dissemination and molecular characterization of...

Research ArticleDissemination and Molecular Characterization ofStaphylococcus aureus at a Tertiary Referral Hospital inXiamen City, China

Yiwen Yu,1,2 Yihui Yao,2 QinyunWeng,3 Jingyi Li,4 Jianwei Huang,3

Yiqun Liao,4 Fu Zhu,3 Qifeng Zhao,2 Xu Shen,2 and Jianjun Niu2

1School of Public Health, Xiamen University, Xiamen, Fujian Province 361102, China2Zhongshan Hospital, Xiamen University, Xiamen, Fujian Province 361004, China3Xiamen Center for Disease Control and Prevention, Xiamen, Fujian Province 361021, China4School of Life Sciences, Xiamen University, Xiamen, Fujian Province 361102, China

Correspondence should be addressed to Jianjun Niu; [email protected]

Received 22 December 2016; Revised 28 March 2017; Accepted 23 April 2017; Published 5 July 2017

Academic Editor: Wen-Jun Li

Copyright © 2017 Yiwen Yu et al.This is an open access article distributed under theCreative CommonsAttribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Staphylococcus aureus is a global epidemic pathogen that causes heavy disease burden. The aim of this study was to determinewhich globally known S. aureus lineages are currently present in a hospital of Xiamen. Therefore, the 426 S. aureus strains weredetected byMeltingCurveAnalysis (MCA) and genotyped byPulsed FieldGel Electrophoresis (PFGE) aswell asMulticolorMeltingCurve Analysis-BasedMultilocusMelt Typing (MLMT). In addition, Multilocus Sequence Typing (MLST) was used to identify 108representative strains. In light of eighteen antibiotics except for Vancomycin (by Broth DilutionMethod), we used the Kirby-Bauerdisc diffusionmethod to assess antibiotic susceptibility of 426 S. aureus strains. Finally, PFGE analysis revealed 14 different patternswith three major patterns (C10, C8, and C11) that accounted for 69.42% of all S. aureus strains, andMT-1∼MT-5 occupied most partof the strains by MLMT. MLST revealed 25 different STs with the predominant types being ST239, ST59, and ST188. There havebeen 8 antibiotics that showed more than 50% resistance of all S. aureus strains. In summary, we found several of the lineages arepredominant in our hospital. And antibiotic resistance is still a severe problem that needs to be controlled in clinic.

1. Introduction

Staphylococcus aureus, the major pathogen of human infec-tious diseases which can cause various lesions by secretinga variety of virulence factors, has been becoming a greatproblem at home and abroad [1]. As we know, S. aureus canbe isolated from patients with different kinds of diseases, andthere are considerable variations in the prevalence of S. aureusaccording to geographic areas. It was proposed by someexperts that most S. aureus strains belong to a few distinctpandemic lineages which were predominant in hospitals orcommunities [2].

In recent years, several typing methods have beenevaluated for S. aureus strains characterization, such asrepetitive sequence based PCR (rep-PCR), Pulsed FieldGel Electrophoresis (PFGE), Multilocus Sequence Typing

(MLST), Multilocus Variable-Number Tandem RepeatsAnalysis (MLVA), Staphylococcal Protein A (spa), andStaphylococcal Cassette Chromosome (SCC-mec) Typing[3]. Historically, PFGE used to be the “gold standard”of S. aureus typing because of convenience and highdiscriminatory power [4]. At the present time, PFGE is stillwidely used for short-term and local epidemiology to identifyoutbreak; however, it is less suitable for long-term and globalepidemiology since it was not permitted to compare betweendifferent research centers. In addition, PFGE data mightchange over time and produceweak bands so that it is difficultfor us to compare over long time periods or avoid a matter ofsubjective interpretation [5]. Therefore, another moleculartyping method, MLST, served as an auxiliary reference.MLST involves the sequencing of seven housekeeping genes(arcC, aroE, glpF, gmK, pta, tpi, and yqiL) and each unique

HindawiBioMed Research InternationalVolume 2017, Article ID 1367179, 9 pageshttps://doi.org/10.1155/2017/1367179

https://doi.org/10.1155/2017/1367179

2 BioMed Research International

allelic profile is assigned a sequence type (ST) [6]. Clonalcomplexes are defined as groups of STs in which every STshares at least five of seven identical alleles with at least oneother ST in the group [7]. In the meantime, as an expensiveand time-consuming method, MLST has limitation of usagesomewhere. So based on MLST, a new genotyping methodcalled Multicolor Melting Curve Analysis-Based MultilocusMelt Typing (MLMT) were proposed by Li et al. [8] fromEngineering Research Center of Molecular Diagnostics,Ministry of Education, State Key Laboratory of CellularStress Biology, School of Life Sciences, Xiamen University.The entireMLMTprocedure could be finishedwithin 3 hourson a real-time PCR machine and the cost was approximately50 times less than MLST. The ability of detecting multipleSNPs in one reaction further helps to simplify the operationand increase the throughput. Moreover, the interpretationof 𝑇𝑚s into binary codes can be easily automated and the

efficiency of detection can be further improved. In thisstudy, all S. aureus strains collected in our hospital had beenanalyzed by PFGE andMLMT from 1 January 2014 to 30 June2015; also the representatives were identified by MLST. Theobjectives of the current study were to combine the PFGEdata with additional typingmethods (MLMT andMLST) andto determine which of the globally known clones were foundin our hospital during a one-year-and-a-half study period.

Except for genotyping, the resistance to antibiotics, espe-cially the emergence of multidrug resistant bacteria, hasbrought great difficulties and challenges to clinical medicine.In 1961, one year after methicillin was applied to clini-cal therapeutics, methicillin-resistant Staphylococcus aureuswere isolated from patients and quickly spread throughoutthe world subsequently. Multiple-drug resistance of MRSAis serious, especially sensitive to glycopeptide antibioticssuch as Vancomycin. However, in recent years, it limitedthe use of glycopeptide antibiotics since the appearanceof Vancomycin-intermediate S. aureus (VISA) or heteroge-nous Vancomycin-intermediate S. aureus (hVISA). In orderto provide the basis for clinical anti-infection treatment,antimicrobial susceptibility should be tested and becomean auxiliary method combined with molecular genotyping.The aims of this investigation were to evaluate the molecu-lar and clinical epidemiology of Staphylococcus aureus andto explore the transmission between different wards andsickbeds.

2. Methods and Materials

This retrospective studywas conducted from 1 January 2014 to30 June 2015 at an 800-bed teaching hospital which providestertiary care, located in the city of Xiamen, China.With strictpersonal information protection as conditions, institutionalreview boards and independent ethics committees of Healthand Family Planning Commission of Xiamen approved thisretrospective study and the retrospective use of the patientinformation.

2.1. Bacterial Strains. A total of 458 consecutive, nondupli-cate Staphylococcus aureus strains were collected includingboth adults and children and detected by Melting Curve

Table 1: Demographics and sources of 426 S. aureus strains.

Characteristics Demographic outcomesN N%

SexMale 243 57.04%Female 183 42.96%

Age (years) mean ± SD 65.96 ± 17.86<30 13 3.05%30∼ 54 15.73%45∼ 76 17.84%60∼ 105 24.65%75 178 41.78%

Patient locationMedical ward 159 37.32%Surgical ward 199 46.71%ICU 68 15.96%

Duration of hospitalstay (days) beforeculture

0∼9 251 58.9%10∼19 83 19.48%>20 92 21.60%

Sources of specimenBlood 13 3.05%Bile 1 0.23%Secreta 35 8.22%Fester 6 1.41%Sputamentum 344 80.75%Pleural effusion 1 0.23%Synovial fluid 1 0.23%Drainage liquid 7 1.64%Throat swab 8 1.88%Midstream urine 4 0.94%Others 6 3.05%

Note. Values are mean ± SD of age.

Analysis (MCA)which is based on RT-q-PCR, if the resultingsignal peak at 79∘C means that the strains carry FemAgene (Staphylococcus aureus) and if the rising signal peak at64∘C showed that the strains carry mecA gene, methicillin-resistant Staphylococcus aureus (MRSA). But 24 strains wereunavailable because of failure to obtain complete and accurateclinical data; also another 8 S. aureus strains were excludeddepending on 5 of poor quality, 2 of collectors’ mistakes,and 1 of loss with efficiency of 93.01%. Thus, there were426 S. aureus strains eligible for enrollment, and all of themwere identified by standard biochemical test in accordancewith Clinical and Laboratory Standards Institute (CLSI) [9].We isolated the strains from patients with infections as wellas from colonized individuals (blood, ascites, bile, secreta,fester, sputamentum, pleural effusion, synovial fluid, drainageliquid, throat swab, and midstream urine, Table 1).

BioMed Research International 3

2.2. PFGETyping. Theclonal relationships of all 426 S. aureusstrains were assessed by PFGE using S-maI as previouslydescribed. The PFGE types were defined according to thecriteria of Tenover et al. [10]. The dice coefficient wasused with 1.25% optimization and 1% tolerance to calculatesimilarities between PFGE patterns. The strains with >75%similarity were clustered in different patterns. The resultswere also analyzed usingBioNumerics version 6.6.4 statisticalsoftware, and dendrograms were generated according to asimple matching coefficient and the unweighted pair groupmethod with the arithmetic mean (UPGMA) algorithm.

2.3. MLMT. The dual-labeled, self-quenched probes aloneare nonfluorescent or weakly fluorescent but become fluo-rescent when hybridizing with the reversely complementarysingle-stranded DNA. After asymmetric PCR, the producedexcess single-stranded amplicons are targets for the dual-labeled, self-quenched probes. Post-PCR Melting CurveAnalysis would generate 𝑇

𝑚values reflecting the sequence

variations in the probe-binding region of the amplicons. Dueto the possible existence of polymorphic SNPs sites in theprobe-binding regions, a series of 𝑇

𝑚rather than a single

𝑇𝑚for one allelic type would be generated. The probe was

designed in such a way that it is complementary with noneof the sequence variants at these polymorphic SNP sites.Consequently, the 𝑇

𝑚values for one allelic type would be

always lower than another allelic type. A series of binarycodes could be obtained when multiple SNPs are genotyped.The concatenated serial binary codes are defined as MTs.TheMTs can be further linked to STs or CCs.

2.4. MLST. MLST was performed on selected representa-tive strains of major PFGE patterns as described previ-ously. MLST was performed on all the strains by sequenc-ing the internal fragments of seven housekeeping genes(arcC, aroE, glpF, gmK, pta, tpi, and yqiL). The lociwere amplified using the primers and conditions recom-mended on http://www.mlst.com/ server. Sequencing wasperformed by BGI (Shenzhen, China). Sequences wereanalyzed online (http://pubmlst.org/vparahaemolyticus/) toassign allele numbers and define STs. The clonal complexes(CCs) of S. aureus were analyzed by goe-BURST [11] ofPhyloviz software (http://www.phyloviz.net/) [12]. Those STsthat share identical alleles at six of the seven MLST loci withat least one other ST were classified as one CC [13].

2.5. Susceptibility Testing. Susceptibility testing was per-formed on all Staphylococcus aureus strains using the Kirby-Bauer disc diffusion method in accordance with the per-formance standards for antimicrobial susceptibility testingrecommended by the Clinical and Laboratory StandardsInstitute (CLSI) to 19 antimicrobial agents [14]. And wealso used MIC method (Broth Dilution Method) to testS. aureus resistance to Vancomycin. Multidrug resistancewas arbitrarily defined as resistance of S. aureus to threeor more distinct antimicrobial classes. MCA were used toensure methicillin-resistant S. aureus (MRSA, defined asmecA-positive strains). MRSA strains isolated in this studywere included in the multidrug resistant (MDR) category

irrespective of their susceptibility profiles. But we did notregard intermediate resistance as the antibiotic resistance.Results were analyzed using WHONET5.4 to determineresistance profiles (http://www.whonet.org.cn/index.html).

3. Data Analysis

Bacteriologic and patient data were compiled in an electronicdatabase using Excel (Microsoft). Quantitative variables weresummarized as mean ± SD and qualitative variables as pro-portions (%). In descriptive statistics, frequency and propor-tions were calculated for categorical variables. BioNumericsversion 6.6.4 was used to cluster the outcomes of PFGEincluding picture processing and antibiotics susceptibility aswell as isolated locations. As for MLMT and MLST analysis,Phyloviz software and Adobe Illustrator were used to dealwith the calculation and picture adjustment together. As forantibiotic susceptibility, we use WHONET 5.4 to analyze theoutcomes of 19 antibiotics.

4. Result

426 strains of S. aureus were conducted by PFGE using S-maI to cut into 15∼20 pieces (20 kb∼1200 kb). According to100% similarity clustering standard, they were divided into108 genotypes (P1∼P108). MLMT resolved 426 strains into 20MTs; among them, the most common MT was MT-1, whichwas composed of 284 strains and accounted for 66.67% ofthe total strains. And then, 108 representative strains wereclustered into 14 clusters with 75% similarity standard (C1∼C14). The similarity of all strains was from 55.8% to 100%,and discriminatory index (D) was 0.748 (Figure 1).

In Figure 1, L1∼L27 represent different sources as followsorderly: L1 department of traumatic orthopedics, L2 depart-ment of ENT, L3 department of obstetrics and gynecology,L4 department of hepatobiliary surgery, L4 department ofcadres health, L5 articular surgery, L6 respiratory medicine,L7 department of neurology, L8 ICU, L9 emergency depart-ment, L10 department of spine surgery, L11 department ofgeriatrics, L12 department of urinary surgery, L13 departmentof endocrinology department, L14 department of derma-tology, L15 department of general surgery, L16 departmentof pediatrics, L17 department of medical oncology, L18neurosurgery, L19 department of pediatrics, L20 departmentof anesthesia, L21 department of gastrointestinal surgery, L22department of gastroenterology, L23 department of cardi-ology, L24 department of thoracic surgery, L25 departmentof vascular surgery, L26 department of hematology, L27department of traditional Chinese medicine. Abbreviationsare as follows: P: Penicillin; LVF: Levofloxacin; OX: Oxacillin;NXN: Norfloxacin; CP: Ciprofloxacin; GM: Gentamicin; RIF:Rifampicin; AZI: Azithromycin; E: Erythromycin; CD: Clin-damycin; MIN: Minocycline; T/S: Trimethoprim and Sul-phamethoxazole; C: Chloramphenicol; FD: Furantoin Tige-cycline; Q/D: Quinupristin/Dalfopristin; TGC: Teicoplanin;LZD: Linezolid; VA: Vancomycin.

As we can see, advantages of clusters are C10, 285/426(66.90%); C8, 75/426 (17.61%); C11, 22/426 (5.16%), whichwere predominated and together accounted for 89.67% of

http://www.mlst.com/

http://pubmlst.org/vparahaemolyticus/

http://www.phyloviz.net/

http://www.whonet.org.cn/index.html


96.8

60 65 70 75 80 85 90 95 100

AZI

OX

FD MXN

T/S

E CP CD QD

RIF

LZD

C MIN

P GM

TGC

TEC

VA LVF

PFGE-Sma1 PFGE-Sma1 Resistance phenotype

PFGE ID Type Counts Cluster Location Date of isolation

60.3

65.0

70.2

75.0

74.4

79.4

84.3

85.8

82.1

83.9

88.9

92.9

95.7

95.7

96.6

96.6

1404231814042626140120091302105414031723140212451406046313011770130310491401141514042200140106471302105313110486130304301404195714010760

P1 1P2 1P3 1P4 1P5 1P6 3P7 2P8 1P9 1

P10 1P11 1P12 2P13 1P14 2P15 1P16 1P17 1

C1 L24C1 L19C1 L18C1 L18C2 L6C3 L4C3 L23C4 L15C5 L23C5 L26C5 L18C5 L18C5 L17C5 L12C6 L5C6 L1C7 L6

2015-4-252015-4-292015-1-232014-2-192015-3-202015-2-202015-6-6

2014-1-242014-3-192015-1-162015-4-242015-1-8

2014-2-192014-11-62014-3-7

2015-4-222015-1-9

87.4

90.7

90.0

94.6

96.6

94.9

96.6

94.6

94.1

1402040714011801130203311312202114051195130116511301185714040040131213011301210913111349140314151301216814050639

P18 1P19 1P20 2P21 2P22 1P23 2P24 4P25 4P26 1P27 7P28 7P29 4P30 2P31 2

C7 L23C7 L6C8 L13C8 L18C8 L5C8 L7C8 L8C8 L9C8 L24C8 L7C8 L17C8 L19C8 L7C8 L6

2015-2-112015-1-212014-2-5

2014-12-252015-5-142014-1-232014-1-252015-4-1

2014-12-162014-1-28

2014-11-152015-3-172014-1-292015-5-8

13011858 P32 3 C8 L18 2014-1-2593.1

83.8

82.7

81.2

96.6

86.6 91.0 94.9

96.6

91.5

89.1

96.0

130305331303018413031316130115081311211014021044130302551404168013012397

P33 1P34 1P35 1P36 1P37 2P38 1P39 1P40 1P41 3

C8 L19C8 L6C8 L27C8 L3C8 L7C8 L8C8 L3C8 L7C8 L13

2014-3-82014-3-4

2014-3-182014-1-21

2014-11-262015-2-182014-3-5

2015-4-182014-1-31

55.8

78.3

86.3

85.7

90.7

96.3

94.2

96.3

140212431312239713021050140110941404195614012012131213041302097013110747

P42 2P43 1P44 1P45 1P46 1P47 1P48 1P49 4P50 1

C8 L25C8 L7C8 L17C8 L19C8 L26C8 L9C8 L3C8 L17C8 L11

2015-2-202014-12-302015-2-192015-1-162015-4-222015-1-23

2014-12-162015-2-182014-11-8

68.6

74.8

92.9

90.8

96.6

96.0

13111035140305081301157814021951

P51 1P52 1P53 2P54 3

C8 L24C8 L5C8 L16C8 L7

2014-11-122015-3-6

2014-1-222015-2-28

74.0

80.0

94.2

96.3

97.0

91.8

90.0

131103511311209614011799140421041401024714010650140118001401075414011907

P55 1P56 1P57 1P58 1P59 1P60 1P61 2P62 2P63 2

C8C8C9C9

C10C10C10C10C10

L11L11L11L6L7L7

L18L18L10

2014-11-52014-11-262015-1-212015-4-232015-1-32015-1-8

2015-1-212015-1-9

2015-1-22

89.0 94.3

96.8

97.0

97.1

1311074614042203130121521405019414011420130210441302131513031382

P64 2P65 1P66 5P67 1P68 2P69 1P70 4P71 1

C10C10C10C10C10C10C10C10

L17L5

L10L22L7

L18L7

L27

2014-11-82015-4-242014-1-292015-5-4

2015-1-162014-2-192014-2-222014-3-19

67.271.0

85.4

96.0

94.6

97.0

92.2 96.3

96.8

131209141402002213011650131202161403050614030190140104501302097213021046

P72P73P74P75P76P77P78P79P80

11

207112141

C10C10C10C10C10C10C10C10C10

L27L27L7L7L8

L18L9

L18L27

2014-12-112015-2-2

2014-1-232014-12-32015-3-62015-3-32015-1-6

2014-2-182014-2-19

70.4

84.9

83.2

88.0

90.6

92.3

91.2

96.8

95.8

94.9

96.3

13111781140608671301216713020767130204461404234714051874131221191402097613122120

P81 2P82 2P83 3P84 1P85 33P86 1P87 1P88 2P89 1P90 4

C10C10C10C10C10C11C11C11C11C11

L17L15L21L9L6L5

L20L18L6L7

2014-11-212015-6-112014-1-292014-2-112014-2-6

2015-4-302015-5-24

2014-12-262015-2-17

2014-12-26

72.6

78.1

81.5

82.0

81.5

86.7

91.8

91.5

89.2

94.3

96.6

94.7

96.6

96.6

140105131403227313021057140314901302026213121832130305301303165114011278131211231401175113030254131107931310222314020709140106511403212214032429

P91 1P92 1P93 1P94 1P95 8P96 1P97 1P98 2P99 2

P100 2P101 1P102 1P103 1P104 2P105 1P106 3P107 1P108 1

C11C11C11C11C11C11C12C12C12C12C12C13C13C14C14C14C14C14

L18L17L18L14L5

L17L17L18L8L1L8L7

L19L3

L22L17L7L8

2015-1-72015-3-262014-2-192015-3-182014-2-4

2014-12-232014-3-8

2014-3-212015-1-15

2014-12-132015-1-202014-3-5

2014-11-142014-10-312015-2-142015-1-8

2015-3-272015-3-27

ResistantAbout resistance phenotypes:

IntermediateSusceptible

Figure 1: The PFGE cluster analysis of 108 representative S. aureus strains.


MT19

1-0.23MT20

MT181-0.23

1-0.23

1-0.23MT17

C4

C13C2

C8

MT13

MT14 MT15

MT16

2-0.47

2-0.47 2-0.47

2-0.47

C1

C7 C5; C9C1; C9

MT10

MT12

MT11

4-0.94

4-0.94

4-0.94

C8

C5-3; C10-1

C5-3; C6-1

MT95-1.17

C3-2; C11-3MT8

MT7

6-1.41C12

8-1.88

C14

MT612-2.82

C5-1;C8-11

MT520-4.69

C8

MT3

MT421-4.93

21-4.93

C8

C8MT2

25-5.87

C3-3; C11

MT1284-66.67

C6-1;C8-1;C10-282

Figure 2: MLMT analysis results of 426 S. aureus strains with PFGE. The number-frequency (%) of each MT is given together with thenumber of the corresponding PFGE clusters and the type and number of PFGE clusters of all the MTs obtained from the 426 strains.The sizeof the pies illustrates the relative number of MTs but not in a true scale.

the strains. The remaining 44 strains belonged to 11 sporadicprofiles that were separated from 27 different sites. Theclone strains distribute into concentrated sites, for instance,the largest number of P74 which belonged to C10 contains206 strains (48.36%) from the neurology or neurosurgerywards, and most of the strains similarity may be up to 100%.Compared with the outcomes of PFGE, MLMT resolved426 strains into 20 MTs (MT1∼MT20); among them, MT-7belonged to C14, MT-13 belonged to C1, MT-14 belonged toC7, MT-19 belonged to C2, MT-17 belonged to C4, andMT-3,MT-4, MT-5, MT-10, and MT-20 belonged to C8 (Figure 2).Association analysis between MT and clusters of PFGE forthe 426 strains showed that MLMT results fully agreed withthe theoretical predication.

In order to further analyze the genotype P74 and othercurrent strains, we used MLST as a combined method tocompare with global prevalence clones. 108 representativeswere typed by MLST (one randomly selected per PFGE typecomposed of more than one strain). MLST revealed a totalof 25 different sequence types. ST239 (PFGE types = 27,containing C6, C10), ST188 (PFGE types = 12, containing C8),and ST59 (PFGE types = 11, containing C11) are accounted formost part of the representative strains (Figure 3).

Different MLST types shared common PFGE patterns(e.g., C3 and C22 both belonged to ST30). Vice versa, severalstrains with different MLST shared the same PFGE patterns(e.g., ST121 and ST837 included C1, Table 2).

As for antibiotic susceptibility, this study also tried tofigure out the correlation between genetic background and

antibiotic susceptibility. Figure 1 shows the antibiograms ofthe 108 representative S. aureus strains. Some clones, such asP6 or P7, showed high heterogeneity in their antibiograms,which belong to the same cluster patterns in the meantime.Of the 426 S. aureus strains, 96.48% showed resistanceto Penicillin, 77.70% to Levofloxacin, 70.89% to Oxacillin,53.05% to Azithromycin, 68.54% to Norfloxacin, 67.84% toCiprofloxacin, 64.32% to Gentamicin, 63.85% to Rifampicin,and 44.13% to Clindamycin (>50% would be recognized asresistant to some antibiotics in China). Ten strains (2.34%)were susceptible to all antibiotics (Table 3).

Multidrug resistance to one, four, five, six, seven, eight,nine, ten, eleven, and twelve antibiotics in addition toOxacillin was observed for 0.7%, 5.87%, 0.94%, 12.21%,15.26%, 5.40%, 17.37%, 9.39%, 3.05%, and 0.7% of the strains,respectively (Table 4). The most prominent combinations ofresistance phenotypes are shown in Figure 1. And in Table 4,we can figure out that most of the MRSA strains isolatedwere resistant to at least four antimicrobial agents (includingOxacillin), emphasizing that antibiotic resistance remains aproblem and underlines the importance of infection controlmeasures.

5. Discussion

Staphylococcus aureus is among themost frequently identifiedantimicrobial drug-resistant pathogens worldwide and hasevolved in a relatively few lineages. It has been demonstratedthat some lineages are ecologically highly successful and


30 45 837

121 59

239

2805

258 9

965

1

15

188

128120

2631

630

5

943

7

6

88

72

22

ST: 30 isolates = 2Cluster: C3, C11

ST: 121 isolates = 3Cluster: C1, C1, C1

ST: 45 isolates = 1Cluster: C4

ST: 837 isolates = 1Cluster: C1ST: 59 isolates = 11Cluster: C3, C11, C11 (10)ST: 25 isolates = 2Cluster: C7 (2)ST: 2805 isolates = 1Cluster: C7ST: 239 isolates = 27Cluster: C6, C10 (26)ST: 8 isolates = 3Cluster: C5 (2), C6ST: 630 isolates = 2Cluster: C5, C9ST: 72 isolates = 1Cluster: C9ST: 22 isolates = 1Cluster: C2ST: 9 isolates = 1Cluster: C13

ST: 15 isolates = 5Cluster: C12 (5)ST: 1 isolates = 3Cluster: C8ST: 188 isolates = 12Cluster: C8 (12)ST: 1281 isolates = 1Cluster: C5ST: 20 isolates = 1Cluster: C5ST: 965 isolates = 1Cluster: C8ST: 5 isolates = 5Cluster: C13, C5, C8 (3)ST: 88 isolates = 5Cluster: C14 (5)ST: 6 isolates = 9Cluster: C8 (9)ST: 7 isolates = 6Cluster: C8 (6)ST: 943 isolates = 3Cluster: C8 (3)ST: 2631 isolates = 1Cluster: C10

Figure 3: MST of 108 representative strains by MLST. MST: minimum spanning tree.

Table 2: MLST, MLMT, and PFGE cluster analysis of 108 S. aureus strains.

MLST MLMT PFGE clusters PFGE types CountsST1 MT-10 C8 P44, 39, 31 3ST5 MT-6 C5, C8, C13 P12; P23, 41, 43; P102 5ST6 MT-1, MT3 C8 P36, 27, 30, 33, 35, 28, 37, 38, 29 9ST7 MT-4 C8 P32, 20, 34, 26, 25, 40 6ST8 MT-12 C5, C6 P9, 10; P16 3ST9 MT-18 C13 P16 1ST15 MT-8, MT-12 C12 P97, 98, 99, 100, 101 5ST20 MT-11 C5 P13 1ST22 MT-19 C2 P5 1ST25 MT-14 C7 P17, 19 2ST30 MT-9 C3, C11 P6; P87 2ST45 MT-17 C4 P8 1ST59 MT-2 C11 P95, 93, 96, 88, 90, 91, 89, 94, 92, 86, 87 11ST72 MT-16 C9 P58 1ST88 MT-7 C14 P104, 105, 106, 107, 108 5ST121 MT-13 C1 P1, 2, 4 3ST188 MT-5 C8 P45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 12

ST239 MT-1 C6, C10P15; P59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 74, 75, 77, 78, 79, 80, 81, 82,

83, 84, 85, 8627

ST630 MT-15 C5; C9 P11; P57 2ST837 MT-13 C1 P3 1ST943 MT-4 C8 P21, 22, 24 3ST965 MT-20 C8 P42 1ST1281 MT-11 C5 P14 1ST2631 MT-11 C10 P76 1ST2805 MT-14 C7 P18 1


Table 3: Antibiotic susceptibility of 426 S. aureus strains (19 antibiotic agents).

Antibiotics Resistant Intermediate Susceptible Resistance ratePenicillin 410 0 15 96.24%Levofloxacin 330 1 95 77.46%Oxacillin 301 0 124 70.66%Norfloxacin 292 3 130 68.54%Ciprofloxacin 288 10 127 67.61%Gentamicin 273 5 147 64.08%Rifampicin 271 1 153 63.62%Azithromycin 225 1 199 52.82%Erythromycin 201 5 199 47.18%Clindamycin 187 6 232 43.90%Minocycline 105 136 184 24.65%Trimethoprim and Sulphamethoxazole 46 207 173 10.80%Chloramphenicol 28 1 396 6.57%Furantoin 9 6 410 2.11%Tigecycline 3 11 411 0.70%Quinupristin/Dalfopristin 2 1 422 0.47%Teicoplanin 2 0 423 0.47%Linezolid 1 0 424 0.23%Vancomycin 0 0 425 -

Table 4: Multiresistance phenotypes of 426 S. aureus strainsresistant toother 18 antibiotics including Oxacillin.

Resistance profile Number (%) of strainsOxacillin + 1 antibiotic 3 (0.7%)Oxacillin + 4 antibiotics 25 (5.87%)Oxacillin + 5 antibiotics 4 (0.94%)Oxacillin + 6 antibiotics 52 (12.21%)Oxacillin + 7 antibiotics 65 (15.26%)Oxacillin + 8 antibiotics 23 (5.40%)Oxacillin + 9 antibiotics 74 (17.37%)Oxacillin + 10 antibiotics 40 (9.39%)Oxacillin + 11 antibiotics 13 (3.05%)Oxacillin + 12 antibiotics 3 (0.70%)

that most strains belong to pandemic clones. Surprisingly,there are not many studies reporting data about all typesof S. aureus infections in a general population of XiamenCity, China, in the last decade. This study provides thefirst comprehensive description about the epidemiology of S.aureus at a tertiary referral hospital in Xiamen.

The bacteriologic data is consistent with the research byHeipel et al. [15]; active surveillance by an infection controlpractitioner together with neurosurgeons may enhance sig-nificantly the sensitivity for detecting surgical site infection(SSI) after neurosurgery. Moreover, in a French national-based surveillance program, the SSI rate showed higherprevalence of S. aureus, and this confirms our study that greatpart of them were from surgical wards which seems to be afactor increasing the risk of S. aureus infection [16].

We characterized the S. aureus strains by using differentmolecular typing tools. After analysis of the data, we foundinteresting clinical and epidemiological findings. First, PFGEtype 74 (C10; MT-1) was dominant among the 426 strains, asituation not described in other countries. In the meantime,we found a high degree of clonality of the strains obtainedin this study, which demonstrates that most strains belong topandemic clones. On the other hand, we found that PFGE,MLMT, and MLST presented a good correlation for most S.aureus strains.

The PFGE analysis revealed 108 different genotypes thatincluded 3 predominant clones (C10, C8, and C11). Moreover,the P74 strains were all from neurology or neurosurgerywards and presented aggregation in the same isolation sites.Many studies have demonstrated that a clonal spreading cancause nosocomial outbreaks, so P74 is a great threat whichwould need more attention. Besides, PFGE is known to be ahighly discriminatory and valuable technique for the typingof S. aureus [17] and has been used for staphylococci for localinvestigations and national surveillance in China. It has beenargued that the stability of PFGE may be insufficient for itsapplication to long-term epidemiological studies due to thehigh degrees of genetic variation that have been observedamong pandemic cloneswith a long evolutionary history [18].However, we have not found that the predominant clones inthis study undergo significant changes during one year andhalf.

According to many reports, ST239, a typical Hungar-ian/Brazilian clone which belonged to clonal complex 8(CC8), is the major endemic clone in many Asian countries,although recent studies show that it is being graduallyreplaced by other emerging clones as was also observed in ourstudy (containing PFGE type 74, with the largest number).


It was absent in other major global S. aureus strains fromdivergent clonal and geographical backgrounds. It is likelythat the South American strains associated with ST239 S.aureus are clustered within a uniform phylogenetic cladehighly distinct compared to the Asian clade. This suggeststhat patients may represent an important reservoir for S.aureus dissemination within the hospital, when admitted asinpatients, and reinforces the observation of Alcoceba et al.[19].

Though several studies reported incidences of ST59prevalence in Chicago of United States, there is a paucity ofdata on themolecular epidemiology of such clones. However,in our study, similar observations were found that ST59 mayvary from other current clones from different locations. Andwe suspect that it might be community-acquired infectionbecause ST59 carry Panton Valentine Leukocidin (PVL)which is associated with major CA-MRSA lineages such asST1, ST30, ST8, and ST59 [20]. Few studies have documentedthe epidemiology of CA-MRSA as cause of health-care-onsetinfections in China and it should become another concernedproblem in our hospital. On the other hand, PVL may be theunusual toxic gene both in community and in hospital.

In fact, the present work provides important epidemio-logical information on S. aureus in this tertiary referral hos-pital. More clinical and sequencing data should be obtainedworldwide to help elucidate the global epidemiology andevolution. To confirm the sources and affinity of these strainsmentioned above, we used three molecular typing methods(PFGE, MLMT, and MLST) to explore the clonal spread ofS. aureus between different hospital wards. And the results ofthis study conclude that the combination of PFGE andMLSTis more discriminatory as compared to using a single methodonly and will allow us to better monitor and recognizechanges in S. aureus epidemiology over time.

In Europe and the United States, certain clinical strainsof S. aureus are sensitive to antibiotics which are commonlyused in clinic except for Penicillin. So the condition ofdrug resistance has not attracted enough attention [21, 22].But in our country, multidrug resistance of S. aureus ishighly reported in recent years, especially Erythromycin,Clindamycin,Oxacillin, andPenicillin [23].Thepresent studyrevealed that 70.19% of the strains were multiresistant andthat 51.24% were resistant to at least 7 antimicrobial agents.Some MDR bacteria have a common drug resistance spec-trum (Levofloxacin, Oxacillin, Norfloxacin, Ciprofloxacin,Gentamicin, Rifampicin, and Azithromycin). As we know, inthese years, the emergence of VISA and hVISA was reportedabroad. Although we did not find any strain resistant toVancomycin until now, it still should be a caution for us touse Vancomycin to cure S. aureus infection in patients. Fromanother perspective, the strains identified to the same clusterseem to have similar resistance phenotypes which enhancethe evidence of the genotyping.

However, the current retrospective study lacks an analysisof hospital environmental specimen and may become thelimitation of our further research. But the affinity analysisof S. aureus provides important information on the hospitalepidemiology and infection control surveillance and mon-itoring. Even for other hospitals in Xiamen, this study is

also important to demonstrate the long-term persistence ofan endemic clone of S. aureus responsible for nosocomialinfections within a hospital. S. aureus has been recognized asa major pathogenic species in the hospitalized patients whichsuggested that further investigations should be performedto elucidate its mode of transmission and the S. aureusreservoir and to identify specific preventive measures thatcould minimize the emergence and spread of such bacteriain the hospital environment, especially in the neurology andICU.

In summary, we have demonstrated that (I) several ofthe lineages that are predominant in the world are presentin our hospital, (II) ST188 may be the new prevalencestrains in this study without reports in other former studies,(III) the combination of PFGE, MLMT, and MLST is morediscriminatory as compared to using a single method onlyand will allow us to better monitor and recognize changesin Staphylococcus aureus epidemiology over time, (IV) clonalcomplex predictions based on PFGE and MLMT could beconfirmed by MLST, and (V) there is still a serious problemabout MDR strains.

Ethical Approval

Ethical approval for this study was obtained from the Healthand Family Planning Commission of Xiamen.

Consent

Informed consent was obtained from all individual partici-pants included in the study.

Disclosure

The sponsors had no role in study design, data collectionand analysis, decision to publish, or preparation of themanuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

This study was financially supported by the Natural ScienceFoundation of the Fujian Province (Grant no. 2015J01529)and Chinese National Natural Science Foundation (Grant no.81271929). Besides, the authors appreciated Dr. Tianci Yang’sgreat work for this study including specimen collecting andmanuscript revision.

References

[1] R. Kock, K. Becker, B. Cookson et al., “Methicillin-resistantstaphylococcus aureus (MRSA): burden of disease and controlchallenges in Europe,” Eurosurveillance, vol. 15, no. 41, pp. 1–9,2010.

[2] Z. Lu, Y. Chen, W. Chen et al., “Characteristics of qacA/B-positive staphylococcus aureus isolated from patients and


a hospital environment in China,” Journal of AntimicrobialChemotherapy, vol. 70, no. 3, pp. 653–657, 2015.

[3] J. A.Otter andG. L. French, “Community-associatedmeticillin-resistant Staphylococcus aureus: the case for a genotypic defini-tion,” Journal of Hospital Infection, vol. 81, no. 3, pp. 143–148,2012.

[4] L. K.McDougal, C. D. Steward, G. E. Killgore, J. M. Chaitram, S.K. McAllister, and F. C. Tenover, “Pulsed-field gel electrophore-sis typing of oxacillin-resistant Staphylococcus aureus isolatesfrom theUnited States: establishing a national database,” Journalof Clinical Microbiology, vol. 41, no. 11, pp. 5113–5120, 2003.

[5] V. Li, L. Chui, L. Louie, A. Simor, G. R. Golding, and M.Louie, “Cost-effectiveness and efficacy of spa, SCCmec, andPVL genotyping of methicillin-resistant staphylococcus aureusas compared to pulsed-field gel electrophoresis,” PLoSONE, vol.8, no. 11, article e79149, 2013.

[6] M. Rodriguez, P. G. Hogan, S. W. Satola et al., “Discriminatoryindices of typingmethods for epidemiologic analysis of contem-porary Staphylococcus aureus strains,” Medicine, vol. 94, no. 37,article e1534, 2015.

[7] D. A. Robinson and M. C. Enright, “Multilocus sequencetyping and the evolution of methicillin-resistant Staphylococcusaureus,” Clinical Microbiology and Infection, vol. 10, no. 2, pp.92–97, 2004.

[8] R. Liu, Z. Liu, Y. Xu et al., “Multicolor melting curve analysis-based multilocus melt typing of vibrio parahaemolyticus,” PLoSONE, vol. 10, no. 9, article e0136998, 2015.

[9] M. C. Enright, D. A. Robinson, G. Randle, E. J. Feil, H. Grund-mann, andB.G. Spratt, “The evolutionary history ofmethicillin-resistant Staphylococcus aureus (MRSA),” Proceedings of theNational Academy of Sciences of the United States of America,vol. 99, no. 11, pp. 7687–7692, 2002.

[10] F. C. Tenover, R. D. Arbeit, and R. V. Goering, “Interpretingchromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing,”Journal of Clinical Microbiology, vol. 33, no. 9, Article ID7494007, p. 2233, 1995.

[11] A. P. Francisco, M. Bugalho, M. Ramirez, and J. A. Carrico,“Global optimal eBURST analysis of multilocus typing datausing a graphic matroid approach,” BMCBioinformatics, vol. 10,article no. 152, 2009.

[12] A. P. Francisco, C. Vaz, P. T. Monteiro, J. Melo-Cristino, M.Ramirez, and J. A. Carrico, “PHYLOViZ: phylogenetic infer-ence and data visualization for sequence based typingmethods,”BMC Bioinformatics, vol. 13, no. 1, article 87, 2012.

[13] E. J. Feil, B. C. Li, D. M. Aanensen, W. P. Hanage, and B.G. Spratt, “eBURST: Inferring patterns of evolutionary descentamong clusters of related bacterial genotypes from multilocussequence typing data,” Journal of Bacteriology, vol. 186, no. 5,pp. 1518–1530, 2004.

[14] P. A. Wayne, Performance Standards for Antimicrobial Suscepti-bility Testing: Twenty-Second informational Supplement, Clinicaland Laboratory Standards Institute, USA, M100-S22, 2012.

[15] D. Heipel, J. F. Ober, M. B. Edmond, and G. M. L. Bearman,“Surgical site infection surveillance for neurosurgical proce-dures: a comparison of passive surveillance by surgeons toactive surveillance by infection control professionals,”AmericanJournal of Infection Control, vol. 35, no. 3, pp. 200–202, 2007.

[16] D. Espadinha, N. A. Faria, M. Miragaia et al., “Extensivedissemination of methicillin-resistant Staphylococcus aureus(MRSA) between the hospital and the community in a country

with a high prevalence of nosocomial MRSA,” PLoS ONE, vol.8, no. 4, article e59960, 2013.

[17] Cookson, D. A. Robinson, A. B. Monk et al., “Evalua-tion ofmolecular typing methods in characterizing a Euro-pean collection of epidemicmethicillin-resistant Staphylococcusaureusstrains: the HARMONY collection,” Journal of ClinicalMicrobiology, vol. 45, pp. 1830–1837, 2007.

[18] D. S. Blanc, P. Francioli, and P.M.Hauser, “Poor value of pulsed-field gel electrophoresis to investigate long-term scale epidemi-ology of methicillin-resistant Staphylococcus aureus,” Infection,Genetics and Evolution, vol. 2, no. 2, pp. 145–148, 2002.

[19] E. Alcoceba, A. Mena, M. Cruz Perez et al., “Molecularepidemiology of methicillin-resistant Staphylococcus aureus inmajorcan hospitals: high prevalence of the epidemic cloneEMRSA-15,” Clinical Microbiology and Infection, vol. 13, no. 6,pp. 599–605, 2007.

[20] G. Kahlmeter, “The 2014 Garrod Lecture: EUCAST-are weheading towards international agreement?” Journal of Antimi-crobial Chemotherapy, vol. 70, no. 9, pp. 2427–2439, 2015.

[21] Y. Liu, H. Wang, N. Du et al., “Molecular evidence for spreadof two major methicillin-resistant Staphylococcus aureus cloneswith a unique geographic distribution in Chinese hospitals,”Antimicrobial Agents and Chemotherapy, vol. 53, no. 2, pp. 512–518, 2009.

[22] B. A. Miko, C. A. Hafer, C. J. Lee et al., “Molecular characteri-zation of Methicillin-susceptible Staphylococcus aureus clinicalisolates in the united states, 2004 to 2010,” Journal of ClinicalMicrobiology, vol. 51, no. 3, pp. 874–879, 2013.

[23] G. Rasmussen, S. Monecke, O. Brus, R. Ehricht, and B.Soderquist, “Long termmolecular epidemiology of methicillin-susceptible Staphylococcus aureus bacteremia isolates in Swe-den,” PLoS ONE, vol. 9, no. 12, article e114276, 2014.

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