Development of homogeneous expression of resistance in methicillin-resistant Staphylococcus aureus clinical strains is functionally associated with a β-lactam-mediated SOS response

Objectives: One of the main characteristics of methicillin-resistant Staphylococcus aureus (MRSA) from both hospitals and community is their heterogeneous expression of resistance. Recently, we reported new heterogeneous MRSA isolates phenotypically susceptible to oxacillin despite being mecA positive. These low-level mecA-mediated resistance MRSA strains are very heterogeneous in expression (HeR) and are likely to be clinically relevant since exposure of such isolates to b-lactams can result in high-level homotypic resistance (HoR). We hypothesized that HeR to HoR selection in these clinically relevant strains may be determined by the pre-existence of a hypermutable population that favours its selection in the presence of oxacillin. Methods: Using established procedures, SA13011 HeR to HoR selection was performed by using subinhibitory concentrations of oxacillin and examined for mutability. Real-time RT-PCR and transcriptional profiling by DNA microarray were used to compare gene expression between both populations and related genetically modified SA13011 strain. Results: We found that HeR/HoR selection by oxacillin was associated with increased mutation rate and oxacillin-mediated SOS response. We determined increased expression of both mecA and SOS response lexA/recA regulators. Mutational inactivation of lexA repressor resulted in a significant decrease in both mutation rate and oxacillin resistance in the HoR cells. Complementation of the lexA mutant strain restored oxacillin resistance to the high levels observed in the corresponding HoR wild-type strain. Conclusions: The present results support the notion that SOS response is mechanistically involved in generating mutations that, in addition to mecA induction, allow the selection of a highly oxacillin-resistant population.Fil: Cuirolo, Arabela Ximena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Plata, Konrad. University of Virginia; Estados UnidosFil: Rosato, Adriana E.. University of Virginia; Estados Unido

Development of multidrug resistance in staphylococci driven by efflux

[P282]Aim: To study the efflux driven response of two major staphylococcal pathogens, S. aureus and S. epi-dermidis to the challenge by non-antibiotic drugs. Methods: We adapted three reference strains to ethidium bromide (EtBr), a broad substrate of bacte-rial efflux pumps. The parental strains, S. aureus ATCC25923, S. epidermidis ATCC12228 and S. epider-midis RP62A were cultured in varying concentrations of EtBr, to obtain their EtBr-adapted derivatives; ATCC25923_EtBr; ATCC12228_EtBr and RP62A_EtBr. Susceptibility of parental and adapted strains to 10 antibiotics and 6 biocides was evaluated by microdilution MIC determination with or without efflux inhibitors. Efflux activity was established by fluorometric assays and the relative expression of the genes coding for the main efflux pumps (EPs) of each species quantified by RT-PCR.Results: For each strain tested, exposure to EtBr resulted in the development of a multidrug resistance (MDR) phenotype, which included resistance to fluoroquinolones and decreased susceptibility to bio-cides, including cetrimide, benzalkonium chloride and tetraphenylphosphonium bromide. Efflux inhib-itors such as verapamil reduced these resistance levels. The EtBr-adapted cultures showed increased efflux activity, which was accompanied by over-expression of distinct EP genes, in a temporal pattern. Conclusion: These results show that both S. aureus and S. epidermidis have the potential to develop efflux driven MDR phenotypes when exposed to a non-antibiotic substrate of multidrug EPs, which can be mediated by distinct efflux pumps, depending on the drug and the bacterial genetic background.publishe

Impact of efflux in the development of multidrug resistance phenotypes in Staphylococcus aureus

WOS: 000363382300003Background: Efflux has been recognized as a resistance mechanism to antimicrobials in Staphylococcus aureus; however its role on the development of clinically relevant resistance is still poorly characterized. This study aimed to examine the impact of efflux on development of resistance to fluoroquinolones and other antimicrobials in S. aureus strains representing relevant phenotypes in terms of antibiotic susceptibility and efflux activity. Methods: Two closely related methicillin- and ciprofloxacin-resistant Staphylococcus aureus clinical strains, with different efflux capacity and the pan-susceptible strain ATCC25923 were exposed to constant concentrations of the efflux pump (EP) substrates ciprofloxacin, ethidium bromide and cetrimide. Parental and exposed strains were tested regarding their susceptibility towards antibiotics, biocides and ethidium bromide, efflux capacity and levels of EP gene expression. Occurrence of resistance-associated mutations was screened by sequencing. Results: Multidrug resistance phenotypes emerged upon exposure, independently of the substrate or its concentration, which were correlated with increased efflux capacity of the exposed strains. The temporal pattern of EP gene expression disclosed an early-response with high expression of several genes, followed by a late-response, characterized by overexpression of specific genes. The overall cell response was more pronounced for strains with an initial basal efflux activity. Remarkably, detection of the IS256 element in the promoter regions of mgrA and norA, in some cases associated with increased gene expression, suggests that these genes may be hot spots for IS256 insertion events. The results obtained with exposure of ATCC25923 to ciprofloxacin were particularly striking, revealing a step-wise development of fluoroquinolone resistance, with a first efflux-mediated response, followed by the occurrence of a mutation in grlA that resulted in phenotypic resistance. Additionally, challenge by non-fluoroquinolone agents, particularly cetrimide, promoted cross resistance to fluoroquinolones, revealing the potential role of biocides as selective pressure for the emergence of resistance to these antibiotics. Conclusions: This study reveals efflux as a significant component of S. aureus resistance to fluoroquinolones and biocides and as a primary mechanism to withstand stress imposed by antimicrobials. This efflux-mediated response can result in the emergence of multidrug resistance in healthcare environments and should be taken into account in the management of this major pathogen.publishersversionpublishe

Virulence potential of biofilm producing Staphylococcus pseudintermedius, Staphylococcus aureus and Staphylococcus coagulans causing skin infections in companion animals

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Identification and molecular epidemiology of methicillin resistant Staphylococcus pseudintermedius strains isolated from canine clinical samples in Argentina

Staphylococcus pseudintermedius is the leading cause of pyoderma in dogs and the frequent use of antimicrobial treatment is associated to the development of resistance to nearly all classes of antibiotics. Despite S. pseudintermedius significance, our understanding of the molecular mechanism of β-lactam resistance and its genetic diversity remains limited. We aimed to: i) determine the phenotypic resistance profile of methicillin resistant Staphylococcus pseudintermedius (MRSP) isolated from infected dogs in three different veterinary hospitals in Buenos Aires, Argentina; ii) identify the SCCmec elements and resistance genes; and iii) analyze the clonal relationship between isolates and in regard of dominant lineages found in the world. In addition to the differential levels of β-lactam resistance, MRSP isolates (n = 10) showed resistance to 5–6 families of antibiotics, and were therefore categorized as multidrug-resistant. All the isolates were variant of SCCmec V homologous to S. aureus; additional SCCmecFinder analysis classified five of the genomes as SCCmec type V (5C2&5) with mecA (encodes for PBP2a), mecRI and mecI and all the genes closely related to the reference SCCmec type V S. aureus TSGH17 strain. In the remaining five strains, mecA was present, although other genes associated with SCCmec V including mecR1 and mecI were missing. PBP2a was inducible in low level resistance strains (MRSP 8151), and constitutively expressed in MRSP 8150, suggesting different mecA regulatory mechanisms. MRSP isolates showed significant genetic diversity: eight PFGE clonal types and six multilocus-sequence typing (MLST) sequence types (STs) (339, 649, 919, 920, 921 and 922), including four new STs genetically distinct from STs reported in other geographic areas. Comparative genomics and phylogenetic analyses of the MRSP showed a correlation between the genetic content and the phenotypes, and established the genetic relationship between the isolates. MRSP could be a threat to animal health due to it concerning level of antimicrobial resistance. Our study highlights genetic and epidemiological aspects of multidrug-resistant MRSP strains from Argentina showing high degree of correlation between the resistance genes and the phenotype of the isolates and, furthermore, they appeared evolutionary closer to major worldwide reported ST68 and ST71.Facultad de Ciencias Veterinaria

Carbapenemase-Producing Extraintestinal Pathogenic Escherichia coli From Argentina: Clonal Diversity and Predominance of Hyperepidemic Clones CC10 and CC131

Extraintestinal pathogenic Escherichia coli (ExPEC) causes infections outside the intestine. Particular ExPEC clones, such as clonal complex (CC)/sequence type (ST)131, have been known to sequentially accumulate antimicrobial resistance that starts with chromosomal mutations against fluoroquinolones, followed with the acquisition of blaCTX–M–15 and, more recently, carbapenemases. Here we aimed to investigate the distribution of global epidemic clones of carbapenemase-producing ExPEC from Argentina in representative clinical isolates recovered between July 2008 and March 2017. Carbapenemase-producing ExPEC (n = 160) were referred to the Argentinean reference laboratory. Of these, 71 were selected for genome sequencing. Phenotypic and microbiological studies confirmed the presence of carbapenemases confirmed as KPC-2 (n = 52), NDM-1 (n = 16), IMP-8 (n = 2), and VIM-1 (n = 1) producers. The isolates had been recovered mainly from urine, blood, and abdominal fluids among others, and some were from screening samples. After analyzing the virulence gene content, 76% of the isolates were considered ExPEC, although non-ExPEC isolates were also obtained from extraintestinal sites. Pan-genome phylogeny and clonal analysis showed great clonal diversity, although the first phylogroup in abundance was phylogroup A, harboring CC10 isolates, followed by phylogroup B2 with CC/ST131, mostly H30Rx, the subclone co-producing CTX-M-15. Phylogroups D, B1, C, F, and E were also detected with fewer strains. CC10 and CC/ST131 were found throughout the country. In addition, CC10 nucleated most metalloenzymes, such as NDM-1. Other relevant international clones were identified, such as CC/ST38, CC155, CC14/ST1193, and CC23. Two isolates co-produced KPC-2 and OXA-163 or OXA-439, a point mutation variant of OXA-163, and three isolates co-produced MCR-1 among other resistance genes. To conclude, in this work, we described the molecular epidemiology of carbapenemase-producing ExPEC in Argentina. Further studies are necessary to determine the plasmid families disseminating carbapenemases in ExPEC in this region.Fil: Sanz, María Belén. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorio e Instituto de Salud ; ArgentinaFil: de Belder, Denise Gisele. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorio e Instituto de Salud ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Mendieta, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorio e Instituto de Salud ; ArgentinaFil: Faccone, Diego Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorio e Instituto de Salud ; ArgentinaFil: Poklepovich, Tomás. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Lucero, Celeste. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorio e Instituto de Salud ; ArgentinaFil: Rapoport, Melina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorio e Instituto de Salud ; ArgentinaFil: Campos, Josefina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Tuduri, Ezequiel. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorio e Instituto de Salud ; ArgentinaFil: Saavedra, Mathew O.. Houston Methodist Hospital; Estados UnidosFil: Van der Ploeg, Claudia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Rogé, Ariel Diego. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Pasteran, Fernando. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Corso, Alejandra. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Rosato, Adriana E.. University of California; Estados UnidosFil: Gómez, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentin

Staphylococcal phenotypes induced by naturally occurring and synthetic membrane-interactive polyphenolic β-lactam resistance modifiers.

Galloyl catechins, in particular (-)-epicatechin gallate (ECg), have the capacity to abrogate β-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA); they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (-)-epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for β-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation

VraSR and Virulence Trait Modulation during Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus Infection

Methicillin-resistant S. aureus continues to develop resistance to antimicrobials, including those in current clinical use as daptomycin (DAP). Resistance to DAP arises by mutations in cell membrane and cell wall genes and/or upregulation of the two-component VraSR system. However, less is known about the connection between the pathogen and virulence traits during DAP resistance development. We provide new insights into VraSR and its regulatory role for virulence factors during DAP resistance, highlighting coordinated interactions that favor the higher persistence of MRSA DAP-resistant strains in the infected host.Methicillin-resistant Staphylococcus aureus (MRSA) threatens human health in hospital and community settings. The lipopeptide antibiotic daptomycin (DAP) is a frequently used treatment option for MRSA infection. DAP exposure can cause bacterial resistance because mutations are induced in genes implicated in cell membrane and cell wall metabolism. Adaptations aimed at surviving antimicrobial pressure can affect bacterial physiology and modify in vivo aptitude and pathogenesis. In this study, clinical DAP-susceptible (DAPs) and DAP-resistant (DAPr) MRSA isolates were used to investigate associations between DAP resistance and staphylococcal virulence. We previously found that VraSR is a critical sensor of cell membrane/wall homeostasis associated with DAP acquisition during MRSA infection. The present study found that DAPr CB1634 and CB5014 MRSA strains with vraSR upregulation were less virulent than their susceptible counterparts, CB1631 and CB5013. Differential gene-transcription profile analysis revealed that DAPr CB1634 had decreased agr two-component system expression, virulence factors, and highly suppressed hemolysis activity. Functional genetic analysis performed in DAPr CB1634 strains using vraSR inactivation followed by gene complementation found that vraSR acted as a transcriptional agrA regulator. These results indicated that VraSR has a broad range of regulatory functions. VraSR also appeared to affect DAPr adherence to epithelial cells, which would affect DAPr strain colonization and survival in the host. The correlation between DAP resistance and decreased virulence was also found in the CB5013 (DAPs) and CB5014 (DAPr) pair. Taken together, these findings are the first evidence that DAP resistance and MRSA virulence are tightly connected and involve compromised expression of regulatory and virulence determinants