Skip to main content

Inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) with different multi-locus sequence types (MLST), SCCmec, and spa types

A Correction to this article was published on 06 February 2023

This article has been updated

Abstract

Background

The inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) are unclear. The main aim is to better understand of inhibitory activities of vitamins K2, multi-locus sequence typing (MLST), SCCmec, and spa typing in clinical isolates of QR-MRSA on those mutation and gene expressions.

Materials and methods

After collecting S. aureus clinical isolates and detecting QR-MRSA, the genes encoding norA, grlA, grlB, gyrA, and gyrB were sequenced. After treating isolates by vitamin K2, isolates were prepared to measure norA, grlA, grlB, gyrA, and gyrB gene expression. The quantitative-real-time PCR was used to measure the expression of efflux pump genes.

Results

QR-MRSA, MDR, and XDR strains were reported in 59.4%, 73.9%, and 37.6% of isolates, respectability. SCCmecIV (36.5%) and SCCmecV (26.8%) had the highest frequency. Thirty-nine spa types were identified, t021, t044, and t267 types most prevalent in QR-MRSA isolates. ST22 and ST30 dominated the invasive, drug-resistant isolates and QR-MRSA. In 24 h incubated isolates, the most noticeable change of gene expression with vitamin K2 was that the norA, gyrA, and grlB genes were highly repressed. However, the down-regulation of grlA at 24 h after being treated by vitamin K2 was more than another gene. Further, a significant decrease was observed in QR-MRSA-treated isolates compared to un-treated isolates. In other words, norA, grlA, grlB, gyrA, and gyrB genes were less suppressed by QR-MRSA (p ≤ 0.01, p ≤ 0.05).

Conclusion

Vitamin K2 has significant inhibitory effects on the genes responsible for resistance to fluoroquinolone antibiotics. However, a subminimum inhibitory concentration (sub-MIC) level of vitamin K2 was delayed but did not completely inhibit norA, grlA, grlB, gyrA, and gyrB genes in MRSA strains.

Background

One of the most notorious antibiotic resistance bacteria is methicillin-resistant Staphylococcus aureus (MRSA), a significant pathogen causing nosocomial infection [1, 2]. Whereas non-resistant S. aureus usually employs three penicillin-binding proteins (PBPs), including PBPs 1, 2, and 3, to catalyze cross-linking of peptidoglycan, MRSA has an additional PBP, PBP2a encoded by mecA [3, 4].

Ciprofloxacin and ofloxacin were the most extensively used fluoroquinolones to treat quinolone-resistant and methicillin-resistant S. aureus (QR-MRSA) [5]. Resistance to this class of agents occurs by two main processes. The first one, caused by mutations in the target enzymes, lowers the drug’s affinity for the DNA topoisomerase complex [5]. Those mutations occur in the cellular targets GyrA/GyrB of DNA gyrase, encoded by genes gyrA/gyrB and GrlA/GrlB of topoisomerase IV, encoded by genes grlA/grlB [5,6,7]. The second one, caused by overexpression of efflux pumps. Although the function and composition of MDR efflux pumps are relatively conserved in different species, their regulatory mechanisms vary significantly [5, 8].

With the increasing utilization of fluoroquinolones to fight against QR-MRSA, emerging resistance to these agents is growing. Available treatments for QR-MRSA infections are expanding chemical compounds such as herbal extracts, mineral composition, and vitamins [8,9,10].

Vitamin K (K1, K2, K3) plays an essential role in blood coagulation and protein synthesis processes in plasma, kidneys, and other tissues [11]. Moreover, the inhibitory effects of vitamin K2 on various neoplastic cells and reduced risk of mutagenic events in rapid cell proliferation in the fetus and newborn were reported [12, 13]. Nevertheless, the modulation of plasma membrane permeability by lipid-soluble compounds was reported. The precise mechanism of these vitamins on the resistance factors associated with QR-MRSA has not been studied [9, 10, 14].

The effect of DNA gyrase and topoisomerase mutations and gene expressions on minimum inhibitory concentrations (MICs) was studied to understand better inhibitory activities of vitamins K2, multi-locus sequence typing (MLST), SCCmec, and spa typing in clinical isolates of QR-MRSA on those mutations and gene expressions.

Thus, this study aimed to determine the effect of DNA gyrase and topoisomerase mutations on minimum inhibitory concentrations (MICs) of vitamins K2. This purpose was destined for a better understanding of inhibitory activities of vitamins K2, multi-locus sequence typing (MLST), SCCmec, and spa typing in clinical isolates of QR-MRSA on those mutation and gene expressions.

Materials and methods

Design of study and bacterial isolates

In this study, the isolates were collected between June 2019 and August 2020 from 460 clinical samples by diagnostic microbiology laboratories. Isolates were collected from specimens such as pus swabs (ear, nose and eye, cervical and wound), catheter tips, sputum, blood, body fluids, urine, and CSF throughout Hamadan hospitals.

Morphological and biochemical testing was performed to confirm S. aureus. For confirmation of S. aureus isolates, white colonies surrounded by halos from Blood agar (Hi-Media, India) with 5% sheep blood after incubation for 24 h at 37 °C were plated onto mannitol salt agar (Hi-Media, India). Reaction on mannitol salt agar was interpreted and recorded as positive or negative based on criteria described by Mahon et al. [15]. Then, conventional identification methods were used, which included colony morphology, mannitol fermentation, catalase reaction, and coagulase reaction. Finally, 69 isolates of S. aureus were collected from different specimens.

Antibiotic resistance profile and MRSA strains

Antimicrobial resistance tests were performed using the standard Kirby Bauer disk diffusion method as recommended by Clinical Laboratory Standards Institute [16]. Antibiotics were selected from different categories, containing gentamicin (10 μg), erythromycin (15 μg), tetracycline (30 μg), ciprofloxacin (5 μg), gatifloxacin (5 μg), norfloxacin (10 μg), ofloxacin (5 μg), rifampin (5 μg), penicillin (10 unit), clindamycin (2 μg), and linezolid (30 μg). All antibiotic disks belonged to the MAST Company (MAST Inc., U.K.). For detection of MRSA strains, S. aureus isolates were subjected to cefoxitin (30 µg) (MAST Inc., U.K.) sensitivity test by the Kirby Bauer disk diffusion method. Isolates resistant to at least one agent in three or more antimicrobial classes were identified as multidrug-resistant (MDR). Isolates resistant to at least one agent in all but two or fewer antimicrobial classes were considered extensively drug-resistant (XDR). Isolates with non-susceptibility to all agents in all antimicrobial classes were referred to as pan drug-resistant (PDR) [17]. S. aureus ATCC 25923 strain was used as quality control.

Minimum inhibitory concentration (MIC) of ciprofloxacin and vitamin K2

Using an E-test strip (Liofilchem, Italy), minimum inhibitory concentration (MIC) of ciprofloxacin was detected in all isolates. Also, to determine the antibacterial properties of vitamin K2, the microdilution method was used. In this method, MIC was determined based on the method described by Tintino et al. [13]. S. aureus ATCC 25923 strain was used as quality control.

Genomic DNA extraction

For genomic DNA extraction in all S. aureus isolates, the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) was used according to the manufacturer's instructions. The DNA concentration was assessed by spectrophotometry (Lengguang Instrument Co., Ltd., Shanghai, China).

Detection mutation of norA, grlA, grlB, gyrA, and gyrB genes

A C1001 thermal cycler machine (Bio-Rad, Hercules, CA, USA) was used for performing polymerase chain reaction (PCR) assays. The norA, grlA, grlB, gyrA, and gyrB genes were amplified using primers described by Tahmasebi et al. and Sierra et al. The amplification reaction contained 2 µl of template DNA in a final volume of 25 µl containing 0.8 µM for the primers with 12.5 µl of Taq DNA Polymerase Master Mix RED 2 × (Ampliqon, Denmark). The thermocycling conditions were set at 94 °C for 5 min followed by 30 cycles of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 75 s. Finally, ‌a 1.5% agarose gel with an 85 V was used to visualized gene amplification [2, 7]. The PISHGAM company (Tehran, Iran) performed DNA sequencing using the sanger DNA sequencing method. The DNA sequences for the norA, grlA, grlB, gyrA, and gyrB genes in S. aureus R83 and SA74 strain were retrieved from the NCBI database. NCBI Blast (URL: http: //blast.ncbi.nlm.nih.gov/Blast.cgi) was used for multiple sequence alignments.

RNA extraction and cDNA synthesis

The RNA of treated and un-treated (according to MIC value) S. aureus were extracted. Total bacteria RNA was extracted using Ribo-Ex Bacterial RNA purification kit (biotech zone Inc., USA). The Eurex synthesis kits (EURx Inc., USA) were used to synthesize cDNA, following the manufacturer’s instructions.

Real-time PCR reaction conditions

Real-time PCR was conducted to confirm the expression changes in the norA, grlA, grlB, gyrA, and gyrB genes. This experiment was performed according to the procedure reported by Tahmasebi et al. [2]. Following the manufacturer's instructions, the reaction was carried out using a 5 × HOT FIREPol® EvaGreen® qPCR Supermix (Solis BioDyne Inc., USA). All reactions in the experiment were performed in technical triplicates utilizing 96-well plates, where the total reaction volume was set at 20 μl per sample. Thermal cycling conditions were as follows: 95 °C for 1 min; amplification: 40 cycles at 95 °C for 10 s, 60 °C for 1 min for denaturation, annealing/elongation, respectively. Melt curve analysis was performed immediately following each amplification, and thermal cycling conditions were 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s (3% ramps). The samples were run in triplicate. Cycle threshold values were determined with the Step-One-Plus Software v2.3 (ABI Inc., USA). The sensitivity and specificity of primers were determined by the standard and melting curves.

SCCmec and spa typing

SCCmec and spa typing were carried out according to Vafaeefar et al. and Goudarzi et al. [18, 19]. SCCmec I, II, III, IV, and V types were determined based on the amplification pattern obtained. Cluster analysis of spa types was performed using the Ridom Staph Type version 2.2.1 (Ridom GmbH, Würzburg, Germany), a built-in feature of the Staph Type software [20].

Multi-locus sequence typing (MLST)

The MLST scheme published by Tahmasebi et al. [2] was used in the present study. Briefly, 400–450 bp fragments of seven housekeeping genes were amplified by conventional PCR using primers. The PCR conditions were as follows: denaturation at 95 °C for 3 min; 34 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min; followed by a final extension of 72 °C for 10 min. The fragments were then sequenced by Pishgam Biotech Company (Pishgam Company, Tehran, Iran).

The phylogenetic inferences were obtained by MEGA version 6.0 and Interactive Tree of Life V6 (iTOL v6; https://itol.embl.de/) [21]. Sequence alignments were performed using Clustal W with default parameters. All columns in the multiple alignment matrix with more than 80% gaps were eliminated.

Statistical analysis

All statistical analyses were performed using the GraphPad Prism software (version 5; GraphPad Software Inc.; La Jolla, CA, USA. The Chi-square statistical (p) test and Pearson's correlation coefficient (r) were chosen to explore the association between categorical variables. The difference between the amounts of antibiotic resistance and the prevalence of genes in the various media was statistically significant when p < 0.05.

The relative expression levels of the genes (at 6, 12, and 24 h.), compared to calibrator at 0 h incubation, were normalized and determined from the expression of the reference gene. Expression levels of the genes utilizing the ∆∆Ct method (the target gene = 2−ΔΔCq (where ΔCq = Cq (target gene)—Cq (reference gene), and ΔΔCq = ΔCq (test)—ΔCq (calibrator)). The primer efficiency calculations were determined utilizing REST software version 2008 as described by Pfaffl et al. [22, 23]. All statistical analyses were performed using the GraphPad Prism software (version 5; GraphPad Software Inc.; La Jolla, CA, USA), and the Student’s T-test (two-tailed and two-sample) was carried out. The Cq value of the reference gene and the stability of expression were analyzed using a T-test, two-way ANOVA, and Wilcoxon signed-rank test. A variation with a p < 0.05 was considered statistically significant.

Results

Bacterial isolates and MIC of antibiotics

Sixteen-nine (69) S. aureus isolates were collected from a different clinical specimen. The predominant one being blood (n = 21), followed by urine (n = 17), burned wound (n = 16), pus swab (n = 9), and catheter tube tips (n = 6). Of these, 38 were from males, whereas 31 were from females.

Prevalence of antibiotic resistance

Figure 1A presents the antimicrobial susceptibility distribution of S. aureus isolates. S. aureus isolates were predominantly sensitive to linezolid and rifampin (n = 49, % = 71.0) and erythromycin (n = 36, % = 52.1). Higher resistance to penicillin (n = 52, % = 75.3), ciprofloxacin (n = 43, % = 62.3), and clindamycin and (n = 41, % = 59.4) were observed. Also, more than half of the isolates (n = 41, % = 59.4) were resistant to cefoxitin and considered MRSA. The drug resistance patterns of S. aureus isolate for QR-MRSA, MDR, and XDR were 59.4% (n = 41), 73.9% (n = 51), and 37.6% (n = 26), respectively. No PDR phenotype was observed.

Fig. 1
figure 1

Antimicrobial resistance patterns, efflux pump gene profile and SCCmec caste of S. aureus based on disk diffusion (a) and PCR methods (b)

MIC of ciprofloxacin and vitamin K2

According to Fig. 2, 42 isolates (60.8%) with a 4 μg/ml MIC and ciprofloxacin (Fig. 2a) resistance were considered. Also, nine isolates were sensitive to vitamin K2 (Fig. 2b), 21 isolates were intermediate, and others were resistant to vitamin K2. All MRSA strains were entirely resistant to vitamin K2.

Fig. 2
figure 2

Ciprofloxacin and vitamin K2 MICs based on E-test strip (a) and microtitre broth dilution (b) methods

Mutation of norA, grlA, grlB, gyrA, and gyrB genes

Based on Table 1 and Fig. 1, out of 41 QR-MRSA isolates, 21 (51.2%) and 13 (31.7%) isolates were showed a T2460G mutation in grlA and gyrA genes, respectively. Nineteen (46.3%), 23 (56.1%), and 12 (29.2%) isolates were showed an A1578G mutation in grlA, grlB, and gyrB genes, respectively. For grlB, gyrA, and gyrB, 18 (43.9%), 7 (17.0%), and 29 (70.7%) isolates showed a T1497C mutation. Seventeen isolates (41.4%) showed a C2402T mutation GyrA.

Table 1 Mutations of grlA, grlB, gyrA and gyrB and resistance phenotypes of QR-MRSA

Measurement of norA, grlA, grlB, gyrA, and gyrB genes activity

This experiment revealed that vitamin K2 decreased the expressions of norA, grlA, and grlB genes by 30, 54- and 21-fold, respectively, compared to the un-treated isolates. In contrast, the addition of vitamin K2 significantly induced the expression of gyrB, which was down-regulated only by 18- and 12-fold, respectively, relative to the control. All results are shown in Figs. 3 and 4.

Fig. 3
figure 3

Differences in gene expression levels of norA, gyrA, grlA, gyrB and grlB genes in clinical isolates of S. aureus. a norA, gyrA, grlA, gyrB and grlB gene expressions before treatment with vitamin K2 in MRSA, MDR and XDR strains of S. aureus. b norA, gyrA, grlA, gyrB and grlB gene expressions after treatment with vitamin K2 in MRSA, MDR and XDR strains of S. aureus. c norA, gyrA, grlA, gyrB and grlB gene expressions before treatment with vitamin K2 in different types of SCCmec. d norA, gyrA, grlA, gyrB and grlB gene expressions after treatment with vitamin K2 in different types of SCCmec. Experiments performed in triplicate Medians are shown. Statistics: Kruskal–Wallis one-way analysis of variance (ANOVA) with uncorrected Dunn’s test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant

Fig. 4
figure 4

Differences in gene expression levels of norA, gyrA, grlA, gyrB and grlB genes in treated S. aureus isolates with vitamin K2. a norA, gyrA, grlA, gyrB and grlB gene expressions in fluroquinolone resistnt strains. b norA, gyrA, grlA, gyrB and grlB gene expressions after treatment with vitamin K2 in MRSA strains. c norA, gyrA, grlA, gyrB and grlB gene expressions in MDR strains. d norA, gyrA, grlA, gyrB and grlB gene expressions in XDR strains. Experiments performed in triplicate Medians are shown. Statistics: Kruskal–Wallis one-way analysis of variance (ANOVA) with uncorrected Dunn’s test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant

According to Fig. 5, high-expression of gyrA, grlA, and grlB genes was observed in S. aureus isolated from wound and urinary tract infections. Despite this, efflux pump gene expression in bacteria isolated from blood and pus swab showed low-expressions levels.

Fig. 5
figure 5

Differences in gene expression levels of norA, gyrA, grlA, gyrB and grlB genes in un-treated and treated S. aureus isolates with vitamin K2 based on clinical specimens. a norA gene expressions in un-treated S. aureus isolates. b gyrA gene expressions in un-treated S. aureus isolates. c grlA gene expressions in un-treated S. aureus isolates. d gyrB gene expressions in un-treated S. aureus isolates. e grlB gene expressions in un-treated S. aureus isolates. f norA gene expressions in treated S. aureus isolates. g gyrA gene expressions in treated S. aureus isolates. h grlA gene expressions in treated S. aureus isolates. i gyrB gene expressions in treated S. aureus isolates. j grlB gene expressions in treated S. aureus isolates. Experiments performed in triplicate Medians are shown. Statistics: Kruskal–Wallis one-way analysis of variance (ANOVA) with uncorrected Dunn’s test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant

SCCmec and spa typing results

As shown in Figs. 1b and 6, out of 41 MRSA strains, 15 isolates (36.5%) carried SCCmecIV, 11 isolates (26.8%) carried SCCmecV, nine isolates (21.9%) carried SCCmecIII, two isolates (4.8%) carried SCCmecII, and three isolates (7.3%) carried SCCmecI. However, based on spa-typing, t021 (21%), t044 (19%), and t267 (14%) most common types. Also, t021 was reported in 8% of MSSA isolates, and t044 was identified in 23% MRSA strains.

Fig. 6
figure 6

Dendrogram derived from MLST patterns, SCCmec and spa types showing the relatedness of S. aureus isolated in Iran, Hamadan. The cluster analysis was performed using the MEGA 6 software and based on the neighbor-joining algorithm with 1,000 bootstrap replications. The bar indicates 5% sequence diversity. MRSA methicillin-resistant Staphylococcus aureus, MDR multidrug-resistant, XDR extreme drug resistance, GEN gentamycin, ER erythromycin, TET tetracycline, CIP ciprofloxacin, GAT gatifloxacin, NOR norfloxacin, LIZ linezolid, RIF rifampin, OFL ofloxacin, P penicillin, CL clindamycin. A maximum-likelihood tree and MLST analysis, presence of efflux pump genes, and SCCmec caste. Red indicator: antibiotic-resistant, green indicator: antibiotic sensitive, and yellow indicator: semi-resistant

MLST results

The MLST results are summarized in Fig. 6. Five sequence types (ST5, ST8, ST22, and ST30) were represented by at least two isolates, each that had been assigned different SCCmec types. Combining ST and the SCCmec type and spa types, 11 different genotypes were identified in our area. Type IV SCCmec was present in 9 STs; ST524, ST766, ST125, ST02, ST200, ST22, ST08, ST05, and ST30. Also, Type V SCCmec was found in 8 different STs: ST220, ST111, ST500, ST303, ST08, ST22, ST30, and ST05.

Type t044 spa was present in 9 STs; ST146, ST76, ST05, ST174, ST500, ST30, ST08, ST22, and ST02. Type t037 and t030 spa were present in 11 (ST02, ST1037, ST600, ST22, ST229, ST44, ST524, ST125, ST111, ST220, and ST30) and 6 (ST839, ST148, ST303, ST22, ST08, and ST30) different STs, respectively.

Statistical analysis results

The statistical analyses of the present study are shown in Table 1. Comparing both un-treated and treated isolates showed a significant correlation between vitamin K2 and antibiotic resistance patterns. In other words, the MIC of vitamin K2 showed a significant difference in MDR, XDR, and antibiotic-sensitive isolates (p < 0.05). Based on χ2 and t-test, a significant association was reported between MIC of vitamin K2 and SCCmec type. Further, a strong correlation between the prevalence of norA, grlA, grlB, gyrA, and gyrB genes and MIC of vitamin K2 was observed (p < 0.05) (p < 0.001). However, a negative correlation between spa typing and MIC of vitamin K2 was observed in this study (p > 0.05).

Based on Figs. 4 and 5, a significant correlation was reported between the expression of norA, grlA, grlB, gyrA, and gyrB genes and resistance to the antibiotic. A clear correlation was observed between norA, grlA, grlB, gyrA, and gyrB genes expression SCCmec typing. Thus, the expression of norA, grlA, grlB, gyrA, and gyrB genes showed a significant decrease in MRSA and MSSA strains (p > 0.001). Still, no good correlation was observed between norA, grlA, grlB, gyrA, gyrB expression levels, and spa typing (p > 0.001). Also, Table 2 and Fig. 5 show a significant association between the clinical specimens and the expression of efflux pump genes.

Table 2 Pearson (r) and χ2 (p) correlation between antibiotic resistance, efflux pump gene expression profile, vitamin K2 and molecular typing in of S. aureus isolates

Discussion

Among the 69 S. aureus isolates obtained in this study, 30.4% were collected from blood, 24.6% from urine, 23.1% from the burned wound, 13% from pus swap, and 8.6% from the catheter. In a study conducted by Kot et al. [24], a high prevalence of blood and wound infection by S. aureus was reported. Most studies have demonstrated that there is a significant association between antibiotic resistance patterns and clinical specimens. In wound infections, the bacteria are more resistant to treatment, consistent with the current study [2, 8].

Most isolates were resistant to penicillin (75.3%) and ciprofloxacin (62.3%). Also, 52.1% and 20.2% of the isolate were MDR and XDR. This observation agrees with Cabrera et al. and Kot et al., similar to that investigated in this study [24, 25]. These findings are in contrast to the data reported from Singapore [26], Nepal [27], and the United States [28].

In the present study, based on QR-MRSA MIC, 2409C, T2460G, T1497C most common mutation in norA, grlA, grlB, gyrA, and gyrB genes. The observations also agree with the results reported by Hassanzadeh et al. [29] and Hashem et al. [30]. SCCmec typing for MRSA isolates showed the predominance of SCCmec type IV (63.4%) followed by type V (56%), type II (53.6%), type III (46.3%), and type I (31.7%). A similar pattern of results was observed in the study of Taherikalani et al. [31]. Moreover, these results were essentially confirmed by some studies from Saudi Arabia [32], Iraq [1], and South Africa [33], which stated that SCCmec IV and V were the most dominant types.

However, spa typing in the present study indicated that t030, t044, and t037 were the most common types, and t267 was a unique type in MRSA strains. In this study, the diversity of spa types in MRSA was more extensive than previously found in S. aureus in Iran [34, 35]. A high prevalence of t044 (31.7%) was detected in MRSA. This finding was also reported in Kuwait and [36], Iran [37] as well as in Europe [38, 39]. On the other hand, in the MSSA strains, t044, t037, and t030 were the most prevalent spa-types, which was not comparable with the findings of Satta et al. [40] and Mazi et al. [41].

Quantitative real-time PCR results showed that norA, grlA, and grlB genes were down-regulated in MSSA after treatment with vitamin K2. Generally, in MRSA strains, at 6 h, norA, grlA, were down-regulated; at 12 h, it was up-regulated, and at 24 h, it was down-regulated. Surprisingly after treating MRSA and MSSA with vitamin K2, the grlB and gyrA gene was up-regulated at 6 h and down-regulated at 12 h (−2.737). In MDR/MRSA strains, the gyrB gene was also up-regulated at 12 h and down-regulated at 24 h (−0.737). This study's results seem to correlate with Tintino et al. [13], where norA was down-regulated at 12 h. The results obtained here may have implications for understanding that methicillin resistance plays a critical role in the function of vitamin K2. However, it can be said that MRSA strains showed more significant changes in the resistance due to the effect of vitamin K2.

According to Table 1 and Fig. 5, the effect of vitamin K2 also showed a significant difference in the clinical specimen types. Different changes in norA, grlA, grlB, and gyrA activity gene expression were obtained in strains isolated from urine culture. The most crucial reason for the difference in vitamin K2 effect on clinical isolates is the typical and high consumption of fluoroquinolones to cure urinary tract infection infections. Hence, special attention should be paid to the isolates' source to inhibit efflux pumps in staphylococcal infections. We found agreement when comparing our observations with results from Brazil [14, 42], Germany [12], and Norway. Our study’s findings show that gene expression increases significantly in blood isolates than in urine and wound isolates; however, some strains are susceptible to fluoroquinolones. Therefore, some studies have demonstrated some effectors on efflux pumps, such as the type of clinical specimens, which should be considered in clinical and laboratory investigations [9, 11, 43].

Tintino et al. [14] and Harakeh et al. [44] confirmed that some fat-soluble vitamins could increase antibiotic penetration in drug-resistant strains. They also suggested that some natural vitamins have a high effect on reducing the activity of β-lactamase enzymes. However, our result indicated the vitamin K2 significantly down-regulated norA, grlA, grlB, gyrA genes giving fold changes of -1.022, -2.611, -1.891, -1.936, and -3.442 at 6, 12, and 24 h. On the other hand, the expression pattern of norA, grlA, grlB, gyrA genes in both MRSA and MSSA was completely different from the control sample.

The current study, Fig. 3, shows that norA, grlA, grlB, and gyrA were down-regulated in SCCmec type IV, V, and III after treatment with vitamin K2. Also, the pattern of expression of norA, grlA, and gyrA in isolates carrying SCCmec IV and V appears very different from that of isolates carrying SCCmec I and II genes. However, in the expression pattern observed before treatment of isolates, norA, grlA, grlB, gyrA genes in all SCCmec types at 24 h were down-regulated. Although similar studies in norA, grlA, grlB, gyrA activity are not available, Choi et al. [45], Yuan et al. [10], and Qu et al.[46] showed the antimicrobial effect of vitamin K on MRSA strains. They also found that vitamin K had different functions in MRSA and drug-sensitive strains. These findings provide further evidence that, during the stationary phase, expression of grlA, grlB, and gyrA decreased by sevenfold in treated MSSA strains in laboratory conditions, independent of the vitamin k2, in fluoroquinolones resistance. This suggested additional regulatory mechanisms for this resistance [9].

Consistent with the findings of other studies [40, 41], we found that t037 and t044 are the essential spa types in MRSA, MDR, and XDR strains. Thus, it was further observed that after the vitamin K2 treatment of S. aureus with t037 and t044 spa typing, all genes except the gyrB were down-regulated in the strains giving fold changes of −1.120 (p = 0.01), −2.690, −1.999 (p = 0.02) and −0.120, −0.152 and 0.251 (p = 0.22) after 12 and 24 h. The grlA, grlB, and gyrA genes were also down-regulated in response to vitamin K2, with an increased expression between 3.0 and 4.2 log2-fold in isolates with spa typing t021, t267, and t030. The norA gene was also significantly down-regulated with a 2.2 log2-fold change in t267 and t030 types.

Finally, our knowledge from the present study confirmed the inhibitory effect of vitamin K2 on the S. aureus efflux pump. Previous studies showed the inhibitory function of vitamin K2 on the norA gene (14). However, we found that fat-soluble vitamins are among the best options for inhibiting fluoroquinolone efflux pumps genes (grlA, grlB, and gyrA) in S. aureus. In the function of vitamin K2 on different strains of S. aureus, special attention should be paid to the type of clinical specimen and drug resistance. Another important factor that accounts for the survival of such mutants in the environment is microbial fitness. NorA-mediated resistance has been described in the apparent absence of mutations in topoisomerase genes. Indigenous microbial populations are made up of different communities of the same bacteria, which co-exist and compete for nutrition, space, and growth factors.

Conclusions

Based on the evidence obtained from the present study, vitamin K2 had a good effect in inhibiting puffy pumps’ activity in S. aureus. It was also found that the function of vitamin K2 is significantly different in MRSA and MSSA strains. Significant differences in the expression of norA, grlA, grlB, and gyrA in different SCCmec types identified this locus's role in the function of vitamin K2. We also found that vitamin K2 was a good option for inhibiting MDR and XDR strains. It should be noted that different types of SCCmec, antibiotic resistance patterns, and clinical specimen types are the essential variables in the treatment of clinical isolates of S. aureus with vitamin K2.

Availability of data and materials

The data can be accessible to the interested researchers by the corresponding authors on reasonable request.

Change history

Abbreviations

MRSA:

Methicillin-resistant Staphylococcus aureus

MSSA:

Methicillin-susceptible Staphylococcus aureus

MDR:

Multidrug-resistant

XDR:

Extensively drug-resistant

SCCmec :

Staphylococcal cassette chromosome mec

MLST:

Multi-locus sequence typing

MIC:

Minimal inhibitory concentration

ST:

Sequence type

CC:

Clonal complex

References

  1. Rasheed NA, Hussein NR. Methicillin-resistant Staphylococcus aureus carriage rate and molecular characterization of the staphylococcal cassette chromosome mec among Syrian refugees in Iraq. Int J Infect Dis. 2020;91:218–22. https://doi.org/10.1016/j.ijid.2019.12.006.

    Article  CAS  Google Scholar 

  2. Tahmasebi H, Dehbashi S, Jahantigh M, Arabestani MR. Relationship between biofilm gene expression with antimicrobial resistance pattern and clinical specimen type based on sequence types (STs) of methicillin-resistant S aureus. Mol Biol Rep. 2020;47:1309–20. https://doi.org/10.1007/s11033-019-05233-4.

    Article  CAS  Google Scholar 

  3. Rasheed NA, Hussein NR. Characterization of different virulent factors in methicillin-resistant Staphylococcus aureus isolates recovered from Iraqis and Syrian refugees in Duhok city. Iraq PLOS ONE. 2020;15:e0237714. https://doi.org/10.1371/journal.pone.0237714.

    Article  CAS  Google Scholar 

  4. Heydari N, Alikhani MY, Jalilian FA, Tahmasebi H, Arabestani MR. Evaluation of real time PCR for detection of clinical isolates of Staphylococcus aureus and methicillin-resistance strains based on melting curve analysis method. Koomesh. 2017;19:877–86.

    Google Scholar 

  5. Hassanzadeh S, Ganjloo S, Pourmand MR, Mashhadi R, Ghazvini K. Epidemiology of efflux pumps genes mediating resistance among Staphylococcus aureus; a systematic review. Microb Pathog. 2020;139:103850. https://doi.org/10.1016/j.micpath.2019.103850.

    Article  CAS  Google Scholar 

  6. Schmitz F-J, Jones ME, Hofmann B, et al. Characterization of grlA, grlB, gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC. Antimicrob Agents Chemother. 1998;42:1249–52.

    Article  CAS  Google Scholar 

  7. Sierra JM, Marco F, Ruiz J, Jiménez de Anta MT, Vila J. Correlation between the activity of different fluoroquinolones and the presence of mechanisms of quinolone resistance in epidemiologically related and unrelated strains of methicillin-susceptible and -resistant Staphylococcus aureus. Clin Microbiol Infect. 2002;8:781–90. https://doi.org/10.1046/j.1469-0691.2002.00400.x.

    Article  CAS  Google Scholar 

  8. Zahedani SS, Tahmasebi H, Jahantigh M. Coexistence of virulence factors and efflux pump genes in clinical isolates of Pseudomonas aeruginosa: analysis of biofilm-forming strains from Iran. Int J Microbiol. 2021. https://doi.org/10.1155/2021/5557361.

    Article  Google Scholar 

  9. Paiva SA, Sepe TE, Booth SL, et al. Interaction between vitamin K nutriture and bacterial overgrowth in hypochlorhydria induced by omeprazole. Am J Clin Nutr. 1998;68:699–704. https://doi.org/10.1093/ajcn/68.3.699.

    Article  CAS  Google Scholar 

  10. Ganjun Yuan XZ, Li P, Zhang Q, Cao J. New activity for old drug: in vitro activities of vitamin K3 and menadione sodium bisulfite against methicillin-resistant Staphylococcus aureus. Afr J Pharm Pharmacol. 2014;18:451–4. https://doi.org/10.5897/AJPP2013.3903.

    Article  CAS  Google Scholar 

  11. Sakamoto N, Nishiike T, Iguchi H, Sakamoto K. The effect of diet on blood vitamin K status and urinary mineral excretion assessed by a food questionnaire. Nutr Health. 1999;13:1–10. https://doi.org/10.1177/026010609901300101.

    Article  CAS  Google Scholar 

  12. Minnja SH, Soraya M, Stefan B, Markus MH. Vitamin E as promising adjunct treatment option in the combat of infectious diseases caused by bacterial including multi-drug resistant pathogens—results from a comprehensive literature survey. Eur J Microbiol Immunol. 2020;10:193–201. https://doi.org/10.1556/1886.2020.00020.

    Article  CAS  Google Scholar 

  13. Tintino SR, Souza VCAd, Silva JMAd, et al. Effect of Vitamin K(3) inhibiting the function of NorA efflux pump and its gene expression on Staphylococcus aureus. Membranes. 2020;10:130. https://doi.org/10.3390/membranes10060130.

    Article  CAS  Google Scholar 

  14. Tintino SR, Oliveira-Tintino CDM, Campina FF, et al. Vitamin K enhances the effect of antibiotics inhibiting the efflux pumps of Staphylococcus aureus strains. Med Chem Res. 2018;27:261–7. https://doi.org/10.1007/s00044-017-2063-y.

    Article  CAS  Google Scholar 

  15. Connie R, Mahon DCL, Manuselis G Jr. Textbook of diagnostic microbiology. 5th ed. Philadelphia, USA: Saunders; 2014.

    Google Scholar 

  16. CLSI. Performance standards for antimicrobial susceptibility testing: 30nd informational supplement CLSI M100–S30. Wayne, PA: CLSI; 2020.

    Google Scholar 

  17. Alkofide H, Alhammad AM, Alruwaili A, et al. Multidrug-resistant and extensively drug-resistant enterobacteriaceae: prevalence, treatments, and outcomes—a retrospective cohort study. Infect Drug Resist. 2020;13:4653–62. https://doi.org/10.2147/IDR.S283488.

    Article  Google Scholar 

  18. Vafaeefar M, Yousef Alikhani M, Tahmasebi H, Arabestani MR. Identification and determination of the relationship between ccr alleles and antibiotic resistance in clinical isolates of methicillin resistant Staphylococcus aureus. J Babol Univ Med Sci. 2017;19:28–35.

    Google Scholar 

  19. Goudarzi M, Seyedjavadi SS, Azad M, Goudarzi H, Azimi H. Distribution of spa types, integrons and associated gene cassettes in Staphylococcus aureus strains isolated from intensive care units of hospitals in Tehran. Iran Arch Clin Infect Dis. 2016;11:e38813. https://doi.org/10.5812/archcid.38813.

    Article  Google Scholar 

  20. Mellmann A, Weniger T, Berssenbrügge C, et al. Based Upon Repeat Pattern (BURP): an algorithm to characterize the long-term evolution of Staphylococcus aureus populations based on spa polymorphisms. BMC Microbiol. 2007;7:98. https://doi.org/10.1186/1471-2180-7-98.

    Article  CAS  Google Scholar 

  21. Novovic K, Mihajlovic S, Vasiljevic Z, Filipic B, Begovic J, Jovcic B. Carbapenem-resistant Acinetobacter baumannii from Serbia: revision of CarO classification. PLoS ONE. 2015;10:e0122793–e0122793. https://doi.org/10.1371/journal.pone.0122793.

    Article  CAS  Google Scholar 

  22. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45. https://doi.org/10.1093/nar/29.9.e45.

    Article  CAS  Google Scholar 

  23. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002;30:e36–e36.

    Article  Google Scholar 

  24. Kot B, Wierzchowska K, Piechota M, Grużewska A. Antimicrobial resistance patterns in methicillin-resistant Staphylococcus aureus from patients hospitalized during 2015–2017 in hospitals in Poland. Med Princ Pract. 2020;29:61–8. https://doi.org/10.1159/000501788.

    Article  Google Scholar 

  25. Cabrera R, Fernández-Barat L, Motos A, et al. Molecular characterization of methicillin-resistant Staphylococcus aureus clinical strains from the endotracheal tubes of patients with nosocomial pneumonia. Antimicrob Resist Infect Control. 2020;9:43. https://doi.org/10.1186/s13756-020-0679-z.

    Article  Google Scholar 

  26. Shankar N, Soe PM, Tam CC. Prevalence and risk of acquisition of methicillin-resistant Staphylococcus aureus among households: a systematic review. Int J Infect Dis. 2020;92:105–13. https://doi.org/10.1016/j.ijid.2020.01.008.

    Article  CAS  Google Scholar 

  27. Arjyal C, Kc J, Neupane S. Prevalence of methicillin-resistant Staphylococcus aureus in Shrines. Int J Microbiol. 2020. https://doi.org/10.1155/2020/7981648.

    Article  Google Scholar 

  28. See I, Mu Y, Albrecht V, et al. Trends in incidence of Methicillin-resistant Staphylococcus aureus bloodstream infections differ by strain type and healthcare exposure, United States, 2005–2013. Clin Infect Dis. 2019;70:19–25. https://doi.org/10.1093/cid/ciz158.

    Article  CAS  Google Scholar 

  29. Hassanzadeh S, Mashhadi R, Yousefi M, Askari E, Saniei M, Pourmand MR. Frequency of efflux pump genes mediating ciprofloxacin and antiseptic resistance in methicillin-resistant Staphylococcus aureus isolates. Microb Pathog. 2017;111:71–4. https://doi.org/10.1016/j.micpath.2017.08.026.

    Article  CAS  Google Scholar 

  30. Hashem RA, Yassin AS, Zedan HH, Amin MA. Fluoroquinolone resistant mechanisms in methicillin-resistant Staphylococcus aureus clinical isolates in Cairo. Egypt J Infect Dev Ctries. 2013;7:796–803. https://doi.org/10.3855/jidc.3105.

    Article  CAS  Google Scholar 

  31. Taherikalani M, Mohammadzad MR, Soroush S, et al. Determining the prevalence of SCCmec polymorphism, virulence and antibiotic resistance genes among methicillin-resistant Staphylococcus aureus (MRSA) isolates collected from selected hospitals in west of Iran. J Chemother. 2016;28:104–9. https://doi.org/10.1179/1973947815Y.0000000018.

    Article  CAS  Google Scholar 

  32. Albarrag A, Shami A, Almutairi A, Alsudairi S, Aldakeel S, Al-Amodi A. Prevalence and molecular genetics of methicillin-resistant Staphylococcus aureus colonization in nursing homes in Saudi Arabia. Can J Infect Dis Med Microbiol. 2020. https://doi.org/10.1155/2020/2434350.

    Article  Google Scholar 

  33. Singh-Moodley A, Lowe M, Mogokotleng R, Perovic O. Diversity of SCCmec elements and spa types in South African Staphylococcus aureus mecA-positive blood culture isolates. BMC Infect Dis. 2020;20:816. https://doi.org/10.1186/s12879-020-05547-w.

    Article  CAS  Google Scholar 

  34. Hashemizadeh Z, Bazargani A, Kalantar-Neyestanaki D, Mohebi S, Hadi N. Determining spa-type of methicillin-resistant Staphylococcus aureus (MRSA) via high-resolution melting (HRM) analysis, Shiraz. Iran BMC Res Notes. 2020;13:97. https://doi.org/10.1186/s13104-020-04948-z.

    Article  CAS  Google Scholar 

  35. Moosavian M, Baratian Dehkordi P, Hashemzadeh M. Characterization of SCCmec, Spa types and multidrug resistant of methicillin-resistant Staphylococcus aureus isolates in Ahvaz. Iran Infect Drug Resist. 2020;13:1033–44. https://doi.org/10.2147/idr.s244896.

    Article  CAS  Google Scholar 

  36. Boswihi SS, Udo EE, AlFouzan W. Antibiotic resistance and typing of the methicillin-resistant Staphylococcus aureus clones in Kuwait hospitals, 2016–2017. BMC Microbiol. 2020;20:314. https://doi.org/10.1186/s12866-020-02009-w.

    Article  CAS  Google Scholar 

  37. Goudarzi M, Razeghi M, Chirani AS, Fazeli M, Tayebi Z, Pouriran R. Characteristics of methicillin-resistant Staphylococcus aureus carrying the toxic shock syndrome toxin gene: high prevalence of clonal complex 22 strains and the emergence of new spa types t223 and t605 in Iran. New Microbes New Infect. 2020;36:100695. https://doi.org/10.1016/j.nmni.2020.100695.

    Article  CAS  Google Scholar 

  38. Mairi A, Touati A, Lavigne JP. Methicillin-resistant Staphylococcus aureus ST80 clone: a systematic review. Toxins (Basel). 2020. https://doi.org/10.3390/toxins12020119.

    Article  Google Scholar 

  39. Strommenger B, Kettlitz C, Weniger T, Harmsen D, Friedrich AW, Witte W. Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing. J Clin Microbiol. 2006;44:2533–40. https://doi.org/10.1128/jcm.00420-06.

    Article  CAS  Google Scholar 

  40. Satta G, Ling CL, Cunningham ES, McHugh TD, Hopkins S. Utility and limitations of Spa-typing in understanding the epidemiology of Staphylococcus aureus bacteraemia isolates in a single University Hospital. BMC Res Notes. 2013;6:398. https://doi.org/10.1186/1756-0500-6-398.

    Article  CAS  Google Scholar 

  41. Mazi W, Sangal V, Sandstrom G, Saeed A, Yu J. Evaluation of spa-typing of methicillin-resistant Staphylococcus aureus using high-resolution melting analysis. Int J Infect Dis. 2015;38:125–8. https://doi.org/10.1016/j.ijid.2015.05.002.

    Article  Google Scholar 

  42. Andrade JC, Morais Braga MFB, Guedes GMM, et al. Menadione (vitamin K) enhances the antibiotic activity of drugs by cell membrane permeabilization mechanism. Saudi J Biol Sci. 2017;24:59–64. https://doi.org/10.1016/j.sjbs.2015.09.004.

    Article  CAS  Google Scholar 

  43. Fusaro M, Gallieni M, Rizzo MA, et al. Vitamin K plasma levels determination in human health. Clin Chem Lab Med. 2017;55:789–99. https://doi.org/10.1515/cclm-2016-0783.

    Article  CAS  Google Scholar 

  44. Harakeh S, Azhar E, Almasaudi S, et al. Effects of a specific nutrient combination on ESBL resistance. Saudi J Biol Sci. 2019;26:1576–80. https://doi.org/10.1016/j.sjbs.2018.10.013.

    Article  CAS  Google Scholar 

  45. Choi S-r, Frandsen J, Narayanasamy P. Novel long-chain compounds with both immunomodulatory and MenA inhibitory activities against Staphylococcus aureus and its biofilm. Sci Rep. 2017;7:40077. https://doi.org/10.1038/srep40077.

    Article  CAS  Google Scholar 

  46. Qu D, Hou Z, Li J, et al. A new coumarin compound DCH combats methicillin-resistant Staphylococcus aureus biofilm by targeting arginine repressor. Sci Adv. 2020;6:eaay9597. https://doi.org/10.1126/sciadv.aay9597.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This article's authors are grateful to Hamadan University of Medical Sciences for their financial support in conducting the research. This work was supported by a research grant from Hamadan University of medical sciences (Grant/Award Number: 9510075757).

Author information

Authors and Affiliations

Authors

Contributions

NKP, SD, and BZ performed the tests, collected and analyzed the data, and performed the data analysis. HT and SD contributed to design and project admiration. MRA designed the project and contributed to all the steps of the project. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Mohammad Reza Arabestani.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Hamadan University of Medical Sciences, and also, about the clinical samples, consent was taken by the ethics committee of Hamadan University of Medical Sciences (Code No: IRUMSHA. REC. 1395.757). We confirm that all methods were performed following the relevant guidelines and regulations.

Competing interests

All authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The original online version of this article was revised: The author’s affiliation which was incorrectly published in the original version has been corrected.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pasandideh, N.K., Tahmasebi, H., Dehbashi, S. et al. Inhibitory activities of vitamins K2 against clinical isolates of quinolone-resistant and methicillin-resistant Staphylococcus aureus (QR-MRSA) with different multi-locus sequence types (MLST), SCCmec, and spa types. Eur J Med Res 27, 295 (2022). https://doi.org/10.1186/s40001-022-00939-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40001-022-00939-x

Keywords

  • Methicillin-resistant Staphylococcus aureus
  • Fluoroquinolone
  • Drug resistance
  • Gene expression
  • Vitamin K
  • Multi-locus sequence typing