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Investigation of mechanisms and molecular epidemiology of linezolid nonsusceptible Enterococcus faecalis isolated from a teaching hospital in China
Journal of Microbiology, Immunology and Infection (2016) 49, 595e599
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BRIEF COMMUNICATION
Investigation of mechanisms and molecular epidemiology of linezolid nonsusceptible Enterococcus faecalis isolated from a teaching hospital in China Bin Li a, Chuan-Ling Ma a, Xiao Yu b, Yao Sun a, Mei-Mei Li a, Jian-Zhong Ye a, Ya-Pei Zhang a, Qing Wu a, Tie-Li Zhou a,* a Department of Clinical Laboratory, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China b School of Laboratory Medicine and life Science, Wenzhou Medical University, Wenzhou, Zhejiang Province, China
Received 23 September 2014; received in revised form 23 April 2015; accepted 21 May 2015
Available online 27 June 2015
KEYWORDS Enterococcus faecalis; Linezolid nonsusceptible; Multilocus sequence typing; Pulsed-field gel electrophoresis; 23S rRNA
Abstract The epidemiological and molecular characteristics of eight linezolid nonsusceptible Enterococcus faecalis isolated from a teaching hospital in China (January to July 2014) were investigated. The target site modifications and cfr gene associated with linezolid resistance were not found. Results of the epidemiological investigation indicated that linezolid resistance possibly occurred on several independent occasions and was often not related to linezolid administration. Copyright ª 2015, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
* Corresponding author. Department of Clinical Laboratory, First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang Street, Ouhai District, Wenzhou, Zhejiang Province, China. E-mail address: [email protected] (T.-L. Zhou). http://dx.doi.org/10.1016/j.jmii.2015.05.010 1684-1182/Copyright ª 2015, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
596
Introduction Enterococci have emerged in the past decade as an increasingly important cause of nosocomial infections. Linezolid, the first member of a new class of antibiotics (oxazolidinones), has been introduced into clinical practice for infections caused by multiresistant Gram-positive bacteria especially vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus.1 However, linezolid resistant isolates had emerged in several countries shortly after the drug appeared on the market, and they have also emerged recently in several hospitals in China.2 Vancomycin-resistant enterococci have caused hospital outbreaks worldwide; the occurrence of linezolid nonsusceptible enterococci, therefore, represents an immediate threat for patient care. A variety of mutations in domain V of 23S rRNA genes, especially G2576U and the multidrug resistance gene cfr, which codes for an adenine methyltransferase that modifies adenosine at position 2503 in the 23S rRNA, play an important role in linezolid resistance of enterococci. Mutations of the bacterial ribosomal proteins L3 and L4 have also been associated with resistance to linezolid,3e5 and active efflux pump systems may also be involved.6 Pulsed-field gel electrophoresis (PFGE) has been considered the “gold standard” for the study of hospital outbreaks because of its high degree of isolate differentiation. Multilocus sequence typing (MLST) offers the possibility to transfer typing data between laboratories or compare results via Internet, thus providing a powerful tool for global epidemiologic studies. The aim of this study was to investigate the epidemiological and molecular characteristics of linezolid nonsusceptible Enterococcus faecalis isolated from a Chinese hospital.
Methods Strain selection Eight E. faecalis clinical strains exhibiting linezolid Minimum inhibitory concentrations (MICs) 4 mg/L were collected from the First Affiliated Hospital of Wenzhou Medical University from January to July 2014. The clinical records of patients with linezolid nonsusceptible E. faecalis were examined retrospectively. Staphylococcus capitis(cfrþ) used for positive control in this study was donated by Rong Zhang, charge technician from the Second Affiliated Hospital of Zhejiang University.
Antimicrobial susceptibility testing Susceptibility testing was determined using the agar dilution method and broth microdilution method (linezolid and Daptomycin) according to Clinical and Laboratory Standards Institute recommendations (CLSI, 2013). E. faecalis ATCC29212 was used as quality control.
Molecular detection of mutations and resistance gene cfr The presence of mutations in domain V of 23S rRNA and ribosomal proteins L3 and L4, and cfr gene were
B. Li et al. investigated using polymerase chain reaction as described previously.3,4 Genomic DNA extracted from E. faecalis ATCC29212 and S. capitis-(cfrþ) were used as negative and positive control for cfr, respectively. The positive polymerase chain reaction products were sequenced by Shanghai Majorbio Bio-Pharm Technology Co. (Shanghai, China). Nucleotide and deduced amino acid sequences were compared with E. faecalis V583 strain and the linezolid susceptible E. faecalis clinical isolates (MIC, 1 mg/ mL) recovered from the same hospital during the study interval.
Effect of efflux pump mechanism The MIC of linezolid was determined using the broth microdilution method in either the presence or absence of reserpine (20 mg/L) or carbonyl cyanide m-chlorophenylhydrazone (CCCP; 5 mg/L), according to Clinical and Laboratory Standards Institute guidelines. An inoculum of 0.5 Mcfarland Standard of each isolate was inoculated into MuellereHinton cation-adjusted broth containing serial dilutions of linezolid. Four-fold or more reduction of linezolid MICs in the presence or the absence of reserpine or CCCP was defined as a positive phenotype for efflux.7
PFGE PFGE was performed on the CHEFeMapper XA PFGE system (Bio-Rad, Hercules, CA, USA) with a running time of 20 hours and pulse duration of 3.5e23.5 seconds. Salmonella serotype Braenderup H9812 DNA marker was used for the standard molecular weight and size determinations. Pattern analysis was carried out using QualityOne software (Bio-Rad Laboratories). Percentage similarities were identified on a dendrogram derived from the unweighted pair group method with arithmetic averages. Band position tolerance and optimization were set at 1.5% and 0.5%, respectively. Isolates showing a similarity coefficient of 80% were considered to be genetically related.8
MLST The linezolid nonsusceptible E. faecalis isolates were characterized by MLST using seven housekeeping genes (gdh, gyd, pstS, gki, aroE, xpt, and yqiL) according to the protocols and sequencing primers available at the E. faecalis database (http://efaecalis.mlst.net/).
Results Clinical characteristics of patients with linezolid nonsusceptible E. faecalis The clinical information of patients is given in Table 1. Two of the strains were isolated from urine, three from vaginal secretions, two from surgical wounds, and one from blood. No patients were treated with linezolid before linezolid nonsusceptible E. faecalis was isolated.
Linezolid nonsusceptible E. faecalis isolates Table 1
597
Clinical characteristics of patients with linezolid nonsusceptible Enterococcus faecalis isolates
Patient No.
Patient
Admission
Underlying disease
Samples
Previous hospitalization
Treatment outcome
LZD MIC (mg/L)
08/2/14
Colorectal cancer
Wound
No
4
GAS
20/2/14
Acute pancreatitis
Urine
No
F
GYN
12/2/14
Uterine leiomyoma
Vaginal fluid
No
33 42 80
F M M
OP OP GS
d d 21/7/14
Vaginal fluid Urine Blood
No No Yes
7(F524)
31
F
GYN
04/6/14
None Epididymitis Pancreatic cancer, OJ, diabetes, hypertension CIN, IUA
Discharge on Day 32 Discharge on Day 8 Discharge on Day 15 d d Relapse
Vaginal fluid
Yes
8(F527)
82
F
DER
04/5/14
Wound
Yes
Age (y)
Sex
Ward
Date
1(F465)
39
M
GS
2(F467)
50
M
3(F471)
48
4(F496) 5(F509) 6(F511)
T2DM, hypertension, gout
Discharge on Day 10 Discharge on Day 17
8 4 8 8 8 8 8
CIN Z cervical intraepithelial neoplasias; DER Z dermatology; F Z female; GAS Z gastroenterology; GS Z gastrointestinal surgery; GYN Z gynecology; IUA Z intrauterine adhesion; LZD Z linezolid; M Z male; OJ Z obstructive jaundice; MIC = minimum inhibitory concentration; OP Z outpatient; T2DM Z type 2 diabetes mellitus.
Antimicrobial susceptibility testing
V of the 23S rRNA genes, and the ribosomal proteins L3 and L4.
The eight E. faecalis strains were resistant to linezolid at a low level (4e8 mg/L), and all showed multidrug-resistant phenotype (resistant to 3 different antibiotics). All (8/8) of these isolates were resistant to quinupristin/dalfopristin, tetracycline, and erythromycin, and high-level gentamycin and ciprofloxacin resistance were present in 87.5% and 62.5% of these isolates, respectively. All E. faecalis were susceptible to vancomycin, teicoplanin, daptomycin, and the newly introduced tigecycline, and only one was resistant to ampicillin, as shown in Table 2.
Resistance genes and mutations Analysis of sequencing and blasted results failed to find the cfr gene and the possible mutations, including domain Table 2 isolates
Effect of the efflux pump mechanism The MICs of these isolates to linezolid were not changed or just decreased at one dilution in the presence of CCCP or reserpine. All strains grew in the presence of only reserpine (20 mg/L) or CCCP (5 mg/L).
Molecular epidemiology analysis PFGE analysis showed seven unrelated genotypes (Types AeG). Type A contained two subtypes (A1, A2), and these were isolated from patients in different wards. MLST data showed that five sequence types (ST16, ST49, ST586, ST587, and ST588) were assigned for these isolates, and the most
Antibiotic susceptibilities and linezolid resistance mechanisms of linezolid nonsusceptible Enterococcus faecalis
Patient no.
MIC (mg/L)
AMP CIP F 1(F465) 8 2(F467) 8 3(F471) 8 4(F496) 8 5(F509) 8 6(F511) 16 7(F524) 8 8(F527) 8
1 16 2 16 16 16 8 1
16 16 16 64 16 16 16 16
Genetic resistance markers
PFGE MLST type type
Q/D TET VAN ERY TGC
TEC DAP GEH LZD LZD þ LZD þ 23S L3/L4 cfr reserpine CCCP rRNA
>4 >4 >4 >4 >4 >4 >4 >4
2 2 2 2 2 4 2 2
16 16 16 16 16 16 16 16
2 2 1 2 2 2 1 2
8 8 8 8 8 8 8 8
0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12
0.5 1 1 1 1 1 0.5 1
R R R R R R S R
4 8 4 8 8 8 8 8
4 4 4 4 4 4 4 4
4 8 4 8 8 8 8 8
/ / / / / / / /
A1 B C D E F G A2
ST16 ST586 ST587 ST588 ST16 ST16 ST49 ST16
AMP Z ampicillin; CCCP Z carbonyl cyanide m-chlorophenylhydrazone; CIP Z ciprofloxacin; DAP Z daptomycin; ERY Z erythromycin; F Z nitrofurantoin; GEH Z high-level gentamycin; LZD Z linezolid; MLST Z multilocus sequence typing; e Z negative; PFGE Z pulsedfield gel electrophoresis; Q/D Z quinupristin/dalfopristin; TEC Z teicoplanin; TET Z tetracycline; TGC Z tigecycline; VAN Z vancomycin; MIC = minimum inhibitory concentration.
598 dominant clone was ST16. ST586, ST587, and ST588 were three novel sequence types detected from these isolates. Isolates with differently related PFGE patterns (e.g., Type A, Type E, and Type F) had the same sequence type (ST16; Table 2). Patterns and dendrogram analysis produced by PFGE are shown in Figure 1.
Discussion According to an epidemiological survey carried out by Chinese bacterial resistance monitoring network (CHINET) in 2012, the resistance rate of E. faecalis to linezolid was 0.3%. This is the first report of linezolid nonsusceptible Gram-positive bacteria in our hospital, and these strains were all isolated in a short period, which is alarming and requires ongoing surveillance. All isolates were multidrug resistant, but still sensitive to glycopeptides. A retrospective analysis of the patients’ treatment history showed no linezolid therapy prior to the linezolid nonsusceptible E. faecalis isolation. These strains were isolated on widely divergent dates and from different departments of the hospital (Table1). The epidemiological investigation also suggested that these isolates were sporadic cases, which indicated that patient-to-patient transmission was not the main cause of rapid emergence of linezolid resistance. It could also have occurred on several independent occasions and was often not related to linezolid administration. According to the clinical records, Patient 6 (F511), an 80-yearold man suffering from various underlying diseases, has been living in the hospital and been treated with various antimicrobials except for linezolid for a long time (from July 2014 to present). We speculated that he carried the linezolid nonsusceptible isolates prior to his administration, and selective pressure from long-term use of other antimicrobials may have contributed to the occurrence of these bacteria in the hospital setting. Alterations in the 23S rRNA target have remained the dominant mechanism detected among linezolid nonsusceptible enterococci, with a limited number of clinical
B. Li et al. isolates also carrying cfr. However, our results did not show any target site modifications or presence of cfr associated with linezolid resistance. Recent studies evaluating the linezolid resistance mechanisms in Gram-positive isolates from Canada have also reported similar results.9,10 Multidrug resistance (MDR) efflux transporters that pump out a wide range of substrates are known as one of the most important mechanisms of antimicrobial resistance in E. faecalis. The efflux pump phenotype experiment is based on the fact that if efflux pump inhibitors (e.g., reserpine or CCCP) significantly reduce the resistance of the MDR strain to antibiotics to which it was initially resistant or renders the MDR clinical strain as susceptible to antibiotics as the wild-type strain, then it is highly probable that the MDR phenotype of that strain is attributable to an overexpression efflux pump system. However, in our study, the MICs of the tested isolates to linezolid were not changed or just decreased at one dilution in the presence of CCCP or reserpine. It was likely that the capacity of the efflux pump to eliminate linezolid was limited. Therefore, these lowlevel resistance phenotypes were most probably due to other mechanisms. Overall, the present MLST results showed the existence of five allelic profiles, of which ST586, ST587, and ST588 were three novel sequence types. This indicates that some stimulus may lead to the emergence of new characteristics of regional strains. Isolates with differently related PFGE patterns (e.g., Type A, Type E, and Type F) had the same sequence type (ST16). However, they were isolated on widely divergent dates and from different departments of the hospital, and MLST is less discriminatory than PFGE when a short timescale is examined. Therefore, these linezolid nonsusceptible isolates represented sporadic cases. In conclusion, we failed to detect the known possible mechanisms of resistance to linezolid in these E. faecalis strains. The epidemiological investigation indicated that linezolid resistance possibly occurred on several independent occasions and was often not related to linezolid administration. A multidisciplinary approach is urgently
Figure 1. PFGE patterns and dendogram analysis of eight linezolid-nonsusceptible Enterococcus faecalis isolates. Lane M Z Salmonella serotype Braenderup strain H9812 Marker (kb); lanes 1e8 Z eight linezolid-nonsusceptible E. faecalis isolates 1e8 (Table 1). Dendogram was generated using QualityOne Software (Bio-Rad Laboratories, Hercules, CA, USA). The phylogenetic tree was constructed using UPGMA clustering. A genetic similarity index scale is shown in the left of the dendogram. PFGE Z pulsedfield gel electrophoresis; UPGMA Z unweighted pair group method with arithmetic mean.
Linezolid nonsusceptible E. faecalis isolates
599
needed to evaluate the risk factors associated with the rapid occurrence and dissemination of linezolid resistance. 4.
Conflicts of interest All contributing authors declare no conflict of interest.
5.
Acknowledgments 6.
This work was supported by research grants from the National Natural Science Foundation of China (no. 81171614), Zhejiang Provincial Natural Science Foundation of China (LY14H190005), and Zhejiang Province Program for the Cultivation of High-level innovative Health talents ((2012) 141).
7.
8.
References 1. Leach KL, Brickner SJ, Noe MC, Miller PF. Linezolid, the first oxazolidinone antibacterial agent. Ann N Y Acad Sci 2011; 1222:49e54. 2. Cai JC, Hu YY, Zhang R, Zhou HW, Chen GX. Linezolid-resistant clinical isolates of methicillin-resistant coagulase-negative staphylococci and Enterococcus faecium from China. J Med Microbiol 2012;61:1568e73. 3. Locke JB, Hilgers M, Shaw KJ. Mutations in ribosomal protein L3 are associated with oxazolidinone resistance in staphylococci
9.
10.
of clinical origin. Antimicrob Agents Chemother 2009;53: 5275e8. 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;46:3334e6. Diaz L, Kiratisin P, Mendes RE, Panesso D, Singh KV, Arias CA. Transferable plasmid-mediated resistance to linezolid due to cfr in a human clinical isolate of Enterococcus faecalis. Antimicrob Agents Chemother 2012;56:3917e22. Escribano I, Rodriguez JC, Llorca B, Garcia-Pachon E, Ruiz M, Royo G. Importance of the efflux pump systems in the resistance of Mycobacterium tuberculosis to fluoroquinolones and linezolid. Chemotherapy 2007;53:397e401. Shi WF, Jiang JP, Xu N, Huang ZM, Wang YY. Inhibitory effects of reserpine and carbonyl cyanidem-chlorophenylhydrazone on fluoroquinolone resistance of Acinetobacter baumannii. Clin Med J (Engl) 2005;118:340e3. Mendes RE, Deshpande LM, Farrell DJ, Spanu T, Fadda G, Jones RN. Assessment of linezolid resistance mechanisms among Staphylococcus epidermidis causing bacteraemia in Rome, Italy. J Antimicrob Chemother 2010;65:2329e35. Patel SN, Memari N, Shahinas D, Toye B, Jamieson FB, Farrell DJ. Linezolid resistance in Enterococcus faecium isolated in Ontario, Canada. Diagn Microbiol Infect Dis 2013;77: 350e3. Livermore DM, Warner M, Mushtaq S, North S, Woodford N. In vitro activity of the oxazolidinone RWJ-416457 against linezolid-resistant and -susceptible staphylococci and enterococci. Antimicrob Agents Chemother 2007;51:1112e4.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.e-jmii.com
BRIEF COMMUNICATION
Investigation of mechanisms and molecular epidemiology of linezolid nonsusceptible Enterococcus faecalis isolated from a teaching hospital in China Bin Li a, Chuan-Ling Ma a, Xiao Yu b, Yao Sun a, Mei-Mei Li a, Jian-Zhong Ye a, Ya-Pei Zhang a, Qing Wu a, Tie-Li Zhou a,* a Department of Clinical Laboratory, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China b School of Laboratory Medicine and life Science, Wenzhou Medical University, Wenzhou, Zhejiang Province, China
Received 23 September 2014; received in revised form 23 April 2015; accepted 21 May 2015
Available online 27 June 2015
KEYWORDS Enterococcus faecalis; Linezolid nonsusceptible; Multilocus sequence typing; Pulsed-field gel electrophoresis; 23S rRNA
Abstract The epidemiological and molecular characteristics of eight linezolid nonsusceptible Enterococcus faecalis isolated from a teaching hospital in China (January to July 2014) were investigated. The target site modifications and cfr gene associated with linezolid resistance were not found. Results of the epidemiological investigation indicated that linezolid resistance possibly occurred on several independent occasions and was often not related to linezolid administration. Copyright ª 2015, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
* Corresponding author. Department of Clinical Laboratory, First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang Street, Ouhai District, Wenzhou, Zhejiang Province, China. E-mail address: [email protected] (T.-L. Zhou). http://dx.doi.org/10.1016/j.jmii.2015.05.010 1684-1182/Copyright ª 2015, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
596
Introduction Enterococci have emerged in the past decade as an increasingly important cause of nosocomial infections. Linezolid, the first member of a new class of antibiotics (oxazolidinones), has been introduced into clinical practice for infections caused by multiresistant Gram-positive bacteria especially vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus.1 However, linezolid resistant isolates had emerged in several countries shortly after the drug appeared on the market, and they have also emerged recently in several hospitals in China.2 Vancomycin-resistant enterococci have caused hospital outbreaks worldwide; the occurrence of linezolid nonsusceptible enterococci, therefore, represents an immediate threat for patient care. A variety of mutations in domain V of 23S rRNA genes, especially G2576U and the multidrug resistance gene cfr, which codes for an adenine methyltransferase that modifies adenosine at position 2503 in the 23S rRNA, play an important role in linezolid resistance of enterococci. Mutations of the bacterial ribosomal proteins L3 and L4 have also been associated with resistance to linezolid,3e5 and active efflux pump systems may also be involved.6 Pulsed-field gel electrophoresis (PFGE) has been considered the “gold standard” for the study of hospital outbreaks because of its high degree of isolate differentiation. Multilocus sequence typing (MLST) offers the possibility to transfer typing data between laboratories or compare results via Internet, thus providing a powerful tool for global epidemiologic studies. The aim of this study was to investigate the epidemiological and molecular characteristics of linezolid nonsusceptible Enterococcus faecalis isolated from a Chinese hospital.
Methods Strain selection Eight E. faecalis clinical strains exhibiting linezolid Minimum inhibitory concentrations (MICs) 4 mg/L were collected from the First Affiliated Hospital of Wenzhou Medical University from January to July 2014. The clinical records of patients with linezolid nonsusceptible E. faecalis were examined retrospectively. Staphylococcus capitis(cfrþ) used for positive control in this study was donated by Rong Zhang, charge technician from the Second Affiliated Hospital of Zhejiang University.
Antimicrobial susceptibility testing Susceptibility testing was determined using the agar dilution method and broth microdilution method (linezolid and Daptomycin) according to Clinical and Laboratory Standards Institute recommendations (CLSI, 2013). E. faecalis ATCC29212 was used as quality control.
Molecular detection of mutations and resistance gene cfr The presence of mutations in domain V of 23S rRNA and ribosomal proteins L3 and L4, and cfr gene were
B. Li et al. investigated using polymerase chain reaction as described previously.3,4 Genomic DNA extracted from E. faecalis ATCC29212 and S. capitis-(cfrþ) were used as negative and positive control for cfr, respectively. The positive polymerase chain reaction products were sequenced by Shanghai Majorbio Bio-Pharm Technology Co. (Shanghai, China). Nucleotide and deduced amino acid sequences were compared with E. faecalis V583 strain and the linezolid susceptible E. faecalis clinical isolates (MIC, 1 mg/ mL) recovered from the same hospital during the study interval.
Effect of efflux pump mechanism The MIC of linezolid was determined using the broth microdilution method in either the presence or absence of reserpine (20 mg/L) or carbonyl cyanide m-chlorophenylhydrazone (CCCP; 5 mg/L), according to Clinical and Laboratory Standards Institute guidelines. An inoculum of 0.5 Mcfarland Standard of each isolate was inoculated into MuellereHinton cation-adjusted broth containing serial dilutions of linezolid. Four-fold or more reduction of linezolid MICs in the presence or the absence of reserpine or CCCP was defined as a positive phenotype for efflux.7
PFGE PFGE was performed on the CHEFeMapper XA PFGE system (Bio-Rad, Hercules, CA, USA) with a running time of 20 hours and pulse duration of 3.5e23.5 seconds. Salmonella serotype Braenderup H9812 DNA marker was used for the standard molecular weight and size determinations. Pattern analysis was carried out using QualityOne software (Bio-Rad Laboratories). Percentage similarities were identified on a dendrogram derived from the unweighted pair group method with arithmetic averages. Band position tolerance and optimization were set at 1.5% and 0.5%, respectively. Isolates showing a similarity coefficient of 80% were considered to be genetically related.8
MLST The linezolid nonsusceptible E. faecalis isolates were characterized by MLST using seven housekeeping genes (gdh, gyd, pstS, gki, aroE, xpt, and yqiL) according to the protocols and sequencing primers available at the E. faecalis database (http://efaecalis.mlst.net/).
Results Clinical characteristics of patients with linezolid nonsusceptible E. faecalis The clinical information of patients is given in Table 1. Two of the strains were isolated from urine, three from vaginal secretions, two from surgical wounds, and one from blood. No patients were treated with linezolid before linezolid nonsusceptible E. faecalis was isolated.
Linezolid nonsusceptible E. faecalis isolates Table 1
597
Clinical characteristics of patients with linezolid nonsusceptible Enterococcus faecalis isolates
Patient No.
Patient
Admission
Underlying disease
Samples
Previous hospitalization
Treatment outcome
LZD MIC (mg/L)
08/2/14
Colorectal cancer
Wound
No
4
GAS
20/2/14
Acute pancreatitis
Urine
No
F
GYN
12/2/14
Uterine leiomyoma
Vaginal fluid
No
33 42 80
F M M
OP OP GS
d d 21/7/14
Vaginal fluid Urine Blood
No No Yes
7(F524)
31
F
GYN
04/6/14
None Epididymitis Pancreatic cancer, OJ, diabetes, hypertension CIN, IUA
Discharge on Day 32 Discharge on Day 8 Discharge on Day 15 d d Relapse
Vaginal fluid
Yes
8(F527)
82
F
DER
04/5/14
Wound
Yes
Age (y)
Sex
Ward
Date
1(F465)
39
M
GS
2(F467)
50
M
3(F471)
48
4(F496) 5(F509) 6(F511)
T2DM, hypertension, gout
Discharge on Day 10 Discharge on Day 17
8 4 8 8 8 8 8
CIN Z cervical intraepithelial neoplasias; DER Z dermatology; F Z female; GAS Z gastroenterology; GS Z gastrointestinal surgery; GYN Z gynecology; IUA Z intrauterine adhesion; LZD Z linezolid; M Z male; OJ Z obstructive jaundice; MIC = minimum inhibitory concentration; OP Z outpatient; T2DM Z type 2 diabetes mellitus.
Antimicrobial susceptibility testing
V of the 23S rRNA genes, and the ribosomal proteins L3 and L4.
The eight E. faecalis strains were resistant to linezolid at a low level (4e8 mg/L), and all showed multidrug-resistant phenotype (resistant to 3 different antibiotics). All (8/8) of these isolates were resistant to quinupristin/dalfopristin, tetracycline, and erythromycin, and high-level gentamycin and ciprofloxacin resistance were present in 87.5% and 62.5% of these isolates, respectively. All E. faecalis were susceptible to vancomycin, teicoplanin, daptomycin, and the newly introduced tigecycline, and only one was resistant to ampicillin, as shown in Table 2.
Resistance genes and mutations Analysis of sequencing and blasted results failed to find the cfr gene and the possible mutations, including domain Table 2 isolates
Effect of the efflux pump mechanism The MICs of these isolates to linezolid were not changed or just decreased at one dilution in the presence of CCCP or reserpine. All strains grew in the presence of only reserpine (20 mg/L) or CCCP (5 mg/L).
Molecular epidemiology analysis PFGE analysis showed seven unrelated genotypes (Types AeG). Type A contained two subtypes (A1, A2), and these were isolated from patients in different wards. MLST data showed that five sequence types (ST16, ST49, ST586, ST587, and ST588) were assigned for these isolates, and the most
Antibiotic susceptibilities and linezolid resistance mechanisms of linezolid nonsusceptible Enterococcus faecalis
Patient no.
MIC (mg/L)
AMP CIP F 1(F465) 8 2(F467) 8 3(F471) 8 4(F496) 8 5(F509) 8 6(F511) 16 7(F524) 8 8(F527) 8
1 16 2 16 16 16 8 1
16 16 16 64 16 16 16 16
Genetic resistance markers
PFGE MLST type type
Q/D TET VAN ERY TGC
TEC DAP GEH LZD LZD þ LZD þ 23S L3/L4 cfr reserpine CCCP rRNA
>4 >4 >4 >4 >4 >4 >4 >4
2 2 2 2 2 4 2 2
16 16 16 16 16 16 16 16
2 2 1 2 2 2 1 2
8 8 8 8 8 8 8 8
0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12
0.5 1 1 1 1 1 0.5 1
R R R R R R S R
4 8 4 8 8 8 8 8
4 4 4 4 4 4 4 4
4 8 4 8 8 8 8 8
/ / / / / / / /
A1 B C D E F G A2
ST16 ST586 ST587 ST588 ST16 ST16 ST49 ST16
AMP Z ampicillin; CCCP Z carbonyl cyanide m-chlorophenylhydrazone; CIP Z ciprofloxacin; DAP Z daptomycin; ERY Z erythromycin; F Z nitrofurantoin; GEH Z high-level gentamycin; LZD Z linezolid; MLST Z multilocus sequence typing; e Z negative; PFGE Z pulsedfield gel electrophoresis; Q/D Z quinupristin/dalfopristin; TEC Z teicoplanin; TET Z tetracycline; TGC Z tigecycline; VAN Z vancomycin; MIC = minimum inhibitory concentration.
598 dominant clone was ST16. ST586, ST587, and ST588 were three novel sequence types detected from these isolates. Isolates with differently related PFGE patterns (e.g., Type A, Type E, and Type F) had the same sequence type (ST16; Table 2). Patterns and dendrogram analysis produced by PFGE are shown in Figure 1.
Discussion According to an epidemiological survey carried out by Chinese bacterial resistance monitoring network (CHINET) in 2012, the resistance rate of E. faecalis to linezolid was 0.3%. This is the first report of linezolid nonsusceptible Gram-positive bacteria in our hospital, and these strains were all isolated in a short period, which is alarming and requires ongoing surveillance. All isolates were multidrug resistant, but still sensitive to glycopeptides. A retrospective analysis of the patients’ treatment history showed no linezolid therapy prior to the linezolid nonsusceptible E. faecalis isolation. These strains were isolated on widely divergent dates and from different departments of the hospital (Table1). The epidemiological investigation also suggested that these isolates were sporadic cases, which indicated that patient-to-patient transmission was not the main cause of rapid emergence of linezolid resistance. It could also have occurred on several independent occasions and was often not related to linezolid administration. According to the clinical records, Patient 6 (F511), an 80-yearold man suffering from various underlying diseases, has been living in the hospital and been treated with various antimicrobials except for linezolid for a long time (from July 2014 to present). We speculated that he carried the linezolid nonsusceptible isolates prior to his administration, and selective pressure from long-term use of other antimicrobials may have contributed to the occurrence of these bacteria in the hospital setting. Alterations in the 23S rRNA target have remained the dominant mechanism detected among linezolid nonsusceptible enterococci, with a limited number of clinical
B. Li et al. isolates also carrying cfr. However, our results did not show any target site modifications or presence of cfr associated with linezolid resistance. Recent studies evaluating the linezolid resistance mechanisms in Gram-positive isolates from Canada have also reported similar results.9,10 Multidrug resistance (MDR) efflux transporters that pump out a wide range of substrates are known as one of the most important mechanisms of antimicrobial resistance in E. faecalis. The efflux pump phenotype experiment is based on the fact that if efflux pump inhibitors (e.g., reserpine or CCCP) significantly reduce the resistance of the MDR strain to antibiotics to which it was initially resistant or renders the MDR clinical strain as susceptible to antibiotics as the wild-type strain, then it is highly probable that the MDR phenotype of that strain is attributable to an overexpression efflux pump system. However, in our study, the MICs of the tested isolates to linezolid were not changed or just decreased at one dilution in the presence of CCCP or reserpine. It was likely that the capacity of the efflux pump to eliminate linezolid was limited. Therefore, these lowlevel resistance phenotypes were most probably due to other mechanisms. Overall, the present MLST results showed the existence of five allelic profiles, of which ST586, ST587, and ST588 were three novel sequence types. This indicates that some stimulus may lead to the emergence of new characteristics of regional strains. Isolates with differently related PFGE patterns (e.g., Type A, Type E, and Type F) had the same sequence type (ST16). However, they were isolated on widely divergent dates and from different departments of the hospital, and MLST is less discriminatory than PFGE when a short timescale is examined. Therefore, these linezolid nonsusceptible isolates represented sporadic cases. In conclusion, we failed to detect the known possible mechanisms of resistance to linezolid in these E. faecalis strains. The epidemiological investigation indicated that linezolid resistance possibly occurred on several independent occasions and was often not related to linezolid administration. A multidisciplinary approach is urgently
Figure 1. PFGE patterns and dendogram analysis of eight linezolid-nonsusceptible Enterococcus faecalis isolates. Lane M Z Salmonella serotype Braenderup strain H9812 Marker (kb); lanes 1e8 Z eight linezolid-nonsusceptible E. faecalis isolates 1e8 (Table 1). Dendogram was generated using QualityOne Software (Bio-Rad Laboratories, Hercules, CA, USA). The phylogenetic tree was constructed using UPGMA clustering. A genetic similarity index scale is shown in the left of the dendogram. PFGE Z pulsedfield gel electrophoresis; UPGMA Z unweighted pair group method with arithmetic mean.
Linezolid nonsusceptible E. faecalis isolates
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needed to evaluate the risk factors associated with the rapid occurrence and dissemination of linezolid resistance. 4.
Conflicts of interest All contributing authors declare no conflict of interest.
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Acknowledgments 6.
This work was supported by research grants from the National Natural Science Foundation of China (no. 81171614), Zhejiang Provincial Natural Science Foundation of China (LY14H190005), and Zhejiang Province Program for the Cultivation of High-level innovative Health talents ((2012) 141).
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