|Year : 2012 | Volume
| Issue : 2 | Page : 215-217
Be alert to the alterations in the biological characteristics in heterogeneous vancomycin-intermediate Staphylococcus aureus
X Zhou, YY Dai, XL Ma
Department of Clinical Laboratory, Anhui Provincial Hospital, Hefei 230001, China
|Date of Submission||08-Oct-2011|
|Date of Acceptance||02-Apr-2011|
|Date of Web Publication||28-May-2012|
X L Ma
Department of Clinical Laboratory, Anhui Provincial Hospital, Hefei 230001
The development of reduced vancomycin susceptibility in Staphylococcus aureus in many cases appears to be associated with characteristic changes. These changes may have pitfall of identifying S. aureus by automated testing methods like Vitek 32. In this study, we retested 24 heterogeneous vancomycin-intermediate Staphylococcus haemolyticus (h-VISH) collected in 2008-2010 at the Department of Clinical Microbiology by conventional biochemical tests and polymerase chain reaction (PCR). The heterogeneous vancomycin-intermediate S. aureus (hVISA) reversion test and electron microscopic examination were also used. Six isolates of 24 h-VISH possessed nuc, coa, and 16S rRNA genes, and could be reversed into S. aureus. It suggested that biochemical and morphological changes in hVISA and vancomycin-intermediate S. aureus (VISA) should be considered, and the detection of S. aureus, especially reduced vancomycin susceptibility isolates, requires more attention and different techniques.
Keywords: Coagulase, heterogeneous vancomycin-intermediate Staphylococcus aureus, heterogeneous vancomycin-intermediate Staphylococcus haemolyticus
|How to cite this article:|
Zhou X, Dai Y Y, Ma X L. Be alert to the alterations in the biological characteristics in heterogeneous vancomycin-intermediate Staphylococcus aureus. Indian J Med Microbiol 2012;30:215-7
|How to cite this URL:|
Zhou X, Dai Y Y, Ma X L. Be alert to the alterations in the biological characteristics in heterogeneous vancomycin-intermediate Staphylococcus aureus. Indian J Med Microbiol [serial online] 2012 [cited 2013 May 22];30:215-7. Available from: http://www.ijmm.org/text.asp?2012/30/2/215/96696
| ~ Introduction|| |
Staphylococcus aureus is one of the most common causative pathogenic bacteria of suppurative skin inflammation, osteomyelitis, meningitis, sepsis and other infections.  The high mortality due to S. aureus was decreased by penicillin in the 1940s, but this was short lived by acquisition of antimicrobial resistance in S. aureus. Methicillin-resistant S. aureus (MRSA) that are cross resistant to all β-lactams, including penicillins and cephalosporins, account for hospital- and community-acquired infections worldwide. The glycopeptide antibiotic, vancomycin, was first introduced in 1958 as the most effective treatment for MRSA infections. Since first being reported in 1997,  several cases of vancomycin-intermediate S. aureus (VISA) and heterogeneous vancomycin-intermediate S. aureus (hVISA) infections have been reported around the world.
VISA and hVISA usually develop changes in morphology and biological characteristics, such as cell wall thickening, slow growth, smaller colonies, decreased pigment formation, less or not haemolytic, reduced coagulase activity, etc.  Coagulase, a major phenotypic determinant of S. aureus, is often used to identify S. aureus.  If the coagulase test is inaccurate, automated testing methods like Vitek 32 (bioMerieux) may have pitfall as Staphylococcus haemolyticus S. aureus possess similar biochemical tests.
| ~ Materials and Methods|| |
A total of 24 h-VISH strains were used in this study by population analysis profiles. These strains were collected in 2008-2010 from clinical specimens including sputum (n=10, 41.7%), prostatic fluid (n=9, 37.5%), blood (n=2, 8.3%), wound secretion (n=1, 4.2%), pus (n=1, 4.2%) and drainage fluids (n=1, 4.2%). The isolate was plated on fresh blood agar and was incubated overnight at 37°C in 5% CO 2 . The colony was identified by coagulase test, catalase test, and further confirmed by Vitek 32. In the tube coagulase test, the organism was mixed with 0.5 ml of rabbit plasma in a test tube and incubated at 37°C for more than 4 h and then at room temperature overnight. Tubes were examined for clot formation at 30-min intervals until 24 h.
These 24 isolates that grew on the blood agar were suspended and then incubated with lysostaphin and proteinase K at 37°C for 1 h, then boiled and centrifuged. The supernatants were used as DNA templates. DNA was amplified by polymerase chain reaction (PCR) with specific primers and optimal conditions as follows. For S. aureus, nuc: Forward, 5´-GGTAGCCATCATTATTGTAGGTGT-3´, reverse, 5´-CTGTTGTTTAGCTTT ATTTTGTGC-3´; coa: Forward, 5´-GTTCAAGGTCCCGATTTTCT-3´, reverse, 5´-CTAGGCCCATATGTCGCAGT-3´; 16S rRNA: Forward, 5´-GGAATTCAAAGAATTGACGGGGGC, reverse, 5´-CGGGATCCCAGGCCCGGGAACGTATTCAC. The reaction mixture was subjected to denaturation at 94°C for 5 min followed by 30 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 30 sec, and then a final extension step of 72°C for 10 min was done. The PCR products were subjected to electrophoresis on 1% agarose gel with ethidium bromide at 100 V for 30 min. Coomassie Blue stained gels were captured by a digital camera system. The strains of positive nuc, coa, and 16S rRNA genes serailly passaged daily on nutrient agar without vancomycin supplementation. After 15 days of passage on nonselective medium, vancomycin-susceptible revertants were obtained as determined by broth dilution minimal inhibitory concentration (MIC). Revertant isolates were identified by conventional biochemical and automated testing methods. Following the conventional method of treating the specimen, transmission electron microscopy (JEM-1230, JEOL, Japan) and scanning electron microscopy (JSM-6700, JEOL, Japan) were used to enlarge 60,000 times for observation. S. aureus ATCC 29213 was used as the reference strain.
| ~ Results|| |
h-VISH strains displayed coagulase-negative character and were identified as S. haemolyticus by Vitek 32 system. By PCR analysis, six strains contained 16S-rRNA, coa and nuc genes. These strains with serial passaging showed different biochemical characters from their respective parent, such as faster growth, bigger colonies, increased pigment formation, haemolytic, coagulase positive, mannose positive, mannitol positive and turanose positive. And they were identified as S. aureus by Vitek 32 system. Scanning electron microscopic photo showed that revertant isolates owned typical cell wall surface smoothness and regularity characteristics. However, their parents' cell wall surface appeared rough with irregular uplifted spots.
| ~ Discussion|| |
Although the first reports of acquired resistance to glycopeptides in coagulase-negative staphylococci (CoNS) appeared in 1979 and 1983, , these reports did not gain much attention as CoNS were considered as relatively avirulent organisms and not thought to cause severe infection. In contrast to CoNS, S. aureus is a dangerous pathogen with well-known virulence, causing serious infections such as pneumonia or bacteraemia.  The first report of decreased susceptibility to vancomycin in S. aureus was published in 1997 and it generated a great deal of attention and public health concern. Even more worrying is the fact that vancomycin-resistant S. aureus will emerge as a nosocomial pathogen with disastrous consequences if the isolates missed identification and there are no effective control measures for preventing nosocomial transmission.
Controversy still exists regarding the mechanisms of vancomycin resistance in S. aureus, while many studies focus on the cell wall changes of reduced vancomycin susceptibility in S. aureus not related to the acquisition of vanA. Several reports , suggested that cell wall thickening in some strains could be obviously detected by electron microscopy, after exposure to vancomycin. Vancomycin inhibits wall synthesis by binding to the C-terminal d-Ala-d-Ala residue of the peptidoglycan precursor in S. aureus.  Nevertheless, the drug would be prevented from diffusing to its active site and the potency is decreased by the thickened cell wall.  Apart from cell wall rearrangements, some biochemical and morphological changes can be found in low-level vancomycin-resistant (hVISA and VISA) isolates. The cultures of hVISA and VISA on blood agar are different from the standard S. aureus in the properties including reduced pigmentation, smaller colony size and reduced haemolytic activity.  Other common features including lower growth rate, larger cell diameter, decreased susceptibility to lysostaphin and an overexpression of PBP2 and PBP2´ were also observed in the resistant mutants.  In this study, six hVISA isolates had the similar biochemical and morphological changes such as cell wall thickening, slow growth, smaller colonies, decreased pigment formation, less haemolytic, coagulase negative, mannose negative, mannitol negative and turanose negative. These changes may interfere with the identification of bacteria.
Coagulase test, a major phenotypic determinant of S. aureus, is often used to identify S. aureus.  However, it has been reported that coagulase activity decreased in vancomycin-resistant S. aureus, occasionally leading to misidentification if coagulase test is incubated for less than 4 h.  In this study, we found that the hVISA strains showed negative plasma coagulation activity even by extending the incubation period to 24 h. Interestingly, vancomycin-susceptible revertants from six hVISA strains displayed coagulase-positive character. The reason of this phenomenon is still unknown. It may due to low expression of plasma coagulation enzyme gene or the enzyme activity affected by the thickened bacterial cell wall. The scanning electron microscope revealed that those vancomycin-susceptible revertants' cell wall surface became smooth and regular. However, their parents' cell wall surface appeared rough with irregular uplifted spots. Also, coagulase test turned from negative to positive simultaneously.
Several studies , reported that reduced susceptibility to vancomycin was more common in S. haemolyticus, and S. haemolyticus was the only successful isolate to show induced vancomycin resistance in in vitro studies in several CoNS species.  S. haemolyticus displayed higher incidence of reduced susceptibility to vancomycin compared to other CoNS species, which even reached 42% as reported by Froggatt et al.  The high prevalence of vancomycin resistance is unclear and fuels controversy. Interestingly, unlike the case for S. haemolyticus, it appears that there are seldom any VISA isolates detected in hospital microbiology laboratories. If S. aureus with vancomycin resistance was misclassified as S. haemolyticus, the great difference in the prevalence of vancomycin resistance between S. haemolyticus and S. aureus may be overstated. In this study, six hVISA had been misidentified as S. haemolyticus by automated testing methods, which suggested that biochemical and morphological changes in hVISA and VISA should be considered. Therefore, the detection of S. aureus, especially vancomycin-resistant S. aureus, requires more attention and different techniques.
| ~ References|| |
|1.||Otto M. Basis of virulence in community-associated methicillin-resistant Staphylococcus aureus. Annu Rev Microbiol 2010;64:143-62. |
|2.||Smith TL, Pearson ML, Wilcox KR, Cruz C, Lancaster MV, Robinson-Dunn B, et al. Emergence of vancomycin resistance in Staphylococcus aureus. Glycopeptide-Intermediate Staphylococcus aureus Working Group. N Engl J Med 1999;340:493-501. |
|3.||Moreira B, Boyle-Vavra S, deJonge BL, Daum RS. Increased production of penicillin-binding protein 2, increased detection of other penicillin-binding proteins, and decreased coagulase activity associated with glycopeptide resistance in Staphylococcus aureus. Antimicrob Agents Chemother 1997;41:1788-93. |
|4.||Wilcox MH, Walker C, Winstanley TG, Limb DI. True identity of control Staphylococcus aureus strains and their performance in the tube coagulase test. J Med Microbiol 1996;44:496-9. |
|5.||Siebert WT, Moreland N, Williams TW Jr. Synergy of vancomycin plus cefazolin or cephalothin against methicillin-resistant Staphylococcus epidermidis. J Infect Dis 1979;139:452-7. |
|6.||Tuazon CU, Miller H. Clinical and microbiologic aspects of serious infections caused by Staphylococcus epidermidis. Scand J Infect Dis 1983;15:347-60. |
|7.||Pfeltz RF, Singh VK, Schmidt JL, Batten MA, Baranyk CS, Nadakavukaren MJ, et al. Characterization of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental backgrounds. Antimicrob Agents Chemother 2000;44:294-303. |
|8.||Cui L, Murakami H, Kuwahara-Arai K, Hanaki H, Hiramatsu K. Contribution of a thickened cell wall and its glutamine nonamidated component to the vancomycin resistance expressed by Staphylococcus aureus Mu50. Antimicrob Agents Chemother 2000;44:2276-85. |
|9.||Pootoolal J, Neu J, Wright GD. Glycopeptide antibiotic resistance. Annu Rev Pharmacol Toxicol 2002;42:381-408. |
|10.||Howden BP, Davies JK, Johnson PD, Stinear TP, Grayson ML. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: Resistance mechanisms, laboratory detection, and clinical implications. Clin Microbiol Rev 2010;23:99-139. |
|11.||Froggatt JW, Johnston JL, Galetto DW, Archer GL. Antimicrobial resistance in nosocomial isolates of Staphylococcus haemolyticus. Antimicrob Agents Chemother 1989;33:460-6. |
|12.||Kristof K, Kocsis E, Szabo D, Kardos S, Cser V, Nagy K, et al. Significance of methicillin-teicoplanin resistant Staphylococcus haemolyticus in bloodstream infections in patients of the Semmelweis University hospitals in Hungary. Eur J Clin Microbiol Infect Dis 2011;30:691-9. |
|13.||Schwalbe RS, Ritz WJ, Verma PR, Barranco EA, Gilligan PH. Selection for vancomycin resistance in clinical isolates of Staphylococcus haemolyticus. J Infect Dis 1990;161:45-51. |