Elsevier

Environmental Pollution

Volume 249, June 2019, Pages 696-702
Environmental Pollution

Antibiotic resistance and heavy metal tolerance in cultured bacteria from hot springs as indicators of environmental intrinsic resistance and tolerance levels

https://doi.org/10.1016/j.envpol.2019.03.059Get rights and content

Highlights

  • Hot spring bacteria were tested against 10 antibiotics and 8 heavy metal salts.

  • Resistance found against ceftriaxone>nalidixic acid>carbenicillin.

  • All isolates grew on ≥2 heavy-metal salts at 10 and 40 mM concentrations.

  • Low antibiotic resistance in pristine sites may reflect baseline antibiotic resistance levels.

Abstract

Antibiotic resistance (AR) in the environment is a growing and global concern for public health, and intrinsic AR from pristine sites untouched by pharmaceutical antibiotics is not commonly studied. Forty aerobic bacteria were isolated from water and sediment samples of hot springs in South Africa. Resistance against ten antibiotics (carbenicillin, gentamicin, kanamycin, streptomycin, tetracycline, chloramphenicol, ceftriaxone, co-trimoxazole, nalidixic acid and norfloxacin) was tested using a standard disk diffusion assay. Resistance to one or two antibiotics were equally found in 37.5%, while the remaining 22% showed complete sensitivity. Intermediate resistance was found for ceftriaxone (52.5%), nalidixic acid (37.5%) and carbenicillin (22.5%), while low levels of resistance were observed for streptomycin (5%) and kanamycin (2.5%), and total sensitivity towards the other antibiotics. Twenty-nine isolates were also tested against eight different heavy-metal salts (Al, Cr, Cu, Fe, Hg, Mn, Ni and Pb) at 10 and 40 mM. All isolates were tolerant and able to grow on ≥2 heavy-metal salts at both concentrations. No association was observed between AR and heavy metal tolerance (HMT). Based on the relatively low AR levels, hot spring sites are pristine environments reflecting baseline levels for comparison to other potentially contaminated groundwater sites.

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.

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