Next Article in Journal
Risk of Antimicrobial Resistant Non-Typhoidal Salmonella during Asymptomatic Infection Passage between Pet Dogs and Their Human Caregivers in Khon Kaen, Thailand
Previous Article in Journal
Antimicrobials as Single and Combination Therapy for Colistin-Resistant Pseudomonas aeruginosa at a University Hospital in Thailand
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antibiotics
Volume 9
Issue 8
10.3390/antibiotics9080476
antibiotics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genetic Determinants for Metal Tolerance and Antimicrobial Resistance Detected in Bacteria Isolated from Soils of Olive Tree Farms

by
Nicolás Glibota
,
Mª José Grande
,
Antonio Galvez
* and
Elena Ortega
Área de Microbiología, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaén, 23071-Jaén, Spain
*
Author to whom correspondence should be addressed.
Antibiotics 2020, 9(8), 476; https://doi.org/10.3390/antibiotics9080476
Submission received: 17 June 2020 / Revised: 18 July 2020 / Accepted: 1 August 2020 / Published: 3 August 2020
Browse Figure
Versions Notes

Abstract

:
Copper-derived compounds are often used in olive tree farms. In a previous study, a collection of bacterial strains isolated from olive tree farms were identified and tested for phenotypic antimicrobial resistance and heavy metal tolerance. The aim of this work was to study the genetic determinants of resistance and to evaluate the co-occurrence of metal tolerance and antibiotic resistance genes. Both metal tolerance and antibiotic resistance genes (including beta-lactamase genes) were detected in the bacterial strains from Cu-treated soils. A high percentage of the strains positive for metal tolerance genes also carried antibiotic resistance genes, especially for genes involved in resistances to beta-lactams and tetracycline. Significant associations were detected between genes involved in copper tolerance and genes coding for beta-lactamases or tetracycline resistance mechanisms. A significant association was also detected between zntA (coding for a Zn(II)-translocating P-type ATPase) and tetC genes. In conclusion, bacteria from soils of Cu-treated olive farms may carry both metal tolerance and antibiotic resistance genes. The positive associations detected between metal tolerance genes and antibiotic resistance genes suggests co-selection of such genetic traits by exposure to metals.

1. Introduction

Antibiotic-resistant (AR) bacteria and their genetic determinants are now being considered as worrisome environmental contaminants, and several studies have shown that natural environments may act as reservoirs of AR bacteria [1,2,3,4]. Not only antibiotics but also other compounds may be responsible for a prolonged pressure that promotes the selection of bacterial antibiotic resistance in different environmental situations, including bioactive compounds such as heavy metals, biocides, detergents, and organic solvents [5,6,7]. Thus, many contaminants could promote antibiotic resistance through co-selection, which could imply co-resistance mechanisms, if different resistance determinants are linked genetically, or cross-resistance, when the same genetic determinant confers resistance to both types of compounds [8]. Among heavy metals, copper has been found to have stronger impacts on antibiotic resistance genes (ARGs) compared to for example iron, lead, nickel and zinc [9,10].
Antibiotic resistance has now become a global issue [11]. The burden of antibiotic resistance may be of greater magnitude when coupled to resistance/tolerance to non-antibiotic compounds. Multidrug-resistant Salmonella [12] and methicillin-resistant Staphylococcus aureus (MRSA) isolated from animals [13,14] may carry both metal (mainly Cu and Zn) and antibiotic resistance genes. The genetic linkage among different resistance genes suggests that metals could co-select antibiotic resistance in diverse bacterial strains, including human pathogens [15,16].
Previous studies highlight the role of copper contamination in the development of AR bacteria in the environment. Cu-resistant bacteria isolated from soils contaminated with copper were resistance to several types of antimicrobials, including ampicillin, olaquindox, chloramphenicol, tetracycline, and nalidixic acid [17]. Main copper resistance mechanisms rely on export systems based on P-type ATPases or periplasmic detoxification systems such as metallochaperones, multicopper oxidases or resistance-nodulation-division (RND) systems [18]. An increase of both antibiotic resistance and ARGs was reported during copper pollution in source water [19]. Thus, it seems important to reduce not only the use of antibiotics but also the factors that contribute to prevalence of AR bacteria in the environment [20].
Application of copper-derived compounds in olive tree farms is a common practice in South Spain for prevention or treatment of infections caused by the fungus Spilocaea oleagina. Phenotypic metal tolerance and AR in bacteria from Cu-contaminated soils in olive tree farms was established in a previous study [21]. The purpose of this study was to evaluate the presence of genetic determinants for metal tolerance or AR in strains isolated from Cu-treated soils in the province of Jaén and classified as resistant to both type of compounds. Possible associations between antimicrobial resistance determinants were also investigated in order to identify gene combinations that may pose a risk for co-selection of resistances.

2. Results

2.1. Genetic Determinants Coding for Heavy Metal Tolerance and Antibiotic Resistance

The results indicated that 67.7% of the strains carried at least one of the metal tolerance genes investigated (Table 1). Positive results were obtained for the multicopper oxidase gene copA (26.04% of strains), the DNA binding repressor protein gene pcoR (21.88%), the copper inner membrane pump pcoD (10.42%), the outer membrane protein copB (2.08%), the periplasmic copper binding protein copC (3.13%) and the multicopper oxidase gene pcoA (3.13% of strains). The copper export ATPase gene tcrB was found in 2.08% of strains, as well as the silver/copper periplasmic metal binding protein gene silE (2.08%).
The P-type ATPase zntA gene (cadmium, lead and zinc resistances) was found in a high percentage of strains (39.58%). Other metal-tolerance genes detected were czcD (13.54%), merA (1.04%), chrB (8.33%), and smtA (9.38%).
Investigation of genes related to antibiotic resistance yielded positive results in 89.58% of the strains (Table 1). 58.3% of the strains presented at least one of the β-lactamase resistance genes studied (blaTEM, blaPSE, blaCTX-M or blaCTX-M-2). blaCTX-M was the most frequent β-lactamase gene detected (30.21% of the strains), followed by blaTEM (15.63%). Genes involved in tetracycline resistance (tetA, B, C, D, E or G) were also found in 53.1% of strains. The most frequent tet genes were tetA (36.46%) and tetC (16.67%). Genes coding for export pumps were also detected in resistant strains, although their prevalence was lower. acrB gene, involved in the expression of the AcrAB-TolC efflux pump, was the more frequently detected determinant, with 37.5% of positive strains. 7.3% of the strains presented the aac(6′)-Ie-aph(2″)-Ia gene encoding the bi-functional aminoglycoside-modifying enzyme. Only 3.1% of strains were positive for dfrA12 gene, coding for sulfonamide and trimethoprim resistance. The qacA/B gene that confers resistance to quaternary ammonium compounds (QACs) and dyes and the deleted qacE∆1 were detected in 8.3% and 3.1%, respectively. The integrase gene intl1 associated with class I integrons carrying antimicrobial resistance genes was found in 14.5% of strains.

2.2. Association between Heavy Metal Tolerance and Genetic Determinants for Antibiotic Resistance

A high percentage of the strains positive for metal tolerance genes also carried antibiotic resistance genes, especially for genes involved in resistances to beta-lactams and tetracycline (Figure 1). Statistically significant associations were detected between copA and tetB/tetC/tetD (p = 0.041) or blaPSE/blaTEM (p = 0.01), as well as between copC and blaPSE/blaTEM (p = 0.008). Significant associations were also found between pcoR and blaCTX−M (p = 0.031). A significant association was also detected between zntA and tetC genes (p = 0.023).

3. Discussion

Heavy metals frequently spread on soils by agricultural practices are ubiquitous contaminants highly stable and resistant to degradation. Even low levels of heavy metals have been described as responsible for emergence and dissemination of antimicrobial resistant (AR) strains through co-selection, cross-resistance, co-regulation or biofilm induction [22]. The presence of even low concentrations of antibiotics or toxic compounds in the environment can select for resistance to different antibiotics and heavy metals through a coupled mechanism of resistance against both types of compounds. Reduced cell permeability, target site modification, efflux pump upregulation, acquisition of a neutralizing enzyme with activity against different classes of compounds, as well as physically linked genes responsible for two or more resistances may trigger resistance to more than one antimicrobial agent [23].
The co-selective potential of metals and biocides and its possible role in transmission of antimicrobial resistance genes to human pathogens in different ecosystems needs to be thoroughly investigated [24]. Cu-treated soils from olive tree agricultural fields may represent a source of bacteria subjected to a long-lasting selective pressure by copper and therefore there is a risk for co-selection of antibiotic resistance genes (ARGs).
A possible link between increased Cu levels and the presence of ARGs in agricultural soils subjected to long-term Cu contamination has been previously described [5,10,25]. Cu presence in soils associates positively with certain ARGs even at relatively low levels of the metal [26], suggesting that this metal can increase ARG prevalence even at subtoxic levels.
The present study revealed that the P-type ATPase zntA gene was the genetic determinant for metal tolerance more frequently detected among the selected isolates, followed by other metal tolerance genes (e.g., copA, pcoR, czcD, pcoD, smtA and chrB). These results illustrate how soil bacteria from agricultural fields can cumulate different metal-tolerance genes. The results obtained also indicated the presence of ARGs in the metal-tolerant strains. β-lactamase and tetracycline resistance genes were also frequently found in the selected strains.
The analysis of genetic determinants involved in metal tolerances and antibiotic resistances also shows significant associations between cop genes and genetic determinants for tetracycline and β-lactam resistances, as well as between pcoR, coding for a DNA binding repressor protein involved in copper tolerance, and genes coding for β-lactamases. A significant association was also detected between zntA, as well as czcD, and genes for tetracycline resistance. Similar relationships between specific metal tolerance and antimicrobial resistance genes were found by Pal et al. [27] from sequence analysis of more than 2500 chromosomes and 4500 plasmids. Both plasmids and genomes with biocide or metal resistance genes more frequently carried antibiotic resistance genes compared to those without biocide or metal resistance genes. Moreover, both types of genes coexisted in bacterial genomes from several environmental sources, including contaminated soils. Copper resistance (pco) and β-lactamases (blaCTX-M) genes coupled in the same plasmid have also been recently described in poultry and pigs in China [28], and copper-tolerant isolates also show the mcr-1 transferrable colistin resistance gene [29]. These results could suggest that copper may co-select for resistance to antimicrobials considered as an alternative for treatment of multiply resistant strains (such as colistin).
On the other hand, co-selection can also be promoted when different resistance genes are controlled by a single regulatory gene [30]. For example, the expression of the CzcCBA efflux pump and OprD porin (involved in cell permeability to carbapenems) is regulated by CzcR [31].
Previous results further illustrate the promiscuity of the nature of antibiotic resistance among Bacillus species and suggest that environmental bacteria may represent a reservoir of resistance genes with the potential to transfer resistance to the food chain or indeed to clinically relevant organisms [32]. Moradali et al. [33] have also summarized several of the well characterized molecular mechanisms which enable Pseudomonas species to survive various hostile conditions such as during pathogenesis and antibiotic treatment. These mechanisms form multiple layers of physiological adaptations correlating with social behavior and lifestyle of bacteria while responding environmental stimuli. Such extraordinary adaptive capability relies on extensive numbers of regulatory or controlling factors within integrated and complex signal processing pathways. These enable bacteria to perceive and process environmental cues in order to orchestrate physiological changes to promote adaptation to unfavorable conditions. The ubiquitous presence of P. aeruginosa as well as its prevalence and persistence in clinical settings including intrinsic resistance to therapeutics are also attributed to its extraordinary capability of survival by recruiting an arsenal of responsive mechanisms. The genes encoding extended-spectrum β-lactamases and carbapenemase are clinically important not only due to their hydrolyzing activity on a wide range of β-lactams such as carbapenems and extended-spectrum cephalosporins, but also for their worldwide prevalence [34]. The global epidemiology of carbapenem-resistant P. aeruginosa was recently analyzed by Hong et al. [35], who reported that the geographical prevalence of extended-spectrum β-lactamases and carbapenemase genes differs from country to country, whereas the genes encoding carbapenemases such as IMP, VIM, and NDM type metallo-β-lactamases have been found in all continents. Recent research [36] points to the environment as an important component for the transmission of resistant bacteria and in the emergence of resistant pathogens. However, a deeper understanding of the evolutionary and ecological processes that lead to clinical appearance of resistance genes is still lacking, which calls for better models of how resistance genes evolve, are mobilized, transferred and disseminated in the environment. Deep analysis of resistance genes in various environmental conditions are of great interest to define the ecological and evolutionary environmental factors that contribute to resistance development and transmission in the community and even in clinical settings.
Resistant isolates of soil origin also efficiently use resistance nodulation-cell division (RND) efflux pumps (EP) [37]. Stenotrophomonas spp. environmental strains had been demonstrated to possess similar ARGs as clinical strains [38,39] and similar action of EP in S. maltophilia and Stenotrophomonas of other species are now described, indicating that the EP present (RND and ABC) are able to confer resistance. Interestingly, it has been found that efflux is also used by Chryseobacterium spp. of soil origin, thought these bacteria were mostly know to be resistant by drug modification mechanisms [40].
Hence, resistance mechanisms studies of the most prevalent groups of cultivable bacteria from soils of different farming systems support the significant role of RND and ABC EPs in mediating resistance. The efficient efflux-mediated mechanisms in soil bacteria, therefore, might represent a source for multidrug resistance spread including horizontal transfer [41,42].
Our results may enhance the understanding of the genetics of co-selection and facilitate the evaluation of possible risk scenarios of resistance dissemination in order to reduce the alarming spread of antibiotic resistance genes in the future.

4. Materials and Methods

4.1. Strain Selection

Heavy metal tolerant bacteria were isolated from soil samples of olive tree farms in a previous study [21]. Strain identification and antibiotic resistance phenotype were established [21] and the 96 strains used in the present study were selected according to soil source and metal tolerance [21].

4.2. PCR Detection of Antimicrobial Resistance Genes

The primers and PCR conditions for amplification of antimicrobial resistance genes are detailed in Supplementary file 1. Efflux pump genes studied included acrB and mdfA [43] and oqxA [44]. Genes qacA/B (encoding resistance to quaternary ammonium compounds, QACs) were determined according to Noguchi et al. [45]. Amplification of qacE and qacE∆1 genes, their association with Class I integrons, and the presence of the integrase intl1 gene were studied according to Chuanchuen et al. [46]. Sulfonamide and trimethoprim resistance genes dfrA12 and dfrA15 [47] and aac(6′)-Ie-aph(2″)-Ia gene involved in aminoglycoside resistance [48] were also investigated.
The beta-lactamase blaPSE [49], blaTEM [47], blaCTX-M and blaCTX-M-2 [50] and tetracycline resistance genes tet(A), tet(B), tet(C), tet(D), tet(E) and tet(G) [51] were also studied.

4.3. Heavy Metals Tolerance Genes

The multicopper oxidase gene from the plasmid-borne operons pcoABCDRSE (from E. coli) and copABCDRS (from Pseudomonas syringae) was amplified according to Kamika and Momba [52] and Badar et al. [53]. pcoD (a copper inner membrane pump) [54], and pcoR, a DNA binding repressor protein gene [55,56] were also determined.
Other copper-resistance genes investigated included the cueAR operon (P1-type ATPase, MerR-type regulatory protein) [57] and tcrB (copper export ATPase) belonging to the tcrYAZB cluster described in Enterococcus [54,58]. The genes silA (silver inner membrane proton/cation antiporter) and silE (silver/copper periplasmic metal binding protein) of the silCFBAPRSE cluster [59], arsB (arsenite transmembrane pump) [60] as well as other genes related to metal tolerance such as merA (mercuric reductase) [61] and terF (tellurite resistance protein) were also studied [60].
The zinc-chromate resistance gene chrB was searched according to Nies et al. [62] and Chihomvu et al. [55] and the czcD gene (regulation of cobalt, cadmium and zinc efflux system) as described by Medardus et al. [12]. Lead resistance fragment smtAB genes were detected according to Naik et al. [63], and pbrA was studied for lead resistance [64]. The ncc operon was amplified as a fragment that spanned the nccA [65]. The Zn(II)-translocating P-type ATPase zntA gene described in E. coli was investigated according to Rensing et al. [66].

4.4. Statistics

Odds ratio (OR) and exact 95% confidence intervals were determined in order to evaluate the association between genetic determinants for heavy metal tolerance genetic and antibiotic resistance. An OR ≤ 1 indicated negative association and OR > 1 positive association.
Differences in the prevalence of antibiotic resistant strains, presence of heavy metal tolerance genes and genetic determinants for antibiotic resistance were analyzed (χ2 test). Significance of the association between heavy metal tolerance genetic determinants and both phenotypic resistance to antibiotics or presence of antibiotic resistance genes was analysed by Fisher’s test (p < 0.05).

5. Conclusions

The present study shows that bacteria from soils of Cu-treated olive farms may carry both metal tolerance and antibiotic resistance genes. The positive associations detected between metal tolerance genes and antibiotic resistance genes suggest co-selection of such genetic traits by exposure to metals.

Supplementary Materials

The following are available online at https://www.mdpi.com/2079-6382/9/8/476/s1, Table S1: Primers and PCR conditions for amplification of antimicrobial resistance genes.

Author Contributions

Conceptualization, E.O. and A.G.; methodology, E.O., A.G. and M.J.G.; software, N.G.; validation, E.O., N.G., M.J.G. and A.G.; formal analysis, N.G.; investigation, N.G. and M.J.G.; writing—original draft preparation, E.O. and N.G.; writing—review and editing, A.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by University of Jaen (grant number RS-AGR230).

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Ashbolt, N.J.; Amezquita, A.; Backhaus, T.; Borriello, P.; Bandt, K.K.; Collignon, P.; Coors, A.; Finley, R.; Gaze, W.H.; Heberer, T.; et al. Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance. Environ. Health Perspect. 2013, 121, 993–1001. [Google Scholar] [CrossRef] [Green Version]
  2. Marti, E.; Variatza, E.; Balcazar, J.L. The role of aquatic ecosystems as reservoirs of antibiotic resistance. Trends Microbiol. 2014, 22, 36–41. [Google Scholar] [CrossRef]
  3. Berendonk, T.U.; Manaia, C.M.; Merlin, C.; Fatta-Kassinos, D.; Cytryn, E.; Walsh, F.; Bürgmann, H.; Sørum, H.; Norström, M.; Pons, M.N.; et al. Tackling antibiotic resistance: The environmental framework. Nat. Rev. Microbiol. 2015, 13, 310–317. [Google Scholar] [CrossRef]
  4. Singer, A.C.; Shaw, H.; Rhodes, V.; Hart, A. Review of antimicrobial resistance in the environment and its relevance to environmental regulators. Front. Microbiol. 2016, 7, 1728. [Google Scholar] [CrossRef] [Green Version]
  5. Knapp, C.W.; McCluskey, S.M.; Singh, B.K.; Campbell, C.D.; Hudson, G.; Graham, D.W. Antibiotic resistance gene abundances correlated with metal and geochemical conditions in archived Scottish soils. PLoS ONE 2011, 6, e27300. [Google Scholar] [CrossRef]
  6. Zhu, Y.G.; Johnson, T.A.; Su, J.Q.; Qiao, M.; Guo, G.X.; Stedtfeld, R.D.; Hashsham, S.A.; Tiedje, J.M. Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc. Nat. Acad. Sci. USA 2013, 110, 3435–3444. [Google Scholar] [CrossRef] [Green Version]
  7. Wales, A.; Davies, R.H. Co-Selection of Resistance to Antibiotics, Biocides and Heavy Metals, and Its Relevance to Foodborne Pathogens. Antibiotics 2015, 4, 567–604. [Google Scholar] [CrossRef] [Green Version]
  8. Icgen, B.; Yilmaz, F. Co-occurrence of antibiotic and heavy metal resistance in Kızılırmak river isolates. Bull. Environ. Contam. Toxicol. 2014, 93, 735–743. [Google Scholar] [CrossRef]
  9. Liu, B.; Li, Y.; Gao, S.; Chen, X. Copper exposure to soil under single and repeated application: Selection for the microbial community tolerance and effects on the dissipation of antibiotics. J. Hazard. Mater. 2017, 325, 129–135. [Google Scholar] [CrossRef]
  10. Poole, K. At the Nexus of Antibiotics and Metals: The Impact of Cu and Zn on Antibiotic Activity and Resistance. Trends Microbiol. 2017, 25, 820–832. [Google Scholar] [CrossRef]
  11. World Health Organization. Antimicrobial Resistance: Global Report on Surveillance; WHO Press: Geneva, Switzerland, 2014. [Google Scholar]
  12. Medardus, J.J.; Molla, B.Z.; Nicol, M.; Morrow, W.M.; Rajala-Schultz, P.J.; Kazwala, R.; Gebreyes, W.A. In-feed use of heavy metal micronutrients in US swine production systems and its role in persistence of multidrug-resistant salmonellae. Appl. Environ. Microbiol. 2014, 80, 2317–2325. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Slifierz, M.J.; Friendship, R.; Weese, J.S. Zinc oxide therapy increases preva-lence and persistence of methicillin-resistant Staphylococcus aureus in pigs: A randomized controlled trial. Zoonoses Public Health 2015, 62, 301–308. [Google Scholar] [CrossRef] [PubMed]
  14. Amachawadi, R.G.; Scott, H.M.; Nitikanchana, S.; Vinasco, J.; Tokach, M.D.; Dritz, S.S.; Nelssen, J.L.; Goodband, R.D.; Nagaraja, T.G. Nasal carriage of mecA-positive methicillin-resistant Staphylococcus aureus in pigs exhibits dose-response to zinc supplementation. Foodborne Pathog. Dis. 2015, 12, 159–163. [Google Scholar] [CrossRef]
  15. Li, L.G.; Xia, Y.; Zhang, T. Co-occurrence of antibiotic and metal resistance genes revealed in complete genome collection. ISME J. 2017, 11, 651–662. [Google Scholar] [CrossRef] [PubMed]
  16. Staehlin, B.M.; Gibbons, J.G.; Rokas, A.; O’Halloran, T.V.; Slot, J.C. Evolution of a heavy metal homeostasis/resistance island reflects increasing copper stress in enterobacteria. Genome Biol. Evol. 2016, 8, 811–826. [Google Scholar] [CrossRef] [Green Version]
  17. Berg, J.; Thorsen, M.K.; Holm, P.E.; Jensen, J.; Nybroe, O.; Brandt, K.K. Cu exposure under field conditions coselects for antibiotic resistance as determined by a novel cultivation-independent bacterial community tolerance assay. Environ. Sci. Technol. 2010, 44, 8724–8728. [Google Scholar] [CrossRef]
  18. Bondarczuk, K.; Piotrowska-Seget, Z. Molecular basis of active copper resistance mechanisms in Gram-negative bacteria. Cell Biol. Toxicol. 2013, 29, 397–405. [Google Scholar] [CrossRef] [Green Version]
  19. Zhang, M.; Chen, L.; Ye, C.; Yu, X. Co-selection of antibiotic resistance via copper shock loading on bacteria from a drinking water bio-filter. Environ. Pollut. 2018, 233, 132–141. [Google Scholar] [CrossRef]
  20. Xu, Y.; Xu, J.; Mao, D.; Luo, Y. Effect of the selective pressure of sub-lethal level of heavy metals on the fate and distribution of ARGs in the catchment scale. Environ. Pollut. 2017, 220, 900–908. [Google Scholar] [CrossRef]
  21. Glibota, N.; Grande Burgos, M.J.; Gálvez, A.; Ortega, E. Copper tolerance and antibiotic resistance in soil bacteria from olive tree agricultural fields routinely treated with copper compounds. J. Sci. Food Agric. 2019, 99, 4677–4685. [Google Scholar] [CrossRef]
  22. Yu, Z.; Gunn, L.; Wall, P.; Fanning, S. Antimicrobial resistance and its association with tolerance to heavy metals in agriculture production. Food Microbiol. 2017, 64, 23–32. [Google Scholar] [CrossRef]
  23. Seiler, C.; Berendonk, T.U. Heavy metal driven co-selection of antibiotic resistance in soil and water bodies impacted by agriculture and aquaculture. Front. Microbiol. 2012, 3, 399. [Google Scholar] [CrossRef] [Green Version]
  24. Fard, R.M.; Heuzenroeder, M.W.; Barton, M.D. Antimicrobial and heavy metal resistance in commensal enterococci isolated from pigs. Vet. Microbiol. 2011, 48, 276–282. [Google Scholar] [CrossRef]
  25. Hu, H.W.; Wang, J.T.; Li, J.; Li, J.J.; Ma, Y.B.; Chen, D.; He, J.Z. Field-based evidence for copper contamination induced changes of antibiotic resistance in agricultural soils. Environ. Microbiol. 2016, 18, 3896–3909. [Google Scholar] [CrossRef]
  26. Knapp, C.W.; Callan, A.C.; Aitken, B.; Shearn, R.; Koenders, A.; Hinwood, A. Relationship between antibiotic resistance genes and metals in residential soil samples from Western Australia. Environ. Sci. Pollut. Res. Int. 2017, 24, 2484–2494. [Google Scholar] [CrossRef]
  27. Pal, C.; Bengtsson-Palme, J.; Kristiansson, E.; Larsson, D.G. Co-occurrence of resistance genes to antibiotics; biocides and metals reveals novel insights into their co-selection potential. BMC Genomics 2015, 16, 964. [Google Scholar] [CrossRef] [Green Version]
  28. Fang, L.; Li, X.; Li, L.; Li, S.; Liao, X.; Sun, J.; Liu, Y. Co-spread of metal and antibiotic resistance within ST3-IncHI2 plasmids from E. coli isolates of food-producing animals. Sci. Rep. 2007, 6, 25312. [Google Scholar]
  29. Campos, J.; Cristino, L.; Peixe, L.; Antunes, P. MCR-1 in multidrug-resistant and copper-tolerant clinically relevant Salmonella 1;4;[5];12:i:- and S. Rissen clones in Portugal; 2011 to 2015. Eurosurveillance 2016, 21, 30270. [Google Scholar] [CrossRef]
  30. Pal, C.; Asiani, K.; Arya, S.; Rensing, C.; Stekel, D.J.; Larsson, D.G.J.; Hobman, J.L. Metal resistance and its association with antibiotic resistance. Adv. Microb. Physiol. 2017, 70, 261–313. [Google Scholar]
  31. Perron, K.; Caille, O.; Rossier, C.; Van Delden, C.; Dumas, J.; Köhler, T. CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa. J. Biol. Chem. 2004, 279, 8761–8768. [Google Scholar] [CrossRef] [Green Version]
  32. Barbosa, T.M.; Phelan, R.W.; Leong, D.; Morrissey, J.P.; Adams, C.; Dobson, A.D.W.; O’Gara, F. A novel erythromycin resistance plasmid from Bacillus sp. strain HS24, isolated from the marine sponge Haliclona simulans. PLoS ONE 2014, 9, e115583. [Google Scholar] [CrossRef] [Green Version]
  33. Moradali, M.F.; Ghods, S.; Rehm, B.H.A. Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence. Front. Cell. Infect. Microbiol. 2017, 7, 39. [Google Scholar] [CrossRef] [Green Version]
  34. Sullivan, R.; Schaus, D.; John, M.; Delport, J.A. Extended spectrum beta-lactamases: A minireview of clinical relevant groups. J. Med. Microb. Diagn. 2016, 4, 203. [Google Scholar] [CrossRef] [Green Version]
  35. Hong, D.J.; Bae, I.K.; Jang, I.H.; Jeong, S.H.; Kang, H.K.; Lee, K. Epidemiology and characteristics of metallo-β-lactamase-producing Pseudomonas aeruginosa. Infect. Chemother. 2015, 47, 81–97. [Google Scholar] [CrossRef]
  36. Bengtsson-Palme, J.; Kristiansson, E.; Larsson, D.G.J. Environmental factors influencing the development and spread of antibiotic resistance. FEMS Microbiol. Rev. 2018, 42, 1. [Google Scholar] [CrossRef]
  37. Armalytė, J.; Skerniškytė, J.; Bakienė, E.; Krasauskas, R.; Šiugždinienė, R.; Kareivienė, V.; Kerzienė, S.; Klimienė, I.; Sužiedėlienė, E.; Ružauskas, M. Microbial Diversity and Antimicrobial Resistance Profile in Microbiota From Soils of Conventional and Organic Farming Systems. Front. Microbiol. 2019, 10, 892. [Google Scholar] [CrossRef]
  38. Youenou, B.; Favre-Bonté, S.; Bodilis, J.; Brothier, E.; Dubost, A.; Muller, D.; Nazaret, S. Comparative genomics of environmental and clinical Stenotrophomonas maltophilia strains with different antibiotic resistance profiles. Genome Biol. Evol. 2015, 7, 2484–2505. [Google Scholar] [CrossRef] [Green Version]
  39. Wang, Y.; He, T.; Shen, Z.; Wu, C. Antimicrobial Resistance in Stenotrophomonas spp. Microbiol. Spectr. 2018, 6, ARBA-0005-2017. [Google Scholar]
  40. Lin, X.-H.; Xu, Y.-H.; Sun, X.-H.; Huang, Y.; Li, J.-B. Genetic diversity analyses of antimicrobial resistance genes in clinical Chryseobacterium meningosepticum isolated from Hefei, China. Int. J. Antimicrob. Agents 2012, 40, 186–188. [Google Scholar] [CrossRef]
  41. Dolejska, M.; Villa, L.; Poirel, L.; Nordmann, P.; Carattoli, A. Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance-nodulation-cell division/multidrug efflux pump. J. Antimicrob. Chemother. 2013, 68, 34–39. [Google Scholar] [CrossRef] [Green Version]
  42. Walsh, F.; Duffy, B. The culturable soil antibiotic resistome: A community of multi-drug resistant bacteria. PLoS ONE 2013, 8, e65567. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Swick, M.C.; Morgan-Linnell, S.K.; Carlson, K.M.; Zechiedrich, L. Expression of multidrug efflux pump genes acrAB-tolC; mdfA; and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance. Antimicrob. Agents Chemother. 2011, 55, 921–924. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Hansen, L.S.; Jensen, L.B.; Sørensen, H.I. Substrate specificity of the OqxAB multidrug resistance pump in Escherichia coli and selected enteric bacteria. J. Antimicrob. Chemother. 2007, 60, 145–147. [Google Scholar] [CrossRef]
  45. Noguchi, N.; Hase, M.; Kitta, M.; Sasatsu, M.; Deguchi, K.; Kono, M. Susceptibilities to antiseptic agents and distribution of antiseptic-resistance genes qacA/B and smr of methicillin-resistant Staphylococcus aureus isolated in Asia during 1998 and 1999. J. Med. Microbiol. 2005, 54, 557–565. [Google Scholar] [CrossRef]
  46. Chuanchuen, R.; Khemtong, S.; Padungtod, P. Occurrence of qacE/qacEDelta1 genes and their correlation with class 1 integrons in Salmonella enterica isolates from poultry and swine. Southeast Asian J. Trop. Med. Public Health 2007, 38, 855–862. [Google Scholar] [PubMed]
  47. Sáenz, Y.; Briñas, L.; Domínguez, E.; Ruiz, J.; Zarazaga, M.; Vila, J.; Torres, C. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human; animal; and food origins. Antimicrob. Agents Chemother. 2004, 48, 3996–4001. [Google Scholar] [CrossRef] [Green Version]
  48. Vakulenko, S.B.; Donabedian, S.M.; Voskresenskiy, A.M.; Zervos, M.J.; Lerner, S.A.; Chow, J.W. Multiplex PCR for detection of aminoglycoside resistance genes in enterococci. Antimicrob. Agents Chemother. 2003, 47, 1423–1426. [Google Scholar] [CrossRef] [Green Version]
  49. Chiu, C.H.; Su, L.H.; Chu, C.H.; Wang, M.H.; Yeh, C.M.; Weill, F.X.; Chu, C. Detection of multidrug-resistant Salmonella enterica serovar Typhimurium phage types DT102; DT104; and U302 by multiplex PCR. J. Clin. Microbiol. 2006, 44, 2354–2358. [Google Scholar] [CrossRef] [Green Version]
  50. Bertrand, S.; Weill, F.X.; Cloeckaert, A.; Vrints, M.; Mairiaux, E.; Praud, K.; Dierick, K.; Wildemauve, C.; Godard, C.; Butaye, P.; et al. Clonal emergence of extended-spectrum β-lactamase (CTX-M-2)-producing Salmonella enterica serovar Virchow isolates with reduced susceptibilities to ciprofloxacin among poultry and humans in Belgium and France (2000 to 2003). J. Clin. Microbiol. 2006, 44, 2897–2903. [Google Scholar] [CrossRef] [Green Version]
  51. Ng, L.K.; Martin, I.; Alfa, M.; Mulvey, M. Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell. Probes. 2001, 15, 209–215. [Google Scholar] [CrossRef]
  52. Kamika, I.; Momba, M.N. Assessing the resistance and bioremediation ability of selected bacterial and protozoan species to heavy metals in metal-rich industrial wastewater. BMC Microbiol. 2013, 13, 28. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Badar, U.; Ahmed, N.; Shoeb, E.; Gadd, G.M. Identification of the pco operon in Enterobacter species isolated from contaminated soil. Int. J. Adv. Res. 2014, 2, 227–233. [Google Scholar]
  54. Mourão, J.; Novais, C.; Machado, J.; Peixe, L.; Antunes, P. Metal tolerance in emerging clinically relevant multidrug-resistant Salmonella enterica serotype 4,[5],12:i:− Clones circulating in Europe. Int. J. Antimicrob. Agents 2015, 45, 610–616. [Google Scholar] [CrossRef] [PubMed]
  55. Chihomvu, P.; Stegmann, P.; Pillay, M. Characterization and Structure Prediction of Partial Length Protein Sequences of pcoA; pcoR and chrB Genes from Heavy Metal Resistant Bacteria from the Klip River; South Africa. Int. J. Mol. Sci. 2015, 16, 7352–7374. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Brown, N.L.; Rouch, D.A.; Lee, B.T. Copper resistance determinants in bacteria. Plasmid 1992, 27, 41–51. [Google Scholar] [CrossRef]
  57. Adaikkalam, V.; Swarup, S. Molecular characterization of an operon; cueAR; encoding a putative P1-type ATPase and a MerR-type regulatory protein involved in copper homeostasis in Pseudomonas putida. Microbiology 2002, 148, 2857–2867. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Hasman, H.; Kempf, I.; Chidaine, B.; Cariolet, R.; Ersbøll, A.K.; Houe, H.; Aarestrup, F.M. Copper resistance in Enterococcus faecium, mediated by the tcrB gene, is selected by supplementation of pig feed with copper sulfate. Appl. Environ. Microbiol. 2006, 72, 5784–5789. [Google Scholar] [CrossRef] [Green Version]
  59. Silver, S. Bacterial silver resistance: Molecular biology and uses and misuses of silver compounds. FEMS Microbiol. Rev. 2003, 27, 341–353. [Google Scholar] [CrossRef] [Green Version]
  60. García Fernández, A.; Cloeckaert, A.; Bertini, A.; Praud, K.; Doublet, B.; Weill, F.X.; Carattoli, A. Comparative analysis of IncHI2 plasmids carrying blaCTX-M-2 or blaCTX-M-9 from Escherichia coli and Salmonella enterica strains isolated from poultry and humans. Antimicrob. Agents Chemother. 2007, 51, 4177–4180. [Google Scholar] [CrossRef] [Green Version]
  61. Liebert, C.A.; Wireman, J.; Smith, T.; Summers, A.O. Phylogeny of mercury resistance (mer) operons of gram-negative bacteria isolated from the fecal flora of primates. Appl. Environ. Microbiol. 1997, 63, 1066–1076. [Google Scholar] [CrossRef] [Green Version]
  62. Nies, A.; Nies, D.H.; Silver, S. Nucleotide sequence and expression of a plasmid-encoded chromate resistance determinant from Alcaligenes eutrophus. J. Biol. Chem. 1990, 265, 5648–5653. [Google Scholar] [PubMed]
  63. Naik, M.M.; Shamim, K.; Dubey, S.K. Biological characterization of lead resistant bacteria to explore role of bacterial metallothionein in lead resistance. Curr. Sci. 2012, 103, 426–429. [Google Scholar]
  64. Borremans, B.; Hobman, J.L.; Provoost, A.; Brown, N.L.; van Der Lelie, D. Cloning and functional analysis of thepbr lead resistance determinant of Ralstonia metallidurans CH34. J. Bacteriol. 2001, 183, 5651–5658. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Abou-Shanab, R.; Van Berkum, P.; Angle, J. Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale. Chemosphere 2007, 68, 360–367. [Google Scholar] [CrossRef]
  66. Rensing, C.; Mitra, B.; Rosen, B.P. The zntA gene of Escherichia coli encodes a Zn (II)-translocating P-type ATPase. Proc. Natl. Acad. Sci. USA 1997, 94, 14326–14331. [Google Scholar] [CrossRef] [Green Version]
Antibiotics 09 00476 g001 550
Figure 1. Association between heavy metal tolerance and antibiotic resistance genetic determinants. The percentage of genetic determinants of antimicrobial resistance found in strains that tested positive for heavy metal tolerance genes cop (a), pco (b), czcD (c) or zntA (d) are shown.
Figure 1. Association between heavy metal tolerance and antibiotic resistance genetic determinants. The percentage of genetic determinants of antimicrobial resistance found in strains that tested positive for heavy metal tolerance genes cop (a), pco (b), czcD (c) or zntA (d) are shown.
Antibiotics 09 00476 g001
Table
Table 1. Genetic determinants of heavy metal tolerant isolates from agricultural soils.
Table 1. Genetic determinants of heavy metal tolerant isolates from agricultural soils.
Species (Isolate)Heavy Metal Tolerance Genetic DeterminantsAntibiotic Resistance Genetic Determinants
Bacillus cereus (T22Pb3)-acrB
Bacillus endophyticus (T37Ni3)-tetA, tetC
Bacillus fordii (T11Ni1)chrB-
Bacillus fordii (T18Ni1)pcoR, copAqacA/B
Bacillus megaterium (T1Ni2)pcoR, chrB, copAintl1, tetA
Bacillus psychrosaccharolyticus (T4Ni3)copAintl1, tetA, tetC, blaPSE
Bacillus sp. (T17Ni3)-tetB, blaPSE, blaTEM
Bacillus sp. (T19Pb3)-blaCTX−M
Bacillus sp. (T20Pb2)pcoDacrB, aac(6′)-Ie-aph(2″)-Ia, tetA, tetE
Bacillus sp. (T21Zn1)pcoAblaCTX−M
Bacillus sp. (T28Pb3)-acrB, blaTEM
Bacillus sp. (T11Pb1)-acrB, blaCTX−M
Bacillus sp. (T12Pb1)-acrB
Bacillus sp. (T13Ni2)-intl1, tetG, blaCTX−M
Bacillus sp. (T13Pb1)-acrB, blaCTX−M
Bacillus sp. (T14Pb1)-intl1
Bacillus sp. (T26Ni3)smtAacrB, qacA/B, intl1, dfrA12, tetG, blaCTX−M, blaCTX−M2
Bacillus sp. (T26Pb2)-acrB
Bacillus sp. (T37Ni2)-dfrA12
Bacillus sp. (T8Pb1)-acrB
Bacillus sp. (T9Ni2)-blaCTX−M, blaCTX−M2
Bacillus sp. (T9Pb1)--
Burkholderia zhejiangensis (T37Cd2)zntAtetA
Burkholderia zhejiangensis (T38Zn3)zntAqacA/B, blaCTX−M
Chryseobacterium formosense (T11Zn1)-tetA, tetC, blaTEM
Chryseobacterium gleum (T14Cd2)-tetC
Chryseobacterium gleum (T4Ni2)-tetA, tetC, blaCTX−M2
Chryseobacterium gleum (T6Pb1)pcoR, zntAacrB, tetC, tetG, blaCTX−M
Chryseobacterium gleum (T6Zn3)pcoR, copBtetG, blaCTX−M, blaCTX−M2
Chryseobacterium hispalense (T3Zn3)-tetA, tetC, blaCTX−M
Chryseobacterium oranimense (E5Zn1)zntAacrB, tetD, blaCTX−M
Chryseobacterium oranimense (T18Cd1)-acrB, qacA/B, tetA, blaCTX−M2
Chryseobacterium oranimense (T30Zn1)-aac(6′)-Ie-aph(2″)-Ia, tetA, tetC
Chryseobacterium oranimense (T31Zn1)pcoR, zntAtetC
Chryseobacterium oranimense (T37Cd3)pcoR-
Chryseobacterium oranimense (T3Cd1)-acrB, tetA, tetC, blaCTX−M
Chryseobacterium oranimense (T3Ni2)pcoRtetA, tetD, blaCTX−M
Chryseobacterium oranimense (T3Zn2)-tetA, tetE, blaCTX−M
Chryseobacterium oranimense (T4Cd1)pcoRacrB, blaCTX−M
Chryseobacterium oranimense (T5Cd1)pcoRtetA, tetC, blaCTX−M
Chryseobacterium oranimense (T5Ni3)pcoRtetA, tetC, blaCTX−M
Chryseobacterium oranimense (T6Cd1)-acrB, intl1, tetA, tetC, blaCTX−M
Chryseobacterium piperi (T8Cd1)-acrB, qacA/B, tetA, tetC, blaCTX−M
Enterobacter (Klebsiella) aerogenes (E1Pb3)copB, copC, pcoD, tcrB, merA, zntAblaTEM
Enterococcus faecalis (T26Zn2)pcoRtetA, tetB, tetD, blaCTX−M
Flavobacterium johnsoniae (E1Zn1)-acrB, qacE∆1, tetA, tetC
Janthinobacterium lividum (T25Cd3)--
Pseudomonas entomophila (E1Cu3)copA, pcoA, czcD, smtA, zntAacrB, intl1, tetA, blaCTX−M
Pseudomonas entomophila (T11Cd1)-tetA, blaCTX−M2, blaTEM
Pseudomonas entomophila (T17Pb1)copA, zntAacrB, tetA, tetG, blaTEM
Pseudomonas entomophila (T23Cu2)copA, zntAacrB, tetA
Pseudomonas entomophila (T34Cu2)copA, zntAacrB, intl1, aac(62)-Ie-aph(2″)-Ia
Pseudomonas entomophila (T5Cu2)copA, smtAacrB, blaCTX−M
Pseudomonas entomophila (T6Cu1)czcD, smtA, zntAacrB, tetA
Pseudomonas entomophila (T7Ni1)copA, pcoD, czcD, smtA, zntA-
Pseudomonas fluorescens (E5Zn2)copA, pcoD, zntAaac(6′)-Ie-aph(2″)-Ia, tetG, blaCTX−M
Pseudomonas fluorescens (E5Zn3)copA, pcoD, zntAacrB, aac(6′)-Ie-aph(2″)-Ia, blaCTX−M, blaPSE
Pseudomonas fluorescens (T12Cu1)pcoR, pcoD, zntAblaCTX−M
Pseudomonas fluorescens (T15Cd1)chrB, czcD, zntAtetA
Pseudomonas fluorescens (T25Cu3)zntAtetB, blaCTX−M2
Pseudomonas fluorescens (T25Ni3)czcDqacA/B, tetG
Pseudomonas fluorescens (T31Cd3)czcD, zntAqacA/B, tetD, blaPSE, blaTEM
Pseudomonas fluorescens (T32Cd3)pcoR, copA, zntAtetA
Pseudomonas fluorescens (T35Cd3)copA, czcD, zntAtetA, blaTEM
Pseudomonas fluorescens (T35Cu2)copA, pcoD, smtAacrB, tetA
Pseudomonas fluorescens (T3Ni3)copA, pcoDtetA, blaTEM
Pseudomonas fluorescens (T5Cd2)pcoR, chrB, czcD, zntAtetA, blaCTX−M, blaTEM
Pseudomonas fluorescens (T7Cd3)pcoR, zntAtetA
Pseudomonas lutea (E3Cu2)czcD, zntAintl1
Pseudomonas lutea (T8Ni2)--
Pseudomonas putida (T1Cu3)copA, copC, czcD, smtAacrB, intl1, tetE, blaCTX−M, blaCTX−M2, blaTEM
Pseudomonas sp. (T15Ni1)copA, zntAdfrA12, tetE
Pseudomonas sp. (T16Ni3)-tetA, blaPSE
Pseudomonas sp. (T15Cu3)copA, zntAaac(6′)-Ie-aph(2″)-Ia, tetE, blaTEM
Pseudomonas sp. (T18Cu3)copA, silE, zntAacrB, intl1, tetA, blaCTX−M2
Pseudomonas sp. (T19Cu2)copA, silE,acrB, intl1
Pseudomonas sp. (T22Cu1)pcoR, pcoD, zntAtetD, tetE, tetG, blaCTX−M
Pseudomonas sp. (T24Cu3)pcoR, zntAacrB, intl1
Pseudomonas sp. (T24Ni1)pcoR, copAacrB
Pseudomonas sp. (T28Cu2)chrB, copA, zntAacrB, blaTEM
Pseudomonas sp. (T36Cu3)chrB, copA, czcD, smtA, zntAintl1
Pseudomonas sp. (T40Cd3)czcD, zntAqacA/B, acrB, tetB, blaCTX−M2
Pseudomonas sp. (T7Cu2)pcoD, zntAacrB
Serratia proteamaculans (T3Cd2)zntAacrB, tetB, tetE, tetG
Serratia proteamaculans (T4Pb1)pcoR, copAaac(6′)-Ie-aph(2″)-Ia, tetB, tetG, blaCTX−M, blaPSE
Sphingobacterium paucimobilis (T30Zn3)pcoR, copA, copCtetC, tetG, blaPSE
Stenotrophomonas maltophilia (T19Zn1)zntAblaTEM
Stenotrophomonas maltophilia (T40Zn1)zntAacrB, blaTEM
Stenotrophomonas rhizophila (E1Zn3)pcoA-
Stenotrophomonas rhizophila (T31Zn2)zntA-
Variovorax paradoxus (E4Zn1)pcoRacrB, tetA
Variovorax paradoxus (T18Zn2)czcD, smtA, zntAtetB
Variovorax paradoxus (T23Zn2)zntA-
Variovorax paradoxus (T32Zn2)chrBtetE
Variovorax paradoxus (T32Zn3)chrB-
Variovorax paradoxus (T7Zn1)-tetA

Share and Cite

MDPI and ACS Style

Glibota, N.; Grande, M.J.; Galvez, A.; Ortega, E. Genetic Determinants for Metal Tolerance and Antimicrobial Resistance Detected in Bacteria Isolated from Soils of Olive Tree Farms. Antibiotics 2020, 9, 476. https://doi.org/10.3390/antibiotics9080476

AMA Style

Glibota N, Grande MJ, Galvez A, Ortega E. Genetic Determinants for Metal Tolerance and Antimicrobial Resistance Detected in Bacteria Isolated from Soils of Olive Tree Farms. Antibiotics. 2020; 9(8):476. https://doi.org/10.3390/antibiotics9080476

Chicago/Turabian Style

Glibota, Nicolás, Mª José Grande, Antonio Galvez, and Elena Ortega. 2020. "Genetic Determinants for Metal Tolerance and Antimicrobial Resistance Detected in Bacteria Isolated from Soils of Olive Tree Farms" Antibiotics 9, no. 8: 476. https://doi.org/10.3390/antibiotics9080476

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop