontal gene transfer to human pathogens of environmental origin [16].

Various solid and liquid wastes or effluents contain pathogenic microbes along with different chemical agents which induce the resistance traits and make them able to escape easily from water treatment plants. Similarly, municipal landfills are major contamination sources for superficial and drinking water systems, particularly when located in urban areas [17, 18]. A better knowledge of landfill- associated microbial communities may provide valuable insights on the bioremediation of wastes and promote pathogen monitoring. Microbial activity inside landfills leads to decomposition of inorganic and organic substances to stabilize the waste by oxidation of ammonium [19], reduction of nitrous oxide [20], and hydrolysis of cellulose [21, 22]. Considering all these facts, we designed the present study to identify the pathogenic microbial population and investigate their antimicrobial traits from different waste samples collected from different areas in Bangladesh.

Method and Materials

Study Area, Sampling and Sample Processing

A total of 16 environmental samples were collected from the different areas of Bangladesh following standard protocol [23, 24]. The samples were prepared for the microbiological assay according to the standard methods as described by Cappuccino & Sherman in 2005. For the identification and enumeration of pathogenic bacteria from solid samples, 10g of each sample was blended with 90 ml of buffer peptone water (pH 7.2 ± 0.2) and for liquid samples, 1 ml sample was mixed with 9ml normal saline then diluted both types of samples up to 10-8 according to the standard guideline.

Microbiological Analysis of Total Viable Bacteria and Fungi

For the enumeration of total viable bacteria (TVB) and the total fungal load, 0.1 ml of each sample from the dilutions 10-2 and 10-4 was inoculated onto the nutrient agar (NA) and Sabouraud’s dextrose agar (SDA) plates, respectively. Plates were incubated at 37°C for 24 hours and at 25°C for 48 hours for total viable bacteria and fungi, respectively [23, 24, 25, 26, 27, 28].

Estimation of Fecal Coliform, Escherichia coli, Klebsiella spp., Staphylococcus spp., and Pseudomonas spp.

From the dilutions 10-2 and 10-4, 0.1 ml of each sample was spread onto the membrane fecal coliform (MFC) agar and MacConkey agar for the enumeration of total fecal coliform (TFC), and coliforms (especially, Escherichia coli and Klebsiella spp.), respectively. Plates were incubated for 24 hours at 44.5°C and 37°C for fecal coliform and coliforms, correspondingly. Likewise, Staphylococcus spp. and Pseudomonas spp. were isolated onto Mannitol Salt Agar (MSA) and Pseudomonas agar, respectively by adding 0.1 ml of diluted sample each, and all the plates were then incubated at 37°C for 24 hours [23, 24, 25, 26, 27, 28].

Isolation of Salmonella spp., Shigella spp. and Vibrio spp.

Ten (10) ml of sample was transferred into 90 ml of selenite cysteine broth (SCB) and alkaline peptone water (APW) for the enrichment of Salmonella, Shigella, and vibrio spp., respectively, and incubated at 37°C for 6 hours. After incubation, the samples were diluted up to 10-6, and then 0.1 ml of samples from each of the 10-3 and 10-5 dilutions were spread onto Salmonella-Shigella (SS) agar and thiosulfate citrate bile salt sucrose (TCBS) agar for the isolation of Salmonella spp. and Shigella spp., and Vibrio spp., consecutively. Plates were incubated at 37°C for 48 hours for the detection of typical colonies. Finally, all the isolates were biochemically examined following standard procedures as described earlier [23, 24, 25, 26, 27, 28].

Biochemical Tests for the Confirmative Identification

Finally, the standard biochemical tests were performed for the identification of all the pathogenic isolates found in the samples by the previously described methods [29].

Preparation and Testing of the Samples for Antimicrobial Assay

Anti-bacterial properties of the tested samples were observed against different pathogenic strains such as Escherichia coli, Pseudomonas spp., Listeria spp., Vibrio spp., Klebsiella spp., Staphylococcus aureus, Bacillus spp., and Salmonella spp. [30]. At first, the lawns of bacterial suspensions (105 cfu or 0.5 OD measured by spectrophotometer) including each of the mentioned bacteria were prepared and 100μl of homogenized samples were introduced into the wells. Absolute ethanol and methanol were used as negative controls while the antibiotic discs of gentamicin (10 μl) were used as a positive control. Plates were incubated at 37°C for 12-18 hours and examined for formation of the zone of inhibitions (mm) [30].

Results & Discussions

Most of the developing countries of Asia are most heavily populated and quickly moving towards urbanization [31]. But due to shortcomings in waste management, financial and some other challenges, contributes to making this situation more critical. According to Das, et al. [32] large amounts of industrial waste are released into the environment without treatment.

Recovery of Microorganisms from the Industrial Waste and Environmental Samples

Most of the samples of both categories (solid and liquid wastes) were found to be highly contaminated by bacterial and fungal flora 102-107 cfu/g or cfu/ml (Table 1). In the case of industrial samples, the contamination rate of total viable bacteria and fungi was noticed within the range of 105-107 and 102-104 cfu/ml, respectively. Whereas in the medical wastes, the total viable bacterial and fungal count were estimated within the range of 103-106 and 102-104 cfu/g or cfu/ml, respectively. On the other hand, the estimated total viable bacterial and fungal count of water samples were 106-107 and 104-105 cfu/ml, respectively. In the case of the agricultural land soil sample, 106 cfu/g bacteria and 104 cfu/g fungi were counted. In garden soil samples, counted viable bacterial and fungal loads were 107 and 104 cfu/g, respectively. Staphylococcus spp. and Pseudomonas spp. were predominantly found in the majority of the samples in an average of 104 cfu/g or mL (Tables 1 & 2). Vibrio spp. was only found in a few solid and liquid industrial waste samples. Some solid and liquid waste and environmental samples were found to contain klebsiella spp. which can contribute to health-related problems if any contaminate our ground and surface water.

However, all the samples were devoid of the growth of E. coli, Salmonella spp., and Shigella spp. (Tables 2&3). Achudume and Olawale, et al. [33] reported the presence of bacterial species including Pseudomonas, Mirococcus, Actinomyces, Neisseria, Bacillus, and Klebsiella in waste dump areas. Park, et al. [34] identified several opportunistic pathogenic bacteria and viruses in medical waste samples. Munshi, et al. [35] tested different waste samples near including hospital, pharmaceutical, domestic and municipal wastes, and found a huge load of pathogenic bacteria. Das, et al. [32] found huge bacterial and pathogenic load in the tannery waste and environmental samples in their study in 2017. Akter, et al. [36] reported a viable bacterial count up to 108 cfu/g in the household waste samples.

SamplesMicrobial load (cfu/g or mL)
TVBFungiKlebsiella spp.Staphylococcus spp.Pseudomonas spp.Vibrio spp.
Solid samplesIndustrial waste 14.5×1073.6×1031.9×1033.5×1031.8×1024.5×102
Industrial waste 28.7×1071.1×1045.8×10308.0×1070
Industrial waste 39.5×1062.0×1045.5×1061.2×1043.6×1040
Medical waste 11.0×1044.5×10302.8×1033.0×1030
Medical waste 22.0×1042.5×10301.2×1031.1×1030
Medical waste 33.1×1041.2×10301.8×1031.2×1030
Garden soil 17.6×1076.0×1042.8×1023.0×1023.0×1030
Garden soil 21.6×1077.0×10401.5×1036.5×1040
Agriculture soil1.2×1061.8×10403.5×1042.8×1040
Liquid samplesIndustrial waste 13.5×1065.0×10405.6×1021.4×1030
Industrial waste 22.8×1058.0×10203.0×1021.8×1030
Industrial waste 33.7×1056.0×102008.8×1032.2×102
Industrial waste 42.9×1053.6×102001.2×1033.2×102
Medical waste 17.0×1067.8×1041.3×1031.3×1034.8×1030
Medical waste 23.7×1041.0×1021.5×102000
Medical waste 35.3×1051.4×10202.5×1023.1×1040
Medical waste 43.2×1035.3×102001.0×1010
Pond water 14.6×1063.0×1043.7×1026.0×1031.5×1030
Pond water 24.8×1071.5×10506.5×1034.0×1020
River water 15.5×1072.4×10502.1×1033.8×1020

Table 1: Microbiological analysis of waste and environmental samples.

TVB – Total Viable Bacteria E. coli, Salmonella spp., and Shigella spp. were absent in all the samples. Table 1: Microbiological analysis of waste and environmental samples.

In vitro Anti-bacterial Activity of the Waste Samples

Among all industrial wastes, only a few solid waste exhibited antimicrobial activity against E. coli and Listeria spp. (Table 3). On the other hand, solid medical waste, control soil, and water samples could not able to retard the growth of laboratory isolates. However, almost all liquid medical samples were found to inhibit the growth of most laboratory organisms. Among them, sample 3 showed the highest zone of inhibition against all the test organisms (Table 3). The presence of antibacterial activity indicates waste samples may contain some degree of antibiotics or other chemicals that might come from untreated hospital wastes. This is very alarming for us because it may increase the antibiotic resistance of pathogenic microbes as well as environmental microbes [37]. Le Page, et al. [38] detected antibiotics in waste and surface water samples. Martin, et al. [39] found antimicrobial potential in the industrial waste samples.

AssumedTSIH S 2IndoleMRVP testCitrateCatalaseOxidase
OrganismslantButtgasreactiontesttesttesttest
Klebsiella spp.YY+---+++-
Pseudomonas spp.RR-----+--
Staphylococcus spp.YR++-+-++-
Vibrio spp.YY--++-+++

Table 2: ** Confirmative biochemical tests for the isolates.

TSI Triple Sugar Iron Test Y Yellow (Acid) R Red (Alkaline) MR Methyl red VP Voges-Proskauer Table 2: Confirmative biochemical tests for the isolates.

SamplesE. coliKlebsiella
spp.		Bacillus
spp.		Pseudomonas
spp.		Vibrio
spp.		Staphylococcus
spp.		Listeria
spp.		Salmonella
spp.
Solid samplesIndustrial waste 100000000
Industrial waste 210 mm0000016 mm0
Industrial waste 300000011 mm0
Medical waste 100000000
Medical waste 200000000
Medical waste 300000000
Garden soil 100000000
Garden soil 200000000
Agriculture soil00000000
Liquid samplesIndustrial waste 100000008 mm0
Industrial waste 200000008 mm0
Industrial waste 300000013 mm0
Industrial waste 400000000
Medical waste 100000000
Medical waste 217 mm017 mm17 mm26 mm21 mm11 mm29 mm
Medical waste 315 mm16 mm14 mm14 mm14 mm16 mm13 mm16 mm
Medical waste 417 mm017 mm17 mm26 mm21 mm11 mm20 mm
Pond water 100000000
Pond water 200000000
River water 100000000

Table 3: Antibacterial activity of waste and environmental samples.

Conclusion

Although industrial wastes might be disposed directly into the environment for decades, the potential environmental impacts are less understood and have only recently become a topic of research interest due to the rapid emergence of diseases and multidrug-resistant bacterial strains. The present study revealed the waste directly exposed to the environment may contain chemical compounds or antibiotics residues. Such activity could be responsible for the multidrug resistance of microbial isolates, which could pose a serious threat to human and animal health. So governments should take responsibility to implement proper rules against open dumping of infectious medical waste as well as municipal waste in unsecured landfills for public health and environmental safety.

Conflicts of Interest

Authors have no potential conflict of interest

Acknowledgements

Stamford University Bangladesh provided the financial and technical supports for the study.

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@article{chakraborty2021,
  title   = {Prevalence of Pathogenic Microorganisms and Determination of Possible Presence of Antimicrobial Residues in Industrial and
Medical Wastes},
  author  = {Chakraborty D, Das KK and Munshi SK},
  journal = {Open Access Journal of Microbiology & Biotechnology},
  year    = {2021},
  volume  = {6},
  number  = {4},
  doi     = {10.23880/oajmb-16000209}
}
Chakraborty D, Das KK and Munshi SK (2021). Prevalence of Pathogenic Microorganisms and Determination of Possible Presence of Antimicrobial Residues in Industrial and
Medical Wastes. Open Access Journal of Microbiology & Biotechnology, 6(4). https://doi.org/10.23880/oajmb-16000209
TY  - JOUR
TI  - Prevalence of Pathogenic Microorganisms and Determination of Possible Presence of Antimicrobial Residues in Industrial and
Medical Wastes
AU  - Chakraborty D, Das KK and Munshi SK
JO  - Open Access Journal of Microbiology & Biotechnology
PY  - 2021
VL  - 6
IS  - 4
DO  - 10.23880/oajmb-16000209
ER  -