Antibiotic resistance and heavy metal tolerance in cultured bacteria from hot springs as indicators of environmental intrinsic resistance and tolerance levels
Graphical abstract
Introduction
Multiple antibiotic resistance (MAR) in bacteria are a huge and escalating problem for public health (Woolhouse et al., 2016). Antibiotic resistance genes (ARGs) via their environmental microbial hosts are capable of being transferred to potentially clinically important pathogens especially in water bodies and wastewater treatment plants (Rizzo et al., 2013). Several studies have reported MAR in aquatic environments (Vaz-Moreira et al., 2014) and it is now regarded as a hazardous pollutant of water (Sanderson et al., 2016).
However, antibiotic resistance (AR) is ancient, occurring in the environment for billions of years, intrinsically and naturally (Allen et al., 2010) reported in pristine antibiotic free locations such as the Antarctic (Miller et al., 2009), remote isolated caves (Bhullar et al., 2012) and remote mountain streams (Lima-Bittencourt et al., 2007). Evidence of genes that provide resistance against antibiotics without gene expression has been reported in 30 000-year-old permafrost sediments (D'Costa et al., 2011) and ancient arctic sediments (Perron et al., 2015). This intrinsic AR differs from AR induced by the presence of external pharmaceutical sources, in function and diversity (Sengupta et al., 2013). There is a greater genetic diversity of intrinsic or natural AR, and as indicated by metagenomics studies, the genes are chromosomally located and often sub-inhibitory (Perron et al., 2015).
Although there are abundant sources of sampling possibilities for microorganisms in the post-antibiotic era, in modern times it is difficult to find a site in the environment that has not been exposed to man-made antibiotics. Hot and cold spring sites are sources of sampling possibilities for microorganisms that have no previous exposure to man-made antibiotics and MAR has been investigated in hot springs in Turkey (Sariözlü et al., 2012), Jordan (Akel et al., 2008) and India (Sen et al., 2010). A correlation between MAR and heavy-metal tolerance (HMT) has been reported in isolates from rivers (Icgen and Yilmaz, 2014). Although hot spring isolates have been reported to carry plasmids (Munster et al., 1985; Khalil et al., 2003), this association has not been well investigated.
The aim of this study was to determine the level of resistance to antibiotics and tolerance to heavy metals of cultured bacteria from hot springs in Limpopo Province, South Africa (SA). The study was used to explore the possible use of hot springs to provide baseline levels of AR that occurs naturally in such a unique environment and to comment on the contribution it has to water safety.
Section snippets
Study site and sample collection
Sampling sites were five hot springs in the Limpopo Province, SA and water and sediment samples were collected as previously described (Jardine et al., 2017). The details are described in Table 1 together with the concentrations of heavy metals found in the hot spring water at different sites.
Isolation of bacteria
Bacteria from water samples were isolated and cultured on four different media according to the manufacturer's instructions as indicated in Appendix A and explained in Jardine et al. (2017). Pure cultures
Isolation and identification of bacteria
Appendix B provides information on the isolates obtained from the five hot springs in Limpopo Province, South Africa including growth temperature, isolation media and comparison of the 16S rDNA sequences with GenBank and accession numbers. In summary, at a growth temperature of 37 °C, one isolate each was identified for the following genera; Kocuria, Arthrobacter, Hafnia, Solibacillus and Aneurinibacillus. Seven isolates from the Bacillus genus grew at 37 °C while seventeen isolates grew at a
Conclusions
This study reports on 40 bacterial isolates from hot springs in Limpopo Province, SA, that were resistant to four antibiotics and up to eight heavy metals. The levels of AR in hot springs were generally low, consistent with other “pristine” environments, and compared with environments involving human activity such as WWTPs where AR levels are very high. The MAR index can be used as an indicator of water quality, and in this study, this value was 2.5% compared with pristine mountain rivers
Acknowledgements
Dr Gill Hendry for her recommendations on statistical analysis, the African Centre for DNA Barcoding and University of Johannesburg, is acknowledged and thanked for assistance in sequencing of isolates. This research project did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors, and was supported by the University of Johannesburg, South Africa.
References (50)
- et al.
Co-selection of antibiotic and metal resistance
Trends Microbiol.
(2006) - et al.
Screening of potential bioremediation enzymes from hot spring bacteria using conventional plate assays and Liquid Chromatography –tandem Mass Spectrophotometry (LC- MS/MS)
J. Environ. Manag.
(2018) - et al.
Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance
Clin. Microbiol. Infect.
(2012) - et al.
Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review
Sci. Total Environ.
(2013) - et al.
Phenotypic and genotypic characterization of three novel halophilic Bacillus strains from Jordanian hot springs
Jordan J. Biol. Sci.
(2008) - et al.
Call of the wild: antibiotic resistance genes in natural environments
Nat. Rev. Microbiol.
(2010) - et al.
Susceptibility testing of Bacillus species
J. Antimicrob. Chemother.
(2002) - et al.
The Murray collection of pre-antibiotic era Enterobacteriacae: a unique research resource
Genome Med.
(2015) - et al.
Occurrence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment
Can. J. Microbiol.
(1991) - et al.
Antibiotic resistance is prevalent in an isolated cave microbiome
PLoS One
(2012)
Heavy Metal Tolerance
Multiple antibiotic resistance indexing of coliforms to identify high risk contamination sites in aquatic environment
Indian J. Microbiol.
Antibiotic susceptibility of Bacillus species
J. Infect. Dis.
Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria
Antimicrob. Agents Chemother.
Metal and antibiotic-resistance in psychrotrophic bacteria from Antarctic marine waters
Ecotoxicology
Antibiotic resistance is ancient
Nature
Co-occurrence of antibiotic and heavy metal resistance in Kızılırmak River isolates
Bull. Environ. Contam. Toxicol.
Phylogenetic analysis and antimicrobial profiles of cultured emerging opportunistic pathogens (phyla Actinobacteria and Proteobacteria) identified in hot springs
Int Journal Environ Res Public Health
Isolation of plasmids present in thermophilic strains from hot springs in Jordan
World J. Microbiol. Biotechnol.
Determination of multiple antibiotic and heavy metal resistance of the bacteria isolated from the Küçükçekmece Lagoon, Turkey
Pol. J. Environ. Stud.
Antibiotic resistance profiles of Escherichia coli isolated from different water sources in the Mmabatho locality, North-West Province, South Africa
South Afr. J. Sci.
Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940
Environ. Sci. Technol.
Antibiotic-resistance profile in environmental bacteria isolated from penicillin production wastewater treatment plant and the receiving river
Environ. Microbiol.
Multiple antimicrobial resistance in Enterobacteriaceae isolates from pristine freshwater
Genet. Mol. Res.
Antibiotic resistance profiles of environmental isolates from Mhlathuze River, KwaZulu-Natal (RSA)
Water SA
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