RESUMO
OBJECTIVES: To assess the prevalence of cryptic silver (Ag+) resistance amongst clinical isolates of Gram-negative bacteria, and to examine how overt Ag+ resistance becomes activated in such strains. METHODS: Established methods were used to determine the susceptibility of 444 recent clinical isolates to Ag+, and to evaluate the potential for overt Ag+ resistance to emerge in susceptible isolates by spontaneous mutation. The genetic basis for Ag+ resistance was investigated using PCR amplification and DNA sequencing. RESULTS: None of the isolates tested displayed overt Ag+ resistance. However, upon silver challenge, high-level Ag+ resistance (silver nitrate MIC >128 mg/L) was selected at high frequency (10-7 to 10-8) in 76% of isolates of Enterobacter spp., â¼58% of isolates of Klebsiella spp. and â¼0.7% of isolates of Escherichia coli. All strains in which Ag+ resistance could be selected harboured the sil operon, with resistance apparently resulting from activation of this system as a consequence of single missense mutations in silS. By contrast, Ag+ resistance was not selected in isolates lacking sil, which included all tested representatives of Pseudomonas aeruginosa, Acinetobacter spp., Citrobacter spp. and Proteus spp. CONCLUSIONS: Whilst overt Ag+ resistance in Gram-negative pathogens is uncommon, cryptic Ag+ resistance pertaining to the sil operon is prevalent and readily activated in particular genera (Enterobacter and Klebsiella).
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Nitrato de Prata/farmacologia , Prata/farmacologia , Enterobacter/efeitos dos fármacos , Escherichia coli/genética , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Klebsiella/efeitos dos fármacos , Mutação de Sentido Incorreto/efeitos dos fármacos , Óperon , PrevalênciaRESUMO
BACKGROUND: The spread of bacterial plasmids is an increasing global problem contributing to the widespread dissemination of antibiotic resistance genes including ß-lactamases. Our understanding of the details of the biological mechanisms by which these natural plasmids are able to persist in bacterial populations and are able to establish themselves in new hosts via conjugative transfer is very poor. We recently identified and sequenced a globally successful plasmid, pCT, conferring ß-lactam resistance. RESULTS: Here, we investigated six plasmid encoded factors (tra and pil loci; rci shufflon recombinase, a putative sigma factor, a putative parB partitioning gene and a pndACB toxin-antitoxin system) hypothesised to contribute to the 'evolutionary success' of plasmid pCT. Using a functional genomics approach, the role of these loci was investigated by systematically inactivating each region and examining the impact on plasmid persistence, conjugation and bacterial host biology. While the tra locus was found to be essential for all pCT conjugative transfer, the second conjugation (pil) locus was found to increase conjugation frequencies in liquid media to particular bacterial host recipients (determined in part by the rci shufflon recombinase). Inactivation of the pCT pndACB system and parB did not reduce the stability of this plasmid. CONCLUSIONS: Our findings suggest the success of pCT may be due to a combination of factors including plasmid stability within a range of bacterial hosts, a lack of a fitness burden and efficient transfer rates to new bacterial hosts rather than the presence of a particular gene or phenotype transferred to the host. The methodology used in our study could be applied to other 'successful' globally distributed plasmids to discover the role of currently unknown plasmid backbone genes or to investigate other factors which allow these elements to persist and spread.
Assuntos
Bactérias/genética , Evolução Molecular , Transferência Genética Horizontal , Genes Bacterianos , Genômica , Plasmídeos , Resistência beta-Lactâmica , Conjugação Genética , Técnicas de Inativação de Genes , Instabilidade Genômica , HumanosRESUMO
The treatment of infections caused by antibiotic-resistant bacteria is one of the great challenges faced by clinicians in the 21st century. Antibiotic resistance genes are often transferred between bacteria by mobile genetic vectors called plasmids. It is commonly believed that removal of antibiotic pressure will reduce the numbers of antibiotic-resistant bacteria due to the perception that carriage of resistance imposes a fitness cost on the bacterium. This study investigated the ability of the plasmid pCT, a globally distributed plasmid that carries an extended-spectrum-ß-lactamase (ESBL) resistance gene (bla(CTX-M-14)), to persist and disseminate in the absence of antibiotic pressure. We investigated key attributes in plasmid success, including conjugation frequencies, bacterial-host growth rates, ability to cause infection, and impact on the fitness of host strains. We also determined the contribution of the bla(CTX-M-14) gene itself to the biology of the plasmid and host bacterium. Carriage of pCT was found to impose no detectable fitness cost on various bacterial hosts. An absence of antibiotic pressure and inactivation of the antibiotic resistance gene also had no effect on plasmid persistence, conjugation frequency, or bacterial-host biology. In conclusion, plasmids such as pCT have evolved to impose little impact on host strains. Therefore, the persistence of antibiotic resistance genes and their vectors is to be expected in the absence of antibiotic selective pressure regardless of antibiotic stewardship. Other means to reduce plasmid stability are needed to prevent the persistence of these vectors and the antibiotic resistance genes they carry.
Assuntos
Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Sequências Repetitivas Dispersas , Plasmídeos/genética , Salmonella typhimurium/genética , beta-Lactamases/genética , Animais , Antibacterianos , Caenorhabditis elegans/microbiologia , Conjugação Genética , Escherichia coli/patogenicidade , Aptidão Genética , Mutagênese Insercional , Plasmídeos/antagonistas & inibidores , Salmonella typhimurium/patogenicidade , Inibidores de beta-LactamasesRESUMO
IncK plasmids encoding CTX-M-14 extended-spectrum ß-lactamase (ESBL) and highly related to plasmid pCT were detected in 13 of 67 (19%) human clinical isolates of Escherichia coli with a group 9 CTX-M-type ESBL from the United Kingdom and in 2 quality assurance isolates. None of these E. coli strains was related to the cattle strain from which pCT was originally characterized.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Plasmídeos/genética , beta-Lactamases/metabolismo , Escherichia coli/classificação , Filogenia , Reino Unido , beta-Lactamases/genéticaRESUMO
Antimicrobial drug resistance is a global challenge for the 21st century with the emergence of resistant bacterial strains worldwide. Transferable resistance to ß-lactam antimicrobial drugs, mediated by production of extended-spectrum ß-lactamases (ESBLs), is of particular concern. In 2004, an ESBL-carrying IncK plasmid (pCT) was isolated from cattle in the United Kingdom. The sequence was a 93,629-bp plasmid encoding a single antimicrobial drug resistance gene, blaCTX-M-14. From this information, PCRs identifying novel features of pCT were designed and applied to isolates from several countries, showing that the plasmid has disseminated worldwide in bacteria from humans and animals. Complete DNA sequences can be used as a platform to develop rapid epidemiologic tools to identify and trace the spread of plasmids in clinically relevant pathogens, thus facilitating a better understanding of their distribution and ability to transfer between bacteria of humans and animals.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Epidemiologia Molecular , Plasmídeos/genética , beta-Lactamases/genética , Animais , Bovinos , Escherichia coli/classificação , Ordem dos Genes , Humanos , Dados de Sequência Molecular , Filogenia , Reino Unido/epidemiologiaRESUMO
The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 lacking a functional ramA or ramR or with plasmid-mediated high-level overexpression of ramA were compared to those of the wild-type parental strain. Inactivation of ramA led to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption of ramA led to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressed ramA led to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, including acrAB, acrEF, and tolC. Decreased expression of 34 Salmonella pathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization of Caenorhabditis elegans were also seen. Disruption of ramR led to the increased expression of ramA, acrAB, and tolC, but not to the same level as when ramA was overexpressed on a plasmid. Inactivation of ramR had a more limited effect on pathogenicity gene expression. In silico analysis of a suggested RamA-binding consensus sequence identified target genes, including ramR, acrA, tolC, sipABC, and ssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Salmonella typhimurium/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Bactérias/genética , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Transporte/genética , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Porinas , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Transativadores/genética , VirulênciaRESUMO
PURPOSE: With an increase in the numbers of bacterial isolates resistant to first-line antibiotics, there has been a revival in the use of older drugs including fosfomycin with novel mechanisms of action. We aimed to investigate the prevalence and genotypic nature of fosfomycin resistance in Escherichia coli from urinary tract infections (UTIs) using the various methods available in the clinical microbiology laboratory. METHODOLOGY: In total, 1000 culture-positive urine samples were assessed for the presence of E. coli and fosfomycin susceptibility was determined using the MAST Uri system, microbroth dilution, agar dilution and E-test strips.Results/Key findings. Initial investigation using breakpoint susceptibility testing on the MAST Uri system identified 62 of 657 (9.5â%) E. coli isolates as fosfomycin-resistant (MIC≥32 µg ml-1). However, on further testing, a lower rate of eight of the 62 (1.3â%) were robustly confirmed to be resistant using microbroth dilution, agar dilution and E-test strips. These true resistant isolates belonged to diverse E. coli multi-locus sequence types and each had a unique set of chromosomal alterations in genes associated with fosfomycin resistance. Fosfomycin-resistant isolates were not multiply drug resistant and did not carry plasmidic fosfomycin resistance genes. Therefore, the use of fosfomycin may be unlikely to drive selection of a particular clone or movement of transferrable resistance genes. CONCLUSION: Fosfomycin remains a viable option for the treatment of E. coli in uncomplicated UTIs; different susceptibility testing platforms can give very different results regarding the prevalence of fosfomycin resistance, with false positives being a potential problem that may unnecessarily limit the use of this agent.
Assuntos
Antibacterianos/farmacologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/efeitos dos fármacos , Fosfomicina/farmacologia , Infecções Urinárias/microbiologia , Aminoácidos/química , Aminoácidos/genética , Antibacterianos/uso terapêutico , Contagem de Colônia Microbiana , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana/genética , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Fosfomicina/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Infecções Urinárias/tratamento farmacológico , Sequenciamento Completo do GenomaAssuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bovinos , Doenças dos Bovinos/tratamento farmacológico , Cefalosporinas/uso terapêutico , Clortetraciclina/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Plasmídeos , Estados UnidosRESUMO
The study objective was to determine the effects of two treatment regimens on quantities of ceftiofur and tetracycline resistance genes in feedlot cattle. The two regimens were ceftiofur crystalline-free acid (CCFA) administered to either one or all steers within a pen and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. A 26-day randomized controlled field trial was conducted on 176 steers. Real-time PCR was used to quantify bla(CMY-2), bla(CTX-M), tet(A), tet(B), and 16S rRNA gene copies/gram of feces from community DNA. A significant increase in ceftiofur resistance and a decrease in tetracycline resistance elements were observed among the treatment groups in which all steers received CCFA treatment, expressed as gene copies/gram of feces. Subsequent chlortetracycline administration led to rapid expansion of both ceftiofur and tetracycline resistance gene copies/gram of feces. Our data suggest that chlortetracycline is contraindicated when attempting to avoid expansion of resistance to critically important third-generation cephalosporins.
Assuntos
Bactérias/genética , Infecções Bacterianas/tratamento farmacológico , Cefalosporinas/administração & dosagem , Metagenoma/efeitos dos fármacos , Resistência a Tetraciclina/genética , Animais , Bactérias/efeitos dos fármacos , Infecções Bacterianas/genética , Infecções Bacterianas/veterinária , Bovinos , Clortetraciclina/administração & dosagem , Fezes/microbiologia , Metagenoma/genética , RNA Ribossômico 16S/genética , Resistência a Tetraciclina/efeitos dos fármacosRESUMO
A randomized controlled field trial was conducted to evaluate the effects of two sets of treatment strategies on ceftiofur and tetracycline resistance in feedlot cattle. The strategies consisted of ceftiofur crystalline-free acid (CCFA) administered to either one or all of the steers within a pen, followed by feeding or not feeding a therapeutic dose of chlortetracycline (CTC). Eighty-eight steers were randomly allocated to eight pens of 11 steers each. Both treatment regimens were randomly assigned to the pens in a two-way full factorial design. Non-type-specific (NTS) E. coli (nâ=â1,050) were isolated from fecal samples gathered on Days 0, 4, 12, and 26. Antimicrobial susceptibility profiles were determined using a microbroth dilution technique. PCR was used to detect tet(A), tet(B), and bla CMY-2 genes within each isolate. Chlortetracycline administration greatly exacerbated the already increased levels of both phenotypic and genotypic ceftiofur resistance conferred by prior CCFA treatment (P<0.05). The four treatment regimens also influenced the phenotypic multidrug resistance count of NTS E. coli populations. Chlortetracycline treatment alone was associated with an increased probability of selecting isolates that harbored tet(B) versus tet(A) (P<0.05); meanwhile, there was an inverse association between finding tet(A) versus tet(B) genes for any given regimen (P<0.05). The presence of a tet(A) gene was associated with an isolate exhibiting reduced phenotypic susceptibility to a higher median number of antimicrobials (nâ=â289, medianâ=â6; 95% CIâ=â4-8) compared with the tet(B) gene (nâ=â208, medianâ=â3; 95% CIâ=â3-4). Results indicate that CTC can exacerbate ceftiofur resistance following CCFA therapy and therefore should be avoided, especially when considering their use in sequence. Further studies are required to establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together.