Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.     

Transport systems encoded by bacterial plasmids

A variety of bacterial functions are encoded on plasmids, extrachromosomal elements. Examples of plasmid-borne functions are antibiotic production and resistance, degradation of recalcitrant chemicals, virulence factors, and plant symbiotic properties. Several transport systems with diverse functions have recently been found to be carried on plasmids. These systems serve to either accumulate or extrude a compound from a cell. The focus of this review is to present a survey on several of these novel plasmid-borne transport systems emphasizing functions, components, and molecular genetics.     

Detection of conjugative plasmids and antibiotic resistance genes in anthropogenic soils from Germany and India

PCR typing methods were used to assess the presence of plasmids of the incompatibility (Inc) groups IncP, IncN, IncW, IncQ and rolling circle plasmids of the pMV158 type in total DNA extracts from anthropogenic soils from India and Germany. Ten different soils from two different locations in Germany, the urban park Berlin Tiergarten and the abandoned sewage field Berlin-Buch, and from four different locations in India were analysed. PCR amplification of the total DNA extracts revealed the prevalence of IncP-specific sequences in Berlin Buch and Indian soil samples. The detected IncP plasmids contained at least one transfer function, the origin of transfer, oriT. In contrast to IncP-specific sequences, IncQ, IncN, IncW and pMV158-specific sequences were never detected. The presence of ampC, tet (O), ermB, SHV-5, mecA, and vanA antibiotic resistance genes was also tested. Three Indian soil samples irrigated with wastewater contained the ampC gene, whereas the other resistance genes were not found in any of the samples. Detection of IncP trfA2 and oriT sequences by PCR amplification and hybridization is a clear indication that IncP plasmids are prevalent in these habitats. Exogenous plasmid isolation revealed conjugative plasmids belonging to the IncPbeta group encoding resistance to ampicillin.     

Conjugative plasmids: vessels of the communal gene pool

Comparative whole-genome analyses have demonstrated that horizontal gene transfer (HGT) provides a significant contribution to prokaryotic genome innovation. The evolution of specific prokaryotes is therefore tightly linked to the environment in which they live and the communal pool of genes available within that environment. Here we use the term supergenome to describe the set of all genes that a prokaryotic ‘individual’ can draw on within a particular environmental setting. Conjugative plasmids can be considered particularly successful entities within the communal pool, which have enabled HGT over large taxonomic distances. These plasmids are collections of discrete regions of genes that function as ‘backbone modules’ to undertake different aspects of overall plasmid maintenance and propagation. Conjugative plasmids often carry suites of ‘accessory elements’ that contribute adaptive traits to the hosts and, potentially, other resident prokaryotes within specific environmental niches. Insight into the evolution of plasmid modules therefore contributes to our knowledge of gene dissemination and evolution within prokaryotic communities. This communal pool provides the prokaryotes with an important mechanistic framework for obtaining adaptability and functional diversity that alleviates the need for large genomes of specialized ‘private genes’.     

变形杆菌的耐药性及接合性R质粒的检测

本文报告了从烧伤病人感染创面分离、鉴定的35株变形杆菌的药敏试验及接合性R质粒的检测结果。35株变形杆菌双测试的6种抗菌药物都具有不同程度的耐药性。其中,对6种药物同时耐受的有26株(74.3％)。对其中的27株做了接合试验,接合性R质粒的检出率为70.4%。     

Persistence of pBR322-related plasmids in Escherichia coli grown in chemostat cultures

Abstract The stability of plasmid pBR322 and a number of close derivatives was examined by continuous culture of Escherichia coli . Cultures were subjected to either glucose, phosphate or magnesium limitation in non-selective medium at a dilution rate of 0.1/h. Under these conditions pBR322 was eventually lost from the population, but only after a distinct lag period. The closely related plasmids pBR325 and especially pBR327 and pBR328, but not pAT153, were lost more rapidly. Three cosmids pHC79, pSJ55 and pJB8 were generally found to be less stable than the pBR322-type plasmids from which they were derived. Chimaeric plasmids containing DNA from yeast and from a thermophilic bacillus were also unstable in E. coli .     

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.     

High levels of multiple metal resistance and its correlation to antibiotic resistance in environmental isolates of Acinetobacter

Forty strains of Acinetobacter were isolated from different environmental sources. All the strains were classified into four genospecies, i.e. A. baumannii (33 isolates), A. calcoaceticus (three isolates), A. junii (three isolates) and A. genospecies3 (one isolate). Susceptibility of these 40 strains to salts of 20 heavy metals and 18 antibiotics was tested by the agar dilution method. All environmental isolates of Acinetobacter were resistant to multiple metal ions (minimum 13 metal ions) while all but one of the strains were resistant to multiple antibiotics (minimum four antibiotics). The maximum number of strains were found to be sensitive to mercury (60% strains) while all strains were resistant to copper, lead, boron and tungsten even at 10 mm concentration. Salts of these four metal ions may be added to the growth medium to facilitate selective isolation of Acinetobacter. Rifampicin and nalidixic acid were the most toxic antibiotics, inhibiting 94.5 and 89.5% of the acinetobacters, respectively. A. genospecies3 was found to be the most resistant species, tolerating high concentrations of all the 20 metal ions and also to a greater number of antibiotics than any other species of Acinetobacter tested. An inhibitory concentration (10 mm) of Ni²⁺ and Zn²⁺ was observed to inhibit the growth of all of the clinical isolates but allowed the growth of the environmental isolates, facilitating the differentiation between pathogenic and non-pathogenic acinetobacters.     

Enhancement of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in soil by dissemination of catabolic plasmids

Few studies have been done to evaluate the transfer of catabolic plasmids from an introduced donor strain to indigenous microbial populations as a means to remediate contaminated soils. In this work we determined the effect of the conjugative transfer of two 2,4-D degradative plasmids to indigenous soil bacterial populations on the rate of 2,4-D degradation in soil. We also assessed the influence of the presence of 2,4-D on the number of transconjugants formed. The two plasmids used, pEMT1k and pEMT3k, encode 2,4-D degradative genes (tfd) that differ in DNA sequence as well as gene organisation, and confer different growth rates to Ralstonia eutropha JMP228 when grown with 2,4-D as a sole carbon source. In an agricultural soil (Ardoyen) treated with 2,4-D (100 ppm) there were ca. 10⁷CFU of transconjugants per gram bearing pEMT1k as well as a high number of pEMT3k bearing transconjugants (ca. 10⁶ CFU/g). In this soil the formation of a high number of 2,4-D degrading transconjugants resulted in faster degradation of 2,4-D as compared to the uninoculated control soil. In contrast, only transconjugants with pEMT1k were detected (at a level of ca. 10³ CFU/g soil) in the untreated Ardoyen soil. High numbers of transconjugants that carried pEMT1k were also found in a second experiment done using forest soil (Lembeke) treated with 100 ppm 2,4-D. However, unlike in the Ardoyen soil, no transconjugants with pEMT3k were detected and the transfer of plasmid pEMT1k to indigenous bacteria did not result in a higher rate of decrease of 2,4-D. This may be because 2,4-D was readily metabolised by indigenous bacteria in this soil. The results indicate that bioaugmentation with catabolic plasmids may be a viable means to enhance the bioremediation of soils which lack an adequate intrinsic ability to degrade a given xenobiotic.     

Positive epistasis between co-infecting plasmids promotes plasmid survival in bacterial populations

Plasmids have a key role in the horizontal transfer of genes among bacteria. Although plasmids are catalysts for bacterial evolution, it is challenging to understand how they can persist in bacterial populations over the long term because of the burden they impose on their hosts (the ‘plasmid paradox''). This paradox is especially perplexing in the case of ‘small'' plasmids, which are unable to self-transfer by conjugation. Here, for the first time, we investigate how interactions between co-infecting plasmids influence plasmid persistence. Using an experimental model system based on interactions between a diverse assemblage of ‘large'' plasmids and a single small plasmid, pNI105, in the pathogenic bacterium Pseudomonas aeruginosa, we demonstrate that positive epistasis minimizes the cost associated with carrying multiple plasmids over the short term and increases the stability of the small plasmid over a longer time scale. In support of these experimental data, bioinformatic analysis showed that associations between small and large plasmids are more common than would be expected owing to chance alone across a range of families of bacteria; more generally, we find that co-infection with multiple plasmids is more common than would be expected owing to chance across a wide range of bacterial phyla. Collectively, these results suggest that positive epistasis promotes plasmid stability in bacterial populations. These findings pave the way for future mechanistic studies aimed at elucidating the molecular mechanisms of plasmid–plasmid interaction, and evolutionary studies aimed at understanding how the coevolution of plasmids drives the spread of plasmid-encoded traits.     

Oligotrophic bacteria from rendzina forest soil

Oligotrophic bacteria were shown to exist abundantly in all layers of a rendzina forest soil throughout the year. Two-hundred-three oligotrophic bacteria were isolated from forest soil (Aoba, Sendai) at different layers (L, F, H and A layers) throughout the year, and their morphological and physiological characteristics were examined. A high proportion (95%) of the isolated oligotrophs were Gram-negative, non-spore forming bacteria. Based on the cell shape, the isolates were divided into four groups: regular rods, curved/spiral bacteria, irregular rods, and buddin and/or prosthecate bacteria. Each group of bacteria is discussed in relation to the physiological characteristics. Notably oligotrophic bacteria of different cell types showed a marked zonal distribution in respect to profile depth.     

Diversity of soil mycobacterium isolates from three sites that degrade polycyclic aromatic hydrocarbons

AIMS: This paper investigates the diversity of polycyclic aromatic hydrocarbon (PAH)-degrading mycobacterium isolates from three different sites within United States: Montana, Texas and Indiana. METHODS AND RESULTS: All five mycobacterium isolates differed in chromosomal restriction enzyme-fragmentation patterns; three isolates possessed linear plasmids. The DNA sequence between the murA and rRNA genes were divergent but the sequence upstream of nidBA genes, encoding a dioxygenase involved in pyrene oxidation, was more highly conserved. Long-chain fatty acid analysis showed most similarity between three isolates from the same Montana site. All isolates were sensitive to rifampicin and isoniazid, used in tuberculosis treatment, and to syringopeptins, produced by plant-associated pseudomonads. Biofilm growth was least for isolate MCS that grew on plate medium as rough-edged colonies. The patterns of substrate utilization in Biolog plates showed clustering of the Montana isolates compared with Mycobacterium vanbaalenii and Mycobacterium gilvum. CONCLUSION: The five PAH-degrading mycobacterium isolates studied differ in genetic and biochemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Different properties with respect to antibiotic susceptibility, substrate utilization and biofilm formation could influence the survival in soil of the microbe and their suitability for use in bioaugmentation.     

Occurrence and diversity of plasmids in populations of streptomycetes in soil

Studies were made of naturally occurring plasmids hosted in Streptomyces strains isolated from two different terrestrial ecosystems: an agricultural field and a protected forest area. Six out of the 147 screened isolates contained plasmids. The strains containing these plasmids were all isolated from the agricultural soil. Plasmids were not found among the strains isolated from the forest area. Cross hybridization of the six newly isolated plasmids revealed very high similarities between four of them. However, no similarities were found between the six newly isolated plasmids and well studied streptomycete plasmids such as pIJ101 and SCP2^*. The host strains of the four similar plasmids belonged to three different species S. anulatus, S. rochei and S. diastaticus. This implies a possible conjugative transfer of these plasmids within the streptomycete population in the agricultural area. The reason for the absence of streptomycete plasmids from the populations derived from the forest area is discussed.     

Arsenical resistance in the IncHI2 plasmids

The IncHI2 plasmid R478, like other arsenic resistance IncH plasmids, provides increased levels of resistance to sodium arsenate (up to 100mM) and sodium arsenite (up to 10mM) to the host cell. An arsenic resistance fragment of R478 was cloned and sequenced revealing four arsenic resistance associated gene homologues, arsR, arsB, arsC, and arsH. Two other open reading frames in the cloned fragment were found to be homologues of sulphate transport associated genes. Both the four gene arsenic resistance operons and the two gene sulphate transport operons have been previously shown to be transposon associated. However, no evidence of transposability was found associated with these operons in R478. Both the R478 associated arsenic and sulphate transport operons were shown to be common to all arsenic resistance IncH plasmids examined by Southern hybridisation and PCR analysis.     

Characterization of a mobile and multiple resistance plasmid isolated from swine manure and its detection in soil after manure application

Aims: To isolate and characterize multiple antibiotic resistance plasmids found in swine manure and test for plasmid‐associated genetic markers in soil following manure application to an agricultural field. Methods and Results: Plasmids were isolated from an erythromycin enrichment culture that used liquid swine manure as an inoculant. Plasmids were transformed into Escherichia coli DH10β for subsequent characterization. We isolated and DNA sequenced a 22 102‐bp plasmid (pMC2) that confers macrolide, and tetracycline resistances, and carries genes predicted to code for mercury and chromium resistance. Conjugation experiments using an pRP4 derivative as a helper plasmid confirm that pMC2 has a functional mobilization unit. PCR was used to detect genetic elements found on pMC2 in DNA extracted from manure amended soil. Conclusions: The pMC2 plasmid has a tetracycline‐resistant core and has acquired additional resistance genes by insertion of an accessory region (12 762 bp) containing macrolide, mercury and chromium resistance genes, which was inserted between the truncated DDE motifs within the Tn903/IS102 mobile element. Significance and Impact of the Study: Liquid swine manure used for manure spreading contains multiple antibiotic resistance plasmids that can be detected in soil following manure application.     

Shotgun proteomics of bacterial pathogens: advances,challenges and clinical implications

Mass spectrometry-based proteomics is increasingly used in analysis of bacterial pathogens. Simple experimental set-ups based on high accuracy mass spectrometry and powerful biochemical and bioinformatics tools are capable of reliably quantifying levels of several thousand bacterial proteins in a single experiment, reaching the analytical capacity to completely map whole proteomes. Here the authors present the state-of-the-art in bacterial pathogen proteomics and discuss challenges that the field is facing, especially in analysis of low abundant, modified proteins from organisms that are difficult to culture. Constant improvements in speed and sensitivity of mass spectrometers, as well as in bioinformatic and biochemical workflows will soon allow for comprehensive analysis of regulatory mechanisms of pathogenicity and enable routine application of proteomics in the clinical setting.     

Metal resistance and plasmid DNA in Thiobacillus ferrooxidans

The minimal inhibitory concentrations of copper and nickel were determined for each of fifteen isolates of T. ferrooxidans native to a Cu/Ni tailings environment. Ten isolates were inhibited by 160 mM Cu,²⁺ or less, and ten were inhibited by 160 mM Ni²⁺or less. The isolates were screened for plasmid DNA using an alkaline lysis method and CCC plasmid forms were confirmed using the Hintermann technique. Two isolates were found to be devoid of plasmid DNA, and only one isolate contained more than two plasmids. Variability existed in plasmid size, although the majority were larger than the standard pBR322 (4.3 kbp). One plasmid was selected for further analysis using restriction endonucleases. EcoRI, HindIII and KpnI all cleaved the plasmid in two locations, and PstI cleaved the plasmid in six locations. PstI-digested fragments of the plasmid were ligated into pBR322, and the recombinant plasmids were transformed into Escherichia coli ATCC 8739. Four genetically-different transformants resulted, and each was grown in media containing 2.0 mM Cu²⁺ and compared to the growth of a control under similar conditions. There was no conferred copper resistance in E. coli, although one recombinant plasmid appeared to decrease the tolerance for E. coli ATCC 8739 to Cu²⁺.