The horizontal flow of the plasmid resistome: clues from inter‐generic similarity networks

By integrating sequence similarity data of plasmid‐encoded antibiotic resistance determinants with those coming from a less transferred molecular marker, we constructed a network in which all the sequences that most likely underwent horizontal gene transfer (HGT) were linked together. The analysis of this network revealed that either geographical barriers or taxonomical distance can often be overcome since phylogenetically unrelated bacteria, and/or those inhabiting distinct environments, were found to share common antibiotic resistance determinants, probably as a result of (one or multiple) HGT event(s). Data obtained also revealed that bacteria viable through multiple environments (ubiquitous) are likely to give a crucial contribution to the spreading of bacterial resistance towards antimicrobial compounds. These analyses represent a first attempt to give an almost global picture of the horizontal flow of antibiotic resistance determinants at the whole bacterial community level, also underlining the power of HGT among bacteria and how this ‘horizontal flow’ is poorly affected by both taxonomy and physical distance. Finally, data presented may be useful in the infections control procedures, suggesting which bacterial species are more likely acting as vectors of antibiotic resistance determinants.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic producers, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.     

Genetic control of tetracycline resistance in bacteria]

Modern data on prevalence, structural and functional organization of the tetracycline resistance determinants in bacteria are reviewed. The three mechanisms of the antibiotic resistance are the tetracycline efflux, the ribosomal protection and the antibiotic modification. The problems of evolution of tetracycline resistance genes are discussed.     

A new look at antibiotic resistance

Abstract Since the discovery of antibiotic resistance in clinically important microbes, attention has focused properly on the profound medical aspects of this problem. However, studies of antibiotic resistance are of great interest in their own right for studies of gene regulation, evolution, chromosome structure, etc.; several resistance genes in clinical isolates are components of classical 'operon' structures. The construction of cloning vectors and gene transfer systems, particularly for interspecies studies, rely heavily on the use of antibiotic resistance genes, since these phenotypes can be used to select for DNA transfer between microbes, plants, and animals. Studies of the role of resistance mechanisms and their genetic determinants in antibiotic-producing organisms have shown that these functions play important roles in biosynthetic pathways and can provide important genetic and biochemical tools for the rational analysis of antibiotic production.     

活性污泥抗生素抗性基因研究进展

抗生素抗性在全球范围内的传播扩散严重威胁人类健康。活性污泥是污水处理系统重要的处理工艺,同时也是抗生素抗性及其发生水平基因转移的一个重要储库和热区。目前,随着研究手段和技术的不断更新,活性污泥中抗生素抗性的研究不断增加,但是仍有许多科学问题亟待解决。本文主要针对活性污泥抗生素抗性的5个主要方面进行深入讨论:(1)活性污泥中抗性基因的丰度和分布的影响因素;(2)污泥抗性基因的研究方法;(3)活性污泥抗性基因的传播与扩散;(4)污泥中抗性基因环境风险评估;(5)研究展望。本综述在活性污泥抗生素抗性研究基础上,阐述了驱动抗生素抗性扩散的基本微生物生态过程研究进展,旨在为污水处理工艺的发展和优化及抗性基因控制政策的制定提供科学基础。     

Genetic determinants of antibiotic resistance in enterobacteria

Antibiotic resistance of enterobacterial strains from population isolated in Krasnodar region is rather often controlled by the "plasmid" genes. The conclusion is based on using the colony hybridization with [32P]-DNA fragments of plasmids, carrying the genetic determinants of antibiotic resistance, as a method for antibiotic resistance, genes screening. Kanamycin resistance in the majority of strains is coded by APH (3') II gene, streptomycin resistance by APH (3") gene, chloramphenicol resistance by CATI, sulphonilamide resistance by DHPS type II gene. Tetracycline resistance of the studied enterobacterial strains is not connected with the widespread genetic determinants of a new class tetracycline resistance.     

Mobile Genetic Element-Encoded Cytolysin Connects Virulence to Methicillin Resistance in MRSA

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.     

Phenotypic diversity and antibiotic resistance in soil bacterial communities

The community structure in two different agricultural soils has been investigated. Phenotypic diversity was assessed by applying BIOLOG-profiles on a total of 208 bacterial isolates. Diversity indices were calculated from cluster analysis of the BIOLOG data. The bacterial isolates were also evaluated for resistance towards six different antibiotics, mercury resistance and the presence of plasmids. The presence of tetracycline-resistant determinants class A to E among Gram-negative bacteria was analysed with DNA probes. The distribution of tetracycline resistance markers among colonies growing on non-selective and tetracycline-selective plates were compared. The phenotypic approach demonstrated some difference in the diversity within the two soils. The frequency of antibiotic resistance isolates was high in both soils, whereas the frequency of mercury resistance differed significantly. We found no correlation between plasmid profiles and antibiotic resistance patterns. We found all the tetracycline resistance determinants except class B, indicating that the diversity of the tetracycline resistance determinants was complex in populations of resident soil bacteria under no apparent selective pressure for the genes in question.     

Plasmid-mediated resistance to tellurite: expressed and cryptic.

The ability of some bacteria to grow in the presence of high concentrations of tellurium compounds has been recognized for almost 100 years. Since then, interest in this phenomenon has generated a slow but steady trickle of literature. In the past few years, the use of modern techniques in molecular biology has led to a dramatic increase in our understanding of the genetics of several bacterial determinants for resistance to tellurium compounds. These determinants are frequently found to be encoded by plasmids which carry multiple antibiotic resistance determinants. Our understanding of the biochemistry of these systems remains limited. In this article, the history of the study of bacterial resistance to tellurium compounds is briefly reviewed. This is followed by an analysis of the recent developments in the study of plasmid-mediated resistance determinants. Finally, preliminary investigations on the possible mechanisms of bacterial resistance to tellurium compounds are presented.     

Persistence of antibiotic resistance in bacterial populations

Unfortunately for mankind, it is very likely that the antibiotic resistance problem we have generated during the last 60 years due to the extensive use and misuse of antibiotics is here to stay for the foreseeable future. This view is based on theoretical arguments, mathematical modeling, experiments and clinical interventions, suggesting that even if we could reduce antibiotic use, resistant clones would remain persistent and only slowly (if at all) be outcompeted by their susceptible relatives. In this review, we discuss the multitude of mechanisms and processes that are involved in causing the persistence of chromosomal and plasmid-borne resistance determinants and how we might use them to our advantage to increase the likelihood of reversing the problem. Of particular interest is the recent demonstration that a very low antibiotic concentration can be enriching for resistant bacteria and the implication that antibiotic release into the environment could contribute to the selection for resistance. Several mechanisms are contributing to the stability of antibiotic resistance in bacterial populations and even if antibiotic use is reduced it is likely that most resistance mechanisms will persist for considerable times.     

Impact of Manure Fertilization on the Abundance of Antibiotic-Resistant Bacteria and Frequency of Detection of Antibiotic Resistance Genes in Soil and on Vegetables at Harvest

Consumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.     

In vitro activity of antibiotic combinations against multidrug-resistant strains of Acinetobacter baumannii and the effects of their antibiotic resistance determinants

Various combinations of antibiotics are reported to show synergy in treating nosocomial infections with multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii). Here, we studied hospital-acquired outbreak strains of MDR A. baumannii to evaluate optimal combinations of antibiotics. One hundred and twenty-one strains were grouped into one major and one minor clonal group based on repetitive PCR amplification. Twenty representative strains were tested for antibiotic synergy using Etest(?). Five strains were further analyzed by analytical isoelectric focusing and PCR to identify β-lactamase genes or other antibiotic resistance determinants. Our investigation showed that the outbreak strains of MDR A. baumannii belonged to two dominant clones. A combination of colistin and doxycycline showed the best result, being additive or synergistic against 70% of tested strains. Antibiotic additivity was observed more frequently than synergy. Strains possessing the same clonality did not necessarily demonstrate the same response to antibiotic combinations in vitro. We conclude that the effect of antibiotic combinations on our outbreak strains of MDR A. baumannii seemed strain-specific. The bacterial response to antibiotic combinations is probably a result of complex interactions between multiple concomitant antibiotic resistance determinants in each strain.     

Pervasive sign epistasis between conjugative plasmids and drug-resistance chromosomal mutations

Multidrug-resistant bacteria arise mostly by the accumulation of plasmids and chromosomal mutations. Typically, these resistant determinants are costly to the bacterial cell. Yet, recently, it has been found that, in Escherichia coli bacterial cells, a mutation conferring resistance to an antibiotic can be advantageous to the bacterial cell if another antibiotic-resistance mutation is already present, a phenomenon called sign epistasis. Here we study the interaction between antibiotic-resistance chromosomal mutations and conjugative (i.e., self-transmissible) plasmids and find many cases of sign epistasis (40%)--including one of reciprocal sign epistasis where the strain carrying both resistance determinants is fitter than the two strains carrying only one of the determinants. This implies that the acquisition of an additional resistance plasmid or of a resistance mutation often increases the fitness of a bacterial strain already resistant to antibiotics. We further show that there is an overall antagonistic interaction between mutations and plasmids (52%). These results further complicate expectations of resistance reversal by interdiction of antibiotic use.     

Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool

The dramatic spread of antibiotic resistance is a crisis in the treatment of infectious diseases that affect humans. Several studies suggest that wastewater treatment plants (WWTP) are reservoirs for diverse mobile antibiotic resistance elements. This review summarizes findings derived from genomic analysis of IncP-1 resistance plasmids isolated from WWTP bacteria. Plasmids that belong to the IncP-1 group are self-transmissible, and transfer to and replicate in a wide range of hosts. Their backbone functions are described with respect to their impact on vegetative replication, stable maintenance and inheritance, mobility and plasmid control. Accessory genetic modules, mainly representing mobile genetic elements, are integrated in-between functional plasmid backbone modules. These elements carry determinants conferring resistance to nearly all clinically relevant antimicrobial drug classes, to heavy metals, and quaternary ammonium compounds used as disinfectants. All plasmids analysed here contain integrons that potentially facilitate integration, exchange and dissemination of resistance gene cassettes. Comparative genomics of accessory modules located on plasmids from WWTP and corresponding modules previously identified in other bacterial genomes revealed that animal, human and plant pathogens and other bacteria isolated from different habitats share a common pool of resistance determinants.     

Antibiotic resistance in the bacteria of a remote upland river catchment

The incidence of antibiotic resistance was determined in a total of 183 heterotrophic bacteria of non-faecal origin isolated from the catchment of a remote upland stream. Some 40% were sensitive to each of the eight antibiotics tested while more than 35% were resistant to at least two compounds. Three isolates each carried seven resistance determinants. Unsuccessful attempts were made, using two curing techniques, to demonstrate that resistance was plasmid-borne. The high incidence of antibiotic resistance, particularly amongst pseudomonads, in an environment virtually free from anthropogenic influence is surprising, and has implications for the assessment of the spread of antibiotic resistance amongst populations of environmental bacteria.     

A novel type of colistin resistance genes selected from random sequence space

Antibiotic resistance is a rapidly increasing medical problem that severely limits the success of antibiotic treatments, and the identification of resistance determinants is key for surveillance and control of resistance dissemination. Horizontal transfer is the dominant mechanism for spread of resistance genes between bacteria but little is known about the original emergence of resistance genes. Here, we examined experimentally if random sequences can generate novel antibiotic resistance determinants de novo. By utilizing highly diverse expression libraries encoding random sequences to select for open reading frames that confer resistance to the last-resort antibiotic colistin in Escherichia coli, six de novo colistin resistance conferring peptides (Dcr) were identified. The peptides act via direct interactions with the sensor kinase PmrB (also termed BasS in E. coli), causing an activation of the PmrAB two-component system (TCS), modification of the lipid A domain of lipopolysaccharide and subsequent colistin resistance. This kinase-activation was extended to other TCS by generation of chimeric sensor kinases. Our results demonstrate that peptides with novel activities mediated via specific peptide-protein interactions in the transmembrane domain of a sensory transducer can be selected de novo, suggesting that the origination of such peptides from non-coding regions is conceivable. In addition, we identified a novel class of resistance determinants for a key antibiotic that is used as a last resort treatment for several significant pathogens. The high-level resistance provided at low expression levels, absence of significant growth defects and the functionality of Dcr peptides across different genera suggest that this class of peptides could potentially evolve as bona fide resistance determinants in natura.     

Characterization of antibiotic resistance determinants in oral biofilms

Oral biofilms contain numerous antibiotic resistance determinants that can be transferred within or outside of the oral cavity. The aim of this study was to evaluate the prevalence and the relative level of antibiotic resistance determinants from oral biofilms. Oral biofilm samples that were collected from healthy subjects and periodontitis patients were subjected to qualitative and quantitative analyses for selected antibiotic resistance determinants using PCR. The prevalence of tet(Q), tet(M), cfxA, and bla _TEM was very high both in the patient and the healthy subject group, with a tendency toward higher values in the patient group, with the exception of erm(F), which was more prevalent in the healthy group. The two extended spectrum β-lactam (ESBL) resistance determinants bla _SHV and bla _TEM showed a dramatic difference, as bla _TEM was present in all of the samples and bla _SHV was not found at all. The aacA-aphD, vanA, and mecA genes were rarely detected, suggesting that they are not common in oral bacteria. A quantitative PCR analysis showed that the relative amount of resistance determinants present in oral biofilms of the patient group was much greater than that of the healthy group, exhibiting 17-, 13-, 145-, and 3-fold increases for tet(Q), tet(M), erm(F), and cfxA, respectively. The results of this study suggest that the oral antibiotic resistome is more diverse and abundant in periodontitis patients than in healthy subjects, suggesting that there is a difference in the diversity and distribution of antibiotic resistance in oral biofilms associated with health and disease.     

Identification of Aminoglycoside and β-Lactam Resistance Genes from within an Infant Gut Functional Metagenomic Library

The infant gut microbiota develops rapidly during the first 2 years of life, acquiring microorganisms from diverse sources. During this time, significant opportunities exist for the infant to acquire antibiotic resistant bacteria, which can become established and constitute the infant gut resistome. With increased antibiotic resistance limiting our ability to treat bacterial infections, investigations into resistance reservoirs are highly pertinent. This study aimed to explore the nascent resistome in antibiotically-naïve infant gut microbiomes, using a combination of metagenomic approaches. Faecal samples from 22 six-month-old infants without previous antibiotic exposure were used to construct a pooled metagenomic library, which was functionally screened for ampicillin and gentamicin resistance. Our library of ∼220Mb contained 0.45 ampicillin resistant hits/Mb and 0.059 gentamicin resistant hits/Mb. PCR-based analysis of fosmid clones and uncloned metagenomic DNA, revealed a diverse and abundant aminoglycoside and β-lactam resistance reservoir within the infant gut, with resistance determinants exhibiting homology to those found in common gut inhabitants, including Escherichia coli, Enterococcus sp., and Clostridium difficile, as well as to genes from cryptic environmental bacteria. Notably, the genes identified differed from those revealed when a sequence-driven PCR-based screen of metagenomic DNA was employed. Carriage of these antibiotic resistance determinants conferred substantial, but varied (2–512x), increases in antibiotic resistance to their bacterial host. These data provide insights into the infant gut resistome, revealing the presence of a varied aminoglycoside and β-lactam resistance reservoir even in the absence of selective pressure, confirming the infant resistome establishes early in life, perhaps even at birth.     

The human gut resistome

In recent decades, the emergence and spread of antibiotic resistance among bacterial pathogens has become a major threat to public health. Bacteria can acquire antibiotic resistance genes by the mobilization and transfer of resistance genes from a donor strain. The human gut contains a densely populated microbial ecosystem, termed the gut microbiota, which offers ample opportunities for the horizontal transfer of genetic material, including antibiotic resistance genes. Recent technological advances allow microbiota-wide studies into the diversity and dynamics of the antibiotic resistance genes that are harboured by the gut microbiota (‘the gut resistome’). Genes conferring resistance to antibiotics are ubiquitously present among the gut microbiota of humans and most resistance genes are harboured by strictly anaerobic gut commensals. The horizontal transfer of genetic material, including antibiotic resistance genes, through conjugation and transduction is a frequent event in the gut microbiota, but mostly involves non-pathogenic gut commensals as these dominate the microbiota of healthy individuals. Resistance gene transfer from commensals to gut-dwelling opportunistic pathogens appears to be a relatively rare event but may contribute to the emergence of multi-drug resistant strains, as is illustrated by the vancomycin resistance determinants that are shared by anaerobic gut commensals and the nosocomial pathogen Enterococcus faecium.     

Co-selection of antibiotic and metal resistance

There is growing concern that metal contamination functions as a selective agent in the proliferation of antibiotic resistance. Documented associations between the types and levels of metal contamination and specific patterns of antibiotic resistance suggest that several mechanisms underlie this co-selection process. These co-selection mechanisms include co-resistance (different resistance determinants present on the same genetic element) and cross-resistance (the same genetic determinant responsible for resistance to antibiotics and metals). Indirect but shared regulatory responses to metal and antibiotic exposure such as biofilm induction also represent potential co-selection mechanisms used by prokaryotes. Metal contamination, therefore, represents a long-standing, widespread and recalcitrant selection pressure with both environmental and clinical importance that potentially contributes to the maintenance and spread of antibiotic resistance factors.