RESUMEN
Vancomycin has proven remarkably durable to resistance evolution by Staphylococcus aureus despite widespread treatment with vancomycin in the clinic. Only 16 cases of vancomycin-resistant S. aureus (VRSA) have been documented in the United States. It is thought that the failure of VRSA to spread is partly due to the fitness cost imposed by the vanA operon, which is the only known means of high-level resistance. Here, we show that the fitness cost of vanA-mediated resistance can be overcome through laboratory evolution of VRSA in the presence of vancomycin. Adaptation to vancomycin imposed a tradeoff such that fitness in the presence of vancomycin increased, while fitness in its absence decreased in evolved lineages. Comparing the genomes of vancomycin-exposed and vancomycin-unexposed lineages pinpointed the D-alanine:D-alanine ligase gene (ddl) as the target of loss-of-function mutations, which were associated with the observed fitness tradeoff. Vancomycin-exposed lineages exhibited vancomycin dependence and abnormal colony morphology in the absence of drug, which were associated with mutations in ddl. However, further evolution of vancomycin-exposed lineages in the absence of vancomycin enabled some evolved lineages to escape this fitness tradeoff. Many vancomycin-exposed lineages maintained resistance in the absence of vancomycin, unlike their ancestral VRSA strains. These results indicate that VRSA might be able to compensate for the fitness deficit associated with vanA-mediated resistance, which may pose a threat to the prolonged durability of vancomycin in the clinic. Our results also suggest vancomycin treatment should be immediately discontinued in patients after VRSA is identified to mitigate potential adaptations.
Asunto(s)
Antibacterianos , Infecciones Estafilocócicas , Staphylococcus aureus Resistente a Vancomicina , Vancomicina , Vancomicina/farmacología , Antibacterianos/farmacología , Humanos , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus Resistente a Vancomicina/genética , Resistencia a la Vancomicina/genética , Pruebas de Sensibilidad Microbiana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , MutaciónRESUMEN
Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.
Asunto(s)
Antibacterianos , Clostridioides difficile , Resistencia a la Vancomicina , Vancomicina , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Resistencia a la Vancomicina/genética , Vancomicina/farmacología , Antibacterianos/farmacología , Aptitud Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Transducción de Señal , Mutación , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/genéticaRESUMEN
Infections caused by vancomycin-resistant enterococci (VRE) are common. Real-time PCR assays targeting vanA and vanB facilitate screening of patients in healthcare settings to limit the risk of dissemination, especially amongst those at high-risk of infection or with limited treatment options. Such assays are commonly performed as reflex testing procedures where they augment phenotypic techniques and shorten turnaround time to benefit timely clinical management. 'Random access' and 'sample-to-result' real-time PCR platforms are suited for this application as they are of low complexity and less technically demanding. Modelled on these attributes, we configured a real-time PCR assay (VRE BD) for detection of vanA/B in clinical isolates of enterococci, adapted for the BD Max System (Becton Dickinson). We applied an unconventional approach by testing suspensions of microorganisms in water to circumvent the traditional pre-analytical genomic extraction process. Our objective of this study was to assess the performance of this assay for detection of VRE in cultures by validating against a traditional real-time PCR assay based on the LightCycler 2.0 platform (Roche, VRE RO). A high level of analytical sensitivity and specificity (≥99.0%) for both genes was obtained when testing suspensions derived from blood agar. Results for suspensions obtained from chromID VRE (Edwards Group) showed a similar level of performance for vanA detection (100%), but not for the vanB target (≥90.9%) where a lesser number of isolates were available for testing. However, our results for VRE detection in isolates from these media were repeatable and reproducible, and equated to positive and negative predictive values of ≥95.2% and ≥97.8%, respectively. Furthermore, the VRE BD assay was also able to accurately detect VRE in clinical and spiked BacT/ALERT (bioMérieux) blood cultures. Thus, the technical simplicity, short turnaround time and robustness of this high performing assay for VRE is suitable for reflex testing. In addition, the format developed for the BD Max platform has potential application for reflex testing other molecular targets of clinical importance.
Asunto(s)
Proteínas Bacterianas , Ligasas de Carbono-Oxígeno , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Enterococos Resistentes a la Vancomicina , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Ligasas de Carbono-Oxígeno/genética , Proteínas Bacterianas/genética , Enterococos Resistentes a la Vancomicina/aislamiento & purificación , Enterococos Resistentes a la Vancomicina/genética , Infecciones por Bacterias Grampositivas/diagnóstico , Infecciones por Bacterias Grampositivas/microbiología , Enterococcus/aislamiento & purificación , Enterococcus/genética , Resistencia a la Vancomicina/genéticaRESUMEN
AIMS: To investigate enterococci carrying linezolid and vancomycin resistance genes from fecal samples recovered from wild boars. METHODS AND RESULTS: Florfenicol- and vancomycin-resistant enterococci, isolated on selective agar plates, were screened by PCR for the presence of linezolid and vancomycin resistance genes. Five isolates carried optrA or poxtA linezolid resistance genes; one strain was resistant to vancomycin for the presence of vanA gene. All isolates were tested for their antibiotic susceptibility and subjected to Whole Genome Sequencing (WGS) analysis. In Enterococcus faecalis (E. faecalis) V1344 and V1676, the optrA was located on the new pV1344-optrA and pV1676-optrA plasmids, respectively, whereas in Enterococcus faecium (E. faecium) V1339 this gene was on a 22 354-bp chromosomal genetic context identical to the one detected in a human E. faecium isolate. In both E. faecium V1682 and E. durans V1343, poxtA was on the p1818-c plasmid previously found in a human E. faecium isolate. In E. faecium V1328, the vanA gene was on the Tn1546 transposon in turn located on a new pV1328-vanA plasmid. Only E. faecium V1682 successfully transferred the poxtA gene to an enterococcal recipient in filter mating assays. CONCLUSIONS: The occurrence of genetic elements carrying linezolid and vancomycin resistance genes in enterococci from wild boars is a matter of concern, moreover, the sharing of plasmids and transposons between isolates from wild animals, human, and environment indicates an exchange of genetic material between these settings.
Asunto(s)
Proteínas Bacterianas , Farmacorresistencia Bacteriana , Enterococcus faecalis , Enterococcus faecium , Sus scrofa , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , Farmacorresistencia Bacteriana/genética , Enterococcus faecalis/genética , Enterococcus faecalis/aislamiento & purificación , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecium/genética , Enterococcus faecium/aislamiento & purificación , Enterococcus faecium/efectos de los fármacos , Heces/microbiología , Genoma Bacteriano , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Italia , Linezolid/farmacología , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Sus scrofa/microbiología , Resistencia a la Vancomicina/genética , Secuenciación Completa del GenomaRESUMEN
Antibiotic resistance is an increasing challenge for the human pathogen Staphylococcus aureus. Methicillin-resistant S. aureus (MRSA) clones have spread globally, and a growing number display decreased susceptibility to vancomycin, the favoured antibiotic for treatment of MRSA infections. These vancomycin-intermediate S. aureus (VISA) or heterogeneous vancomycin-intermediate S. aureus (hVISA) strains arise from accumulation of a variety of point mutations, leading to cell wall thickening and reduced vancomycin binding to the cell wall building block, Lipid II, at the septum. They display only minor changes in vancomycin susceptibility, with varying tolerance between cells in a population, and therefore, they can be difficult to detect. In this review, we summarize current knowledge of VISA and hVISA. We discuss the role of genetic strain background or epistasis for VISA development and the possibility of strains being 'transient' VISA with gene expression changes mediated by, for example, VraTSR, GraXSR, or WalRK signal transduction systems, leading to temporary vancomycin tolerance. Additionally, we address collateral susceptibility to other antibiotics than vancomycin. Specifically, we estimate how mutations in rpoB, encoding the ß-subunit of the RNA polymerase, affect overall protein structure and compare changes with rifampicin resistance. Ultimately, such in-depth analysis of VISA and hVISA strains in terms of genetic and transcriptional changes, as well as changes in protein structures, may pave the way for improved detection and guide antibiotic therapy by revealing strains at risk of VISA development. Such tools will be valuable for keeping vancomycin an asset also in the future.
Asunto(s)
Antibacterianos , Resistencia a la Vancomicina , Vancomicina , Vancomicina/farmacología , Antibacterianos/farmacología , Humanos , Resistencia a la Vancomicina/genética , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Adaptación Fisiológica , Staphylococcus aureus Resistente a Vancomicina/genética , Staphylococcus aureus Resistente a Vancomicina/efectos de los fármacos , Staphylococcus aureus Resistente a Vancomicina/metabolismo , Mutación , Transducción de SeñalRESUMEN
BACKGROUND: This study analyzed the genetic traits and fitness costs of vancomycin-resistant Enterococcus faecium (VREfm) blood isolates carrying Tn1546-type transposons harboring the vanA operon. METHODS: All E. faecium blood isolates were collected from eight general hospitals in South Korea during one-year study period. Antimicrobial susceptibility testing and vanA and vanB PCR were performed. Growth rates of E. faecium isolates were determined. The vanA-positive isolates were subjected to whole genome sequencing and conjugation experiments. RESULTS: Among 308 E. faecium isolates, 132 (42.9%) were positive for vanA. All Tn1546-type transposons harboring the vanA operon located on the plasmids, but on the chromosome in seven isolates. The plasmids harboring the vanA operon were grouped into four types; two types of circular, nonconjugative plasmids (Type A, n = 50; Type B, n = 46), and two types of putative linear, conjugative plasmids (Type C, n = 16; Type D, n = 5). Growth rates of vanA-positive E. faecium isolates were significantly lower than those of vanA-negative isolates (P < 0.001), and reduction in growth rate under vancomycin pressure was significantly larger in isolates harboring putative linear plasmids than in those harboring circular plasmids (P = 0.020). CONCLUSIONS: The possession of vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination. Fitness burden to bacterial hosts was varied by type and size of the vanA operon-harboring plasmid.
Asunto(s)
Antibacterianos , Proteínas Bacterianas , Ligasas de Carbono-Oxígeno , Elementos Transponibles de ADN , Enterococcus faecium , Pruebas de Sensibilidad Microbiana , Operón , Plásmidos , Plásmidos/genética , Enterococcus faecium/genética , Humanos , Proteínas Bacterianas/genética , República de Corea , Ligasas de Carbono-Oxígeno/genética , Antibacterianos/farmacología , Secuenciación Completa del Genoma , Infecciones por Bacterias Grampositivas/microbiología , Enterococos Resistentes a la Vancomicina/genética , Resistencia a la Vancomicina/genética , Aptitud Genética , Vancomicina/farmacología , Conjugación GenéticaRESUMEN
BACKGROUND: This study investigates the distribution and characteristics of linezolid and vancomycin susceptibilities among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and explores the underlying resistance mechanisms. METHODS: A total of 2842 Enterococcus clinical isolates from patients were retrospectively collected, and their clinical data were further analyzed. The minimum inhibitory concentrations (MICs) of vancomycin and linezolid were validated by broth dilution method. The resistance genes optrA, cfr, vanA, vanB and vanM were investigated using polymerase chain reaction (PCR). Housekeeping genes and resistance genes were obtianed through whole-genome sequencing (WGS). RESULTS: Of the 2842 Enterococcus isolates, 88.5% (2516) originated from urine, with E. faecium accounted for 60.1% of these. The vanA gene was identified in 27/28 vancomycin resistant Enterococcus (VRE) isolates, 4 of which carried both vanA and vanM genes. The remaining strain was vanM positive. The optrA gene was identified in all E. faecalis isolates among linezolid resistant Enterococcus (LRE). E. faecium showed a higher multiple antibiotic resistance index (MAR index) compared to E. faecalis. The multi-locus sequence typing (MLST) showed the sequence type of E. faecium mainly belongs to clonal complex (CC) 17, nearly E. faecalis isolates analyzed were differentiated into 7 characteristics of sequence types (STs), among which ST16 of CC16 were the major lineage. CONCLUSION: Urine was the primary source of VRE and LRE isolates in this study. E. faecium showed higher levels of resistance compared to E. faecalis. OptrA gene was detected in 91.6% of LRE, which could explain linezolid resistance, and van genes were detected in all vancomycin resistant Enterococcus strains, while vanA was a key resistance mechanism in VRE identified in this study.
Asunto(s)
Enterococcus faecium , Infecciones por Bacterias Grampositivas , Linezolid , Pruebas de Sensibilidad Microbiana , Linezolid/farmacología , Humanos , China/epidemiología , Enterococcus faecium/genética , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/aislamiento & purificación , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Enterococcus faecalis/genética , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/aislamiento & purificación , Femenino , Vancomicina/farmacología , Antibacterianos/farmacología , Epidemiología Molecular , Adulto , Resistencia a la Vancomicina/genética , Anciano , Estudios Retrospectivos , Enterococos Resistentes a la Vancomicina/genética , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Enterococos Resistentes a la Vancomicina/aislamiento & purificación , Adulto Joven , Enterococcus/genética , Enterococcus/efectos de los fármacos , Enterococcus/aislamiento & purificaciónRESUMEN
BackgroundVancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible.AimThe aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE.MethodsFrom 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance.ResultsIn total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E.â¯faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm.ConclusionFrom 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.
Asunto(s)
Antibacterianos , Proteínas Bacterianas , Ligasas de Carbono-Oxígeno , Enterococcus faecium , Infecciones por Bacterias Grampositivas , Linezolid , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Resistencia a la Vancomicina , Enterococos Resistentes a la Vancomicina , Enterococos Resistentes a la Vancomicina/genética , Enterococos Resistentes a la Vancomicina/aislamiento & purificación , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Enterococcus faecium/genética , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/aislamiento & purificación , Humanos , Dinamarca/epidemiología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , Linezolid/farmacología , Resistencia a la Vancomicina/genética , Secuenciación Completa del Genoma , Vancomicina/farmacología , Vancomicina/uso terapéutico , GenotipoRESUMEN
Staphylococcus aureus is a notorious pathogen responsible for various severe diseases. Due to the emergence of drug-resistant strains, the prevention and treatment of S. aureus infections have become increasingly challenging. Vancomycin is considered to be one of the last-resort drugs for treating most methicillin-resistant S. aureus (MRSA), so it is of great significance to further reveal the mechanism of vancomycin resistance. VraFG is one of the few important ABC (ATP-binding cassette) transporters in S. aureus that can form TCS (two-component systems)/ABC transporter modules. ABC transporters can couple the energy released from ATP hydrolysis to translocate solutes across the cell membrane. In this study, we obtained a strain with decreased vancomycin susceptibility after serial passaging and selection. Subsequently, whole-genome sequencing was performed on this laboratory-derived strain MWA2 and a novel single point mutation was discovered in vraF gene, leading to decreased sensitivity to vancomycin and daptomycin. Furthermore, the mutation reduces autolysis of S. aureus and downregulates the expression of lytM, isaA, and atlA. Additionally, we observed that the mutant has a less net negative surface charge than wild-type strain. We also noted an increase in the expression of the dlt operon and mprF gene, which are associated with cell surface charge and serve to hinder the binding of cationic peptides by promoting electrostatic repulsion. Moreover, this mutation has been shown to enhance hemolytic activity, expand subcutaneous abscesses, reflecting an increased virulence. This study confirms the impact of a point mutation of VraF on S. aureus antibiotic resistance and virulence, contributing to a broader understanding of ABC transporter function and providing new targets for treating S. aureus infections.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Antibacterianos , Proteínas Bacterianas , Infecciones Estafilocócicas , Staphylococcus aureus , Vancomicina , Virulencia/genética , Infecciones Estafilocócicas/microbiología , Antibacterianos/farmacología , Vancomicina/farmacología , Animales , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/metabolismo , Pruebas de Sensibilidad Microbiana , Resistencia a la Vancomicina/genética , Secuenciación Completa del Genoma , Daptomicina/farmacología , Ratones , Autólisis , Humanos , Mutación Puntual , Mutación , FemeninoRESUMEN
Staphylococcus aureus is a leading cause of healthcare-associated infections globally. Vancomycin-resistant S. aureus (VRSA), those with high-level resistance [minimum inhibitory concentration (MIC) of 16-32 µg/mL vancomycin], are uncommon, whereas vancomycin-intermediate S. aureus (VISA; MIC of 4-8 µg/mL), are isolated more frequently and develop during long-term and/or repeated use of the antibiotic. VISA can be difficult to eradicate and infections may persist. Our knowledge of mechanisms that underlie the development of VISA is incomplete. We used a genomics approach to investigate the VISA phenotype in three prominent S. aureus lineages. All VISA clinical isolates tested had increased cell wall thickness compared with vancomycin-susceptible S. aureus strains. Growth rates of clonal complex (CC) 5, CC8, and CC45 clinical isolates were reduced in 2 µg/mL vancomycin compared to media alone. Culture in 2 and 4 µg/mL vancomycin sequentially for two weeks reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin in a majority of CC5, CC8, and CC45 isolates tested. We identified alleles reported previously to contribute to the VISA phenotype, but unexpectedly, these alleles were unique to each CC. A subtherapeutic concentration of vancomycin elicited changes in the VISA transcriptome-common and unique-among the three CCs tested. Multiple genes, including those encoding a glycerate kinase, an M50 family metallopeptidase, and an uncharacterized membrane protein, were upregulated among all three lineages and not reported previously as associated with VISA. Although there are lineage-specific changes in DNA sequence, our findings suggest changes in the VISA transcriptome constitute a general response to stress that confers reduced susceptibility to multiple antibiotics. IMPORTANCE: Our understanding of the mechanisms that underlie the development of vancomycin-intermediate Staphylococcus aureus (VISA) is incomplete. To provide a more comprehensive view of this process, we compared genome sequences of clonal complex (CC) 5, CC8, and CC45 VISA clinical isolates and measured changes in the transcriptomes of these isolates during culture with a subtherapeutic concentration of vancomycin. Notably, we identified differentially expressed genes that were lineage-specific or common to the lineages tested, including genes that have not been previously reported to contribute to a VISA phenotype. Changes in gene expression were accompanied by reduced growth rate, increased cell wall thickness, and reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin. Our results provide support to the idea that changes in gene expression contribute to the development of VISA among three CCs that are a prominent cause of human infections.
Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas , Staphylococcus aureus , Resistencia a la Vancomicina , Vancomicina , Vancomicina/farmacología , Antibacterianos/farmacología , Humanos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Infecciones Estafilocócicas/microbiología , Resistencia a la Vancomicina/genética , Staphylococcus aureus Resistente a Vancomicina/genética , Staphylococcus aureus Resistente a Vancomicina/efectos de los fármacos , Staphylococcus aureus Resistente a Vancomicina/metabolismo , Daptomicina/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
BACKGROUND: The glycopeptide vancomycin is the antimicrobial agent-of-choice for the treatment of severe non-gastrointestinal infections with members of Bacillus cereus sensu lato (s.l.). Recently, sporadic detection of vancomycin-resistant phenotypes emerged, mostly for agar diffusion testing such as the disc diffusion method or gradient test (e.g. Etest®) method. RESULTS: In this work, we were able to disprove a preliminarily assumed high resistance to vancomycin in an isolate of B. cereus s.l. using broth microdilution and agar dilution. Microscopic imaging during vancomycin susceptibility testing showed spreading towards the inhibition zone, which strongly suggested sliding motility. Furthermore, transcriptomic analysis using RNA-Seq on the nanopore platform revealed several key genes of biofilm formation (e.g. calY, tasA, krsEABC) to be up-regulated in pseudo-resistant cells, substantiating that bacterial sliding is responsible for the observed mobility. Down-regulation of virulence (e.g. hblABCD, nheABC, plcR) and flagellar genes compared with swarming cells also confirmed the non-swarming phenotype of the pseudo-resistant isolate. CONCLUSIONS: The results highlight an insufficiency of agar diffusion testing for vancomycin susceptibility in the B. cereus group, and reference methods like broth microdilution are strongly recommended. As currently no guideline mentions interfering phenotypes in antimicrobial susceptibility testing of B. cereus s.l., this knowledge is essential to obtain reliable results on vancomycin susceptibility. In addition, this is the first report of sliding motility undermining accurate antimicrobial susceptibility testing in B. cereus s.l. and may serve as a basis for future studies on bacterial motility in susceptibility testing and its potential impact on treatment efficacy.
Asunto(s)
Antibacterianos , Bacillus cereus , Pruebas de Sensibilidad Microbiana , Resistencia a la Vancomicina , Vancomicina , Bacillus cereus/efectos de los fármacos , Bacillus cereus/genética , Pruebas de Sensibilidad Microbiana/métodos , Vancomicina/farmacología , Antibacterianos/farmacología , Resistencia a la Vancomicina/genética , Biopelículas/efectos de los fármacos , Humanos , Perfilación de la Expresión GénicaRESUMEN
The emergence of resistance against the last-resort antibiotic vancomycin in staphylococcal infections is a serious concern for human health. Although various drug-resistant pathogens of diverse genetic backgrounds show higher virulence potential, the underlying mechanism behind this is not yet clear due to variability in their genetic dispositions. In this study, we investigated the correlation between resistance and virulence in adaptively evolved isogenic strains. The vancomycin-susceptible Staphylococcus aureus USA300 was exposed to various concentrations of vancomycin repeatedly as a mimic of the clinical regimen to obtain mutation(s)-accrued-clonally-selected (MACS) strains. The phenotypic analyses followed by expression of the representative genes responsible for virulence and resistance of MACS strains were investigated. MACS strains obtained under 2 and 8 µg/ml vancomycin, named Van2 and Van8, respectively; showed enhanced vancomycin minimal inhibitory concentrations (MIC) to 4 and 16 µg/ml, respectively. The cell adhesion and invasion of MACS strains increased in proportion to their MICs. The correlation between resistance and virulence potential was partially explained by the differential expression of genes known to be involved in both virulence and resistance in MACS strains compared to parent S. aureus USA300. Repeated treatment of vancomycin against vancomycin-susceptible S. aureus (VSSA) leads to the emergence of vancomycin-resistant strains with variable levels of enhanced virulence potentials.
Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas , Staphylococcus aureus , Resistencia a la Vancomicina , Vancomicina , Vancomicina/farmacología , Antibacterianos/farmacología , Virulencia/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Infecciones Estafilocócicas/microbiología , Resistencia a la Vancomicina/genética , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mutación , Factores de Virulencia/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacosRESUMEN
Background: Antibiotic resistance is a global public health concern that has been exacerbated by the overuse and misuse of antibiotics, leading to the emergence of resistant bacteria. The gut microbiota, often influenced by antibiotic usage, plays a crucial role in overall health. Therefore, this study aimed to investigate the prevalence of antibiotic resistant genes in the gut microbiota of Indonesian coastal and highland populations, as well as to identify vancomycin-resistant bacteria and their resistant genes. Methods: Stool samples were collected from 22 individuals residing in Pacet, Mojokerto, and Kenjeran, Surabaya Indonesia in 2022. The read count of antibiotic resistant genes was analyzed in the collected samples, and the bacterium concentration was counted by plating on the antibiotic-containing agar plate. Vancomycin-resistant strains were further isolated, and the presence of vancomycin-resistant genes was detected using a multiplex polymerase chain reaction (PCR). Results: The antibiotic resistant genes for tetracycline, aminoglycosides, macrolides, beta-lactams, and vancomycin were found in high frequency in all stool samples (100%) of the gut microbiota. Meanwhile, those meant for chloramphenicol and sulfonamides were found in 86% and 16% of the samples, respectively. Notably, vancomycin-resistant genes were found in 16 intrinsically resistant Gram-negative bacterial strains. Among the detected vancomycin-resistant genes, vanG was the most prevalent (27.3%), while vanA was the least prevalent (4.5%). Conclusion: The presence of multiple vancomycin resistance genes in intrinsically resistant Gram-negative bacterial strains demonstrated the importance of the gut microbiota as a reservoir and hub for the horizontal transfer of antibiotic resistant genes.
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Microbioma Gastrointestinal , Humanos , Microbioma Gastrointestinal/efectos de los fármacos , Indonesia , Resistencia a la Vancomicina/genética , Vancomicina/farmacología , Vancomicina/uso terapéutico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Heces/microbiología , Masculino , Femenino , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/clasificación , Adulto , Genes BacterianosRESUMEN
The dipeptide D-Ala-D-Ala is an essential component of peptidoglycan and the target of vancomycin. Most Clostridioides difficile strains possess the vanG operon responsible for the synthesis of D-Ala-D-Ser, which can replace D-Ala-D-Ala in peptidoglycan. The C. difficile vanG operon is regulated by a two-component system, VanRS, but is not induced sufficiently by vancomycin to confer resistance to this antibiotic. Surprisingly, in the absence of the VanS histidine kinase (HK), the vanG operon is still induced by vancomycin and also by another antibiotic, ramoplanin, in a VanR-dependent manner. This suggested the cross-regulation of VanR by another HK or kinases that are activated in the presence of certain lipid II-targeting antibiotics. We identified these HKs as CD35990 and CD22880. However, mutations in either or both HKs did not affect the regulation of the vanG operon in wild-type cells suggesting that intact VanS prevents the cross-activation of VanR by non-cognate HKs. Overproduction of VanR in the absence of VanS, CD35990, and CD22880 led to high expression of the vanG operon indicating that VanR can potentially utilize at least one more phosphate donor for its activation. Candidate targets of CD35990- and CD22880-mediated regulation in the presence of vancomycin or ramoplanin were identified by RNA-Seq.
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Antibacterianos , Proteínas Bacterianas , Clostridioides difficile , Regulación Bacteriana de la Expresión Génica , Histidina Quinasa , Operón , Resistencia a la Vancomicina , Vancomicina , Operón/genética , Clostridioides difficile/genética , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/metabolismo , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vancomicina/farmacología , Resistencia a la Vancomicina/genética , Antibacterianos/farmacología , Depsipéptidos/farmacología , Factores de TranscripciónRESUMEN
Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.
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Proteínas Bacterianas , Multimerización de Proteína , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Dominio Catalítico , Metaloendopeptidasas/química , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/química , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/fisiología , Resistencia a la Vancomicina/genética , Staphylococcus aureus Resistente a Meticilina/enzimología , Staphylococcus aureus Resistente a Meticilina/metabolismoRESUMEN
BACKGROUND: Glycopeptide antibiotic vancomycin is a bactericidal antibiotic available for the infection to Staphylococcus aureus (SA), however, SA has a strong adaptive capacity and thereby acquires resistance to vancomycin. This study aims to illuminate the possible molecular mechanism of vancomycin resistance of SA based on the 16S rRNA sequencing data and microarray profiling data. METHODS: 16S rRNA sequencing data of control samples and urinary tract infection samples were retrieved from the EMBL-EBI (European Molecular Biology Laboratory - European Bioinformatics Institute) database. Correlation of gut flora and clinical indicators was evaluated. The possible targets regulated by SA were predicted by microarray profiling and subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. CXCL10 gene knockout and overexpression were introduced to evaluate the effect of CXCL10 on the virulence of SA and the resistance to vancomycin. SA strains were co-cultured with urethral epithelial cells in vitro. The presence of SA virulence factors was detected using PCR. Biofilm formation of SA strains was assessed using the microtiter plate method. Furthermore, the antibiotic sensitivity of SA strains was evaluated through vancomycin testing. RESULTS: Gut flora and its species abundance had significant difference between urinary tract infection and control samples. SA was significantly differentially expressed in urinary tract infection samples. Resistance of SA to vancomycin mainly linked to the D-alanine metabolism pathway. SA may participate in the occurrence of urinary tract infection by upregulating CXCL10. In addition, CXCL10 mainly affected the SA resistance to vancomycin through the TLR signaling pathway. In vitro experimental results further confirmed that the overexpression of CXCL10 in SA increased SA virulence and decreased its susceptibility to vancomycin. In vitro experimental validation demonstrated that the knockout of CXCL10 in urethral epithelial cells enhanced the sensitivity of Staphylococcus aureus (SA) to vancomycin. CONCLUSION: SA upregulates the expression of CXCL10 in urethral epithelial cells, thereby activating the TLR signaling pathway and promoting resistance to glycopeptide antibiotics in SA.
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Antibacterianos , Quimiocina CXCL10 , Infecciones Estafilocócicas , Staphylococcus aureus , Infecciones Urinarias , Resistencia a la Vancomicina , Vancomicina , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Vancomicina/farmacología , Humanos , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Antibacterianos/farmacología , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/genética , Resistencia a la Vancomicina/genética , Infecciones Urinarias/microbiología , Infecciones Urinarias/tratamiento farmacológico , Biopelículas/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , ARN Ribosómico 16S/genética , Células Epiteliales/microbiología , Células Epiteliales/efectos de los fármacos , Femenino , MasculinoRESUMEN
The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.
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Proteínas Bacterianas , Ligasas de Carbono-Oxígeno , ARN sin Sentido , Enterococos Resistentes a la Vancomicina , Vancomicina , Enterococos Resistentes a la Vancomicina/genética , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Ligasas de Carbono-Oxígeno/genética , ARN sin Sentido/genética , Proteínas Bacterianas/genética , Humanos , Vancomicina/farmacología , Plásmidos/genética , Resistencia a la Vancomicina/genética , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Silenciador del GenRESUMEN
BACKGROUND: Vancomycin is frequently used as a last line of defence against infections due to multidrug-resistant Staphylococcus aureus (S. aureus). A recent finding described the acquisition of vancomycin-resistant S. aureus strains by the integration of an enterococcal plasmid containing the vanA operon into the S. aureus chromosome via homologous recombination involving a specific integration site called locus L2. METHODS: To characterise all mechanisms of acquisition of vanA, this study analysed the 15 706 S. aureus genomes to look for vanA and described its genetic environment. RESULTS: A complete vanA operon was found in 25 S. aureus strains isolated from 12 patients, including nine co-isolated with vancomycin-resistant Enterococcus strains. VanA was found within transposon Tn1546-like elements on 17 plasmids and eight chromosomes. VanA might be acquired through conjugation of enterococcal and staphylococcal plasmids, transposition of Tn1546 carrying vanA and plasmid integration into the chromosome. Further, L2 was detected in 2087 genomes (13.3%) of S. aureus strains across different continents. Six potential chromosomal hotspots for integration of the entire vanA-containing enterococcal plasmid were identified by homologous recombination via L2. CONCLUSIONS: These findings suggest that the recently described scenario in a New York patient could be reproduced anywhere. Surveillance of this possibility is mandatory, especially in patients with vancomycin-resistant Enterococcus infection or colonisation.
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Proteínas Bacterianas , Ligasas de Carbono-Oxígeno , Elementos Transponibles de ADN , Genoma Bacteriano , Operón , Plásmidos , Infecciones Estafilocócicas , Staphylococcus aureus , Resistencia a la Vancomicina , Humanos , Plásmidos/genética , Resistencia a la Vancomicina/genética , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Elementos Transponibles de ADN/genética , Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , Genoma Bacteriano/genética , Infecciones Estafilocócicas/microbiología , Antibacterianos/farmacología , Vancomicina/farmacologíaRESUMEN
Vancomycin variable Enterococcus (VVE) bacteria are phenotypically susceptible to vancomycin, but they harbor the vanA gene. We aimed to ascertain the prevalence of VVE among clinically isolated vancomycin-susceptible Enterococcus faecium (VSE) isolates, as well as elucidate the molecular characteristics of the vanA gene cluster within these isolates. Notably, we investigated the prevalence and structure of the vanA gene cluster of VVE. Between June 2021 and May 2022, we collected consecutive, non-duplicated vancomycin-susceptible Enterococcus faecium (VSE) samples. Real-time PCR was performed to detect the presence of vanA, vanB, and vanC. Overlapping PCR with sequencing and whole-genome sequencing were performed for structural analysis. Sequence types (STs) were determined by multilocus sequence typing. Exposure testing was performed to assess the ability of the isolates to acquire vancomycin resistance. Among 282 VSE isolates tested, 20 isolates (7.1%) were VVE. Among them, 17 isolates had partial deletions in the IS1216 or IS1542 sequences in vanS (N=10), vanR (N=5), or vanH (N=2). All VVE isolates belonged to the CC17 complex and comprised five STs, namely ST17 (40.0%), ST1421 (25.0%), ST80 (25.0%), ST787 (5.0%), and ST981 (5.0%). Most isolates were related to three hospital-associated clones (ST17, ST1421, and ST80). After vancomycin exposure, 18 of the 20 VVEs acquired vancomycin resistance. Considering the high reversion rate, detecting VVE by screening VSE for vanA is critical for appropriate treatment and infection control.
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Antibacterianos , Proteínas Bacterianas , Enterococcus faecium , Infecciones por Bacterias Grampositivas , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Resistencia a la Vancomicina , Vancomicina , Enterococcus faecium/genética , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/aislamiento & purificación , Humanos , Vancomicina/farmacología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/diagnóstico , Resistencia a la Vancomicina/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , Secuenciación Completa del Genoma , Reacción en Cadena en Tiempo Real de la Polimerasa , Prevalencia , Familia de MultigenesRESUMEN
BACKGROUND: VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA, vanB, vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. METHODS: Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. RESULTS: All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16â minutes, respectively). CONCLUSIONS: The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE.