Research Article The Characteristics of Staphylococcus...

Research ArticleThe Characteristics of Staphylococcus aureusSmall Colony Variant Isolated from Chronic Mastitis ata Dairy Farm in Yunnan Province China

Li-li Zhu Feng-cai Zou Yu-lin Yan Qi-hui Wang Yong-qiang Shi and Wei-jie Qu

Institute of Animal Science and Technology Yunnan Agricultural University Kunming 650051 China

Correspondence should be addressed to Wei-jie Qu 928701531qqcom

Received 26 November 2015 Revised 20 January 2016 Accepted 20 January 2016

Academic Editor Hassan Zaraket

Copyright copy 2016 Li-li Zhu et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Staphylococcus aureus is a major causative agent leading to bovine mastitis and has specific phonotypical characteristics includingsmall colony slow growth and decreased hemolysis therefore named as the small colony variants (SCVs) Out of 30 tested samplesof the chronic S aureus cases one strain of SCVs (S aureus SCV22) was isolated along with its parental strains (S aureus11) Saureus SCV22 showed a slow growth rate when it is compared with the parental strain However their resistant patterns weresimilar Meanwhile S aureus SCV22 depicted the lower rate of apoptosis in bovine mammary epithelial cells These findings ofthe present study presented the unique characteristics of S aureus SCV22 for the first time in Yunnan province which provided aprophase foundation for further study about the pathogenesis of S aureus SCVs in chronic mastitis

1 Introduction

Bovine mastitis is wildly prevalent among dairy cows mainlycaused by the pathogenic Staphylococcus aureus with a hugeeconomic loss in dairy production A hypothesis first pro-posed by Proctor et al has showed that chronic or recurrentinfection was associated with S aureus SCVs [1] Before the Saureus SCVs were found S aureus was generally consideredto be extracellular bacteria However many new reportsshowed that S aureus SCVs owned the ability to persist intra-cellularly in nonprofessional phagocytes such as epithelialcells fibroblasts osteoblasts and endothelial cells [2 3]

To the best of our knowledge no reports have been doc-umented for the isolation of S aureus SCVs from dairy cowsin Yunnan province southwest China as well as the relatedresearch The aim of this study was to explore intracellu-lar persistence apoptosis of mammary epithelial cells andestablishment of mastitis model in S aureus SCVs and theresults would provide prophase foundation to better know theinfectionmechanisms of S aureus SCVs in chronicmastitis infurther study

2 Materials and Methods

21 Isolation and Identification of S aureus and SCVs fromMilk Raw milk samples (119899 = 30) were aseptically gatheredfrom scattered-feeding cows at a dairy farm in Kunming cityof Yunnan province Accordingly all samples were culturedon Trypticase Soy Agar (TSA HUANKAI GuangzhouChina) complemented with 5 sheep blood (RuiteGuangzhou China) and then cultivated at 37∘C for 24 h and48 h respectively S aureus was identified according to thephonotypical characteristics of being large creamy andforcefully hemolytic on TSA with 5 sheep blood PotentialSCVs colonies of tiny nonpigmented and nonhemolyticcolonies on TSA with 5 sheep blood were also collected[4 5] Isolates of potential SCVs were subcultured on TSAfor ten generations to assess their stability SCVs and theirparental strains were primarily tested for routine biochemicalproperties then the potential SCVs were identified as Saureus SCVs with the gene (nuc nucA and 16srDNA) bymultiple PCR amplification (Table 1)The identified S aureusSCV and its parental strains were termed as S aureus SCV22

Hindawi Publishing Corporatione Scientific World JournalVolume 2016 Article ID 9157605 8 pageshttpdxdoiorg10115520169157605

2 The Scientific World Journal

Table 1 Primer information

Purpose gene Sequence Fragment length (bp) 119879119898

16SrDNA Upstream primer 51015840-GGCGTTGCTCCGTCAGGCTT-31015840 375

54∘C

Downstream primers 51015840-CGCTGGCGGCGTGCCTAAT-31015840

nucA Upstream primer 51015840-CGCTTGCTATGATTGTGGTAGCC-31015840 239Downstream primers 51015840-TTCGGTTTCACCGTTTCTGGCG-31015840

nuc Upstream primer 51015840-TCGTCAAGGCTTGGCTAAAGTTGC-31015840 126Downstream primers 51015840-TCAGCGTTGTCTTCGCTCCAAA-31015840

and S aureus11 respectively All isolates were stored at minus80∘Cin Trypticase Soy Broth (TSB HUANKAI) complementedwith 25 volumes of glycerin (HUANKAI) for furtherinvestigation

22 Auxotrophism Assay Auxotrophy test of the SCVs wasperformed on Mueller-Hinton agar (MHA Difco BD Bei-jing China) with 1 120583gmL of hemin (Sigma ShanghaiChina) 1120583gmL of thymidine (Sigma) 1 120583gmL of mena-dione sodiumbisulfite (Sigma) and 100 120583gmL of thiamine asdescribed previously [6 7] Auxotrophism SCVs were identi-fiedwhich showed the growth similar to their parental strainsafter incubating overnight at 37∘C

23 Bacterial Growth Curves SCVs and their parental strainswere analyzed in triplicate for growth curve features [1 8]Thecryopreserved strains were inoculated into 10mL TSB brothovernight at 37∘C 1mL of overnight culture was transferredto a flask containing 200mL TSB and then incubated at 37∘Con a rotary shaker (150 rpm) Bacterial growth was deter-mined by measuring the OD 600 nm per 4 hours

24 Cell Culture All reagents were purchased from Biolog-ical Industries (BI Israel) The primary bovine mammaryepithelial cells (Procell Wuhan China) were cultured inepidermal cell growth medium supplemented with 10 fetalcalf serum (FCS Sigma-Aldrich Deisenhofen Germany)epidermal growth factor (01 ngmL) pig insulin and hydro-cortisoneThe frozen cells were resuscitated in the cell cultureplate with cell climbing piece in advance The MECs wereused after 3 passages for respective experiments

25 Infection Experiments For infection experiments [9]bacteria of S aureus SCV22 and S aureus11 were grownovernight at 37∘C on TSA complemented with 5 sheepblood picking a single colony to cultivate in liquid mediumfor 4 h then collecting bacteria suspending them withDMEMF12 (sigma) and then adjusting the thalli concentra-tion to 108 cfumL When the cells were grown to 80 onecell was infected using 100 bacteria The infection test wasterminated after 3 h

26 Apoptosis The infected cells were dyed with Annexin V-FITCPI kit (Beyotime Shanghai China) [10] The amountof apoptosis cells was measured by flow cytometry (BD FAC-SCalibur USA) and analyzed with Cellquest Pro software

27 Scanning Electron Microscopy The infected cells werewashed 5 times with PBS and the cells were fixed withglutaraldehyde phosphate buffer Then cell dehydration wasperformed for 15min in each solution using a graded ethanolfrom 30 to 100 The cell climbing was dried according tothe critical point drying method before being sputter coatedwith gold in aE-1010 Ion Sputter Coater (Hitachi Japan)Finally the change of mammary epithelial cells was observedbyAS-3000N scanning electronmicroscopy (Hitachi Japan)

28 The Intracellular Persistence Assay The infection exper-iment was conducted again and incubated for 3 h at 37∘Cwith 5 CO

2to allow for the phagocytosis and adhesion of

S aureus SCV22 and S aureus11 The tested cell plate waswashed with sterile PBS and 100mgmL of gentamicin and50mgmLof penicillinwere added respectivelyThe cell platewas washed again and lysostaphin was added to eliminateextracellular bacteria and then incubated for 30min 3 h9 h and 12 h respectively At each time point the cells werewashed thrice with PBS to remove lysostaphin which wasfollowed by addition of 50 120583L 025 Triton X-100 to scatterepithelial cells and release intracellular bacteria The celllysing reagent was diluted with sterile water 100mL diluentwas spread on the AGAR plate The number of intracellularcolony-forming units (CFU) was counted after S aureusSCV22 and SASCV11 had grown overnight at 37∘C at eachtime point

29 Antimicrobial Susceptibility Testing Antimicrobial sus-ceptibility of S aureus SCV22 was determined strictlywith 10 antibiotics according to the drug susceptibility kit(Tianhe) instruction [11] while the standard S aureus strain(ATCC29213 Tianhe Hangzhou China) was used as qualitycontrol (QC)

3 Results

31 Isolation and Identification of S aureus11 and S aureusSCV22 fromMilk Out of 30 samples collected fromHolsteindairy cows 8 isolates of suspected typical S aureus11 werefound They were subcultured at 37∘C for 16 h on TSA with5 sheep blood The result showed that all isolates haveshown the typical morphology characteristics like (4mm)120573 hemolytic and creamy colonies (Figure 1(b)) and bacteriawere Gram-positive coccus and arranged as grape-like clus-ters under light microscope In addition catalase test on pureTSA incubated at 37∘C for 16 hwas strong positive Prevalence

The Scientific World Journal 3

(a) (b)

Figure 1 Colony morphological of S aureus SCV22 with pinpoint colonies on Trypticase Soy Agar with 5 sheep blood (a) compared toStaphylococcus aureus11 (b)

of coagulase was 100 by the commercial kit (Staphylase TestKit Oxoid Beijing China)

Out of eight S aureus isolates one was putative SCV22Colonies of SCV11 with pinpoint (03mm) were nonpig-mented and failed to present an extremely slight hemolysisuntil incubated for 24 h at 37∘C on TSA with 5 sheep blood(Figure 1(a)) Under the light microscope these potentialSCV11 appeared to be identical to typical S aureus ForSCV11 catalase test was also positive while coagulase was notdetected In the course of a continuous passage for ten genera-tions these results were stable until the 10-generation passage

All isolates were positive to multiple PCR test (Figure 2)The sequence analysis of 16S rRNA showed that gene of Saureus SCV22 and its parental strains are homologous withpublished S aureus sequence

32 Auxotrophism Assay The auxotrophism analysis of Saureus SCV22 revealed a thymidine dependence the aux-otrophism strainswere close to their parental strains onMHAwith 1 120583gmL of thymidine However the S aureus SCV22was negative for hemin menadione and thiamine (Figure 3)

33 Bacterial GrowthCurves According to the growth curvesof S aureus SCV22 and their parental strains (hereafter Saureus11) cultured in pure TSB S aureus11 showed a typicalbacterial growth curve In contrast S aureus SCV22 showed alinear growth curve which had an extended lag phase but theboundary between the lag phase and log phase could not bedistinguished because of none breakpoint shown in Figure 4

34 Intracellular Assay Assays for S aureus SCV22 and Saureus11 were done in triplicate to compare and quantifytheir ability to persist intracellularly within primary bovinemammary epithelial cells line Measurement of CFU was

100

250

500

1 2M(bp)

Figure 2 Molecular typing of S aureus11 and S aureus SCV22isolated from cases of bovine mastitis by multiple Multiplex PCRMolecular markers 50 and 100 bp ladder (Takara Japan)

conducted at the time points of 05 h 3 h 9 h and 12 h respec-tively (Figure 5) Hereby prevalence of persistence fromthe time point of 3 h was becoming different between Saureus SCV22 and S aureus11 and the CFU number of Saureus SCV22 was relatively stable (Figure 6)

35 Apoptosis Compared to the control group apoptosisrates of primary bovinemammary epithelial cells in infectiongroups of S aureus SCV22 and S aureus11 increased signif-icantly (119875 lt 005) and the difference between the S aureus


(a) (b)

(c)

Figure 3 Auxotrophism assay small colony variants S aureus SCV22 (a) their parental strains (b) and auxotrophism strains (c)

OD600nm

S aureus11S aureus SCV22

10 20 30 400

Time (h)

0

1

2

3

Figure 4 Growth curves of small colony variants S aureus SCV22in Trypticase Soy Broth compared to their parental strains

SCV22 groups and the S aureus11 groups was also statisticallysignificant (119875 lt 001) (Figure 7)

36 Scanning Electron Microscopy Under the electronmicroscopy control cells were grown normally In the

S aureus11 infection groups cells became round pieceand flocked together after falling off their cytomembraneruptured and the nucleus were unable to distinguish becauseof being shriveled Cells of S aureus SCV22 infection groupstended to flake but cellular membrane was intact and noextravasation of cytoplasm was found (Figure 8)

37 Antimicrobial Susceptibility Testing TheATCC25923 wassensitive to 10 antibiotics On the contrary the S aureusSCV22 had a resistance to six antibiotics the same resultswere given in repeated test (Table 2)

4 Discussions

Bovine mastitis is wildly common among most of the cowfarms caused by S aureus especially the chronic mastitisMany reports showed that a small colony variant fromS aureus with special phonotypical characteristics was themajor pathogenic bacteria leading to chronicmastitis [12ndash14]In the present study although S aureus SCV22 and S aureus11had different phenotypic characteristics and growth curvestheir targeted fragments were the same by multiple PCRamplification

Since SCVs phenomenon was found from Eberthellatyphosa strains by Jacobsen in 1910 [15] various SCVs fromdifferent bacteria have been reported [16ndash19] Among them


(a1) (b1)

(a2) (b2)

(a3) (b3)

(a4) (b4)

Figure 5 (40x) Morphological changes indicating cell damage detachment and rounding of primary bovine mammary epithelial cellsinfected with small colony variants S aureus SCV22 and their parental strains after 05 hours (a1 b1) 3 hours (a2 b2) 9 hours (a3 b3) and12 hours (a4 b4) S aureus11 was presented in (a1) (a2) (a3) and (a4) S aureus SCV22 was presented in (b1) (b2) (b3) and (B4)

S aureus SCVswere wildly studied theirmain characteristicsare small colony slow growth forming biofilms survivingin host cells chronically and even reducing the sensitivity toantibiotics which are consistent to our results of which smallphenotype and slow growth of SCVs are mainly caused bymetabolic defects [10] and two types can be divided includ-ing electron transfer defect and thymine synthesis defectTheformer is the typical auxotroph the normal phenotype can beformed by supplying menadione or hemin In this study theresult showed that S aureus SCV22 was thymine-dependentS aureus SCVs this type had been reported from patientswith cystic fibrosis (CF) previously

In the present study S aureus SCV22 had a high resis-tance to SXT while their parental strains were sensitive SXTcan interfere with the synthesis of tetrahydrofolic acid toinhibit the growth of S aureus [4] In order to resist SXT

thymine-dependent SCVs absorbed the thymine in extracel-lular and survived ultimately This phenomenon of thymine-dependent SCVs may be related to gene disruption mutationof thyA because the thymidylic acid synthetase and tetrahy-drofolic acid encoded by gene are important coenzyme forthymidylic acid synthetase

In this study the results of intracellular assay apoptosistest and scanning electron microscopy indicated that Saureus SCVs persisted longer in nonprofessional phagocyteswithout profound damage as previously reported [20ndash22]This feature successfully avoided the host immunity andantibiotic therapy that partly explained its antibiotic resis-tance and the failure of antibody-mediated immune response(AMIR)

In conclusion chronic mastitis is widespread in cowfarms in Yunnan probability due to free-ranging outdated



50 1510

Time (h)

0

5

10

15

M C

FUm

L

Figure 6 S aureus SCV22 M CFUmL (1 million colony-forming unitsmL) recovered from primary bovine mammary epithelial cells atseveral time points compared to S aureus11

Prop

idiu

m io

dide

S aureus11Data007

035 2054

5124 2787

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

S aureus SCV22

057 1573

6891 1478

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

022 1155

7634 1190

Control

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

(a)

S aureus SCV22Control

lowast

lowastlowast

S aureus11

0

20

40

60

Apop

tosis

rate

()

(b)

Figure 7 Small colony variants S aureus SCV22 and their parental strain S aureus11 induced apoptosis of primary bovinemammary epithelialcells (a b) lowastTherewere significant differences between S aureus SCV22 and control lowastlowastThere is a very significant difference between S aureus11and control


(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 8 Representative scanning electronmicrographs displayed the various effects of apoptosis (controls (g h and i) small colony variantsS aureus SCV22 (d e and f) and their parental strain S aureus11 (a b and c) 5000x (c f and i) 2000x (b e and h) and 500x (a d and g))Arrows in (b c e f h and i) represented the morphology of cytomembrane

Table 2 Susceptibility test of ATCC 25923 and S aureus SCV22isolates

Antibiotic ATCC 25923 S aureus SCV22Fosfomycin S SSXT S ROxacillin S SAmpicillin S RVancomycin S RStreptomycin S RVancomycin S SAmpicillin S ROxacillin S SGentamicin S RS sensitive R resistantSXT sulfamethoxazole trimethoprim

production technology lack of technical personnel andabuse of antibiotics In the study S aureus SCVs isolated from

dairy cows were reported in Yunnan for the first time whichare strongly connected with chronic mastitis [23ndash25] Todate there is no treatment to exhaustively eliminate chronicmastitis in production therefore further study should becarried out to better know the infection mechanisms of Saureus SCVs in chronic mastitis

Conflict of Interests

There is no conflict of interests

Acknowledgment

This research was supported by the Chinese National NaturalScience Foundation Project (31260629)

References

[1] R A Proctor P Van Langevelde M Kristjansson J N Maslowand R D Arbeit ldquoPersistent and relapsing infections associated


with small-colony variants of Staphylococcus aureusrdquo ClinicalInfectious Diseases vol 20 no 1 pp 95ndash102 1995

[2] B KahlMHerrmannA S Everding et al ldquoPersistent infectionwith small colony variant strains of Staphylococcus aureus inpatients with cystic fibrosisrdquo Journal of Infectious Diseases vol177 no 4 pp 1023ndash1029 1998

[3] O Vesga M C Groeschel M F Otten D W Brar J M Vannand R A Proctor ldquoStaphylococcus aureus small colony variantsare induced by the endothelial cell intracellular milieurdquo Journalof Infectious Diseases vol 173 no 3 pp 739ndash742 1996

[4] M L Kaplan and W E Dye ldquoGrowth requirements of somesmall-colony-forming variants of Staphylococcus aureusrdquo Jour-nal of Clinical Microbiology vol 4 no 4 pp 343ndash348 1976

[5] J Lannergard C von Eiff G Sander et al ldquoIdentification of thegenetic basis for clinical menadione-auxotrophic small-colonyvariant isolates of Staphylococcus aureusrdquo Antimicrobial Agentsand Chemotherapy vol 52 no 11 pp 4017ndash4022 2008

[6] C Kohler C von Eiff G Peters R A ProctorMHecker and SEngelmann ldquoPhysiological characterization of a heme-deficientmutant of Staphylococcus aureus by a proteomic approachrdquoJournal of Bacteriology vol 185 no 23 pp 6928ndash6937 2003

[7] J G Malone T Jaeger C Spangler et al ldquoYfiBNR mediatescyclic di-GMP dependent small colony variant formation andpersistence in Pseudomonas aeruginosardquo PLoS Pathogens vol6 no 3 Article ID e1000804 2010

[8] A Oliver R Canton P Campo F Baquero and J BlazquezldquoHigh frequency of hypermutable Pseudomonas aeruginosa incystic fibrosis lung infectionrdquo Science vol 288 no 5469 pp1251ndash1253 2000

[9] P G Quie ldquoMicrocolonies (G variants) of Staphylococcusaureusrdquo Yale Journal of Biology and Medicine vol 41 no 5 pp394ndash403 1969

[10] R A Proctor P van Langevelde M Kristjansson J N Maslowand R D Arbeit ldquoPersistent and relapsing infections associatedwith small-colony variants of Staphylococcus aureusrdquo ClinicalInfectious Diseases vol 20 no 1 pp 95ndash102 1995

[11] F Schaaff G Bierbaum N Baumert P Bartmann and H-GSahl ldquoMutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureusrdquo Inter-national Journal of Medical Microbiology vol 293 no 6 pp427ndash435 2003

[12] H Atalla C Gyles C L Jacob H Moisan F Malouin and BMallard ldquoCharacterization of a Staphylococcus aureus smallcolony variant (SCV) associated with persistent bovine masti-tisrdquo Foodborne Pathogens and Disease vol 5 no 6 pp 785ndash7992008

[13] M Schneider KMuhlemann S Droz S Couzinet C Casaultaand S Zimmerli ldquoClinical characteristics associated with iso-lation of small-colony variants of Staphylococcus aureus andPseudomonas aeruginosa from respiratory secretions of patientswith cystic fibrosisrdquo Journal of Clinical Microbiology vol 46 no5 pp 1832ndash1834 2008

[14] P C YWoo A S P Leung KW Leung and K Y Yuen ldquoIden-tification of slide coagulase positive tube coagulase negativeStaphylococcus aureus by 16S ribosomal RNA gene sequencingrdquoMolecular Pathology vol 54 no 4 pp 244ndash247 2001

[15] H E Morton and J Shoemaker ldquoThe identification ofNeisseriagonorrhoeae bymeans of bacterial variation and the detection ofsmall colony forms in clinical materialrdquo Journal of Bacteriologyvol 50 pp 585ndash587 1945

[16] R C Massey A Buckling and S J Peacock ldquoPhenotypicswitching of antibiotic resistance circumvents permanent costs

in Staphylococcus aureusrdquo Current Biology vol 11 no 22 pp1810ndash1814 2001

[17] B E Menzies and D S Kernodle ldquoSite-directed mutagenesis ofthe alpha-toxin gene of Staphylococcus aureus role of histidinesin toxin activity in vitro and in a murine modelrdquo Infection andImmunity vol 62 no 5 pp 1843ndash1847 1994

[18] R A Proctor C von Eiff B C Kahl et al ldquoSmall colony vari-ants a pathogenic form of bacteria that facilitates persistent andrecurrent infectionsrdquoNature ReviewsMicrobiology vol 4 no 4pp 295ndash305 2006

[19] A Sabat N Malachowa J Miedzobrodzki andW HryniewiczldquoComparison of PCR-based methods for typing Staphylococcusaureus isolatesrdquo Journal of Clinical Microbiology vol 44 no 10pp 3804ndash3807 2006

[20] F Kipp B C Kahl K Becker et al ldquoEvaluation of twochromogenic agar media for recovery and identification ofStaphylococcus aureus small-colony variantsrdquo Journal of ClinicalMicrobiology vol 43 no 4 pp 1956ndash1959 2005

[21] R W Lacey and A A Mitchell ldquoGentamicin-resistant Staphy-lococcus aureusrdquo The Lancet vol 294 no 7635 pp 1425ndash14261969

[22] J B Lyczak C L Cannon and G B Pier ldquoLung infectionsassociated with cystic fibrosisrdquo Clinical Microbiology Reviewsvol 15 no 2 pp 194ndash222 2002

[23] F Kipp K Becker G Peters and C Von Eiff ldquoEvaluationof different methods to detect methicillin resistance in small-colony variants of Staphylococcus aureusrdquo Journal of ClinicalMicrobiology vol 42 no 3 pp 1277ndash1279 2004

[24] R W Lacey ldquoDwarf-colony variants of Staphylococcus aureusresistant to aminoglucoside antibiotics and to a fatty acidrdquoJournal of Medical Microbiology vol 2 no 3 pp 187ndash197 1969

[25] G Lina Y Piemont F Godail-Gamot et al ldquoInvolvement ofPanton-Valentine leukocidin-producing Staphylococcus aureusin primary skin infections and pneumoniardquo Clinical InfectiousDiseases vol 29 no 5 pp 1128ndash1132 1999

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evolutionary BiologyInternational Journal of


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Applied ampEnvironmentalSoil Science

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Agronomy




Journal of Parasitology Research

Hindawi Publishing Corporation httpwwwhindawicom


Volume 2014

Zoology

GenomicsInternational Journal of


InsectsJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


VirusesJournal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of



Case Reports in Veterinary Medicine


Table 1 Primer information

Purpose gene Sequence Fragment length (bp) 119879119898

16SrDNA Upstream primer 51015840-GGCGTTGCTCCGTCAGGCTT-31015840 375

54∘C

Downstream primers 51015840-CGCTGGCGGCGTGCCTAAT-31015840

nucA Upstream primer 51015840-CGCTTGCTATGATTGTGGTAGCC-31015840 239Downstream primers 51015840-TTCGGTTTCACCGTTTCTGGCG-31015840

nuc Upstream primer 51015840-TCGTCAAGGCTTGGCTAAAGTTGC-31015840 126Downstream primers 51015840-TCAGCGTTGTCTTCGCTCCAAA-31015840

and S aureus11 respectively All isolates were stored at minus80∘Cin Trypticase Soy Broth (TSB HUANKAI) complementedwith 25 volumes of glycerin (HUANKAI) for furtherinvestigation

22 Auxotrophism Assay Auxotrophy test of the SCVs wasperformed on Mueller-Hinton agar (MHA Difco BD Bei-jing China) with 1 120583gmL of hemin (Sigma ShanghaiChina) 1120583gmL of thymidine (Sigma) 1 120583gmL of mena-dione sodiumbisulfite (Sigma) and 100 120583gmL of thiamine asdescribed previously [6 7] Auxotrophism SCVs were identi-fiedwhich showed the growth similar to their parental strainsafter incubating overnight at 37∘C

23 Bacterial Growth Curves SCVs and their parental strainswere analyzed in triplicate for growth curve features [1 8]Thecryopreserved strains were inoculated into 10mL TSB brothovernight at 37∘C 1mL of overnight culture was transferredto a flask containing 200mL TSB and then incubated at 37∘Con a rotary shaker (150 rpm) Bacterial growth was deter-mined by measuring the OD 600 nm per 4 hours

24 Cell Culture All reagents were purchased from Biolog-ical Industries (BI Israel) The primary bovine mammaryepithelial cells (Procell Wuhan China) were cultured inepidermal cell growth medium supplemented with 10 fetalcalf serum (FCS Sigma-Aldrich Deisenhofen Germany)epidermal growth factor (01 ngmL) pig insulin and hydro-cortisoneThe frozen cells were resuscitated in the cell cultureplate with cell climbing piece in advance The MECs wereused after 3 passages for respective experiments

25 Infection Experiments For infection experiments [9]bacteria of S aureus SCV22 and S aureus11 were grownovernight at 37∘C on TSA complemented with 5 sheepblood picking a single colony to cultivate in liquid mediumfor 4 h then collecting bacteria suspending them withDMEMF12 (sigma) and then adjusting the thalli concentra-tion to 108 cfumL When the cells were grown to 80 onecell was infected using 100 bacteria The infection test wasterminated after 3 h

26 Apoptosis The infected cells were dyed with Annexin V-FITCPI kit (Beyotime Shanghai China) [10] The amountof apoptosis cells was measured by flow cytometry (BD FAC-SCalibur USA) and analyzed with Cellquest Pro software

27 Scanning Electron Microscopy The infected cells werewashed 5 times with PBS and the cells were fixed withglutaraldehyde phosphate buffer Then cell dehydration wasperformed for 15min in each solution using a graded ethanolfrom 30 to 100 The cell climbing was dried according tothe critical point drying method before being sputter coatedwith gold in aE-1010 Ion Sputter Coater (Hitachi Japan)Finally the change of mammary epithelial cells was observedbyAS-3000N scanning electronmicroscopy (Hitachi Japan)

28 The Intracellular Persistence Assay The infection exper-iment was conducted again and incubated for 3 h at 37∘Cwith 5 CO

2to allow for the phagocytosis and adhesion of

S aureus SCV22 and S aureus11 The tested cell plate waswashed with sterile PBS and 100mgmL of gentamicin and50mgmLof penicillinwere added respectivelyThe cell platewas washed again and lysostaphin was added to eliminateextracellular bacteria and then incubated for 30min 3 h9 h and 12 h respectively At each time point the cells werewashed thrice with PBS to remove lysostaphin which wasfollowed by addition of 50 120583L 025 Triton X-100 to scatterepithelial cells and release intracellular bacteria The celllysing reagent was diluted with sterile water 100mL diluentwas spread on the AGAR plate The number of intracellularcolony-forming units (CFU) was counted after S aureusSCV22 and SASCV11 had grown overnight at 37∘C at eachtime point

29 Antimicrobial Susceptibility Testing Antimicrobial sus-ceptibility of S aureus SCV22 was determined strictlywith 10 antibiotics according to the drug susceptibility kit(Tianhe) instruction [11] while the standard S aureus strain(ATCC29213 Tianhe Hangzhou China) was used as qualitycontrol (QC)

3 Results

31 Isolation and Identification of S aureus11 and S aureusSCV22 fromMilk Out of 30 samples collected fromHolsteindairy cows 8 isolates of suspected typical S aureus11 werefound They were subcultured at 37∘C for 16 h on TSA with5 sheep blood The result showed that all isolates haveshown the typical morphology characteristics like (4mm)120573 hemolytic and creamy colonies (Figure 1(b)) and bacteriawere Gram-positive coccus and arranged as grape-like clus-ters under light microscope In addition catalase test on pureTSA incubated at 37∘C for 16 hwas strong positive Prevalence


(a) (b)








100

250

500

1 2M(bp)





(a) (b)

(c)


OD600nm


10 20 30 400

Time (h)

0

1

2

3






4 Discussions




(a1) (b1)

(a2) (b2)

(a3) (b3)

(a4) (b4)









50 1510

Time (h)

0

5

10

15

M C

FUm

L


Prop

idiu

m io

dide

S aureus11Data007

035 2054

5124 2787

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

S aureus SCV22

057 1573

6891 1478

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

022 1155

7634 1190

Control

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

(a)


lowast

lowastlowast

S aureus11

0

20

40

60

Apop

tosis

rate

()

(b)



(a) (b) (c)

(d) (e) (f)

(g) (h) (i)








Acknowledgment


References




































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







(a) (b)








100

250

500

1 2M(bp)





(a) (b)

(c)


OD600nm


10 20 30 400

Time (h)

0

1

2

3






4 Discussions




(a1) (b1)

(a2) (b2)

(a3) (b3)

(a4) (b4)









50 1510

Time (h)

0

5

10

15

M C

FUm

L


Prop

idiu

m io

dide

S aureus11Data007

035 2054

5124 2787

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

S aureus SCV22

057 1573

6891 1478

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

022 1155

7634 1190

Control

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

(a)


lowast

lowastlowast

S aureus11

0

20

40

60

Apop

tosis

rate

()

(b)



(a) (b) (c)

(d) (e) (f)

(g) (h) (i)








Acknowledgment


References




































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







(a) (b)

(c)


OD600nm


10 20 30 400

Time (h)

0

1

2

3






4 Discussions




(a1) (b1)

(a2) (b2)

(a3) (b3)

(a4) (b4)









50 1510

Time (h)

0

5

10

15

M C

FUm

L


Prop

idiu

m io

dide

S aureus11Data007

035 2054

5124 2787

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

S aureus SCV22

057 1573

6891 1478

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

022 1155

7634 1190

Control

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

(a)


lowast

lowastlowast

S aureus11

0

20

40

60

Apop

tosis

rate

()

(b)



(a) (b) (c)

(d) (e) (f)

(g) (h) (i)








Acknowledgment


References




































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







(a1) (b1)

(a2) (b2)

(a3) (b3)

(a4) (b4)









50 1510

Time (h)

0

5

10

15

M C

FUm

L


Prop

idiu

m io

dide

S aureus11Data007

035 2054

5124 2787

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

S aureus SCV22

057 1573

6891 1478

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

022 1155

7634 1190

Control

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

(a)


lowast

lowastlowast

S aureus11

0

20

40

60

Apop

tosis

rate

()

(b)



(a) (b) (c)

(d) (e) (f)

(g) (h) (i)








Acknowledgment


References




































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of








50 1510

Time (h)

0

5

10

15

M C

FUm

L


Prop

idiu

m io

dide

S aureus11Data007

035 2054

5124 2787

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

S aureus SCV22

057 1573

6891 1478

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

Prop

idiu

m io

dide

022 1155

7634 1190

Control

101

102

103

104

100

Annexin VFITC

100

101

102

103

104

(a)


lowast

lowastlowast

S aureus11

0

20

40

60

Apop

tosis

rate

()

(b)



(a) (b) (c)

(d) (e) (f)

(g) (h) (i)








Acknowledgment


References




































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







(a) (b) (c)

(d) (e) (f)

(g) (h) (i)








Acknowledgment


References




































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of








































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






Research Article The Characteristics of Staphylococcus...

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