Plasmids and genetic determinants of antibiotic resistance of gram-negative bacteria

Distribution of plasmids and genetic determinants of antibiotic resistance was studied in 129 strains of Pseudomonas, Klebsiella, Serratia and Enterobacter isolated from oncological patients. It was shown that 56 isolates contained the plasmids, 9 conjugative plasmids being plasmids with broad bacterial host spectrum. A significant part of the strains contained genes controlling production of APH (3"), type II APH (3'), type I and II DHPS and type type II DHFR. Genetic determinants of tetracycline resistance of classes D and E were detected for the first time in the strains of Klebsiella, Serratia and Pseudomonas aeruginosa.     

Antibiotic resistance in clinical strains of Pseudomonas aeruginosa isolated from 1979-1984

The levels and spectra of drug resistance were determined in 530 strains of P. aeruginosa isolated in hospitals of three cities of the USSR within 1979-1984. Their conjugative R plasmids were searched for and distribution of various type resistance determinants in the composition of these plasmids was investigated. The results were compared with the findings of analogous studies on clinical strains of P. aeruginosa isolated within 1976-1979. It was shown that there were a rise in the relative number of the strains resistant to kanamycin and a decrease in the occurrence of the P. aeruginosa strains resistant to streptomycin, tetracycline and sulfanilamides. The frequency of the kanamycin, carbenicillin and gentamicin resistance genes in the composition of the detected conjugative R plasmids increased. Hybridization of 32P-labeled probes containing various type antibiotic resistance determinants with strains of P. aeruginosa ML (PAO) containing conjugative R plasmids was indicative of wide spread of genes determining APH(3')II and APH(3") and determinants of classes A and C in the composition of the studied plasmids.     

Promoters active in mammalian cells (pSV40) and in plants (pNOS) permit expression in yeast of the Tn5 APH(3') II gene

Abstract Four plasmids were constructed by associating Escherichia coli and yeast selection markers and replication origins to a structural gene coding for aminoglycoside phosphotransferase (APH(3')) controlled by different flanking sequences. We used the two bacterial genes of Tn5 (APH(3')II) and Tn903 (APH(3')I) as such and the chimeric pSVneo (APH(3')II) and pNOSneo (APH(3')II) constructs, functional in mammalian and plant cells, respectively. Yeast clones resistant to G418 were obtained with all plasmids except with that bearing the bacterial APH(3')II gene. The three plasmids harbouring the functional APH genes, however, conferred different levels of G418 resistance to yeast.     

Dissemination of IncF-type plasmids in multiresistant CTX-M-15-producing Enterobacteriaceae isolates from surgical-site infections in Bangui,Central African Republic

Background

Surgical-site infection is the most frequent health care-associated infection in the developing world, with a strikingly higher prevalence than in developed countries We studied the prevalence of resistance to antibiotics in Enterobacteriaceae isolates from surgical-site infections collected in three major tertiary care centres in Bangui, Central African Republic. We also studied the genetic basis for antibiotic resistance and the genetic background of third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae.

Results

Between April 2011 and April 2012, 195 patients with nosocomial surgical-site infections were consecutively recruited into the study at five surgical departments in three major tertiary care centres. Of the 165 bacterial isolates collected, most were Enterobacteriaceae (102/165, 61.8%). Of these, 65/102 (63.7%) were 3GC-R, which were characterized for resistance gene determinants and genetic background. The bla_CTX-M-15 and aac(6′)-Ib-cr genes were detected in all strains, usually associated with qnr genes (98.5%). Escherichia coli, the most commonly recovered species (33/65, 50.8%), occurred in six different sequence types, including the pandemic B2-O25b-ST131 group (12/33, 36.4%). Resistance transfer was studied in one representative strain of the resistance gene content in each repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequence-PCR banding pattern. Plasmids were characterized by PCR-based replicon typing and sub-typing schemes. In most isolates (18/27, 66.7%), bla_CTX-M-15 genes were found in incompatibility groups F/F31:A4:B1 and F/F36:A4:B1 conjugative plasmids. Horizontal transfer of both plasmids is probably an important mechanism for the spread of bla_CTX-M-15 among Enterobacteriaceae species and hospitals. The presence of sets of antibiotic resistance genes in these two plasmids indicates their capacity for gene rearrangement and their evolution into new variants.

Conclusions

Diverse modes are involved in transmission of resistance, plasmid dissemination probably playing a major role.     

Prospects for using DNA probes for rapid diagnosis of antibiotic resistance in clinical strains of enteric bacteria

Aminoglucoside resistance patterns of clinical strains of enteric bacteria isolated from inpatients of Moscow clinics were determined. APH(3')-I and AAC(3)-II were shown to be the most frequent. The aphA1 and aacC2 genes encoding the enzymes were cloned from the R plasmid of the transconjugant of the E. coli clinical strains. DNA probes based on the determined nucleotide sequences of the cloned genes were constructed and used in DNA-DNA hybridization experiments. The results on the occurrence of APH(3')-I and AAC(3)-II in the strains tested were confirmed by the DNA-DNA hybridization. Prospects for developing a set of DNA probes for rapid diagnosis of antibiotic resistance are discussed.     

Mapping of the gene specifying aminoglycoside 3''-phosphotransferase II on the Pseudomonas aeruginosa chromosome.

We examined the aminoglycoside inactivation enzymes in Pseudomonas aeruginosa strains, seven clinical isolates and seven laboratory strains without plasmids. All strains were found to possess the enzyme aminoglycoside 3'-phosphotransferase II [APH(3')-II]. We isolated an APH(3')-II-deficient mutant from a PAO strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. By plasmid (FP5 or R68.45)-mediated conjugation, we determined the locus of the gene specifying the APH(3')-II between trp-6 and pro-82 on the PAO chromosome and designated this gene aphA. It was concluded that the intrinsic resistance of P. aeruginosa to kanamycins, neomycins, paromomycins, ribostamycin, and butirosins was due to this newly determined gene.     

The detection of diverse aminoglycoside phosphotransferases within natural populations of actinomycetes

The conserved nature of the genes that code for actinomycete secondary metabolite biosynthetic pathways suggests a common evolutionary ancestor and incidences of lateral gene transfer. Resistance genes associated with these biosynthetic pathways also display a high degree of similarity. Actinomycete aminoglycoside phosphotransferase antibiotic resistance enzymes (APH) are coded for by such genes and are therefore good targets for evaluating the bioactive potential of actinomycetes. A set of universal PCR primers for APH encoding genes was used to probe genomic DNA from three collections of actinomycetes to determine the utility of molecular screening. An additional monitoring of populations for the predominance of specific classes of enzymes to predict the potential of environmental sites for providing isolates with interesting metabolic profiles. Approximately one-fifth of all isolates screened gave a positive result by PCR. The PCR products obtained were sequenced and compared to existing APH family members. Sequence analysis resolved the family into nine groups of which six had recognizable phenotypes: 6′-phosphotransferase (APH(6)), 3′-phosphotransferase (APH(3)), hydroxyurea phosphotransferase (HUR), peptide phosphotransferase, hygromycin B phosphotransferase (APH(7″)) and oxidoreductase. The actinomycetes screened fell into seven groups, including three novel groups with unknown phenotypes. The strains clustered according to the environmental site from where they were obtained, providing evidence for the movement of these genes within populations. The value of this as a method for obtaining novel compounds and the significance to the ecology of antibiotic biosynthesis are discussed. Journal of Industrial Microbiology & Biotechnology (2002) 29, 60–69 doi:10.1038/sj.jim.7000260 Received 25 June 2001/ Accepted in revised form 26 March 2002     

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.     

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.     

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

Transformation of sugarcane protoplasts by direct uptake of a selectable chimaeric gene

Sugarcane protoplasts were transformed to kanamycin resistance at a frequency of approximately 8 in 10⁷ following PEG-induced uptake of Sma1 linearised pABD1 plasmid. DNA-treated protoplasts were cultured in agarose droplets, and protoplast-derived transformants selected on 80 g ml^–1 kanamycin. Transformed tissues maintained on this level of antibiotic expressed APH(3)II activity, and contained DNA that hybridised to a probe with the APH(3)II gene.Abbreviations APH(3)II aminoglycoside phosphotransferase - PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid     

Carbapenem resistance in Enterobacteriaceae: here is the storm!

The current worldwide emergence of resistance to the powerful antibiotic carbapenem in Enterobacteriaceae constitutes an important growing public health threat. Sporadic outbreaks or endemic situations with enterobacterial isolates not susceptible to carbapenems are now reported not only in hospital settings but also in the community. Acquired class A (KPC), class B (IMP, VIM, NDM), or class D (OXA-48, OXA-181) carbapenemases, are the most important determinants sustaining resistance to carbapenems. The corresponding genes are mostly plasmid-located and associated with various mobile genetic structures (insertion sequences, integrons, transposons), further enhancing their spread. This review summarizes the current knowledge on carbapenem resistance in Enterobacteriaceae, including activity, distribution, clinical impact, and possible novel antibiotic pathways.     

Microarray analysis of erythromycin resistance determinants

AIMS: To develop a DNA microarray for analysis of genes encoding resistance determinants to erythromycin and the related macrolide, lincosamide and streptogramin B (MLS) compounds. METHODS AND RESULTS: We developed an oligonucleotide microarray containing seven oligonucleotide probes (oligoprobes) for each of the six genes (ermA, ermB, ermC, ereA, ereB and msrA/B) that account for more than 98% of MLS resistance in Staphylococcus aureus clinical isolates. The microarray was used to test reference and clinical S. aureus and Streptococcus pyrogenes strains. Target genes from clinical strains were amplified and fluorescently labelled using multiplex PCR target amplification. The microarray assay correctly identified the MLS resistance genes in the reference strains and clinical isolates of S. aureus, and the results were confirmed by direct DNA sequence analysis. Of 18 S. aureus clinical strains tested, 11 isolates carry MLS determinants. One gene (ermC) was found in all 11 clinical isolates tested, and two others, ermA and msrA/B, were found in five or more isolates. Indeed, eight (72%) of 11 clinical isolate strains contained two or three MLS resistance genes, in one of the three combinations (ermA with ermC, ermC with msrA/B, ermA with ermC and msrA/B). CONCLUSIONS: Oligonucleotide microarray can detect and identify the six MLS resistance determinants analysed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that microarray-based detection of microbial antibiotic resistance genes might be a useful tool for identifying antibiotic resistance determinants in a wide range of bacterial strains, given the high homology among microbial MLS resistance genes.     

Molecular tools to detect the IncJ elements: a family of integrating, antibiotic resistant mobile genetic elements

The IncJ group of enterobacterial mobile genetic elements, which include R391, R392, R705, R997 and pMERPH, have been shown to be site-specific integrating elements encoding variable antibiotic and heavy metal resistance genes. They insert into a specific 17-bp site located in the prfC gene, encoding peptide release factor 3, in Escherichia coli and other hosts. A key feature of known IncJ elements is the presence of a site-specific recombination module consisting of an attachment site on the element and an integrase-encoding gene of the tyrosine recombinase class, which promotes integration between the attachment site on the element and a similar site on the host chromosome. We have cloned and sequenced the integrases from a number of known IncJ elements and designed PCR primers for specific amplification of this gene. Using conserved regions of enterobacterial prfC genes upstream and downstream of the insertion site, and conserved sequences at the ends of the integrated IncJ elements, we have designed specific primers to amplify across the integrated IncJ attL and attR junction fragments. Alignment of over 30 enterobacterial prfC-like genes indicates that the primers designed to amplify attR junction would amplify IncJ element: host junctions from a wide variety of hosts. The IncJ elements have been shown to sensitise recA(+)E. coli K12 strains to UV irradiation. A simple and rapid procedure for demonstrating this effect is described. These tools should enable the rapid detection of such elements in clinical and environmental settings.     

Aminoglycoside 3'-phosphotransferase gene: its cloning, characteristics and use as a molecular probe

Aminoglycoside resistance genes were cloned from transconjugates of aminoglycoside resistant clinical strains of gramnegative bacteria. The resistance determinant cloned from the strains of E. coli transferred kanamycin, monomycin and neomycin resistance to laboratory strains. It was shown that the cloned resistance gene encoded the type I 3'-aminoglycoside phosphotransferase. The results of spot and blot hybridization of the gramnegative bacteria clinical strains with the Bam HI-Pst I fragment of the cloned resistance determinant were indicative of wide-spread distribution of the APH 3' (I) and closely related genes in clinical microbial populations.     

‘To be,or not to be’—The dilemma of ‘silent’ antimicrobial resistance genes in bacteria

Antimicrobial resistance is a serious threat to public health that dramatically undermines our ability to treat bacterial infections. Microorganisms exhibit resistance to different drug classes by acquiring resistance determinants through multiple mechanisms including horizontal gene transfer. The presence of drug resistance genotypes is mostly associated with corresponding phenotypic resistance against the particular antibiotic. However, bacterial communities harbouring silent antimicrobial resistance genes—genes whose presence is not associated with a corresponding resistant phenotype do exist. Under suitable conditions, the expression pattern of such genes often revert and regain resistance and could potentially lead to therapeutic failure. We often miss the presence of silent genes, since the current experimental paradigms are focused on resistant strains. Therefore, the knowledge on the prevalence, importance and mechanism of silent antibiotic resistance genes in bacterial pathogens are very limited. Silent genes, therefore, provide an additional level of complexity in the war against drug-resistant bacteria, reminding us that not only phenotypically resistant strains but also susceptible strains should be carefully investigated. In this review, we discuss the presence of silent antimicrobial resistance genes in bacteria, their relevance and their importance in public health.     

Chimeric genes as dominant selectable markers in plant cells

Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx^R) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx^R gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.