Brazilian Journal of Microbiology 45, 2, 661-665 (2014)
ISSN 1678-4405
Copyright © 2014, Sociedade Brasileira de Microbiologia
www.sbmicrobiologia.org.br
Short Communication
Genotypic and phenotypic detection of efflux pump in Rhodococcus equi
Letícia Trevisan Gressler1, Agueda Castagna de Vargas1, Mateus Matiuzzi da Costa2,
Luciana Pötter3, Bibiana Petri da Silveira1, Luis Antônio Sangioni1,
Sônia de Avila Botton1
1
Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria,
Santa Maria, RS, Brazil.
2
Laboratório de Imunologia e Microbiologia, Universidade Federal do Vale do São Francisco,
Petrolina, PE, Brazil.
3
Departamento de Zootecnia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.
Submitted: April 29, 2013; Approved: March 14, 2014.
Abstract
The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi
isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates
using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in
R. equi.
Key words: ethidium bromide, efflux pump, multidrug resistance, Rhodococcus equi, vapA.
Efflux systems or efflux pumps are transporter proteins of toxic compounds classified into five families according to their primary structure and energy-coupling
mechanism: i. Major Facilitator Superfamily - MFS, ii.
Adenosine Triphosphate-Binding Cassette - ABC, iii.
Small Multidrug Resistance Family - SMR, iv. Resistance-Nodulation-Cell Division Superfamily - RND, and v.
Multidrug and Toxic Compound Extrusion Family MATE (Kumar and Schweizer, 2005). These proteins were
firstly described in Escherichia coli associated with tetracycline resistance profile (Mcmurray et al., 1980).
Although several genes encoding efflux pumps can
be found on plasmids, there are other efflux pump genes located on the chromosome, which provide for the bacterium
an intrinsic mechanism that allows survival in a hostile environment (e.g. the presence of antimicrobials) (Webber
and Piddock, 2003). Then, the efflux pumps are related to
acquired and intrinsic resistance to several antimicrobials
in different bacterial species (Li and Nikaido, 2004).
Cross-resistance was already described in efflux systems,
i.e. the exposure to any one agent that belongs to the substrate profile of an efflux pump can allows its overexpression and consequent cross-resistance to different
substrates (Webber and Piddock, 2003). These systems are
also considered as a major mechanism of resistance among
multidrug-resistant pathogenic microorganisms in veterinary medicine (Paulsen, 2003).
Rhodococcus equi is a facultative intracellular pathogen that infects animals and immunocompromised human
patients (Takai et al., 1991; Takai et al., 2000). This bacterium is the etiological agent of rhodococcosis, an important
disease that typically affects foals less than six months of
age, causing severe lesions including pyogranulomatous
pneumonia and mesenteric lymphadenitis (Prescott, 1991).
In R. equi, the most important virulence factor is associated
with the presence of virulence proteins (Vap) encode by
large plasmids (Takai et al., 1995), which have been used to
classify isolates as virulent (VapA); intermediately virulent
(VapB) and avirulent (without virulence plasmid) (Ribeiro
et al., 2005).
Currently, the treatment of rhodococcosis usually
consists of a combined application of macrolides, such as
erythromycin, azithromycin or clarithromycin, associated
with rifampicin (Muscatello, 2012). However, the increasing resistance in R. equi isolates to various antimicrobials,
including the macrolides group, has been reported (Takai et
al., 1997; Asoh et al., 2003; Buckley and Stanbridge, 2007;
Venner et al., 2012). The microorganisms’ resistance to
Send correspondence to S.A. Botton. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Avenida Roraima 1000,
Prédio 44, Sala 5007, 97105-900, Santa Maria, RS, Brazil. E-mail: sabott20@gmail.com.
662
macrolides has been associated to basically three events:
presence of methylases, efflux pump activity and enzymatic inactivation (Sutcliffe et al., 1996).
In R. equi, the presence of four putative multidrug
efflux proteins was mentioned in the reference strain R.
equi 103S (Letek et al., 2010). This study reported the sequence of a chromosomal gene known as req_39680,
which encodes a protein with 308 amino acids, related to
putative cation efflux system. Similar efflux mechanism
was described in mycobacteria being related to the development of high level drug-resistance (Schmalstieg et al.,
2012); however, studies about efflux systems activity have
not been described in R. equi. Herein, we identified the
presence of the req_39680 gene, as well as verified the
phenotypic expression of efflux mechanism in soil, feces of
healthy animals and clinical R. equi isolates.
R. equi isolates were obtained from ten horsebreeding farms, located in the Brazilian states of Rio
Grande do Sul (RS) and Paraná (PR). The isolates were obtained from soil (n = 30), feces of healthy animals (n = 30)
and foals clinical (n = 30) samples from 1991 to 2012. The
clinical samples were recovered from post-mortem pulmonary lesions subsequent to antimicrobial treatment. The reference strains R. equi ATCC 33701P + (vapA positive), R.
equi ATCC 33701P - (vapA negative) and Staphylococcus
aureus ATCC 25923 were used as control in both
phenotypic and genotypic assays. Mycobacterium goodii
SB314/96 and Nocardia sp. SB57/2008 were used as negative control in the genotypic assays.
Among 90 bacterial isolates analyzed, 70 were identified phenotypic and genotypically previously by Monego
et al. (2009), being classified as vapA positive R. equi
(n = 26) and vapA negative R. equi (n = 44). These isolates
were lyophilized and stored at -20 °C until the tests were
performed. Additionally, 20 soil samples (» 10 g) were collected, especially for this study, in January of 2012 in three
different horse-breeding farms in RS. These samples were
collected from mare-foal paddocks (n = 20) and stored in
sterile plastic bags until the bacteriology culturing. The soil
samples were growing in nalidixic acid-novobiocin-actidione (cycloheximide)-potassium tellurite (NANAT) selective medium described by Woolcock et al. (1980).
Colonies with R. equi profile were submitted to phenotypic
identification (Quinn et al., 1994) and genotypic analysis
for genus, species and vapA gene (Takai et al., 1995). All
the R. equi soil isolates were classified as vapA negative
and also were lyophilized and stored at -20 °C until testing.
In order to evaluate the characteristics of the protein
encoded by req_39680 gene prior to the genotypic tests, it
was performed a bioinformatics analysis. The sequence of
the protein was recovered from R. equi 103S (GenBank access YP_004008630.1) and submitted to analysis by the
Phyre V 2.0 (Protein Homology/Analogy Recognition Engine)
program
Gressler et al.
(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index).
Following, the polymerase chain reaction (PCR)
method was used to detect the req_39680 gene in the R.
equi isolates. Primarily, each strain was grown on Müeller
Hinton agar (Himedia® Laboratories) and incubated at
37 °C for 24 h. Three to five colonies were suspended in
500 mL of Milli-Q water and submitted to DNA extraction
by cethyltrimethylammonium bromide (CTAB) protocol
(Sambrook and Russell, 2001). The extracted DNA was
measured using the Picopet 01 DNA Calculator (Cambridge, England). The PCR primers employed were: forward - RE1 (5’-CCGCGATCCCTCGACACACG-3’) and
reverse - RE2 (5’-CCCACCCGCATCCGCAAGAT-3’).
Both primers were designed using Primer-BLAST program
(http://www.ncbi. nlm.nih.gov/tools/primer-blast/) based
on DNA sequence of the reference strain R. equi 103S
(GenBank
access:
NC_014659.1)
(http://www.ncbi.nlm.nih.gov) in order to amplify the complete gene. The PCR was carried out in a total volume of
25 mL containing: 10 mmoles of each primer, 200 mM of
deoxynucleotides (dNTPs, Invitrogen®), 1 U of DNA polymerase (GoTaq, Invitrogen®), 1X of the 5X enzyme buffer,
and 60 ng of DNA sample. The amplifications were performed using PTC-100 Programmable Thermal Controller
(MJ Research) with the following cycling profile: initial
denaturation at 94°C/1min, 35 cycles of 94 °C/30 s,
63 °C/30 s and 72 °C/1 min 30 s, and a final extension at
72 °C/5 min. PCR products (10 mL) were run in 1% agarose
gel, stained with ethidium bromide, visualized under UV
light and photo documented by L-PIX ST (Loccus® Brazil).
All PCR products were purified, in quadruplicate, with the
PureLink PCR Purification Kit (Invitrogen®) and sent for
DNA sequencing with the same primers for req_39680
gene. DNA sequencing was performed in an automated sequencer ABI-PRISM 3100 Genetic Analyzer (ACT Gene
Molecular Analysis Ltd., Biotechnology Center/UFRGS,
Porto Alegre, RS). DNA consensus sequences were generated by Gap program of the Staden package 4 software
(Staden et al., 2000) and analyzed by Basic Local Alignment Search Tool (BLAST) (NCBI/DNASIS software version 2.5, Software Engineering Co. Ltd., SanBruno,
California, US).
The phenotypic expression of efflux mechanism was
performed in agar containing ethidium bromide (EtBr).
This assay was based on the use of a fluorescent cationic
dye (EtBr) to visualize the efflux mechanism in bacterial
cells (adapted from Martins et al., 2006). Six R. equi isolates from clinical (n = 2), feces (n = 2) and soil (n = 2) samples were tested with different concentrations of EtBr (0.2,
0.4, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mg/mL) in order to determine the toxicity level of this dye for cells. Each group
had an isolate presenting req_39680 gene. The reference
strain S. aureus ATCC 25923 was used as positive control
according Couto et al. (2008). All microorganisms were
Efflux pump in R. equi
cultivated in tryptic soy agar (TSA) medium (Himedia®
Laboratories) for 24 h at 37 °C. A single colony of each
bacterium was transferred to tryptic soy broth (TSB) medium (Himedia® Laboratories) and maintained at 37 °C
overnight. For each microorganism, an inoculum suspension was prepared in 0.9% saline, adjusted to the turbidly of
0.5 on the McFarland’ scale, and absorbance readings were
performed in a spectrophotometer at 600 nm-wavelength.
Subsequently, dilutions in TSB were carried out to obtain
an inoculum containing approximately 1,000 CFU/mL.
This inoculum was applied with swab on plates containing
EtBr incorporated into the TSA and incubated at 37 °C for
48 h under aerobic conditions. Afterward, these plates were
visualized on UV light. Both viability of cells and capacity
of detection of efflux systems at the different concentrations were observed. The 0.2 mg/mL EtBr concentration
was selected to be used in all isolates (90), since provided
the adequate physiological conditions to development of
the R. equi. All tests were performed in duplicate. After the
visualization of the results on UV light the plates were
photo documented by PIX-L ST (Loccus® Brazil). Bacteria
were considered positive to efflux system expression when
the fluorescent colonies were not observed under UV light
and negative when the colonies showed fluorescence.
The data were analyzed by SAS statistical software
(SAS, 2001). The nonparametric Chi-square test was used
to calculate the difference in the req_39680 gene frequency
among R. equi isolates from different sources. Likewise, it
was used the Spearman correlation analysis in order to
evaluate the correlation between the presence of this gene
with the phenotypic expression of efflux mechanism, as
well as the occurrence of vapA gene. The minimum significance level considered was p < 0.05.
The protein encoded by req_39680 gene demonstrated 100% confidence and 96% coverage with MATE
family transporter protein; it was classified as multi
antimicrobial extrusion protein and characterized as cation-bound multidrug and toxin compound extrusion protein
when analyzed by Phyre V 2.0. This finding is very relevant
663
since MATE family transporter proteins are categorized
among the multidrug efflux transporter families present in
various pathogenic microorganisms (Kuroda and Tsuchiya,
2009). Based on these results we selected the req_39680
gene to perform this study using R. equi isolates.
The req_39680 gene was detected in 60% (54/90) of
R. equi isolates tested (Table 1), as well as in R. equi ATCC
33701. This result corroborates with the described by
Rahman et al. (2003), which demonstrated that R. equi
ATCC 33701 has 12 putative genes encoding efflux proteins, as well as 25 proteins of the ABC family, some of
which are related to drug resistance. All PCR- products amplified a specific DNA fragment of approximately 840 bp
(Figure 1). The identity of R. equi PCR products was confirmed by DNA sequence consensus analyses, which
showed 100% of identity with the reference strain R. equi
103S (GenBank access NC_014659.1). A DNA sequence
from a Brazilian R. equi isolate (SB 54/97) was deposited in
GenBank (access JX_512957).
The req_39680 gene was detected in R. equi isolates
from clinical (66.7%), soil (66.7%) and feces (46.7%) samples. No difference was observed in the frequency of the
gene among R. equi isolates from different sources, thus it
Table 1 - Molecular detection of the gene req_39680 and phenotypic expression of efflux mechanism in agar containing EtBr in clinical, feces and
soil R. equi isolates (n = 90).
Positive/total of R. equi isolates
Source
req_39680 gene1
Phenotypic expression of
efflux mechanism
Clinical
20/30*
11/30*
Feces
14/30
03/30
Soil
20/30
04/30
Total
54/90
18/90
1
Presence of req_39680 gene detected by PCR assay.
A high correlation index (0.66; p < 0.0001) was verified between the presence of the req_39680 gene and phenotypic expression of efflux mechanism by Spearman correlation analysis.
*
Figure 1 - PCR amplification patterns of req_39680 gene in R. equi isolates. Amplicons were generated from a single PCR assay with R. equi req_39680
gene-specific primers (approx. 840 bp). Line 1: Molecular weight marker, 100 bp-DNA ladder (Ludwig Biotech®, Brazil); Lines 2-4: Negative controls
(Ultra-pure water, Mycobacterium goodii SB314/962, Nocardia sp., respectively); Line 5: reference strain R. equi ATCC 33701 (positive control); Lines
6-8: Samples from clinical (SB54/97), feces (SB490/95) and soil (SB20/12) isolates R. equi.
664
is present in a comparable proportion of isolates recovered
from all three sources. We suppose that the presence of the
req_39680 gene is not an adaptation to the host but may be
just as relevant to its saprophytic status, given that aerosols
from soil or fecal derived strains are the most likely source
of rhodoccocal pneumonia (Muscatello et al., 2006). Likewise, according Letek et al. (2010) some antimicrobial resistance genes may be associated to efflux systems in
clinical isolates as well as other environmental rhodococci.
Other members of the genus Rhodococcus, such as
Rhodococcus erythropolis and Rhodococcus fascians, assure their survival in the environment through the efflux
pumps (Desomer et al., 1992; Nagy et al., 1997). According Martinez (2009) drug resistance determinants present
in soil organisms typically may have an important impact
on the clinical management of microbial infections
The assessment of phenotypic expression of efflux
mechanism by methodologies using EtBr as substrate is
widely employed in phenotypic studies to verify efflux
pumps (Li and Nikaido, 2004); however, it is based on
qualitative results and have also show limitation due to
physiological characteristics of each microorganism (Martins et al., 2006). In this sense, we firstly standardized the
phenotypic expression of efflux mechanism assay with different EtBr concentrations, in order to know the concentration that did not affect the best growth of the bacterial cells.
We suggest that this methodological approach could be a
useful to further screening assays of efflux systems in R.
equi, furthermore, this is an instrument-free method for the
demonstration of efflux pump activity of bacteria (Martins
et al., 2006).
The efflux mechanism by extruding of EtBr was detected in 20% (18/90) of R. equi isolates tested, including:
61.1% (11/18) clinical, 22.2% (4/18) soil and 16.7% (3/18)
feces isolates (Table 1). Although the req_39680 gene has
been detected in all positive samples for efflux mechanism
by extruding of EtBr, we cannot establish a relationship between efflux and presence or absence of req_39680 gene.
On the other hand, 66.6% (36/54) of req_39680 gene positive R. equi isolates did not show phenotypic expression of
efflux mechanism. We suppose that this mechanism may be
expressed by these isolates in other circumstances to be
evaluated. In this respect, Viveiros et al. (2005) demonstrated that an E. coli strain sensitive to tetracycline became
resistant due to the efflux mechanism induced after slow
and gradual exposure to the antibiotic and this expression
was reverted by serial transfer to drug-free medium or by
exposure to inhibitors of efflux pumps.
A high correlation index (0.66; p < 0.0001) was verified between the presence of the req_39680 gene and
phenotypic expression of efflux mechanism in clinical isolates. However, no correlation was observed in R. equi isolates from feces and soil. We believe that the fact of these
clinical samples have been recovered from foals postmortem pulmonary lesions, subsequent to antimicrobial
Gressler et al.
treatment against R. equi, may be driving an expression of
the efflux mechanism in the host. The treatment failures observed in infections by R. equi may be associated to the extrusion of chemical compounds (e.g. antimicrobials) once
resistance mechanisms to macrolides also include two families of efflux pumps (Roberts et al., 1999), increasing the
chances of failure in infection control.
According to Letek et al. (2010) the correlation between the presence of virulence and antimicrobial resistance genes in R. equi is not well documented. As stated by
these same authors, it is possible the occurrence of a direct
regulatory interaction or only a random effect between
these genes. In this study, no correlation was observed between the presence of both vapA and req_39680 genes in R.
equi isolates. This finding may be better explained by the
location of req_39680 gene, since it has a chromosomal locus and the vapA is a plasmidial gene.
In conclusion, the req_39680 gene is equally distributed among R. equi isolates from different sources and a
high correlation index between phenotypic expression of
efflux mechanism and presence of req_39680 gene was observed in clinical samples. Thus, there is a potential possibility of efflux systems to be an emerging form of
adaptation to pathogenic and saprophytic life by R. equi
isolates; however, additional studies are required to confirm this hypothesis.
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