Detection of efflux pump activity among clinical isolates of Staphylococcus and Micrococcus species

Purpose: To detect efflux pump activity (EPA) and screening a suspected efflux pump inhibitor (EPI) [1(3-(trifluoromethyl)benzyl]-piperazine (TFMBP)], which could help in reducing multi-drug resistance (MDR). Methods: Eighteen isolates, viz, 14 S. aureus, 2 S. lentus, 1 S. xylosus and 1 Micrococcus species from various hospital infections of admitted patients were screened for antibiotics susceptibility to 11 classes of antibiotics including oxacillin and β-lactamase production. Efflux pump activity (EPA) was determined by minimum inhibitory concentration (MIC) technique in the presence and absence of TFMBP, the isolates were also screened for MDR genes. Results: All the isolates were resistant to ampicillin (10 μg) and penicillin (10 μg), but sensitive to bacitracin (10 μg). Majority of the isolates were MDR 12/18 (66.7 %), 10 (55.6 %) were inducible βlactamase producers and 3 (16.7 %) were intrinsic β-lactamase producers. Seven (38.9 %) were resistant to oxacillin and also produced carbapenemase enzyme. Eight (66.7 %) of the 12 MDR isolates gave evidence of EPA with TFMBP. However, no MDR genes were detected. Conclusion: Staphylococcus and Micrococcus species exhibit EPA in antibiotic resistance while a suitable EPI such as TFMBP when combined with specific antibiotics could help combat this menace.


INTRODUCTION
Antibiotic drug development has dropped by about 75 % since the 1980s [1,2] especially between 1962 and 2000.Despite the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) reported in Nigerian secondary and tertiary hospitals [3][4][5], there is still a dearth of information on prevalence and types of multidrug-resistant (MDR) strains among Staphylococcus species, in Nigeria and sub-saharan Africa [6].With more insight into the knowledge of the mechanisms of resistance in MDR bacteria, new initiatives in research into formulation of newer antibiotics or fortification of the existing ones for combating multi-drug resistant organisms will develop.
The typical phenotype of MRSA is multiple resistance to penicillin, oxacillin, cefuroxime, ciprofloxacin, erythromycin, clindamycin, sulphamethoxazole, and gentamicin, amongst other classes of antibiotics [7].MDR exhibited by bacterial organisms to chemically-unrelated antibacterial agents are caused by overexpression of MDR efflux pumps (which are transport proteins).This process leads to the extrusion of toxic substances including clinicallyrelevant antibiotics (which help in combating the pathogenic organisms) into the external environment.These pump proteins are found in both Gram-positive and Gram-negative bacteria, as well as in eukaryotic organisms [8].
Bohnert and Kern [8], studied a number of arylpiperazines for their ability to reverse multidrug resistance in E. coli over-expressing RND (Resistance Nodulation-Division) efflux pumps.Among them, 1-[3-(trifluoromethyl) benzyl]piperazine (TFMBP) (200 µg/mL) was found to lower the effective MIC of levofloxacin four-fold.In recent times, antimicrobial resistance phenotypes are predicted by identifying and characterising the known genes that encode the specific resistance mechanisms.The methods of Polymerase Chain Reaction (PCR) and DNA sequencing are known to offer the promise of increased sensitivity, specificity and speed in the detection of such specific known resistance genes [9,10].Therefore, this study was conducted to identify and determine the genes responsible for efflux pump activity in different isolates of Staphylococcus and Micrococcus species using PCR.

EXPERIMENTAL Microbial isolates
Eighteen clinical isolates comprising of 14 S. aureus, 2 S. lentus, 1 S. xylosus and 1 Micrococcus spp.from infections of urinary tract, upper respiratory tract, ear and eye swab, and blood culture all were identified by the analytical profile index (API) and the analytical profile index web (APIWeb) were used.S. aureus (ATCC 25923) was used as the standard strain in the study.
For further testing, all strains found to be resistant to four or more classes of antibiotics were classified as MDR.

β-Lactamase assay
All isolates showing resistance to the β-lactam antibiotics were further tested for the production of β-lactamase enzyme using nitrocefin (NF), a "colour shift substrate" as described by Livermore and Brown [12].The standard strains, E. coli A (non β-lactamase producing) and E. coli B (β-lactamase producing) used were collected from McNeil Science and Technology culture collection Centre (Usciences, Philadelphia).The tests were performed as follows; In the blank assay, 0.25 mL sterile Mueller Hinton Broth (MHB) and 0.25 mL of NF assay solution was added to the 0.5 mL of 0.01 M Na-HEPES, while in the reaction assay the 0.25 mL sterile MHB was replaced with 0.25 mL of broth culture.A color change from yellow to red in the reaction solution within 10 minutes compared with the blank was indicative of the presence of intrinsic β-lactamase producer [12].While, strains showing the color change after 10 min were considered as inducible β-lactamase producers [12].

Efflux pump activity (EPA) test
MDR isolates were further tested for EPA.The MICs of the test drugs were determined in the presence and absence of TFMBP as EPI.A reduction in MIC in the presence of the EPI indicates resistance due to EPA in the MDR isolates [8].
Eleven of the isolates tested (of which seven were found to be Oxacillin resistant S. aureus ORSA from the MDDST), were picked for this test.The four drugs used in this test were amoxicillin, cefuroxime, ciprofloxacin and tetracycline.These represent four classes of antibiotics to which the MDR organisms were commonly resistant.A total of 37 tests were performed using amoxicillin -11 isolates, cefuroxime -7 isolates, ciprofloxacin -8 isolates, and tetracycline -11 isolates.

Method 1: Broth macro-dilution
The broth macro-dilution (tube method) was used to determine initial range of the MICs of these drugs against the isolates.Four milliliters of sterile Mueller Hinton broth (MHB) containing serially diluted antibiotic in each of 11 test tubes, beginning with 1000 µg/mL of test antibiotic in tube no. 1 to 0.9765 µg/mL in the 11 th tube were set up for the test.Tubes 12 and 13 containing 4 mL of MHB were also set up as growth and sterile controls.Overnight fresh broth (18 h) culture of each isolate was diluted to give a 1:10 3 dilution in normal saline (equivalent to 0.5 McFarland standard), and 0.1 mL of this was introduced into each tube from the second tube to the 12 th tube.The twelfth tube, which contained no antibiotic was the growth control, while the 13 th tube with only 4 mL of sterile MHB served as the sterility control.All of the tubes from the 2 nd to the 13 th were incubated at 35 o C for 18 h.
The MIC was thereafter determined by measuring optical density (OD) at 550 nm in a Spectronic Thermo 20 Genesys spectrophotometer against an un-inoculated MHB as the reference blank.The tube with the lowest concentration that completely inhibited growth was taken as the MIC.A similar procedure was carried out for each of the antibiotics tested.

Method 2: Broth micro-dilution
MDR isolates were subjected to another round of MIC tests employing the micro-dilution assay method in micro-titer plates.The 96-well microtiter plates were used to perform the MIC tests at different concentrations of the antibiotics against the bacterial strains based on the result of the initial macro-dilution assay.
The desired range of concentrations of each antibiotic for the MIC tests was initially obtained through serial double-dilution of each antibiotic in a twelve-channel basin.Sterile phosphate buffer, pH 6.0 was used as diluent for amoxicillin and cefuroxime while sterile water was used for tetracycline and ciprofloxacin.In the case of each antibiotic, starting with a stock concentration of 2000 µg/mL in channel 1 of a twelve-channel basin, the serial double dilution was carried out up to a final concentration of 0.97663 µg/mL in the 12 th channel.
One micro-titer plate was used to test two different drugs (in duplicates) at a time and at ten different concentration levels from well A1 to well H10.The 11th column of wells (A11-H11) was for the growth control of the strain, while the 12 th column of wells (A12-H12) was for a sterility control of the process (Table 1).

Preparation of microdilutions in microtiter plate
EPA test for each drug was done in duplicate in the micro-titer plate.For example, rows A and B were for amoxicillin with TFMBP, C and D for amoxicillin without TFMBP, E and F for cefuroxime with TFMBP, and G and H for cefuroxime without TFMBP.This was done for all the drugs used.
Table 1 shows materials required per column in each row of wells for MIC determination in the presence of the EPI.
For determination of MICs in the absence of the EPI, 34 µL of make-up volume of sterile phosphate buffer or sterile water (for water soluble antibiotics) was added instead.
Thirty-seven EPA tests were carried out as specified in Table 1.The plates were then incubated at 35 o C for 18 h.after which the optical density (OD) of each well in the plates, were read at 650 nm in a micro-titer plate reader, Spectramax plus (384).

Molecular analysis
The DNA of the isolates were extracted and the genotyping was done by Randomly-Amplified Polymorphic DNA (RAPD)-PCR and the PCR products were analysed using the agarose gel electrophoresis [14].DNA quantification and check for purity was also done as described in literature [14].For each chromosomal DNA sample, a PCR mix of 25 µL containing magnesium chloride (6 %; 1.5 µL), forward and reverse primers (0.8 %; 0.2 µL each) for each of the genes to be screened was used.The PCR was conducted by an initial denaturation at 95 o C for 5 min.followed by 30 cycles of denaturation at 95 o C for 30 sec.
The annealing step was done for 60 sec.at various temperatures to suit each primer pair followed by elongation at 72 o C for 60 sec according to the manufacturer's specification.The final elongation step was done at 72 o C for 10 min.The primers used for the PCR were as listed in Table 2 for Nor-A (efflux), Nor-A (resistance), mecA and luk-Pvl resistant genes.

Data analysis
Data entry, coding, cleaning and analysis were done using Excel 2013 software.Descriptive statistics such as mean, maximum and minimum were summarized.

EPA with TFMBP
Of the 12 MDR isolates tested, nine (75.0 %) gave evidence of EPA against at least one of the tested antibiotics.The only Micrococcus sp.tested showed EPA against three drugs and a 16-fold reduction in MIC for only ciprofloxacin when TFMBP was used (Table 4).

MDR and EPA genes
No MDR and EPA genes were detected in the screened isolates in this study (Figure 2).There were no amplifications for any gene detection from all the primers used such as NorA (Resistance), NorA (efflux) and MecA used in this study.

DISCUSSION
All the ceftriaxone-resistant strains studied were MDR.Some of these were ORSA but were susceptible to sulphamethoxazole/trimethoprim.This proves that sulphamethoxazole/trimethoprim has a good spectrum of activity against S. aureus, including several ORSA strains [15].
The frequency of MRSA among the clinical isolates tested corroborates the findings of Zinn et al in Southern European countries, some parts of the United States, Asia and South Africa [16].Though this frequency is lower than the one recorded by Hong et al [17], from tertiary hospitals in Korea, it is a trend that needs to be surveyed regularly and checked.In this study, not all of the MDR isolates were sensitive to lincomycin, suggesting that lincosamides have a moderate spectrum of activity for S. aureus, including some MRSA while all of the Staphylococcus spp.were found to be resistant to both ampicillin and penicillin suggesting all of the be penicillinase-producing.
However, studies conducted in hospitals in Northern Europe, United States, New Zealand, and Australia recorded a much lower frequency [16] than was observed in this study.This may suggest effective control of antibiotics use compared to our environment, where there is less control.
When compared with this study, Shittu et al [6] recorded a lower percentage (16 %) of ORSA from clinical and non-clinical sources from different parts of Nigeria.They also recorded lower resistance to tetracycline compared with the results from this study.Our results may be evidence of widespread exposure to antibiotics in the hospital environment, suggestive of hospitalacquired resistance traits compared to nonclinical environments.
All of the ORSA strains were confirmed as MDR S. aureus, which were resistant to at least four classes of antibiotics similar to other findings [18].Some of the clinical isolates that turned out to be MDR and ORSA strains may not pose any clinical risk on their own.The fact that they are isolated from the vicinity of infections and around patients pose the danger of dissemination of dangerous genetic traits to less harmful pathogenic strains around them [19].This can lead to complications in therapy of otherwise easily-treatable disease conditions.Therefore, the occurrence of such MDR and ORSA traits in the supposed non-pathogenic organisms cannot be overlooked.
As observed in this study, the EPI (TFMBP), with an intrinsic MIC > 400 µg/mL, is unlikely to have any appreciable antibacterial activity of its own [20].It was found to appreciably lower MICs of up to two or three drugs in some of the MDR isolates tested.This is consistent with other reports [21,22] that a single pump is able to confer resistance to multiple compounds.The efflux pump inhibition in organisms from the various clinical sources tested further underscores the suggestion that the use of such inhibitors, in association with substrate antibiotics, may be useful for increasing both the activity and the range of species for which such drugs may be effective [23].Therefore, it is important to do more research on EPIs in combating MDR strains.This may help in reducing the threat of EPA in resistance mechanisms organisms.It is pertinent to mention that the findings in this study are in support of the prediction that the use of bacterial resistance modifiers, such as EPIs, could facilitate the re-introduction of therapeutically-ineffective antibiotics [22], such as tetracycline, amoxicillin, and others, back into clinical use and might even suppress the emergence of MDR strains, if effectively managed.
In this study, all the S. aureus isolates with phenotypic resistance to oxacillin were tested for the carriage of the mecA, Luk-PV, Nor-A (efflux) and Nor-A (resistance) genes.None of these genes were detected in the tested isolates.This is similar to the result obtained in other works [24] in which mecA gene was not found in the tested MRSAs.Other genes such as the Luk-PV for the PVL gene in the ORSAs were not present in the isolates tested, this is corroborated in the result obtained from other workers in which PVL in MRSA was not isolated but PVL in MSSA was observed [25].
From this study, it may be suggested that MDR Staphylococcus and Micrococcus obtained from clinical isolates should be tested to determine the type of resistance involved.This in turn will be a guide for proper selection of the antibiotics for the treatment regimen.
This study also confirms that clinical isolates from the hospital environment could possess the EPA mechanism of drug resistance which could be tackled with a suitable EPI.Non-detection of Luk-PV and mecA in the MRSA isolates might be due to the absence of the genes in these isolates or that the genes tested may not be indigenous to our environment suggesting the possibility of other genes yet unknown to be responsible for the resistance detected among these isolates.

Figure 1 :
Figure 1: Types of resistance traits exhibited by the clinical isolates tested

Figure 2 :
Figure 2: Agarose gel electrophoresis of NorA(Res), NorA(efflux) and MecA amplicons.Key: M = Molecular size marker (100 bp DNA ladder).The samples were on the lanes labelled with Arabic numerals

Table 1 :
Composition of microtiter wells for MIC determination in the presence of 200 µg/mL of TFMBP

Table 3 :
Antibacterial susceptibility of the clinical isolates

Table 4 :
Summary of EPA findings on MDR isolates with TFMBP (200µg/ml) as EPI

Isolate Source Efflux pump activity with TFMBP (-fold reduction in MIC)
Trifluoro-methyl, benzyl piperazine, EPA: Efflux pump activity, EPI: Efflux pump inhibitor; -Less than 2 fold reduction in MIC; * total number of bacterial strains with reduced EPA