Autolysis activity of Vancomycin Resistance Staphylococcus epidermidis

 

Mariam K. Abdrabaa1, May T. Flayyih2

1Department of Medical Lab.Technology, Al-Esraa University College

2Department of Biology, College of Science, University of Baghdad. Baghdad, Iraq

*Corresponding Author E-mail: mariam_khamees@yahoo.com

 

ABSTRACT:

The effect of vancomycin resistant on cell autolysis activity of Staphylococcus epidermidis was detected by whole cell autolytic assay. Three isolates of S. epidermidis, vancomycin sensitive (VSSE), vancomycin resistance (VRSE) and vancomycin intermediate (VISE) were tested. The results revealed that there was significant difference among three isolates, the VSSE isolate (S.epidermidis 22) have the highest autolytic activity in the presence of antibiotic , followed by the VRSE isolate (S. epidermidis 1) and the VISE isolate (S. epidermidis14) which was the lowest autolytic activity with the presence of antibiotic. The result of transmission electron microscope (TEM) showed that the VRSE isolates (S. epidermidis 1) have thicker cell wall followed by VISE (S. epidermidis 14) isolates. However, the VSSE (S. epidermidis 22) didn't showed any cell wall thickening

 

KEYWORDS: Staphylococcus epidermidis, vancomycin resistant, autolysin, VRSE, VISE, VSSE.

 

 


INTRODUCTION:

The first reports of vancomycin resistance in coagulase-negative staphylococci were in 1979 and 19831-2. Though a cause for concern, these reports did not generate a great deal of attention as coagulase-negative staphylococci are generally considered to be relatively a virulent organisms3.

 

Some reports have pointed out reduced susceptibility of glycopeptides within Staphylococcus epidermidis in various European countries, suggesting that such strains might be highly disseminated in the community and in hospitals4-6.

 

Alterations of autolytic activities are always associated with vancomycin resistance in Staphylococci. A slight increased whole cell autolytic activitiy was observed for the presence of vancomycin in the assay buffer. Reduction in autolytic activity may allow the cell to tolerate vancomycin that might otherwise cause lysis-inducing effects. This might be an important step in the development of vancomycin resistance in Staphylococcus aureus7-8. A key feature of the resistance mechanism in this mutant appears to be some alternation of cell wall structure such that it allows capture of the glycopeptide molecules at the periphery of the cells distant from sites of cell wall biosynthesis, which thus become protected from the antibiotic9. this study aims to Detect the presence of Autolysin gene(aae) in clinical isolates community–acquired Vancomycin resistant Staphylococcus epidermidis from patients in Baghdad by PCR technique and Studying the effect of Vancomycin on bacterial autolysis activity.

 

MATERIALS AND METHODS:

Bacterial isolates:

Thirty Staphylococcus epidermidis isolates were isolated from clinical specimens of patients attending baghdad teaching Hospital, Laboratery teaching of Madienat AL- Teb and AL-kindy teaching Hospital for the period from August 2013 to December 2013. The isolates were identified depending on the morphological features on culture media and biochemical tests according to Bergey’s Manual. S. epidermidis was identified according to the morphological features on culture medium and biochemical test with the use of API Staph system10-11. Minimum inhibitory concentrations of vancomycin to thirty Staphylococcus epidermidis isolates were determined .This test was achieved according to Morello et al.12.

 

DNA Extraction from S.epidermidis isolates by using Wizard® Genomic DNA Purification Kit:

1.    All S. epidermidis isolates were cultured on mannitol salt agar, after incubation for 24 hr. at 37ºC. The bacterial isolates were inoculated in tubes contained 5 ml of sterile brain heart infusion broth and incubated overnight 18 hr. at 37 ºC.

2.    Bacterial growth 1.5 ml was transferred to a 2ml microcentrifuge tubes and centrifuged at 14000g for 2 min to pellet the cells. The supernatant was removed.

3.    The pellet cells was resuspended in 480μl of 50mM EDTA, 120 μl of lysozyme was added to the resuspended cells and gently the pipetting to mix, after incubation at 37ºC for 2hr. in water bath, the microcentrifuge tubes were centrifuged for 2 minutes at 14000g and the supernatant was removed.

4.    Nuclei Lysis Solution 600μl was added to the pellet cells and pipetting gently to mix then incubate in water bath at 80ºC for 10 min, the microcentrifuge tubes allowed to cool at room temperature.

5.    RNase solution 3µl was added to the cell lysate, the microfuge tubes were inverted gently to mix, the tubes were incubated in water bath for 60 min. at 37ºC.

6.    Protein Precipitation Solution 200 µl was added to the RNase treated cell lysate, Vortex vigorously at high speed for 20 sec. to mix the Protein Precipitation Solution with the cell lysate, incubation the microfuge tubes in ice bath for 10 min.

7.    The microcentrifuge tubes were centrifuged at 14000g for 3 min.

8.    The supernatant contained DNA was transferred to new clean 1.5 microfuge tubes containing 600 µl of room temperature isopropanol, gently mix by inversion until the thread-like strands of DNA form a visible mass, centrifuged the tubes at 14000g for 2 min.

9.    The supernatant carefully poured off and the tubes drained on clean absorbant paper, Room temperature 70% ethanol was added to wash the DNA pellet, centrifuged the tubes at 14000g for 2 min. and the ethanol was aspirated.

10. DNA Rehydration Solution 100μl was added to the DNA in the microfuge tubes and rehydrate through incubation at room temperature overnight.

11. The DNA was stored in -20ºC.

 

The DNA concentration and purity were determined by using Nano drop instrument from ACT gene (China). The quality of the extracted DNA was checked by 0.8 % agarose gel electrophoresis.

 

Polymerase Chain Reaction (PCR) Technique:

The polymerase chain reaction (PCR) is an in vitro amplification of target DNA with a pair of primers and a DNA polymerase, resulting in several million fold amplification of the target sequence within few hr13. PCR assay was performed in a monoplex patterns in order to amplify different fragments of genes under study in a single tube for detecting of aae Autolysine.

 

The primers listed in table (1) were selected for this study; these primers were provided in a lyophilized form, dissolved in sterile distilled water to give a final concentration of 100 pmol∕μL and stored in deep freezer until used in PCR amplification.

 

Table 1: The primers and their sequences used in conventional PCR for detection of Staphylococcus epidermidis

Gene

Primer

Name

Sequence 5' 3'

Size

(bp)

Length

References

aae

AF

GAG GAG GAT

TTT AAA GTG C

19

 

 

858

 

 

Heilmann et al.(2003)

Gazzola and Cocconcelli (2008)

aae

AR

AAC ATG ACC

ATA GTA ACC-

18

 

 

858

 

 

 

PCR Amplification:

The extracted DNA, primers and PCR premix (Bionner), were thawed at 4ᵒC, vortex and centrifuged briefly to bring the contents to the bottom of the tubes.PCR mixture was set up in a total volume of 20 μL included 5μL of PCR premix 2 μL of each primer, 5 μL of template DNA have been used and 1.5 μL DMSO. The rest volume was completed with sterile de-ionized distilled water, then vortexed and finally 5 μL of template DNA was added. Negative control contained all material except template DNA, so instead that distilled water was added.

 

PCR reaction tubes were centrifuged briefly to mix and bring the contents to the bottom of the tubes, and placed into thermocycler PCR instrument where DNA was amplified as indicating in the table (2). The extracted DNA from staphylococcal isolates were checked for their concentration and purity, and thereafter were analyzed by PCR and the results confirmed by using 1.5 % agarose gel electrophoresis14.

 

Table (2): Program was processed in PCR amplification of aae gene according to Gazzola and Cocconcelli 2007

Stage

Temperature

Time

Cycle

Initial denaturation

95 ºC

1 min

30

Denaturation

94 ºC

30sec

Annealing

52 ºC

1 min

Extension

72 ºC

1min

Final Extension

72 ºC

10min

 

Cell autolysis assays.:

Autolysis assays for S. epidermidis strains were performed as described by Qin et al.15, cell samples (50 ml) were collected from exponential-phase cultures growing in TSB medium (OD580=0.7) containing 1 M NaCl, and cells were pelleted by centrifugation. The cells were washed twice with 50 ml ice-cold water and resuspended in 50 ml 0.05 M Tris/HCl (pH 7.2) containing 0.05 % (v/v) Triton X-100. The cells were then incubated at 30 °C with shaking, and OD580 was measured at 30 min intervals.

 

Transmission electron microscope (TEM):

Transmission electron microscope was made according to Paul et al.16 by using negative stain.

 

Statistical Analysis:

The Statistical Analysis System- SAS17 was used to effect of different factors in study parameters. Least significant difference –LSD test was used to significant compare between means in this study.

 

RESULTS AND DISCUSION:

Vancomycin susceptibility was determined by the minimum inhibitory concentration (MIC) for 30 S. epidermidis isolates, according to Clinical and Laboratory Standards Institute18, the results showed that 12 isolates were resistant to Vancomycin (VRSE), 4 isolates were intermediate resistant (VISE)and 14 isolates were sensitive (VSSE).

 

Aae gene amplification by monoplex PCR technique:

To detect the virulence factors (autolysin, adhesin), staphyloccal surface protein, aae gene in S. epidermidis was used, thirty isolates S. epidermidis were subjected to PCR technique in a monoplex pattern. All S. epidermidis isolates gave positive result for the presence of aae gene as shown in figure (1a,b). The result demonstrated that the 30 (100%) of S. epidermidis isolates were obtained from clinical samples found to has the aae gene. The results of present study showed that aae gene band detected at 858 bp region, all S. epidermidis isolates have aae gene as virulence factor with adhesive and bacteriolytic properties.

 

The positive result of aae gene was confirmed by 1.5% agarose gel electrophoresis stained with ethidium bromide, electrophoresed in 70 volt for 1.5 hrs. and photographed under ultraviolet (UV) trans illuminator.

 

Heilmann et al13. was reported Aae, a novel autolysin/ adhesion from S. epidermidis having both bacteriolytic and adhesive properties also, the same result with Widerström19 in the amplification reaction revealed a PCR product of 858 bp.

 

 

Figure(1 a): Gel electrophoresis of amplified PCR product of aae gene in monoplex pattern, all strains well positive to it, agarose (1.5%), TBE buffer (1x), 70 volt for 1.5 hrs. stained with ethidium bromide. M: DNA ladder (100 bp), line from 1-15 indicate aae gene bands.

 

 

Figure (1 b): Gel electrophoresis of amplified PCR product of aae gene in monoplex pattern all strains well positive to it. Agarose (1.5%), TBE buffer (1x), 70 volt for 1.5 hrs. stained with ethidium bromide. M: DNA ladder (250 bp), line from 16-30 indicate aae gene bands.

 

The function role of Atl (Aae) was bind to fibrinogen, fibronectin and vitronectin19. The bind vitronectin, indicating atl plays a role in binding of the cell not only to naked polystyrene surface during early stage of adherence but also to plasma protein –coated polymer surface during later stage of adherence. Atl has a role in mediating the attachment of bacterial cells to polymersurface, representing the prerequisite for biofilm formation20. In conclusion, Aae is a surface-associated protein with bacteriolytic and adhesive properties representing a new member of the staphylococcal autolysin/adhesins potentially involved in colonization. Other reported autolysis genes, such as the cidABC operon, arlRS and mgrA genes of S.epidermidis, the expression of the atl gene increased in the vancomycin resistance- induced S. haemolyticus, but the expression of the other autolysis-related genes – lrgAB, lytS and sarA – did not change the correlation of expression of atl and autolytic activity has been investigated in vancomycin resistant S. aureus strains, and some showed increased atl gene expression or lower autolysis activity than that of parent isolates21 and the S. epidermidis contained the gene atlA coding for the AtlA autolysin, which has an adhesive function that is involved in the first phase of biofilm formation, as described by Helimann et al.13

 

Autolysis assay:

Whole cell autolysis:

To examine the effects of Vancomycin on whole cell autolytic properties of laboratory-derived (VRSE, VISE, VSSE) isolates, the drug at a concentration of one half of MIC was included in the assay buffer before the OD580 was taken. The whole cell autolytic activities of S. epidermidis 1(VRSA) isolate was shown in figure (2).

 

 

Figure (2): Whole cell autolytic activity profile of VRSE isolate. Profile of S. epidermidis 1. (P) present of Vancomycin;(A) Absence of Vancomycin.

 

The results showed that VRSE isolates (S. epidermidis 1) had low autolytic activity in presence of antibiotic compare to its in absence of antibiotic. There is a significant difference in OD between present and absent of vancomycin (S.epidermidis 1).

 

In presence of vancomycin, the autolytic activity increased inVISE isolates (S. epidermidis 14) compare to its in absence of antibiotic. As shown in figure (3). There was significant difference in OD between present and absence of Vancomycin.

 

In presence of vancomycin the autolytic activity increased in VSSE (S. epidermidis 22). As showed in figure (4). There was significant difference in OD between present and absence of Vancomycin .

 

 

Figure (3): Whole cell autolytic activity profile of laboratory-drvied VISE isolate. Profile of S. epidermidis 14. (P) present of Vancomycin;(A) Absence of Vancomycin.

 

 

Figure (4): Whole cell autolytic activity profile of VSSE isolate. Profile of S. epidermidis 22. (P) present of Vancomycin;(A) Absence of Vancomycin

 

Finally, The results of comparision between autolysis activities of three S.epidermidis isolates revealed that there was significant difference among these isolates (VRSE,VISE and VSSE) in presence and absence of antibiotic (Vancomycin ) ,as shown in table (3), the VSSE isolate (S.epidermidis 22) have the highest autolysis activity in the presence of antibiotic , followed by the VRSE isolate (S. epidermidis 1) and the VISE isolate (S. epidermidis14) which was the lowest autolysis activity with the presence of antibiotic. Calculations of autolysis rate were calculated according to Kim et al.22 as in formula.

 

Percent lysis at time t=[(OD0- ODt)/OD0]×100

 

 

The vancomycin resistant S. epidermidis isolate (VRSE MIC= 256), exhibited autolysis rates was (58%), VSSE and VISE which were (70%), (64%) respectively in absence of vancomycin similar to result of Kim et al. (2012). The autolysis rates increased in the presence of vancomycin in each of S. epidermidis isolate (VSSE 22 and VISSE 14) were (85%), (57%), and decreased in VRSE was (44%) same as Kim et al.22, also came with study of Gazzola and Cocconcelli5.

 

Autolysis is linked to the process of cell division, and is therefore related to the growth of the cell and the expression of autolysins, which hydrolyse cell wall components. The autolysis rates of S. epidermidis with induced vancomycin resistance were less than those of the sensitive isolate. This suggests that resistance to autolysis indicates reduction of cell wall turnover7,23.

 

Table 3: Comparision between autolytic activities of 3 S. epidermidis isolates

Time (min.)

OD (mean ± SE)

T-test value

Absence

Present

VRSE

0.507 ± 0.039

0.404 ± 0.045

0.094 *

VISE

0.513 ± 0.052

0.404 ± 0.064

0.091 *

VSSE

0.401 ± 0.047

0.555 ± 0.027

0.102 *

LSD value

0.078 *

0.094 *

---

* (P<0.05).

 

Transmission electron microscopy (TEM):

TEM was performed on S. epidermidis isolates (VRSE MIC=256, VISE MIC=16, VSSE MIC= 4) to assess cell wall thickness. The result of this study showed that the VRSE contain thicker cell wall as shown in figure (5), than VISE and VSSE as shown in figure (6A andB) by staining cells by negative stain . Cell wall thickening is a common feature of vancomycin resistant staphylococci. TEM revealed that cell wall thickness of vancomycin sensitive Staphylococcue hemolyticus, VSSH were 21.48 nm, increase in thickness of VRSH, VISH 28% and 89 %, respectively by Kim et al.22 The results demonstrated a characteristic change in cell wall thickness similar to that reported in previous studies on with induced vancomycin resistance5,22-24. In the study of Kim et al.22 , the cell wall thickness of the isolate with induced vancomycin resistance was almost 1.9-fold higher than that of its sensetive isolate . In S. aureus, the atl gene plays a fundamental role in cell division and separation. Decreased atl gene expression in vancomycin-resistant S. aureus may produce a build-up of peptidoglycan layers contributing to a thickened cell wall. However, only atl gene expression was upregulated in the vancomycin-resistance-induced S. epidermidis isolates, while cell wall thickness was much greater than that in the parent strain7. Vancomycin-resistance-induced S. epidermidis exhibited a typically thick cell wall, decreased cell growth phenotype, decreased autolysis activity23. Because S. epidermidis is prevalent in hospitals and is regarded as an important nosocomial pathogen with a tendency to develop multiple resistance, further understanding of the mechanisms underlying vancomycin resistance should be acquired through future detailed studies.

 

 

Figure (5): VRSE isolate with MIC= 256 μg\ml, under transmission of electron microscope 13000x(up), 34000x(mid) and 64000 x(down)

 

Figure (6): S. epidermidis under transmission of electron microscope 34000x(up) and 64000x(down).

A- VISE isolate with MIC= 16 μg\ml

B- VSSE isolate with MIC= 4 μg\ml

 

CONCLUSION:

In this work the results showed that there were significant differences in autolytic activity between three S. epidermidis isolates (VRSE, VISE and VSSE) in presence and absence of vancomycin, the VSSE isolate (S. epidermidis 22) have the highest autolysis activity in the presence of antibiotic, followed by the VRSE isolate (S. epidermidis 1) and the VISE isolate (S. epidermidis14) which was the lowest autolysis activity with the presence of antibiotic. vancomycin has effect on autolytic activity but don’t have any effect on presence of aae genes as showed in PCR. The cell wall of VRSE was thicker in compared with VSSE as showed in TSM.

 

 

REFERENCE:

1.     W. T. Siebert, N. Moreland, T. W. Williams, Synergy of vancomycin plus cefazolin or cephalothin against methicillin-resistance Staphylococcus epidermidis. J Infect Dis, 1979, 139,452–457.

2.     C. U. Tuazon, H. Miller, Clinical and microbiologic aspects of serious infections caused by Staphylococcus epidermidis. Scand J Infect Dis, 1983, 15,347-360.

3.     A. Srinivasan,J. D. Dick,T. M. Perl, Vancomycin resistance in staphylococci. Clin Microbiol Rev, 2002, 15,430–438.

4.     F. Trueba, E. Garrabe, R. Hadef, R. Fabre, J. D. Cavallo, K. Tsvetkova, O. Chesneau, High prevalence of teicoplanin resistance among Staphylococcus epidermidis strains in a 5-year retrospective study. J Clin Microbiol, 2006,44,1922–1923.

5.     S. Gazzola, P.S. Cocconcelli, Vancomycin heteroresistance and biofilm formation in Staphylococcus epidermidis from food. Microbiol, 2008, 154, 3224-3231.

6.     S.Natoli, C. Fontana,M. Favaro, Characterization of coagulase-negative staphylococcal isolates from blood with reduced susceptibility to glycopeptides and therapeutic options. BMC Infectious Diseases,2009, 9(83), 90-101.

7.     R.F. Pfeltz, V.K. Singh, J.L. Schmidt, M.A. Batten, C.S. Baranyk, M.J. Nadakavukaren, R.K. Jayaswal, B.J. Wilkinson, Characterization of Passage-Selected Vancomycin-Resistant Staphylococ-cus Aureus Strains of Diverse Parental Backgrounds. Antimicrob Agents Chemother, 2000, 440, 294-303.

8.     J.L. Koehl, A. Muthaiyan, R. K. Jayaswal, K. Ehlert, H. Labischinski, B.J. Wilkinson, Cell Wall Composition and Decresed Autolytic Activity and Lysostaphin Susceptibility of Glycopeptide-Intermediate Staphylococcus aureus. Antimicrob Agents Chemother,2004, 48, 3749-3757.

9.     K. Sieradzki, A. Tomasz, Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus. J Bacteriol, 1997,40,550-560.

10.   J.F. MacFaddin, Biochemical Tests for Identification of Medical Bacteria(3rd ed.), Lippinocott Williams and Wilkins, USA. 2000.

11.   J. P. Harley,L. M. Prescott,Laboratory Exercises in Microbiology (5th ed.), The McGraw-Hill Companies, Inc.,New York.2002.

12.   J.A.Morello, H.E. Mizer, P.A. Granato, Laboratory Manual and Workbook in Microbiology Applications to Patient Care. McGraw Hill, Boston.2006.

13.   C. Heilmann,G. Thumm, G.S. Chhatwal, J. Hartleib, A. Uekotter, G. (2003). Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis. Microbiol,2003, 149,2769–2778.

14.   F.H. Stephenson, Calculations for Molecular Biology and Biotechnology: A guide to mathematics in the laboratory. Elsevier Science. USA.2003.

15.   Z. Qin, X. Yang, L. Yang, J. Jiang, Y. Ou, S. Molin, D. Qu, Formation and properties of in vitro biofilms of icanegative Staphylococcus epidermidis clinical isolates. J Med Microbiol,2007, 56, 83–93.

16.   R. Paul, H. Hazelton, R. Gelderblom, Electron Microscopy for Rapid Diagnosis of Infectious Agents in Emergent Situations.1, 2003,61,530-541.

17.   Statistical Analysis System, User's Guide. Statistical. Version 9. (1th ed.), SAS. Inst. Inc. Cary. N.C. USA.2012.

18.   Clinical Laboratory Standards Institute (CLSI), Performance Standard for Antimicrobial Disk Susceptibility Tests. 2011,31 (1).

19.   M.Widerstrom, Molecular epidemiology of coagulase negative staphylococci in hospitals and in the community Print and Media, Umeå University, Umeå, Sweden.2010.

20.   C. Heilmann, M. Hussain, G. Peters, F. Gotz, Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol,1997, 24,1013–1024.

21.   M.Wootton, P.M. Bennett, A.P. MacGowan, T.R. Walsh, Reduced expression of the atl autolysin gene and susceptibility to autolysis in clinical heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) and GISA strains. J Antimicrob Chemother ,2005,56,944–947.

22.   J.W. Kim, G.T. Chung, J.S. Yoo, Y.S. Lee, J. Yoo, Autolytic activity and molecular characteristics of Staphylococcus haemolyticus strains with induced vancomycin resistance. J Med Microbiol,2012, 61,1428–1434.

23.   A.P. Nunes, L.M. Teixeira, N. L. Iorio, Heterogeneous resistance to vancomycin in Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri clinical strains: characterisation of glycopeptide susceptibility profiles and cell wall thickening. Int J Antimicrob Agents,2006, 27,307-315.

24.   L. Cui, X. Ma, K. Sato, K. Okuma, F.C. Tenover, E.M. Mamizuka, C.G. Gemmell, M.N. Kim, M. C. Ploy, Cell wall thickening is a common feature of vancomycin resistance in Staphylococcus aureus. J Clin Microbiol, 2003,38,530-542.

 

 

 

 

 

Received on 29.08.2018       Accepted on 30.09.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2018; 8(4): 225-230.

DOI: 10.5958/2231-5691.2018.00038.2