- Email: [email protected]
Linezolid activity against clinical Gram-positive cocci with advanced antimicrobial drug resistance in Iran
Accepted Manuscript Title: Linezolid activity against clinical Gram-positive cocci with advanced antimicrobial drug resistance in Iran Authors: Hamidreza Houri, Hossein Kazemian, Hadi Sedigh Ebrahim-Saraie, Asieh Taji, Zahra Tayebi, Hamid Heidari PII: DOI: Reference:
S2213-7165(17)30114-5 http://dx.doi.org/doi:10.1016/j.jgar.2017.06.002 JGAR 438
To appear in: Received date: Revised date: Accepted date:
27-2-2017 28-5-2017 12-6-2017
Please cite this article as: Hamidreza Houri, Hossein Kazemian, Hadi Sedigh EbrahimSaraie, Asieh Taji, Zahra Tayebi, Hamid Heidari, Linezolid activity against clinical Gram-positive cocci with advanced antimicrobial drug resistance in Iran (2010), http://dx.doi.org/10.1016/j.jgar.2017.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Linezolid activity against clinical Gram-positive cocci with advanced antimicrobial drug resistance in Iran
Running Title: Linezolid activity against Gram positive cocci
Hamidreza Houria, Hossein Kazemianb,c, Hadi Sedigh Ebrahim-Saraied, Asieh Tajid, Zahra Tayebie, Hamid Heidarid,* a
Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical
Sciences, Tehran, Iran b
Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran
c
Department of Medical Microbiology, School of Medicine, Tehran University of Medical
Sciences, Tehran, Iran d
Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical
Sciences, Shiraz, Iran e
Microbiology Department, Tehran Medical Sciences Branch, Islamic Azad University,
Tehran, Iran
*Corresponding author: Hamid Heidari, Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Zand St., Imam Hossein Sq., Shiraz, Iran. Tel: +98-9386312941, Fax: +98-7132304356, E-mail: [email protected]
1
Highlights
A collection of MRSA, VRE, PNSP and GBS strains were isolated from various clinical samples.
Antimicrobial susceptibility patterns and multidrug resistant (MDR) strains were determined.
Antibacterial activity of linezolid against isolates was investigated by the standard broth microdilution method.
Resistance to linezolid was not seen among the clinical multidrug resistant isolates.
Abstract Objectives: The aim of the study was to investigate the linezolid activity against clinical Grampositive cocci with advanced antimicrobial drug resistance. Methods: A collection of methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), penicillin non-susceptible Streptococcus pneumoniae (PNSP), and group B streptococci (GBS) were isolated from various clinical samples. Antimicrobial susceptibility tests were done using standard methods. Subsequently, we investigated linezolid antibacterial activities, the first approved oxazolidinone against isolates by the standard broth microdilution method. Results: According to our results, MRSA and PNSP isolates were multidrug resistant, and almost half of the VRE isolates were high level gentamicin resistant (HLGR). Furthermore, resistance to linezolid was not seen among the isolates. The MIC90 values for MRSA, VRE, PNSP and GBS isolates were 4 µg/ml, 2 µg/ml, 1 µg/ml, and 0.5 µg/ml, respectively. Only
2
6.25% of vancomycin resistant enterococci showed intermediate susceptibility to this antibiotic. Conclusions: These findings indicate that linezolid has an excellent activity against clinical drug resistant Gram-positive isolates in Iran. Constant monitoring and surveillance of linezolid MIC distribution allows the researchers to assess and detect gradual upward MIC drifts.
Keywords: Linezolid, MRSA, VRE, PNSP, GBS, Iran
1. Introduction Severe infections caused by Gram-positive cocci remain to be a significant threat to public health [1]. The growing emergence of antimicrobial resistance in these organisms has increased the clinical demand for novel and effective therapeutic antibiotics [2]. Methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE) are considered as the two most important causes of nosocomial infections that have created clinical request for more effective antimicrobial agents [3]. In recent years vancomycin resistant or intermediate S. aureus (VRSA/VISA) has emerged sporadically, thus, compromising the usefulness of vancomycin as a last resort [4]. In addition, penicillin nonsusceptible strains of Streptococcus pneumoniae (penicillin non-susceptible pneumococci or PNSP) causes a significant proportion of community-acquired pneumonia (CAP), and invasive pneumococcal diseases (IPDs) [5,6]. Furthermore, the increasing rates of resistance to clindamycin and erythromycin among Streptococcus agalactiae (group B streptococci or GBS) strains has raised concerns about the use of the antimicrobial agents for the prophylaxis or treatment of GBS infections. 3
Over the past decade, numerous novel antibacterial agents tried to eradicate drug resistant Gram-positive pathogens [7]. Among these agents, linezolid, the first clinically available member of the class oxazolidinones, was approved in 2000 for the treatment of hospital- and community-acquired pneumonia, and complicated skin infections [8]. It is prescribed for severe and life threatening drug resistant infections in Iran. Since there is limited data available on linezolid activity against bacterial pathogens, this study was designed to investigate the linezolid activity against drug resistant Gram-positive cocci including clinical isolates of MRSA, VRE, PNSP and GBS.
2. Materials and Methods 2.1. Bacterial isolates and antimicrobial resistance confirmation Bacterial isolates were obtained from three teaching hospitals and a children’s hospital in Tehran from April 2013 to April 2015. Methicillin-resistant S. aureus (MRSA) We collected 31 multidrug-resistant MRSA isolates from the clinical samples. Amplification of the femA gene was used for molecular confirmation [9]. Methicillin resistance was verified using cefoxitin (30 μg) disc (Mast Group Ltd., UK) by the disc diffusion method on the Mueller- Hinton Agar (Merck Co., Germany), according to Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. The isolates were further tested for the presence of the mecA gene by PCR, as described previously [9]. Additionally, antimicrobial susceptibility tests were performed by the disc diffusion method on the Mueller- Hinton Agar (Merck Co., Germany) for gentamicin (10 µg), ciprofloxacin (5 µg), erythromycin (15 µg), doxycycline (30 µg), rifampin (5 µg), chloramphenicol (30 µg) and co4
trimoxazole (1.25/2375 µg) (Mast Group Ltd., UK). Susceptibility test was also performed by the E-test method on the Mueller- Hinton Agar (Merck Co., Germany) for vancomycin (Liofilchem Co, Italy). S. aureus ATCC 25923 was used as reference strain for antibiotic susceptibility testing. Resistance to three or more agents was considered as multidrug resistant S. aureus (MDRSA) [11]. Vancomycin resistant enterococci (VRE) Thirty-two VRE strains were isolated from urine and wound samples. Verification of genus was done using conventional microbiological tests as described previously [12]. Resistance to vancomycin was confirmed via measurement of minimal inhibitory concentration (MICs) by the standard agar dilution method on the Brain Heart Infusion (BHI) agar (Merck Co., Germany). Furthermore, high-level gentamicin resistant (HLGR) isolates were determined using the standard broth microdilution method. E. faecalis ATCC 29212 was used as standard strain for antibiotic susceptibility testing. Penicillin non-susceptible pneumococci (PNSP) A collection of 15 invasive PNSPs were obtained from pediatric patients with invasive pneumococcal diseases (IPD). In order to confirm the pneumococcal diagnosis, lytA gene was targeted by PCR as described previously [13]. To verify non-susceptibility to penicillin and to determine antimicrobial resistance, we carried out MICs measurement of antibiotics (Sigma-Aldrich Co., USA) by the broth microdilution method, using cation-adjusted Mueller-Hinton broth (Merck Co., Germany) with 2.5-5% lysed horse blood (LHB). In addition to penicillin, antimicrobial susceptibility testing for cefotaxime, ceftriaxone, tetracycline, erythromycin, clindamycin, co-trimoxazole, levofloxacin, and vancomycin was performed using broth microdilution method. S. pneumoniae ATCC 49619 5
was the control strain used to measure the MICs of the tested antimicrobial agents. Multidrug resistance S. pneumoniae (MDRSP) was defined as resistance to three or more agents [11]. Group B streptococci (GBS) Twenty-eight GBS strains were isolated from pediatric and adult patients with meningitis and septicemia. Confirmation of the isolates was performed by conventional laboratory tests, such as negative reaction to catalase, positive reaction to CAMP test, and finally resistance to bacitracin and SXT. Antimicrobial susceptibility tests were done by disc diffusion method on the Mueller-Hinton Agar (Merck Co., Germany) with 5% sheep blood for erythromycin (15 µg), clindamycin (10 µg), tetracycline (30 µg), levofloxacin (5 µg), and vancomycin (30 µg) (Mast Group Ltd., UK). The reference strain for antibiotic susceptibility testing was S. pneumoniae ATCC 49619. All of antimicrobial susceptibility tests and MIC measurements were performed according to CLSI guidelines [10]. 2.2. Determining the minimal inhibitory concentration (MIC) of linezolid Linezolid (Sigma-Aldrich Co., USA) MIC was determined by standard broth micro dilution based on CLSI guidelines [10]. For staphylococci and enterococci, Mueller-Hinton broth (Merck Co., Germany) was used. Cation-adjusted Mueller-Hinton broth (Merck Co., Germany) with supplement of 2.5-5% LHB was used to test pneumococci and GBS [10]. The MIC tests were done twice for each isolate. The MIC50 and MIC90 values were defined as the MIC inhibited 50% and 90% of the isolates, respectively. 3. Results All MRSA were resistant to gentamicin, erythromycin and chloramphenicol. Resistance to ciprofloxacin, doxycycline, rifampin and co-trimoxazole was 93.5%, 90.3%, 61.3% and 6
41.9%, respectively. Vancomycin resistance was not observed among the isolates. By the mentioned definition, all of the MRSA isolates were considered as MDRSA strains. All of the enterococcal isolates were resistant to vancomycin(MIC ≥ 32 µg/ml). Furthermore, 43.7% of them were high level gentamicin resistant (HLGR) (MIC ≥ 512 µg/ml). All PNSP were resistant to penicillin (MIC ≥ 8 µg/ml), co-trimoxazole and tetracycline. The rates of resistance to erythromycin and clindamycin were 80% followed by cefotaxime, and ceftriaxone 26.7 %. Only one isolate revealed intermediate susceptibility to levofloxacin (MIC = 4 µg/ml), and the others were susceptible. Also, all of the isolates were susceptible to vancomycin. Moreover, by the mentioned definition, all the PNSP isolates were considered as MDRSP strains. Over 28 GBS isolates, the most prevalent resistance was related to tetracycline (82.1%). Resistance to erythromycin, clindamycin and levofloxacin was more than 32%, 21% and 10%, respectively. Furthermore, all of the isolates were susceptible to vancomycin. Susceptibility patterns of strains to linezolid are shown in Figure 1. Resistance to linezolid was not observed among the isolates. The MIC50 value for MRSA, VRE was 2 µg/ml and it was 0.5 µg/ml for PNSP and GBS strains. The MIC90 values for MRSA, VRE, PNSP and GBS isolates were also 4 µg/ml, 2 µg/ml, 1 µg/ml, and 0.5 µg/ml, respectively. 4. Discussion In the present study, the effectiveness of linezolid against drug resistant Gram positive cocci was investigated. In accordance with other reports, our results showed that linezolid has excellent properties against clinical MRSA isolates with resistance to a wide range of antibiotics, invasive isolates of MDR penicillin non-susceptible pneumococci, clinical isolates of HLGR, and vancomycin resistant enterococci, and also group B streptococci invasive 7
isolates. Probable mechanism that could lead to linezolid resistance is mutations of the 23S ribosomal RNA (23SrRNA) gene [14]. Gram-positive bacteria contain multiple 23SrRNA alleles (4-6 copies) and occurrence of resistance requires mutation in more than one allele [15]. This probably explains the low frequency of linezolid resistance among Gram positive bacteria, even among those that are MDR strains. In our study, all MRSA isolates that showed resistance to gentamicin, erythromycin, chloramphenicol, and at least one other class of antibiotics, were susceptible to linezolid. In previous studies, linezolid non-susceptible S. aureus were reported sporadically, which were usually related to long-term linezolid therapy in patients with unremovable infected prostheses [1,16]. In the present study, all PNSP were also multi drug resistant, but none of them showed decreased susceptibility to linezolid. The MIC50/90 (0.5/1) were the same as the results of the LEADER (a national surveillance initiative specifically designed to analyze linezolid activity in US) surveillance program in 2004 [11]. All MRSA, GBS and PNSP isolates were vancomycin susceptible, however vancomycin prescription may increase the risk of resistant cocci. Furthermore, in previous studies various adverse events and antimicrobial interventions such as acute renal failure with vancomycin, was reported [17,18]. Only one pneumococcal isolate revealed intermediate susceptibility to levofloxacin (MIC = 4 µg/ml), and the others were susceptible. Some findings suggest that using fluoroquinolones to treat multidrug-resistant pathogens is a risk factor for the clonal spread of levofloxacin resistant pneumococci [19].
8
Even though only 2 isolates (6.25%) of VRE showed intermediate susceptibility to linezolid (MIC = 4 µg/ml), the others were completely susceptible to this antibiotic. In a study conducted by Yasliani et al., in Iran, 4 clinical isolates of linezolid and vancomycin resistant Enterococcus were detected [20]. Decreased susceptibility and resistance to linezolid could spread amongst VRE isolates due to nosocomial spread. Since resistance to linezolid is probable, careful monitoring of MIC distributions to recognize gradual upward changes can deliver early warnings to increasing mutations amongst susceptible bacterial populations before changes to an absolute resistance rate [16]. In conclusion, our results showed the excellent activity of linezolid against drug resistant Gram-positive pathogens in Iran, which may prove to be useful in the treatment of such drug resistant infections.
Acknowledgment The authors wish to thank Mr. Hossein Argasi at the Research Consolation Centre (RCC) at Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript. Especial thanks to the Microbiology Department of Shahid Beheshti University of Medical Sciences for their outmost cooperation.
Declarations Funding: No funding Competing Interests: All authors declare no conflicts of interest. Ethical Approval: Not required
9
References 1. Tian Y, Li T, Zhu Y, Wang B, Zou X, Li M. Mechanisms of linezolid resistance in staphylococci and enterococci isolated from two teaching hospitals in Shanghai, China. BMC microbiol 2014;14:292. 2. Falagas ME, Roussos N, Gkegkes ID, Rafailidis PI, Karageorgopoulos DE. Fosfomycin for the treatment of infections caused by Gram-positive cocci with advanced antimicrobial drug resistance: a review of microbiological, animal and clinical studies. Expert Opin Investig Drugs 2009;18:921-44. 3. Zirakzadeh A, Patel R. Vancomycin-resistant enterococci: colonization, infection, detection, and treatment. Mayo Clin Proc 2006;81:529-36. 4. Gardete S, Tomasz A. Mechanisms of vancomycin resistance in Staphylococcus aureus. J Clin Inves 2014;124:2836-40. 5. Aguiar SI, Serrano I, Pinto FR, Melo-Cristino J, Ramirez M. Changes in Streptococcus pneumoniae serotypes causing invasive disease with non-universal vaccination coverage of the seven-valent conjugate vaccine. Clin Microbiol Infect 2008;14:835-43. 6. Richter SS, Heilmann KP, Dohrn CL, Riahi F, Beekmann SE, Doern GV. Changing epidemiology of antimicrobial-resistant Streptococcus pneumoniae in the United States, 20042005. Clin Infect Dis 2009 ;48:e23-33. 7. Korbila IP, Falagas ME. Investigational antimicrobial drugs for bloodstream infections. Curr Opin Investig Drugs 2008;9:871-8. 8. Swaney SM, Aoki H, Ganoza MC, Shinabarger DL. The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrob Agents Chemother 1998;42:3251-5. 9. Bhutia KO, Singh TS, Biswas S, Adhikari L. Evaluation of phenotypic with genotypic methods for species identification and detection of methicillin resistant in Staphylococcus aureus. Int J Appl Basic Med Res 2012;2:84-91. 10.
Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial
Susceptibility Testing; Twenty-fifth Informational Supplement,. 2015 (m100-s25). 11. Draghi DC, Sheehan DJ, Hogan P, Sahm DF. In vitro activity of linezolid against key gram-positive organisms isolated in the united states: results of the LEADER 2004 surveillance program. Antimicrob Agents Chemother 2005;49:5024-32. 12. Honarm H, Falah Ghavidel M, Nikokar I, Rahbar Taromsari M. Evaluation of a PCR assay to detect enterococcusfaecalis in blood and determine glycopeptides resistance genes: van a and van B. Iranian journal of medical sciences. Iran J Med Sci 2012;37:194-9. 10
13. Sa-Leao R, Simoes AS, Nunes S, Sousa NG, Frazao N, de Lencastre H. Identification, prevalence and population structure of non-typable Streptococcus pneumoniae in carriage samples isolated from preschoolers attending day-care centres. Microbiology 2006;152:36776. 14. Mendes RE, Deshpande LM, Jones RN. Linezolid update: stable in vitro activity following more than a decade of clinical use and summary of associated resistance mechanisms. Drug Resist Updat 2014;17:1-12. 15. Marshall SH, Donskey CJ, Hutton-Thomas R, Salata RA, Rice LB. Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis. Antimicrob Agents Chemother 2002;46:3334-6. 16. Meka VG, Gold HS. Antimicrobial resistance to linezolid. Clin Infect Dis 2004;39:1010-5. 17. Wong-Beringer A, Joo J, Tse E, Beringer P. Vancomycin-associated nephrotoxicity: a critical appraisal of risk with high-dose therapy. Int J Antimicrob Agents 2011;37:95-101. 18. Moenster RP, Linneman TW, Finnegan PM, Hand S, Thomas Z, McDonald JR. Acute renal failure associated with vancomycin and beta-lactams for the treatment of osteomyelitis in diabetics: piperacillin-tazobactam as compared with cefepime. Clin Microbiol Infect 2014;20:O384-9. 19. Isea-Pena MC, Sanz-Moreno JC, Esteban J, Fernandez-Roblas R, Fernandez-Guerrero ML. Risk factors and clinical significance of invasive infections caused by levofloxacinresistant Streptococcus pneumoniae. Infection 2013;41:935-9. 20. Yasliani S, Mohabati Mobarez A, Hosseini Doust R, Satari M, Teymornejad O. Linezolid vancomycin resistant Enterococcus isolated from clinical samples in Tehran hospitals. Indian J Med Sci 2009;63:297-302.
11
Figure 1. Minimal inhibitory concentrations (MICs) of linezolid against studied strains. MRSA, Methicillin-resistant S. aureus; VRE, Vancomycin resistant enterococci; PNSP, Penicillin nonsusceptible pneumococci; GBS, Group B streptococci.
12
S2213-7165(17)30114-5 http://dx.doi.org/doi:10.1016/j.jgar.2017.06.002 JGAR 438
To appear in: Received date: Revised date: Accepted date:
27-2-2017 28-5-2017 12-6-2017
Please cite this article as: Hamidreza Houri, Hossein Kazemian, Hadi Sedigh EbrahimSaraie, Asieh Taji, Zahra Tayebi, Hamid Heidari, Linezolid activity against clinical Gram-positive cocci with advanced antimicrobial drug resistance in Iran (2010), http://dx.doi.org/10.1016/j.jgar.2017.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Linezolid activity against clinical Gram-positive cocci with advanced antimicrobial drug resistance in Iran
Running Title: Linezolid activity against Gram positive cocci
Hamidreza Houria, Hossein Kazemianb,c, Hadi Sedigh Ebrahim-Saraied, Asieh Tajid, Zahra Tayebie, Hamid Heidarid,* a
Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical
Sciences, Tehran, Iran b
Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran
c
Department of Medical Microbiology, School of Medicine, Tehran University of Medical
Sciences, Tehran, Iran d
Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical
Sciences, Shiraz, Iran e
Microbiology Department, Tehran Medical Sciences Branch, Islamic Azad University,
Tehran, Iran
*Corresponding author: Hamid Heidari, Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Zand St., Imam Hossein Sq., Shiraz, Iran. Tel: +98-9386312941, Fax: +98-7132304356, E-mail: [email protected]
1
Highlights
A collection of MRSA, VRE, PNSP and GBS strains were isolated from various clinical samples.
Antimicrobial susceptibility patterns and multidrug resistant (MDR) strains were determined.
Antibacterial activity of linezolid against isolates was investigated by the standard broth microdilution method.
Resistance to linezolid was not seen among the clinical multidrug resistant isolates.
Abstract Objectives: The aim of the study was to investigate the linezolid activity against clinical Grampositive cocci with advanced antimicrobial drug resistance. Methods: A collection of methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), penicillin non-susceptible Streptococcus pneumoniae (PNSP), and group B streptococci (GBS) were isolated from various clinical samples. Antimicrobial susceptibility tests were done using standard methods. Subsequently, we investigated linezolid antibacterial activities, the first approved oxazolidinone against isolates by the standard broth microdilution method. Results: According to our results, MRSA and PNSP isolates were multidrug resistant, and almost half of the VRE isolates were high level gentamicin resistant (HLGR). Furthermore, resistance to linezolid was not seen among the isolates. The MIC90 values for MRSA, VRE, PNSP and GBS isolates were 4 µg/ml, 2 µg/ml, 1 µg/ml, and 0.5 µg/ml, respectively. Only
2
6.25% of vancomycin resistant enterococci showed intermediate susceptibility to this antibiotic. Conclusions: These findings indicate that linezolid has an excellent activity against clinical drug resistant Gram-positive isolates in Iran. Constant monitoring and surveillance of linezolid MIC distribution allows the researchers to assess and detect gradual upward MIC drifts.
Keywords: Linezolid, MRSA, VRE, PNSP, GBS, Iran
1. Introduction Severe infections caused by Gram-positive cocci remain to be a significant threat to public health [1]. The growing emergence of antimicrobial resistance in these organisms has increased the clinical demand for novel and effective therapeutic antibiotics [2]. Methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE) are considered as the two most important causes of nosocomial infections that have created clinical request for more effective antimicrobial agents [3]. In recent years vancomycin resistant or intermediate S. aureus (VRSA/VISA) has emerged sporadically, thus, compromising the usefulness of vancomycin as a last resort [4]. In addition, penicillin nonsusceptible strains of Streptococcus pneumoniae (penicillin non-susceptible pneumococci or PNSP) causes a significant proportion of community-acquired pneumonia (CAP), and invasive pneumococcal diseases (IPDs) [5,6]. Furthermore, the increasing rates of resistance to clindamycin and erythromycin among Streptococcus agalactiae (group B streptococci or GBS) strains has raised concerns about the use of the antimicrobial agents for the prophylaxis or treatment of GBS infections. 3
Over the past decade, numerous novel antibacterial agents tried to eradicate drug resistant Gram-positive pathogens [7]. Among these agents, linezolid, the first clinically available member of the class oxazolidinones, was approved in 2000 for the treatment of hospital- and community-acquired pneumonia, and complicated skin infections [8]. It is prescribed for severe and life threatening drug resistant infections in Iran. Since there is limited data available on linezolid activity against bacterial pathogens, this study was designed to investigate the linezolid activity against drug resistant Gram-positive cocci including clinical isolates of MRSA, VRE, PNSP and GBS.
2. Materials and Methods 2.1. Bacterial isolates and antimicrobial resistance confirmation Bacterial isolates were obtained from three teaching hospitals and a children’s hospital in Tehran from April 2013 to April 2015. Methicillin-resistant S. aureus (MRSA) We collected 31 multidrug-resistant MRSA isolates from the clinical samples. Amplification of the femA gene was used for molecular confirmation [9]. Methicillin resistance was verified using cefoxitin (30 μg) disc (Mast Group Ltd., UK) by the disc diffusion method on the Mueller- Hinton Agar (Merck Co., Germany), according to Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. The isolates were further tested for the presence of the mecA gene by PCR, as described previously [9]. Additionally, antimicrobial susceptibility tests were performed by the disc diffusion method on the Mueller- Hinton Agar (Merck Co., Germany) for gentamicin (10 µg), ciprofloxacin (5 µg), erythromycin (15 µg), doxycycline (30 µg), rifampin (5 µg), chloramphenicol (30 µg) and co4
trimoxazole (1.25/2375 µg) (Mast Group Ltd., UK). Susceptibility test was also performed by the E-test method on the Mueller- Hinton Agar (Merck Co., Germany) for vancomycin (Liofilchem Co, Italy). S. aureus ATCC 25923 was used as reference strain for antibiotic susceptibility testing. Resistance to three or more agents was considered as multidrug resistant S. aureus (MDRSA) [11]. Vancomycin resistant enterococci (VRE) Thirty-two VRE strains were isolated from urine and wound samples. Verification of genus was done using conventional microbiological tests as described previously [12]. Resistance to vancomycin was confirmed via measurement of minimal inhibitory concentration (MICs) by the standard agar dilution method on the Brain Heart Infusion (BHI) agar (Merck Co., Germany). Furthermore, high-level gentamicin resistant (HLGR) isolates were determined using the standard broth microdilution method. E. faecalis ATCC 29212 was used as standard strain for antibiotic susceptibility testing. Penicillin non-susceptible pneumococci (PNSP) A collection of 15 invasive PNSPs were obtained from pediatric patients with invasive pneumococcal diseases (IPD). In order to confirm the pneumococcal diagnosis, lytA gene was targeted by PCR as described previously [13]. To verify non-susceptibility to penicillin and to determine antimicrobial resistance, we carried out MICs measurement of antibiotics (Sigma-Aldrich Co., USA) by the broth microdilution method, using cation-adjusted Mueller-Hinton broth (Merck Co., Germany) with 2.5-5% lysed horse blood (LHB). In addition to penicillin, antimicrobial susceptibility testing for cefotaxime, ceftriaxone, tetracycline, erythromycin, clindamycin, co-trimoxazole, levofloxacin, and vancomycin was performed using broth microdilution method. S. pneumoniae ATCC 49619 5
was the control strain used to measure the MICs of the tested antimicrobial agents. Multidrug resistance S. pneumoniae (MDRSP) was defined as resistance to three or more agents [11]. Group B streptococci (GBS) Twenty-eight GBS strains were isolated from pediatric and adult patients with meningitis and septicemia. Confirmation of the isolates was performed by conventional laboratory tests, such as negative reaction to catalase, positive reaction to CAMP test, and finally resistance to bacitracin and SXT. Antimicrobial susceptibility tests were done by disc diffusion method on the Mueller-Hinton Agar (Merck Co., Germany) with 5% sheep blood for erythromycin (15 µg), clindamycin (10 µg), tetracycline (30 µg), levofloxacin (5 µg), and vancomycin (30 µg) (Mast Group Ltd., UK). The reference strain for antibiotic susceptibility testing was S. pneumoniae ATCC 49619. All of antimicrobial susceptibility tests and MIC measurements were performed according to CLSI guidelines [10]. 2.2. Determining the minimal inhibitory concentration (MIC) of linezolid Linezolid (Sigma-Aldrich Co., USA) MIC was determined by standard broth micro dilution based on CLSI guidelines [10]. For staphylococci and enterococci, Mueller-Hinton broth (Merck Co., Germany) was used. Cation-adjusted Mueller-Hinton broth (Merck Co., Germany) with supplement of 2.5-5% LHB was used to test pneumococci and GBS [10]. The MIC tests were done twice for each isolate. The MIC50 and MIC90 values were defined as the MIC inhibited 50% and 90% of the isolates, respectively. 3. Results All MRSA were resistant to gentamicin, erythromycin and chloramphenicol. Resistance to ciprofloxacin, doxycycline, rifampin and co-trimoxazole was 93.5%, 90.3%, 61.3% and 6
41.9%, respectively. Vancomycin resistance was not observed among the isolates. By the mentioned definition, all of the MRSA isolates were considered as MDRSA strains. All of the enterococcal isolates were resistant to vancomycin(MIC ≥ 32 µg/ml). Furthermore, 43.7% of them were high level gentamicin resistant (HLGR) (MIC ≥ 512 µg/ml). All PNSP were resistant to penicillin (MIC ≥ 8 µg/ml), co-trimoxazole and tetracycline. The rates of resistance to erythromycin and clindamycin were 80% followed by cefotaxime, and ceftriaxone 26.7 %. Only one isolate revealed intermediate susceptibility to levofloxacin (MIC = 4 µg/ml), and the others were susceptible. Also, all of the isolates were susceptible to vancomycin. Moreover, by the mentioned definition, all the PNSP isolates were considered as MDRSP strains. Over 28 GBS isolates, the most prevalent resistance was related to tetracycline (82.1%). Resistance to erythromycin, clindamycin and levofloxacin was more than 32%, 21% and 10%, respectively. Furthermore, all of the isolates were susceptible to vancomycin. Susceptibility patterns of strains to linezolid are shown in Figure 1. Resistance to linezolid was not observed among the isolates. The MIC50 value for MRSA, VRE was 2 µg/ml and it was 0.5 µg/ml for PNSP and GBS strains. The MIC90 values for MRSA, VRE, PNSP and GBS isolates were also 4 µg/ml, 2 µg/ml, 1 µg/ml, and 0.5 µg/ml, respectively. 4. Discussion In the present study, the effectiveness of linezolid against drug resistant Gram positive cocci was investigated. In accordance with other reports, our results showed that linezolid has excellent properties against clinical MRSA isolates with resistance to a wide range of antibiotics, invasive isolates of MDR penicillin non-susceptible pneumococci, clinical isolates of HLGR, and vancomycin resistant enterococci, and also group B streptococci invasive 7
isolates. Probable mechanism that could lead to linezolid resistance is mutations of the 23S ribosomal RNA (23SrRNA) gene [14]. Gram-positive bacteria contain multiple 23SrRNA alleles (4-6 copies) and occurrence of resistance requires mutation in more than one allele [15]. This probably explains the low frequency of linezolid resistance among Gram positive bacteria, even among those that are MDR strains. In our study, all MRSA isolates that showed resistance to gentamicin, erythromycin, chloramphenicol, and at least one other class of antibiotics, were susceptible to linezolid. In previous studies, linezolid non-susceptible S. aureus were reported sporadically, which were usually related to long-term linezolid therapy in patients with unremovable infected prostheses [1,16]. In the present study, all PNSP were also multi drug resistant, but none of them showed decreased susceptibility to linezolid. The MIC50/90 (0.5/1) were the same as the results of the LEADER (a national surveillance initiative specifically designed to analyze linezolid activity in US) surveillance program in 2004 [11]. All MRSA, GBS and PNSP isolates were vancomycin susceptible, however vancomycin prescription may increase the risk of resistant cocci. Furthermore, in previous studies various adverse events and antimicrobial interventions such as acute renal failure with vancomycin, was reported [17,18]. Only one pneumococcal isolate revealed intermediate susceptibility to levofloxacin (MIC = 4 µg/ml), and the others were susceptible. Some findings suggest that using fluoroquinolones to treat multidrug-resistant pathogens is a risk factor for the clonal spread of levofloxacin resistant pneumococci [19].
8
Even though only 2 isolates (6.25%) of VRE showed intermediate susceptibility to linezolid (MIC = 4 µg/ml), the others were completely susceptible to this antibiotic. In a study conducted by Yasliani et al., in Iran, 4 clinical isolates of linezolid and vancomycin resistant Enterococcus were detected [20]. Decreased susceptibility and resistance to linezolid could spread amongst VRE isolates due to nosocomial spread. Since resistance to linezolid is probable, careful monitoring of MIC distributions to recognize gradual upward changes can deliver early warnings to increasing mutations amongst susceptible bacterial populations before changes to an absolute resistance rate [16]. In conclusion, our results showed the excellent activity of linezolid against drug resistant Gram-positive pathogens in Iran, which may prove to be useful in the treatment of such drug resistant infections.
Acknowledgment The authors wish to thank Mr. Hossein Argasi at the Research Consolation Centre (RCC) at Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript. Especial thanks to the Microbiology Department of Shahid Beheshti University of Medical Sciences for their outmost cooperation.
Declarations Funding: No funding Competing Interests: All authors declare no conflicts of interest. Ethical Approval: Not required
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Figure 1. Minimal inhibitory concentrations (MICs) of linezolid against studied strains. MRSA, Methicillin-resistant S. aureus; VRE, Vancomycin resistant enterococci; PNSP, Penicillin nonsusceptible pneumococci; GBS, Group B streptococci.
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