Pål Jarle Johnsen

Factors affecting the reversal of antimicrobial-drug resistance.

The persistence or loss of acquired antimicrobial-drug resistance in bacterial populations previously exposed to drug-selective pressure depends on several biological processes. We review mechanisms promoting or preventing the loss of resistance, including rates of reacquisition, effects of resistance traits on bacterial fitness, linked selection, and segregational stability of resistance determinants. As a case study, we discuss the persistence of glycopeptide-resistant enterococci in Norwegian and Danish poultry farms 12 years after the ban of the animal growth promoter avoparcin. We conclude that complete eradication of antimicrobial resistance in bacterial populations following relaxed drug-selective pressures is not straightforward. Resistance determinants may persist at low, but detectable, levels for many years in the absence of the corresponding drugs.

Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species

We have investigated to what extent natural transformation acting on free DNA substrates can facilitate transfer of mobile elements including transposons, integrons and/or gene cassettes between bacterial species. Naturally transformable cells of Acinetobacter baylyi were exposed to DNA from integron-carrying strains of the genera Acinetobacter, Citrobacter, Enterobacter, Escherichia, Pseudomonas, and Salmonella to determine the nature and frequency of transfer. Exposure to the various DNA sources resulted in acquisition of antibiotic resistance traits as well as entire integrons and transposons, over a 24 h exposure period. DNA incorporation was not solely dependent on integrase functions or the genetic relatedness between species. DNA sequence analyses revealed that several mechanisms facilitated stable integration in the recipient genome depending on the nature of the donor DNA; homologous or heterologous recombination and various types of transposition (Tn21-like and IS26-like). Both donor strains and transformed isolates were extensively characterized by antimicrobial susceptibility testing, integron- and cassette-specific PCRs, DNA sequencing, pulsed field gel electrophoreses (PFGE), Southern blot hybridizations, and by re-transformation assays. Two transformant strains were also genome-sequenced. Our data demonstrate that natural transformation facilitates interspecies transfer of genetic elements, suggesting that the transient presence of DNA in the cytoplasm may be sufficient for genomic integration to occur. Our study provides a plausible explanation for why sequence-conserved transposons, IS elements and integrons can be found disseminated among bacterial species. Moreover, natural transformation of integron harboring populations of competent bacteria revealed that interspecies exchange of gene cassettes can be highly efficient, and independent on genetic relatedness between donor and recipient. In conclusion, natural transformation provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits from divergent species than previously assumed.

PCR-based plasmid typing in Enterococcus faecium strains reveals widely distributed pRE25-, pRUM-, pIP501- and pHTβ-related replicons associated with glycopeptide resistance and stabilizing toxin–antitoxin systems

A PCR-based typing scheme was applied to identify plasmids in an epidemiologically and geographically diverse strain collection of Enterococcus faecium (n=93). Replicon types of pRE25 (n=56), pRUM (n=41), pIP501 (n=17) and pHTbeta (n=14) were observed in 83% of the strains, while pS86, pCF10, pAM373, pMBB1 or pEF418 were not detected. Furthermore, 61% of the strains contained the axe-txe (n=42) or/and the omega-epsilon-zeta (n=18) plasmid stabilization loci. Sequence analyses divided the omega-epsilon-zeta operon into two distinct phylogenetic groups. The present typing scheme accounted for about 60% of the total number of plasmids detected by S1 nuclease analyses, which revealed zero to seven plasmids (10 kb to >200 kb) per isolate. Interestingly, strains belonging to the clinically important clonal complex 17 (CC17) yielded a significantly higher number of plasmids (3.1) and pRUM replicons (74%) than non-CC17 strains (2.2% and 35%, respectively). A prevalent genetic linkage between the pRUM-replicon type and axe-txe was demonstrated by cohybridization analyses. The vanA resistance determinant was associated with all four replicon types, but we also confirmed the genetic linkage of vanA to unknown transferable replicons. PCR-based replicon typing, linked to the detection of other important plasmid-encoded traits, seems to be a feasible tool for tracing disseminating resistance plasmids stably maintained in various environments.

Prevalence, persistence, and molecular characterization of glycopeptide-resistant enterococci in Norwegian poultry and poultry farmers 3 to 8 years after the ban on avoparcin.

ABSTRACT Environmental reservoirs of glycopeptide-resistant enterococci (GRE) in Norway have been linked to former growth promoting use of the glycopeptide avoparcin in poultry production. We have examined the prevalence of fecal GRE in poultry and poultry farmers 3 to 8 years after the Norwegian avoparcin ban in 1995 and performed molecular analyses of the GRE population. Fecal samples from poultry farmers and their flocks on 29 previously avoparcin-exposed farms were collected on five occasions during the study period (1998 to 2003). All flocks (100%) were GRE positive in 1998. Throughout the study period, 78.5% of the poultry samples were GRE positive. Glycopeptide-resistant Enterococcus faecium (GREF) was isolated from 27.6% of the farmer samples in 1998 and from 27.8% of the samples collected between 1998 and 2003. The prevalence of fecal GRE in poultry declined significantly during the study period, but prevalence in samples from the farmers did not decline. PCR analysis revealed a specific Tn1546-plasmid junction fragment in 93.9% of E. faecium isolates. A putative postsegregation killing (PSK) system linked to Tn1546 was detected in 97.1% of the isolates examined. Multilocus sequence typing of glycopeptide-susceptible (n = 10) and -resistant (n = 10) E. faecium isolates from humans (n = 10) and poultry (n = 10) on two farms displayed 17 different sequence types. The study confirms the continuing persistence of a widespread common plasmid-mediated vanA-pRE25-PSK element within a heterogeneous GRE population on Norwegian poultry farms 8 years after the avoparcin ban. Moreover, it suggests an important role of PSK systems in the maintenance of antimicrobial resistance determinants in reservoirs without apparent antimicrobial selection.

Bacterial natural transformation by highly fragmented and damaged DNA

Significance Short and damaged DNA is ubiquitous in most environments and can survive more than half a million years. We show that naturally competent environmental bacteria can take up such degraded DNA and incorporate it into their genomes, including DNA from a 43,000-y-old woolly mammoth bone. The process occurs as part of cellular DNA replication and may resemble the earliest forms of horizontal gene transfer. Our findings suggest that natural genetic exchange of DNA from dead and even extinct organisms to contemporary bacteria can take place over hundreds of thousands of years. Hence damaged and degraded DNA may be a previous unrecognized driver of bacterial evolution with implications for evolutionary theory. DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old.

Persistence of Animal and Human Glycopeptide-Resistant Enterococci on Two Norwegian Poultry Farms Formerly Exposed to Avoparcin Is Associated with a Widespread Plasmid-Mediated vanA Element within a Polyclonal Enterococcus faecium Population

ABSTRACT The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that “plasmid addiction systems” may contribute to the persistence of GREF in nonselective environments.

Stability, persistence, and evolution of plasmid-encoded VanA glycopeptide resistance in enterococci in the absence of antibiotic selection in vitro and in gnotobiotic mice.

Long-term persistence of VanA glycopeptide-resistant enterococci (GRE) has been observed in the absence of antibiotic selection. In the present study, we examined fitness parameters of a glycopeptide-susceptible Enterococcus faecium parent strain and its plasmid-mediated, VanA-resistant derivative before and after 1,000 generations in serial transfer broth cultures with or without antibiotic selection. With the exception of the vanA-containing plasmid, the strains were otherwise isogenic. The stability of the plasmid-encoded vanA resistance determinant was also investigated in vitro and in gnotobiotic mice. Competition experiments revealed that GRE with newly acquired VanA resistance had a 4% reduction in fitness relative to their susceptible parental counterpart. The relative difference in competitive fitness between resistant and susceptible strains was not significantly changed after 1,000 generations. Environmental adaptation was observed in all strains and exceeded the biological cost of resistance. Thus, the evolved VanA-resistant E. faecium populations out-numbered their unevolved ancestral susceptible E. faecium strain in mixed cultures, but remained less competitive than the evolved parent. The glycopeptide resistance determinant was similarly stably maintained during long-term colonization in gnotobiotic mice without antibiotic selection. In vivo vanA plasmid transfer was observed. The results suggest that environmental adaptation, in vivo gene transfer, and plasmid maintenance system(s) favor long-term VanA GRE persistence without antibiotic selection and compensate for the biological costs of possessing the resistance genes.

Complete sequence of Enterococcus faecium pVEF3 and the detection of an ω-ε-ζ toxin–antitoxin module and an ABC transporter

Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

Episodic Selection and the Maintenance of Competence and Natural Transformation in Bacillus subtilis

We present a new hypothesis for the selective pressures responsible for maintaining natural competence and transformation. Our hypothesis is based in part on the observation that in Bacillus subtilis, where transformation is widespread, competence is associated with periods of nongrowth in otherwise growing populations. As postulated for the phenomenon of persistence, the short-term fitness cost associated with the production of transiently nongrowing bacteria can be compensated for and the capacity to produce these competent cells can be favored due to episodes where the population encounters conditions that kill dividing bacteria. With the aid of a mathematical model, we demonstrate that under realistic conditions this “episodic selection” for transiently nongrowing (persisting) bacteria can maintain competence for the uptake and expression of exogenous DNA transformation. We also show that these conditions for maintaining competence are dramatically augmented even by rare episodes where selection favors transformants. Using experimental populations of B. subtilis and antibiotic-mediated episodic selection, we test and provide support for the validity of the assumptions behind this model and the predictions generated from our analysis of its properties. We discuss the potential generality of episodic selection for the maintenance of competence in other naturally transforming species of bacteria and critically evaluate other hypotheses for the maintenance (and evolution) of competence and their relationship to this hypothesis.

Comparative DNA Analysis of Two vanA Plasmids from Enterococcus faecium Strains Isolated from Poultry and a Poultry Farmer in Norway

ABSTRACT The DNA sequences of two plasmids carrying vanA, pVEF1 (39,626 bp) and pVEF2 (39,714 bp), were determined. Forty-three shared coding sequences were identified, and the only nucleotide difference was an 88-bp indel. A postsegregational killing system was identified. This system possibly explains the persistence of the vanA gene cluster in Norwegian poultry farms.