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Selection of Resistance at very low Antibiotic Concentrations
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria.

In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection.

These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 86 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1053
Keyword [en]
Antibiotic resistance, Selection, Antibiotic resistant bacteria, Minimal inhibitory concentration, Heavy metals, Conjugative plasmid, ESBL
National Category
Microbiology in the medical area
Research subject
Microbiology; Biology with specialization in Microbiology; Biology with specialization in Molecular Evolution
Identifiers
URN: urn:nbn:se:uu:diva-235225ISBN: 978-91-554-9101-7 (print)OAI: oai:DiVA.org:uu-235225DiVA: diva2:759386
Public defence
2014-12-17, A1:111a, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2014-11-24 Created: 2014-10-29 Last updated: 2015-02-03
List of papers
1. Activation of cryptic aminoglycoside resistance in Salmonella enterica
Open this publication in new window or tab >>Activation of cryptic aminoglycoside resistance in Salmonella enterica
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2011 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 80, no 6, 1464-1478 p.Article in journal (Refereed) Published
Abstract [en]

Aminoglycoside resistance in bacteria can be acquired by several mechanisms, including drug modification, target alteration, reduced uptake and increased efflux. Here we demonstrate that increased resistance to the aminoglycosides streptomycin and spectinomycin in Salmonella enterica can be conferred by increased expression of an aminoglycoside adenyl transferase encoded by the cryptic, chromo-somally located aadA gene. During growth in rich medium the wild-type strain was susceptible but mutations that impaired electron transport and conferred a small colony variant (SCV) phenotype or growth in glucose/glycerol minimal media resulted in activation of the aadA gene and aminoglycoside resistance. Expression of the aadA gene was positively regulated by the stringent response regulator guanosine penta/tetraphosphate ((p) ppGpp). SCV mutants carrying stop codon mutations in the hemA and ubiA genes showed a streptomycin pseudo-dependent phenotype, where growth was stimulated by streptomycin. Our data suggest that this phenotype is due to streptomycin-induced readthrough of the stop codons, a resulting increase in HemA/UbiA levels and improved electron transport and growth. Our results demonstrate that environmental and mutational activation of a cryptic resistance gene can confer clinically significant resistance and that a streptomycin-pseudo-dependent phenotype can be generated via a novel mechanism that does not involve the classical rpsL mutations.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-156096 (URN)10.1111/j.1365-2958.2011.07657.x (DOI)000292106100006 ()
Available from: 2011-07-11 Created: 2011-07-11 Last updated: 2017-12-11
2. Selection of Resistant Bacteria at Very Low Antibiotic Concentrations
Open this publication in new window or tab >>Selection of Resistant Bacteria at Very Low Antibiotic Concentrations
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2011 (English)In: PLoS pathogens, ISSN 1553-7366, Vol. 7, no 7, e1002158- p.Article in journal (Refereed) Published
Abstract [en]

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-157045 (URN)10.1371/journal.ppat.1002158 (DOI)000293339300042 ()
Available from: 2011-08-15 Created: 2011-08-15 Last updated: 2015-02-03Bibliographically approved
3. Selection of a multidrug resistance plasmid by sublethal levels of antibiotics and heavy metals
Open this publication in new window or tab >>Selection of a multidrug resistance plasmid by sublethal levels of antibiotics and heavy metals
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2014 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 5, e01918-14- p.Article in journal (Refereed) Published
Abstract [en]

How sublethal levels of antibiotics and heavy metals select for clinically important multidrug resistance plasmids is largely unknown. Carriage of plasmids generally confers substantial fitness costs, implying that for the plasmid-carrying bacteria to be maintained in the population, the plasmid cost needs to be balanced by a selective pressure conferred by, for example, antibiotics or heavy metals. We studied the effects of low levels of antibiotics and heavy metals on the selective maintenance of a 220-kbp extended-spectrum β-lactamase (ESBL) plasmid identified in a hospital outbreak of Klebsiella pneumoniae and Escherichia coli. The concentrations of antibiotics and heavy metals required to maintain plasmid-carrying bacteria, the minimal selective concentrations (MSCs), were in all cases below (almost up to 140-fold) the MIC of the plasmid-free susceptible bacteria. This finding indicates that the very low antibiotic and heavy metal levels found in polluted environments and in treated humans and animals might be sufficiently high to maintain multiresistance plasmids. When resistance genes were moved from the plasmid to the chromosome, the MSC decreased, showing that MSC for a specific resistance conditionally depends on genetic context. This finding suggests that a cost-free resistance could be maintained in a population by an infinitesimally low concentration of antibiotic. By studying the effect of combinations of several compounds, it was observed that for certain combinations of drugs each new compound added lowered the minimal selective concentration of the others. This combination effect could be a significant factor in the selection of multidrug resistance plasmids/bacterial clones in complex multidrug environments.

IMPORTANCE: Antibiotic resistance is in many pathogenic bacteria caused by genes that are carried on large conjugative plasmids. These plasmids typically contain multiple antibiotic resistance genes as well as genes that confer resistance to biocides and heavy metals. In this report, we show that very low concentrations of single antibiotics and heavy metals or combinations of compounds can select for a large plasmid that carries resistance to aminoglycosides, β-lactams, tetracycline, macrolides, trimethoprim, sulfonamide, silver, copper, and arsenic. Our findings suggest that the low levels of antibiotics and heavy metals present in polluted external environments and in treated animals and humans could allow for selection and enrichment of bacteria with multiresistance plasmids and thereby contribute to the emergence, maintenance, and transmission of antibiotic-resistant disease-causing bacteria.

National Category
Microbiology in the medical area
Research subject
Microbiology; Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-235222 (URN)10.1128/mBio.01918-14 (DOI)000345459000067 ()25293762 (PubMedID)
Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2017-12-05Bibliographically approved
4. Evolution of Resistance at Non-Lethal (Sub-MIC) Levels of Antibiotics
Open this publication in new window or tab >>Evolution of Resistance at Non-Lethal (Sub-MIC) Levels of Antibiotics
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-235223 (URN)
Available from: 2014-10-29 Created: 2014-10-29 Last updated: 2015-02-03

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