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Revista da Sociedade Brasileira de Medicina Tropical

Print version ISSN 0037-8682On-line version ISSN 1678-9849

Rev. Soc. Bras. Med. Trop. vol.48 no.5 Uberaba Sept./Oct. 2015

http://dx.doi.org/10.1590/0037-8682-0017-2015 

Short Communications

Influence of a subinhibitory concentration of vancomycin on the in vitro expression of virulence-related genes in the vancomycin-resistant Enterococcus faecalis

Tiane Martin de Moura1 

Fabrício Souza Campos2 

Juliana Caierão1 

Ana Claudia Franco2 

Paulo Michel Roehe2 

Pedro Alves d'Azevedo1 

Jeverson Frazzon3 

Ana Paula Guedes Frazzon2 

1Departamento Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil.

2Departamento de Microbiologia, Parasitologia e Imunologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.

3Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.


ABSTRACT

INTRODUCTION:

Exposure to subinhibitory concentrations (SICs) of antimicrobials may alter the bacterial transcriptome.

METHODS

: Here, we evaluated the expression of nine virulence-related genes in vancomycin-resistant enterococci (VRE) urinary tract infection isolates grown at SICs of vancomycin.

RESULTS:

A Subinhibitory concentrations of vancomycin interferes with gene modulation, but does not affect the phenotype of a VRE strain in vitro .

CONCLUSIONS:

Subinhibitory concentrations of vancomycin may regulate the expression of virulence factors in vivo or contribute to the selection of vancomycin-resistant strains.

Keywords: VRE; Biofilm; Expression

Enterococcus faecalis , an important microorganism that causes healthcare-associated infections, is part of the normal human microbiota. Enterococcus faecalis is the fifth most common pathogen that causes catheter-associated urinary tract infections (UTIs) 1 . Several different virulence factors have been reported in clinical strains of E. faecalis , including biofilm formation and the expression of surface adhesion components. The ability of E. faecalis to adhere to medical devices such as ureteral stents and catheters and to develop biofilms is likely associated with its pathogenicity 2 .

The biofilm on plastic (BOP) operon is essential for biofilm formation 3 . Adhesion of collagen of enterococci (ACE) and aggregation substance (ASC10) are proteins that are also crucial for biofilm production 4 5 . The sulfur assimilation system (SUF) is responsible for iron-sulfur cluster biogenesis and is essential for cellular survival, particularly during aerobic growth or oxidative stress. The SUF operon, which is associated with virulence, is controlled by the regulatory protein sensor for oxidative stress (OxyR) and the ferric-uptake regulator (FUR) 6 .

Vancomycin-resistant enterococci (VRE) have emerged as important nosocomial pathogens worldwide. The VanA phenotype (associated with vanA ) is responsible for high levels of resistance to vancomycin 7 . The clinical use of vancomycin is a subject of major concern, because continuous monitoring is required to maintain a concentration of approximately 15mg/L of the drug in patients' sera. Occasionally, because of the complex pharmacokinetics of vancomycin, this value is not achieved and patients are subjected to subinhibitory concentrations (SICs) of the drug. Consequently, frequent incidences of vancomycin failure and poor outcomes have been observed recently. Under these conditions, there is a high probability of selecting resistant or heteroresistant isolates 8 .

Exposure to SICs of antimicrobials may alter the transcriptomic and phenotypic responses of pathogenic bacteria 9 10 ; however, the effect of SICs of antimicrobials on virulence remains unclear. The aim of this study was to evaluate the expression of virulence-related genes in an E. faecalis VRE strain isolated from a urinary tract infection and grown under SICs of vancomycin. The bacterial strains used in this study are listed in Table 1 . The growth capacities of four E. faecalis strains were examined, and the strain with the highest optical density (OD) value of biofilm formation was selected for transcriptional profiling. All the E. faecalis strains were streaked onto Tryptic Soy Broth (TSB) agar and incubated at 37°C overnight (ON). Four individual colonies were used to inoculate the same tube of 5mL of 2× YT broth (1% yeast extract, 1.6% tryptone, and 1% NaCl) and grown ON at 37°C. Urine from six healthy men and women volunteers with no history of UTIs or antibiotic use in the last 6 months was collected, pooled, centrifuged, sterilized twice by filtration, and added to 2× YT broth to make nine different sample concentrations: 50%, 25%, 12%, 10%, 8%, 6%, 4%, and 2% (2×YT-U50-2×YT-U2) and pure urine (100%). The positive control contained only 2× YT broth. The concentration of vancomycin chosen for the assays (64µg/mL) was based on the range 64-1,000µg/mL, which has been shown to produce high levels of resistance 7 .

Table 1: Bacterial strains used in this study. 

Van: vancomycin; MIC: minimal inhibitory concentration; UTI: urinary tract infections; PSC: Pronto Socorro Central ; PoA: Porto Alegre; HSR: Hospital Santa Rita; E.: Enterococcus. a ddl E. faecalis gene; bvancomycin resistance gene A-type; cMinimal inhibitory concentration by broth microdilution method.

All the strains were used to inoculate plates containing 2× YT agar and incubated at 35°C ON. All the isolates were then evaluated for their ability to form biofilms in ten different media: 2× YT broth (control), eight different concentrations of urine (2-50%) and 100% urine under two conditions - drug-free condition (DFC) and vancomycin condition (VC) - by using the crystal violet assay 11 . Four to eight colonies were diluted in 0.9% sterile saline (w/v) until the turbidity matched that of a 0.5 McFarland standard [approximately 1 × 10 8 colony-forming units per milliliter (CFU/ml)]. Eight wells of a 96-well flat-bottomed microplate were filled with 180μL of each media type and 20µL of bacterial suspension. The optical density (OD) was measured at 492nm in a spectrophotometer, and the OD of each strain was determined by comparing the arithmetic mean of the absorbance of the wells with the mean absorbance of the negative controls. The strains were categorized on the basis of their ODs into the following groups: non-adherent, weak biofilm producers, moderate biofilm producers, and strong biofilm producers 11 . Non-inoculated 2× YT wells were used as negative controls, and Staphylococcus epidermidis ATCC 35984 was used as a positive control. The OD 492 of each well was measured; all tests were carried out in triplicate. The effects of the different media on bacterial growth and biofilm formation were evaluated using the paired t-test (level of significance = 0.05).

Eleven genes ( vanA, bopA, bopB, bopC, bopD, ace, asc10, fur, oxyR , tuf , and 23S ) were analyzed by quantitative polymerase chain reaction (qPCR). The oligonucleotides used in this study are listed in Table 2 . Only one strain, exhibiting the maximum OD value, was selected for transcriptional profiling. The qPCR analyses were performed at least three times. Five-hundred microliters of ON culture were inoculated in 49.5mL of 2× YT-U10 in the absence (DFC) and presence (VC) of SICs of vancomycin. Aliquots were collected and extracted at the early-exponential phase (OD 650 0.3), total ribonucleic acid (RNA) was purified and quantified, and cDNAs were synthesized 12. Quantitative PCRs were performed as uniplex reactions in final volumes of 20µL with 100pg of cDNA using the Eco TM Real Time PCR System (Illumina (r) San Diego, USA) and universal cycling protocols.

Table 2: List of genes and oligonucleotides used for qPCR analyses. 

qPCR: quantitative polymerase chain reaction; RNA: ribonucleic acid; LacI: lactose; FUR: ferric-uptake regulator; OxyR: oxidative stress regulator; tuf: elongation factor Tu; ace: adhesion of collagen of enterococci; asc10: aggregation substance; bop: biofilm on plastic; vanA: vancomycin resistance gene A-type; F: primer sense, R: primer antisense. The oligonucleotides were designed using bioinformatics tools (https://www.genscript.com/ssl-bin/app/primer)

We analyzed the effect of different concentrations of urine on biofilm formation by phenotypic characterization and on the basis of the OD values. Biofilm formation in the VRE strains was negatively influenced by the presence of pure urine at 35 °C (no growth was observed). With urine concentrations of 2-50%, all the strains showed growth (OD, 0.016 ± 0.004); however, they were unable to form biofilms under both conditions. The UTI-2389 isolate produced the highest OD value (0.021 ± 0.004). No significant association was observed between biofilm formation and urine concentration. These findings might be a consequence of the in vitro environment, which lacked essential components of the in vivo urinary system such as urothelial cells, glucose, mineral salts, and albumin. Moreover, it has been suggested that biological signals in human urine play an important role in modulating virulence at enterococci infection sites 13 . On the basis of our biofilm assay, all the strains could be classified as non-adherent according to both DFC and VC. Although maximal biofilm induction has been observed at a concentration of 3/4 th the minimal inhibitory concentration (MIC) 10 , in other studies, vancomycin at concentrations ≤ 1/2 the MIC 9 had little or no effect on S. epidermidis biofilm formation. Thus, we concluded that the low concentration of vancomycin used in our study (1/6 th the MIC) produced no effect on biofilm formation, which is consistent with the OD values observed in our study.

On the basis of the results of our biofilm assays, we selected the UTI-2389 strain and 10% urine (2×YT-U10) for subsequent qPCR analysis. The relative quantification of the messenger ribonucleic acid (mRNA) concentrations is shown in Table 3 . Significant upregulation of vanA , which is vancomycin-inducible 7 , was detected under the VC condition. We used this result to investigate the up/downregulation of the other selected genes. Genes of the bop operon were partially regulated in the presence of SICs of vancomycin. Treatment with SICs of vancomycin significantly upregulated bopA and bopC and slightly downregulated bopB and bopD . Similar partial regulation of the bop operon in a VRE strain has been reported previously 14 under growth conditions lacking antimicrobial agents, which suggests that this operon behaves erratically, even under normal growth conditions. Under the conditions tested, the expression of ace was significantly downregulated. However, in a previous study, ace expression was scarcely detected 15 in cells at the mid- and late exponential phases and was not detectable in cells during the stationary phase, suggesting that ace transcription is very low under standard in vitro growth conditions. The expression of acs10 was induced slightly by SICs of vancomycin. ASC10 is expressed in the presence of an intercellular signal cCF10, which is a peptide pheromone 4 . Moreover, antibiotics can act as signaling molecules; therefore, vancomycin could have induced asc10 expression. We also detected minor downregulation of oxyR . During intracellular oxidative stress, the E. faecalis SUF system is induced by OxyR and requires the expression of the integration host factor 6 . In this in vitro study, no hydrogen peroxide production was observed, which may explain the lack of stimulation of oxyR . In contrast to oxyR , the expression of the ferric uptake regulator fur was significantly upregulated in response to the stress caused by vancomycin, indicating that this gene is not regulated in the same manner as oxyR . For example, fur may operate in response to a cofactor in cell homeostasis, because the SUF system is the housekeeping machinery for E. faecalis 6 .

Table 3: Delta cycle threshold (ΔCt) and ratio values of the expression of virulence related genes in a vancomycin-resistant enterococcus strain grown in 2× YT + 10% urine in absence (DFC) and presence (VC) of subinhibitory concentrations of vancomycin. 

ΔCt: cycle threshold variation; YT: yeast extract, tryptone and NaCl broth; DFC: drug-free condition; VC: vancomycin condition; E: efficiency of the reaction; R: the relative expression ratio is the average value of the normalized based on the reference genes; vanA: vancomycin resistance gene A-type; bop: biofilm on plastic; ace: adhesion of collagen of enterococci; asc10: aggregation substance; fur: ferric-uptake regulator; oxyR: oxidative stress regulator; tuf: elongation factor Tu; 23S ribosomal RNA; na: not applicable; *Level of significance = 0.05.

In summary, we present here, for the first time, data on the behavior of VRE from a UTI strain in the presence of SICs of vancomycin. The nine genes evaluated in this study are related to virulence factors that are often present in Enterococcus faecalis. Although there was an increase in the expression of several VRE genes in the presence of SICs of vancomycin, no differences in their phenotypes were observed in vitro. Although the data presented here are from in vitro assays, SICs of vancomycin may contribute to a similar increase in the expression of virulence factors in vivo , leading to adverse clinical outcomes and to the selection of vancomycin-resistant strains. In-vitro experiments may not effectively represent the complex in vivo conditions during enterococcal urinary tract infection. Therefore, additional in vitro and in vivostudies should be carried out to corroborate our findings. The current study is limited because the strains tested were isolated at local hospitals before the study commenced, meaning that we have no patient records detailing their treatments or clinical outcomes. Our main interest was to investigate the behavioral response of a clinical VRE strain against SICs of vancomycin. Therefore, we did not perform tests with other antimicrobials or compare our results to a reference strain.

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Received: January 19, 2015; Accepted: May 18, 2015

Corresponding author : Dra. Tiane Martin de Moura. Depto. Ciências Básicas da Saúde/UFCSPA. Rua Sarmento Leite 245, sala 204, 90050-170 Porto Alegre, Rio Grande do Sul, Brasil. Phone: 55 51 3303-8825 e-mail: tianedemoura@gmail.com

The authors declare that there is no conflict of interest.

Creative Commons License This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.