The Case Study of the Environmental Superbug: Pedobacter


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Meet Pedobacter, a gram-negative bacteria mostly found in soil (Shaya, 2006). Although these species are mainly separated from soils, it can be found also in drinking water, certain seafood, refrigerated food, decayed organic material, glaciers, mud, and other extreme environments. Studies have shown that Pedobacter lusitanus, other closely related species, and the genus type strain are all multidrug resistance or MDR including resistance to colistin, ciprofloxacin, and beta-lactams. MDR gram-negative bacteria can cause serious health problems including severe infections. Treatments for MDR gram-negative bacteria infection are very limited which includes the use of colistin, the last resort antibiotic. Colistin is an effective antibiotic in killing gram-negative bacteria but highly toxic in kidneys (Falagas, 2008). Pedobacter belongs under the phylum Bacteroidetes mostly dominant in soils. P. lusitanus was found from the muds of an abandoned uranium mine. It is resistant to various antibiotics including aminoglycosides, tetracycline, colistin, chloramphenicol, and beta-lactams. A group of researchers at the University of Aveiro in Portugal did a study to understand the resistant feature of the Pedobacter or if it was strain specific. Pedobacter species can produce diverse antibiotic resistance arsenal making them one of the environmental superbugs.

The strains used in this study include Pedobacter lusitanus, Pedobacter himalayensis, Pedobacter hartonius, Pedobacter cryoconitis, and Pedobacter westerhofensis, all are closely related species (Viana, 2018). All strains were cultured in an agar medium that contains casein extract, soy peptone, sodium chloride at 20 degrees Celsius for 48 hours. Disc diffusion was assessed to test the strains for antibiotic resistance. This test, the disc diffusion, is also known as Kirby-Bauer test and it is one of the oldest techniques in microbiology that is commonly used (Science Direct). The discs were saturated with the concentration of different antibiotics and were placed onto the agar’s surface. After the incubation period, a clear zone of no growth around the vicinity of a disc indicates sensitivity to that antibiotics. The size of zone should indicate whether the strain is sensitive (S), intermediate (I), or resistant (R) to that antibiotic. The result of the disc diffusion test shows all Pedobacter strains were resistant to 18 antibiotics but only sensitive or intermediate to the 2 other medicines. However, none of strains were resistant to imipenem. P. heparinus was the most susceptible whereas P. cryoconitis and P. himalayensis were the most resistant. See Table 1 for the summary of the disc diffusion result.

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Table 1. Disc Diffusion Result. Strains: 1 – P. lusitanus; 2 – P. hartonius; 3 – P. himalayensis; 4 – P. heparinus; 5 – P. cryoconitis; 6 – P. westerhofensis. R – resistant; I – intermediate; S – sensitive. The table shows that all strains were resistant to 18 antibiotics and sensitive to one antibiotics (Sulfamethoxazole).

The minimal inhibitory concentration or MICs of antibiotics were determined by broth microdilution. MICs is defined as the least amount of concentration of antibiotic that will impede the growth of bacteria after incubation for one night (Andrews, 2001). Diagnostic laboratories use MICs to confirm bacterial resistance or used as a tool for research of new antibiotics. Broth microdilution is used to test the sensitivity of bacteria to antibiotics (Engelkirk 2008). Basically, the process involves multiple microtiter plates filled with varying concentration of the antibiotics and the bacteria to be tested. The antibiotic solutions were sterilized with cellulose acetate filters before dilution in broth. Bacterial mixtures were added to each dilution of the antibiotics for testing. The plates were incubated 24 hours at 20 degrees Celsius for the bacteria to grow. Bacterial growth was confirmed when the broth became cloudy or a layer of cells formed at the bottom of the broth. The lowest concentration of antibiotics that impedes bacterial growth were reported in MICs. The result classified the strains as resistant, intermediate, and sensitive according to clinical breakpoints. Most strains are resistant to high concentration of some antibiotics which includes ampicillin, apramycin, ceftazidime, colistin, kanamycin, and streptomycin. See Table 2 for the summary of MICs result.

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Table 2. MICs Result (mg/L). Strains: 1 – P. lusitanus; 2 – P. hartonius; 3 – P. himalayensis; 4 – P. heparinus; 5 – P. cryoconitis; 6 – P. westerhofensis. R – resistant; I – intermediate; S – sensitive. The table shows that most strains are resistant to 6 antibiotics at high concentration.      

The beta-lactamases in Pedobacter were identified through UniProt, a database of protein sequence and functional information (Wikipedia). Beta-lactamase is an enzyme present in bacteria that disable beta-lactam antibiotics like penicillin and cephalosporin by hydrolysis (Merriam-Webster). Using the sequence similarity network, Pedobacter beta-lactamase was referenced with known structures in the Beta-Lactamase Database using visualization through Cytoscape. There are 550 genes in Pedobacter that encodes beta-lactamases and Uniprot has information for 37 Pedobacter genomes all similar to beta-lactamases.

The genomes of the Pedobacter strains in this study were analyzed using Comprehensive Antibiotic Resistance Database or CARD, a database that organizes information on antibiotic resistance genes, protein, and phenotype (Jia, 2017). The goal of the analysis is to determine genes possible for antibiotic resistance encoding. Six genes were selected and organized into different groups namely inactivation enzymes, target modification, target replacement, target protection, efflux system, and others. Of the total genes of these Pedobacter strain, about 6% to 8% are antibiotic resistant gene. Most of the Pedobacter genes encode for antibiotic efflux mechanism. No plasmids were detected in all Pedobacter strain thus all the antibiotic resistant genes are found in the chromosomes. See Figure 1 for the CARD analysis.

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Figure 1. CARD Analysis. Number of genes in the genomes of Pedobacter strains possibly involved in antibiotic resistance. Pedobacter strains encode mostly on the efflux system mechanism.             

P. lusitanus has two beta-lactamases encoding gene that were selected for further studies. These genes have been selected because they were not similar with beta-lactamase available in the database and was considered novel enzymes. The two genes were named LUS-1 and PLN-1. Within the Pedobacter genus, P. cryoconitis and P. antarticus have encoding protein genes similar to LUS-1. The other Pedobacter species were analyzed for the presence of gene similar to PLN-1. P. hartonius, P. ginsengisoli, P.terrae, P. suwonensis, P. kyungheensis, and P. antarticus have protein encoding gene similar to PLN-1. See Figure 2 for the comparison of genomic environment.

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Figure 2. Genomic Environment Comparison. A: The gene blaLUS-1 are similar within the three Pedobacter species. B: The gene blaPLN-1 are similar within the 7 Pedobacter species.

Pedobacter strains have multiple mechanisms that may have contributed to their intrinsic multidrug resistant characteristics apart from beta-lactamases. It is most probably that these bacteria have evolved due to the continued presence of antibiotic in the environment. Pedobacter bacteria will be an excellent microbe for the foundations of antibiotic resistant studies.

Although everyone can study bacteria that cause life-threatening disease, studying ordinary and unknown bacteria matters. I chose this article because it is interesting to know that other frivolous bacteria can already be an antibiotic resistant. We know that DNA mutation and bacterial transformation are inevitable and can happen to any types of bacteria including non-pathogens that were already multidrug resistant. It may only take a small amount of DNA mutation for a bacterium to become pathogenic thus making an MDR bacteria a worldwide outbreak cause. This case study gives us one reason to study microbiology because although microbes does not seem to impact our life, they are found everywhere. We need scientists to constantly check, monitor, and study these bacteria for they have already outsmarted us in the research field.

References

Viana, Ana Teresa et. Al. (2018, October). “Environmental superbugs: The case study of Pedobacter spp.” Environmental Pollution. Volume 241: 1048-1055. Accessed October 7, 2019 https://www-sciencedirect-com.ezproxy.palomar.edu/science/article/pii/S0269749118319663

Science Direct. Disk Diffusion. Accessed October 7, 2019 https://www.sciencedirect.com/topics/immunology-and-microbiology/disk-diffusion

Shaya, D (2006). “Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product.” J. Biol. Chem. 281 (22): 15525-35. Accessed October 7, 2019 https://en.wikipedia.org/wiki/Pedobacter

Andrews, JM. (2001, July). “Determination of Minimum Inhibitory Concentrations.” NCBI. Accessed October 7, 2019 https://www.ncbi.nlm.nih.gov/pubmed/11420333

Merriam-Webster. Beta-lactamase. Accessed October 7, 2019 https://www.merriam-webster.com/dictionary/beta-lactamase

Jia, B et. Al. (2017, January). “CARD 2017: Expansion and Model-Centric Curation of the Comprehensive Antibiotic Resistance Database.” Nucleic Acids Research. Page 45. Accessed October 7, 2019 https://en.wikipedia.org/wiki/Comprehensive_Antibiotic_Resistance_Database

Wikipedia. UniProt. Accessed October 7, 2019 https://en.wikipedia.org/wiki/UniProt

Engelkirk, Paul (2008). “Laboratory Diagnosis of Infectious Diseases.” Lippincott Williams & Wilkins. Page 168. Accessed October 7, 2019 https://en.wikipedia.org/wiki/Broth_microdilution#cite_note-a-1

Falagas, ME (October 2008). “Potential of Old-Generation Antibiotics to Address Current Need for New Antibiotics.” Expert Review of Anti-infective Therapy. Accessed October 7, 2019 https://en.wikipedia.org/wiki/Colistin#cite_note-1


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