View of Characterization of Vancomycin Resistant Staphylococcus Aureus Isolated from Outpatients in Iraq

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Characterization of Vancomycin Resistant Staphylococcus Aureus Isolated from Outpatients in Iraq

Khairallah AS Mohammed, Amel K Yaqoob and Zahraa H Abdulkareem

Department of Medical Lab Technology, College of Health and Medical Technology, Southern Technical University, Basrah, Iraq

Running title: VRSA among CA-MRSA

Corresponding author;

Khairallah A S Mohammed Telephone number: +964 781 752 1597

[email protected]

Abstract

Increasing the prevalence of vancomycin resistant Staphylococcus aureus (VRSA) among the community and in healthcare settings has become a global public health concern. This study aimed to investigate the prevalence and antimicrobial susceptibilities of VRSA among outpatients. Seventy-nine isolates of methicillin-resistant S. aureus were collected from 380 various clinical specimens. Antimicrobial susceptibilities were investigated by disk diffusion method and vancomycin MIC was determined by agar dilution method. Molecular characterization of tested isolates was performed by PCR technique to detect the mecA and vanA/vanB genes. The results showed the highest resistance to cefoxitin (79 isolates, 100%), augmentin (73 isolates, 92.4%) and moderate resistance to erythromycin (42 isolates, 53.2%), tetracycline (40 isolates, 50.6%) and clindamycin (30 isolates, 38%). While high susceptibility was found to imipenem, gentamicin and vancomycin which had 78.67%, 82.66% and 91%, respectively. Two strains were resistant to vancomycin (MIC 32 mg/L), and six strains were intermediate to vancomycin (MIC 2 – 4 mg/L). All tested isolates were found positive for mecA but none of them have demonstrated vanA/vanB gene by PCR. The study concluded that the emergence of vancomycin resistance in MRSA from outpatients portends the health authorities to detect and identify these organisms and to find an alternative treatment.

Key Words: PCR, antibiotic resistance, vancomycin-resistant Staphylococcus aureus, Iraq.

Introduction

S aureus is one of the most common microbes associated with nosocomial community-acquired infections, being one of the five most common pathogens to reside in the skin and nasal flora,

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occurring in roughly 25–30% of all healthy individuals (Kuroda et al. 2001; Tong et al. 2015;

David et al. 2010). It can cause a range of repercussions from mild to severely life-threatening infections such as wound infections, abscess formation endovascular diseases, toxic shock syndrome (TSS) and staphylococcal scalded skin syndrome (SSSS) (David et al. 2010).

Treatment of these infections has become more difficult because of the emergence of methicillin (MRSA) and vancomycin resistant (VRSA) strains (Mathews et al. 2010). Existence of MRSA infections and irrational use of Glycopeptides, especially vancomycin, led to the emergence of VRSA phenotype among MRSA (Tenover et al. 2001; 12 Perwaiz et al. 2007). Vancomycin- resistant Staphylococcus aureus (VRSA) strains may harbor different vancomycin resistance genes such as vanA and vanB gene (lark et al. 2005), which can be acquired via plasmid from enterococci (Khanam et al. 2016]. Also, vancomycin resistance phenotype could be due to the thickness of cell wall or overproduction of D-ala-D-ala caused by a sequential mutation (Billot- Klein et al. 1996, Sieradzki and Tomasz, 1999). Initially, the emergence of vancomycin- intermediate Staphylococcus aureus was reported from Japan in the year 1997 (Hiramatsu et al.

1997), then followed by the first existence of vancomycin-resistant S. aureus in the United States in 2002 (CDC, 2002). Thereafter, several reports of vancomycin resistance emerging were raised from throughout the world (Shariati et al. 2020).

In Iraq, limited information is available regarding the occurrence and antimicrobial profiles of methicillin-resistant S. aureus (MRSA) and Vancomycin-resistant Staphylococcus aureus (VRSA), in particular those associated with community infections. It has been reported that the incidence of MRSA has substantially increased over time in Iraq (Kareem et al. 2015; Kareem et al. 2020). Data from homegrown antimicrobial resistance in S. aureus could be beneficial and have important implications for controlling and managing MRSA infections. Our study aimed to investigate the prevalence andthe antimicrobial profile of vancomycin-resistant Staphylococcus aureus (VRSA) isolated from outpatients or patients upon admission into hospitals.

Material and Methods

A total of 79 MRSA isolates were isolated from 380 clinical samples over a period of 25 months from October 2018 to December 2020. They were isolated from urine samples, tonsil swabs, nasal swabs, wound swabs, burn swabs, blood samples, vagina swabs, and sputum. Specimens were collected from outpatients or patients upon admission into hospital in South of Iraq.

Patients who were recently admitted hospital, have recent surgical operation, on hemodialysis, or have intravenous cannula at time of swab taking were excluded.

The S. aureus clinical isolates were initially identified using standard microbiological techniques (Gaillot et al. 2000; Merlino et al. 2000), and then this identification was confirmed by PCR using species-specific 16S rRNA primer pairs. MRSA strains were detected by using cefoxitin (30 µg) disk diffusion and amplification of mecA gene (Skov et al. 2003, Kuroda et al. 2001).

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Antibiotic susceptibility test

Antibiotic susceptibility testing was carried out on Mueller-Hinton agar (Oxoid Limited, Hampshire, England) using the Kirby-Bauer disk diffusion method, according to the recommendations by the Clinical and Laboratory standard Institute (CLS, 2018). The discs of antibiotics (Mast Group, UK) including cefoxitin (30 µg), augmentin (30 µg), clindamycin (2 µg), erythromycin (15 µg), gentamicin (10 µg), imipenem (10 µg), tetracycline (30 µg) and vancomycin (30 µg).

Determination of vancomycin MIC

Vancomycin MIC was determined by agar dilution method according to the procedures recommended by Clinical Laboratory and Standard Institute (CLSI, 20187). Antibiotic dilutions were prepared as described by Andrews (2001). Range of dilutions (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25. 0 mg / L) were prepared in Muller Hinton agar (Andrews, 2001). The density of S.

aureus inoculum was adjusted to 0.5 McFarland. The Muller Hinton agar plates were spot inoculated with the test MRSA isolates and incubated at 37°C for 18 hours. Results were interpreted as the presence or absence of growth, where MIC of a strain was determined based on the lowest concentration of the antibiotic at which there was no visible growth.

Amplification of 16S rRNA, Methicillin, and Vancomycin Resistance Genes

The total DNAs were extracted by using a commercial Kit GeneAll (South Korea). The extracted DNAs were then subjected to simplex PCR to amplify a 228 bp region of the 16S rRNA gene fragment of S. aureus, which is highly conserved in the species, and to detect mecA, Van A and B genes by using specific primers (Table 1). The reaction mix had a final volume of 25 µl consisting of 2 µl (50-100 ng) DNA, 1 µl (20 pmol) of each primer, 12.5 µl of master mix (Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers; Promega) and 8.5 µl of nuclease free water. The PCR amplification program was as follows: an initial denaturation step at 94ôC for 4 min followed by 35 cycles of denaturation at 94ôC for 1 min, an annealing step at 56ôC for 1 min (annealing at 60 ◦C for 1 min for vanA and vanB), and an extension step at 72ôC for 2 min, followed by a final extension at 72ôC for 10 min. The amplified products were then visualized by agarose gel electrophoresis.

Table 1. Primers used in this study.

Primer Primer sequencing 5’ to 3’ Size (bp) References

16S rRNA F: GTA GGT GGC AAG CGT TAT CC R: CGCACATCAGCGTCAG

228 Geha, et al.

(1994) MecA MecA1 GTA GAA ATG ACT GAA CGT CCG ATAA

MecA2 CCA ATT CCA CAT TGT TTC GGT CTAA

310 Monday and Bohach 1999

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VanA F: CAT GAA TAG AAT AAA AGT TGC AAT A R: CCC CTT TAA CGC TAA TAC GAT CAA

1,030 Even et al.

(1993) VanB F: GTG ACA AAC CGG AGG CGA GGA

R: CCG CCA TCC TCC TGC AAA AAA

433 Handwerger, et al.

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Results

Seventy-nine MRSA isolates were collected from 380 clinical samples. All MRSA isolates (n=

79) were phenotypically identified as S. aureus, and this identification was confirmed by PCR using species-specific primers (Table 1). All the MRSA (100%) exhibited positive results for the 16S rRNA gene and possessed the mecA gene (Fig 1). All the isolates 79 (100%) strains were resistant to cefoxitin. The source distribution of MRSA isolates was as follows: tonsil 22 (27.5%), urine 23 (28.8), nasal 12 (15%) wound 11 (13.8%), while burn 6 (7.5%), blood 2 (2.5%), vagina 2 (2.5%), and sputum 1 (1.3%).

Figure 1. The simplex PCR was performed, and products were separated on an agarose gel. Lane 1 and 8, marker; lane 2 - 6, MRSA positive with the 310-bp mecA amplicons.

Antimicrobial susceptibility The MRSA isolates showed the highest resistance to cefoxitin (79 samples, 100%), augmentin (73 samples, 92.4%) and moderate resistance to erythromycin (42 samples, 53.2%), tetracycline (40 samples, 50.6%) and clindamycin (30 sample, 38%). While high susceptibility was found to imipenem, gentamicin and vancomycin which had 78.67%, 82.66% and 91%, respectively.

All 79 (100%) MRSA isolates showed multiple antibiotic-resistance patterns (resistant to three or more antibiotics).

Antibiotic Susceptibility Pattern of VRSA/VISA Isolates

The vancomycin-resistant phenotype of these isolates (VRSA / VISA) was investigated by using agar dilution method, the results showed that 2 isolates (2.5%) with MIC 32 mg/L (VRSA) and 6 isolates (7.6%) had MICs in the range of 2 – 4 mg/L (VISA). The VRSA strains were isolated from nasal and burn samples, whereas the VISA strains were isolated from nasal samples (2 strains), tonsil sample (2 strains) and 1 strain from each burn and urine samples. The antibiotic

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pattern of VRSA/VISA isolates shown in table 2.

Table 2. Antibiotic susceptibility pattern of VRSA (n=2) / VISA (n = 6).

Antibiotics VRSA/VISA Resistance (%) Sensitive (%)

Cefoxitin

VRSA 2 (100) -

VISA 6 (100)

Augmentin

VRSA 2 (100)

VISA 6(100)

Erythromycin

VRSA 2(100)

VISA 6(100)

Tetracycline

VRSA 2(100)

VISA 6(100)

Clindamycin

VRSA 2(100) -

VISA 6(100)

Imipenem

VRSA 2(100)

VISA - 6(100)-

Gentamicin

VRSA - 2 (100)

VISA 2(33.3) 4(66.7)

Amplification of vanA and vanB

The resistance genes vanA and vanB were amplified by polymerase chain reaction using gene- specific primers as described in Table 1. six isolates which were phenotypically resistant to vancomycin and cefoxitin with mecA positive were selected for the identification of vancomycin resistance genes. None of the tested isolates showed positive results for vanA and vanB genes.

Discussion

MRSA is one of the most common pathogens associated with nosocomial and community acquired infections (David et al. 2010). Hence, studying the prevalence and antibiotic susceptibility of MRSA, has great value for the treatment and management of S. aureus infections. In the present study, out of 380 clinical samples, 79 (20.8%) isolates were identified as methicillin resistant S. aureus. These results are comparable to the incidence found in northern Iraq (22.3%), (Hussein et al. Online ahead of Print) and Palestine (24%), (Adwan et al. 2013) but this is lower than the results reported by Huang et al., in the USA (42%), (17 Huang et al. 2006).

The present results showed a high resistance to augmentin (92.4%), which is higher than the findings reported in Nigeria (69.6%), (Onanuga et al. 2012). Most of the MRSA isolates were sensitive to gentamycin (82.7%), imipenem (78.7%) and vancomycin (91%); this result is similar to those obtained in Eritrea (24 Garoy et al., 2019) and Nepal (Baral et al. 2011, 25). These antimicrobials could be more effective alternatives for treatment in our community.

In general, our results revealed that the MRSA isolates were also resistant to other β-lactam antibiotics. However, this resistance differs from one antibiotic to another, which may be due to

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the type of antibiotic or how much of each antibiotic is used among the patients in the community. In addition, the resistance towards any antibiotic depends on the amount of β- lactamase enzyme or the amount of PBB 2a produced by each strain of bacteria. All these factors could be contributors to the variations in the rate of resistance.

Vancomycin was the drug of choice to treat MRSA related infections. Emergence of vancomycin resistant (VRSA) strains among MRSA has made the treatment of the infections caused by these pathogens more difficult. The present results revealed that 2 strains (2.5%) of the tested MRSA were VRSA and 6 strains (7.6%) were VISA. None of the VRSA and VISA, which identified in the 1`present study, have demonstrated vanA/vanB gene by PCR. Thus, the absence of vanA/B genes in the present isolates dose not exclude that these isolates are not VRSA or VISA. It was primarily thought that vancomycin resistance phenotype in S. aureus could occur as a result of acquiring van genes that encode vancomycin resistance in Enterococcus species (Boyce et al. 1994 7). This can occur through transferring of plasmid containing van genes from Enterococcus species into S. aureus. However, this was not the case in our study, as all VRSA and VISA lack van genes. Our findings suggest that vancomycin resistant phenotypes observed in our study could be produced by spontaneous mutation led to alert cell well structure, which may result in prevention vancomycin from entering into cells (Billot-Klein et al. 1996, Sieradzki and Tomasz, 1999). It could be due to the increased usage of vancomycin in hospitals.

Comparing the prevalence rate of VRSA (2.5%) and (7.6%) obtained in the present study with other local study showed variable differences. Al-Dahbi and Al-Mathkhury (2013) reported that the prevalence of VRAS and VISA was 3.8% and 32.1% respectively. Al-Geobory. (2011) and Assafi et al. (2020) reported that the rate of resistance and intermediate to vancomycin was 2.27% and 3% respectively. On the other hand, Al Hossainy (2010) reported that the prevalence of VRSA was 20% VRSA of tested S. aureus. These differences between different studies may be due to methods employed to detect vancomycin resistance phenotype, as most results in those studies were based on disc diffusion method which is not a reliable method.

The prevalence rate of VRSA/VISA obtained in the present study is comparable with the average of the global prevalence rate of VRS (2.4%) and higher than VISA rate (4.3%). According to data reported by Shariati et al. (2020) through their meta-analysis study, the present study showed that the prevalence rate of VRSA among our community is higher than the average rates reported in Iran (1.3%), India (1.6%), and less than the average rates reported in Pakistan (3.3%), Bangladesh (4.5%), Jordan (4%), and Egypt (5.5%). Furthermore, the average rates of VIRSA in our study was higher than the average of the global rate (4.3%), and the average rates in Iran (3.6%), India (4.6%), Pakistan (5.6%), but much lower than the prevalence of VIRSA in Saudi Arabia (18%), (Shariati et al. 2020).

The existence of vancomycin-resistant staphylococci among our community portends the health authorities to detect and identify these organisms as well as finding an alternative treatment.

Furthermore, these findings suggest that vancomycin resistance has the potential to become a

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widespread problem in MRSA strains. Existence of VRSA/VISA could be due to irrational use of Glycopeptides, especially vancomycin or the selective pressure of vancomycin, which is the drug of choice to treat serious infection caused by MRSA. Although these findings provide vital information on CA-MRSA in Iraq, there were some limitations. The sources and the number of clinical samples were not enough to generalise the conclusions to the entire country. Further studies are required to investigate more clinical samples and to detect and identify the VRSA/VISA isolates in addition to tracing the origin of these isolates.

Data Availability: The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest: No conflicts of interest to declare Funding Statement No funding is associated with this study.

Acknowledgment: The authors thank Mr Ali N Alsharefi, for providing part of the clinical specimens.

References

1. Adwan K, Jarrar N, Abu-Hijleh A, Adwan G, Awwad E, Salameh Y. (2013). Molecular analysis and susceptibility patterns of methicillin-resistant Staphylococcus aureus strains causing community- and health care-associated infections in the northern region of Palestine. Am J In-fect Control., 41(3):195–8.

2. Al-Dahbi Ali M and Harith J Al-Mathkhury (2013). Distribution of Methicillin Resistant Staphylococcus aureus in Iraqi patients and Healthcare Workers. Iraqi Journal of Science. Vol 54.

No.2. pp. 293-300.

3. Al-Geobory H A (2011). Comparative study between Methicillin resistant Staphylococcus aureus (MRSA) and Methicillin sensitive Staphylococcus aureus (MSSA), and detect the antimicrobial effects of some plant extracts on them. Msc. Thesis. College of Science/ Baghdad University. Iraq 4. Al-Hossainy D J M. (2007). Isolating and diagnosis staphylococcus aureus bacteria from patients infection of urinary tract infection in Al-Diwanyia city. Al-Qadisiyah Journal for Science Veterinary Medicine.6 (1), pp:52-57

5. Andrews JM. (2001). Determination of minimum inhibitory concentrations. J Antimicrob Chemother., Jul;48 Suppl 1:5-16. doi: 10.1093/jac/48.suppl_1.5.

6. Assafi M S, Hado H A, and Abdulrahman I. S. (2020). Detection of methicillin-resistant Staphylococcus aureus in broiler and broilers farm works in Duhok, Iraq by using conventional and PCR techniques. Iraqi Journal of Veterinary Sciences, Vol. 34, No. 1, 2020 (15-22).

7. Baral R, Khanal B, Acharya A: (2011). Antimicrobial susceptibility patterns of clinical isolates of Staphylococcus aureus in Eastern Nepal. HREN., 9 (2): 78-82.

8. Billot-Klein D, Gutmann L, Bryant D, Bell D, Van Heijenoort J, Grewal J, and Shlaes D M.

(1996). Peptidoglycan synthesis and structure in Staphylococcus haemolyticus expressing increasing levels of resistance to glycopeptide antibiotics. J. Bacteriol., 178:4696–4703.

9. Boyce J M, Opal S M, Chow J W, Zervos M J, Potter-Bynoe G, Sherman C B, Romulo R L, Fortna S, and Medeiros A A. (994). Outbreak of multidrug-resistant Enterococcus faecium with

(8)

transferable vanB class vancomycin resistance. J. Clin. Microbiol., 32:1148–1153.

10. Centers for Disease Control and Prevention (2002). Staphylococcus aureus resistant to vancomycin – United States. Morb Mortal Wkly Rep., 51:565–567

11. Clark N C, Weigel L M, Patel J B, Tenover F C. (2005). Comparison of Tn1546-like elements in vancomycin-resistant Staphylococcus aureus isolates from Michigan and Pennsylvania.

Antimicrob. Agent Chemother, 49, 470–472. [CrossRef] [PubMed]

12. CLSI. Clinical and Laboratory Standards Institute, PA, USA (2018). Performance standards for antimicrobial susceptibility testing (28th edition). CLSI supplement M100: M100-S22. Accessed:

January 6, 2020: https://clsi.org/media/1930/m100ed28_sample.pd

13. David MZ, Daum RS. (2010). Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev., Jul;23(3):616-87.

14. Even S, Sahm D F, and Courvalin P. (1993). The vanB gene of vancomycin-resistant Enterococcus faecalis V583 is structurally related to genes encoding D-Ala:D-Ala ligases and glycopeptide-resistance proteins VanA and VanC. Gene 124:143-144.

15. Gaillot O, Wetsch M, Fortineau N, Berche P. (2000). Evaluation of CHROM agar S. aureus, a new chromogenic medium, for isolation and presumptive identification of Staphylococcus aureus from human clinical specimens. J Clin Microbiol., Apr;38(4):1587-91.

16. Garoy EY, Gebreab YB, Achila OO, Tekeste DG, Kesete R, Ghirmay R, Kiflay R, Tesfu, (2019).

Methicillin-Resistant Staphylococcus aureus (MRSA): Prevalence and Antimicrobial Sensitivity Pattern among Patients-A Multicenter Study in Asmara, Eritrea. Can J Infect Dis Med Microbiol., Feb; 8321834.

17. Geha DJ, Uhl JR, Gustaferro CA, Persing DH. (1994). Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory. J Clin Microbiol., Jul;32(7):1768-72.

18. Handwerger S, Perlman D C, Altarac D, and McAuliffe V. (1992). Concomitant high-level vancomycin and penicillin resistance in clinical isolates of enterococci. Clin. Infect. Dis., 14:655- 661.

19. Hiramatsu K, Aritaka N, Hanaki H. et al. (1997). Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet., 350 (9092):1670–

1673.

20. Huang H, Flynn NM, King JH, Monchaud C, Morita M, Cohen SH. (2006). Comparisons of community-associated methicillin-resistant Staphylococcus aureus (MRSA) and hospital associated MSRA infections in Sacramento, California. J Clin Microbiol., Jul;44(7):2423-7.

21. Hussein N, Salih R S, Rasheed N A. Prevalence of Methicillin-esistant Staphylococcus aureus in Hospitals and Community in Duhok, Kurdistan Region of Iraq, Int J Infect. Online ahead of Print

; 6(2):e89636.

22. Kareem SM, Al-Jubori SS, Ali MR. (2015). Prevalence of erm genes among methicillin resistant Staphylococcus aureus MRSA Iraqi isolates. Int J Curr Microbiol Appl Sci., 4(5):575–585.

23. Kareem SM, Aljubori SS, Ali MR. (2020). Novel determination of spa gene diversity and its molecular typing among Staphylococcus aureus Iraqi isolates obtained from different clinical samples. New Microbes New Infect. Jan 29;34:100653.

24. Khanam S, Haq J A, Shamsuzzaman S, Rahman, M M, Mamun, K Z. (2016). Emergence of Vancomycin Resistant Staphylococcus aureus during Hospital Admission at a Tertiary Care

(9)

Hospital in Bangladesh. Bangladesh J. Infec. Dis., 3, 11–16.

25. Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H, Kobayashi I, Cui L, Oguchi A, Aoki, K-I, Nagai Y. (2001). Whole genome sequencing of meticillin-resistant Staphylococcus aureus.

Lancet, 357, 1225–1240.

26. Mathews A A, Thomas M, Appalaraju B, Jayalakshmi J. (2010). Evaluation and comparison of tests to detect methicillin resistant Staphylococcus aureus. Indian J Pathol Microbiol., 53(1): 79- 82.

27. Merlino J, Leroi M, Bradbury R, Veal D, Harbour C. (2000). New chromogenic identification and detection of Staphylococcus aureus and methicillin-resistant S. aureus. J Clin Microbiol. Jun;

38(6): 2378-80.

28. Monday SR and Bohach GA. (1999) “Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in Staphylococcal isolates. J Clin Microbiol. vol. 37, no. 10, pp.

3411–3414.

29. Onanuga A, Awhowho GO. Antimicrobial resistance of Staphylococcus aureus strains from patients with urinary tract infections in Yenagoa, Nigeria. J Pharm Bioallied Sci. Jul;4(3):226-30.

30. Perwaiz S, Barakzi Q, Farooqi B J, Khursheed N, Sabir N. (2007). Antimicrobial susceptibility pattern of clinical isolates of methicillin resistant Staphylococcus aureus. J. Pak. Med. Assoc., 57, 2.

31. Shariati A, Dadashi M, Moghadam M T. et al. (2020). Global prevalence and distribution of vancomycin resistant, vancomycin intermediate and heterogeneously vancomycin intermediate Staphylococcus aureus clinical isolates: a systematic review and meta-analysis. Sci Rep 10, 12689.

32. Sieradzki, K, and Tomasz A. (1999). Gradual alterations in cell wall structure and metabolism in vancomycin-resistant mutants of Staphylococcus aureus. J. Bacteriol. 181:7566–7570.

33. Skov R, Smyth R, Clausen M, Larsen AR, Frimodt-Moller N, Olssen-Liljequist B,et al. (2003).

Evaluation of a cefoxitin 30 micro disc on Iso-Sensitest agar for detection of methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother. 52(2):204-7.

34. Pebriani, R. ., Jafar, N. ., Wahiduddin, Hidayanti, H. ., Burhanuddin, & Ummu Salamah. (2021).

The Effect of Extract of Canarian Nuts on Reduction of Total Cholesterol Levels of Hyperglicemic Rat. Journal of Scientific Research in Medical and Biological Sciences, 2(1), 19- 29. https://doi.org/10.47631/jsrmbs.v2i1.128

35. Tenover F C, Biddle J W, Lancaster M V. (2001). Increasing resistance to vancomycin and other glycopeptides in Staphylococcus aureus. Emerg. Infect. Dis., 7, 327.

36. Tong S Y, Davis J S, Eichenberger E, Holland T L, Fowler V G. (2015). Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clin.

Mcrobiol. Rev., 28, 603–661.