Mu50 glycopeptide-resistant Staphylococcus aureus: the case of the missing penicillinase

Richard W. Ellis*

Department of Microbiology, South Tyneside District Hospital, South Shields, Tyne and Wear, UK

Keywords: Mu50 Staphylococcus aureus, GISA, penicillinase, ß-lactamase

Sir,

Mu50 glycopeptide-resistant Staphylococcus aureus, first described by Hiramatsu et al.,1 remains the prototype of an increasing number of intermediately susceptible strains of S. aureus. The mode of resistance has yet to be completely described, but seems to be mediated by abnormalities in cell-wall synthesis, with incomplete transamidation of peptidoglycan possibly being important.2,3 The organism is known to be methicillin resistant, but in all the literature pertaining to this organism, no mention has been made of any resistance to the prototypical ß-lactam, penicillin.

Experiments in vitro and in silico exploring the susceptibility of Mu50 S. aureus to penicillin were undertaken.

Identification of the organism as S. aureus was confirmed by the detection of free coagulase with conventional laboratory methods and by biochemical reactions (‘api Staph’; bioMérieux, Basingstoke, UK).

Penicillin Etesting was carried out on Mu50 S. aureus, suspending the organism in sterile distilled water to give a turbidity equivalent to 0.5 McFarland units. The resultant suspension was spread on Iso-Sensitest agar (Oxoid, Basingstoke, UK) and a penicillin Etest strip (AB Biodisk, Dalvägen, Sweden) laid on the surface of the agar. Incubation at 37°C for 18 h in air was carried out and the penicillin MIC read by observing the intersection of bacterial growth with the Etest strip. The MIC was found to be 0.094 mg/L. Testing for the presence of penicillinase using ‘Cefinase’ discs (Oxoid) using growth immediately adjacent to the zone of inhibition and with appropriate controls showed that Mu50 S. aureus was not phenotypically expressing a penicillinase. The breakpoint MIC for S. aureus is defined by the British Society for Antimicrobial Chemotherapy as 0.12 mg/L. Clearly, Mu50 is sensitive to penicillin in vitro, probably by reason of lacking a type-I ß-lactamase (penicillinase).

Using the European Bioinformatics Institute (EMBL) database, BlastN and BlastP searches4 of the published DNA/translated protein sequence of Mu505 for a ß-lactamase gene/amino-acid sequence were undertaken. The search DNA/amino-acid sequence was the N315 S. aureus type I ß-lactamase (accession number AP003139_11, protein identification BAB43879). No ß-lactamase DNA or amino-acid sequences with homology e-values <101 were found in the Mu50 S. aureus genome. Text string searching using the term ‘penicillinase (and) Mu50’ revealed no sequences, but similar searching with text string ‘lactamase (and) Mu50’ revealed three sequences in the Mu50 genome (Table 1).


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Table 1.  Potential ß-lactamase genes in Mu50 S. aureus
 
It is concluded that Mu50 S. aureus does not possess the phenotypic capability to produce a type I ß-lactamase under the conditions of the experiment, probably explaining its in vitro susceptibility to penicillin. The potential of Mu50 to produce three enzymes similar to ß-lactamases is present in the genome, but definitive incontrovertible functions for these genes have not yet been ascribed.

The methicillin resistance of Mu50 S. aureus is not in doubt since it possesses and phenotypically expresses the genes necessary for methicillin resistance. Penicillin may therefore be a suitable and effective therapeutic option for infection due to S. aureus strain Mu50.

Recent work by Avison et al.3 has given weight to the hypothesis that vancomycin resistance in Mu50 is due to fundamental changes that affect the biochemical pathways leading to peptidoglycan synthesis. They also propose that abnormalities in L-glutamine synthesis in Mu50 may lead to defective transamidation of peptidoglycan, thus leading to increased terminal D-alanine (D-Ala) dimers in the cell wall that ‘trap’ vancomycin before it can reach its intracellular target.

It is known that transpeptidases and carboxypeptidases may also have a ß-lactamase function in cell-wall synthesis.6 If the reverse is true, then a simple penicillinase may have a contributory role in peptidoglycan synthesis; the absence of which in Mu50 may then partly explain defective transamidation or indeed the lack of carboxypeptidase-mediated D-Ala dimeric cleavage, both of which may lead to glycopeptide resistance. The findings outlined above may thus be compatible with the findings of dysregulated peptidoglycan synthesis described by Avison et al.3

Nevertheless, many non-methicillin-resistant S. aureus clinical strains do not possess a penicillinase, yet are not considered glycopeptide resistant. To clarify the situation, further work is in progress to determine whether external supplementation of penicillinase and penicillinase gene insertion reverses vancomycin resistance-associated phenotypes in S. aureus strain Mu50.

Acknowledgements

I wish to thank Professor R. Wise at the Department of Microbiology, City Hospital, Birmingham and Dr A. McGowan at the Department of Microbiology, Southmead Hospital, Bristol for providing the organisms for investigation.

Footnotes

* Tel: +44-191-4548888; Fax: +44-191-2024145; E-mail: richard.ellis{at}sthct.nhs.uk Back

References

1 . Hiramatsu, K., Hanaki, H., Ino, T., Yabuta, K., Oguri, T. & Tenover, F. C. (1997). Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. Journal of Antimicrobial Chemotherapy 40, 135–6.[Free Full Text]

2 . Hanaki, H., Kuwahara-Arai, K., Boyle-Vavra, S., Daum, R. S., Labischinski, H. & Hiramatsu, K. (1998). Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50. Journal of Antimicrobial Chemotherapy 42, 199–209.[Abstract]

3 . Avison, M. B., Bennett, P. M., Howe, R. A. & Walsh, T. R. (2002). Preliminary analysis of the genetic basis for vancomycin resistance in Staphylococcus aureus strain Mu50. Journal of Antimicrobial Chemotherapy 49, 255–60.[Abstract/Free Full Text]

4 . Stoesser, G., Baker, W., van den Broek, A., Camon, E., Garcia-Pastor, M., Kanz, C. et al. (2002). The EMBL Nucleotide Sequence Database. Nucleic Acids Research 30, 21–6.[Abstract/Free Full Text]

5 . Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., Kobayashi, I. et al. (2001). Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 1225.[CrossRef][ISI][Medline]

6 . Kozarich, J. W. & Strominger, J. L. (1978). A membrane enzyme from Staphylococcus aureus which catalyzes transpeptidase, carboxypeptidase, and penicillinase activities. Journal of Biological Chemistry 253, 1272–8.[Abstract]





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