Restricted accessResearch articleFirst published online 2021-05
Penicillin-Binding Proteins and Associated Protein Mutations Confer Oxacillin/Cefoxitin Tolerance in Borderline Oxacillin-Resistant Staphylococcus aureus
Among clinical isolates of Staphylococcus aureus, borderline oxacillin-resistant S. aureus (BORSA), which is mildly resistant to oxacillin (OXA) without harboring the mecA or mecC gene, is considered a risk factor for further resistance against multiple antibiotics. In this study, BORSA isolates and their derivatives were characterized through antibiotic susceptibility testing and mutation analysis of the genes encoding penicillin-binding proteins (PBPs) and their related proteins, including the promoter region. Eight BORSA isolates were confirmed to harbor the blaZ gene, and hyperproduction of blaZ-encoded penicillinase was predicted based on the minimum inhibitory concentrations (MICs). Of these, four derivative strains that were spontaneously selected based on viability on media containing high concentrations of OXA showed higher MICs than the parent isolates. The minimum bactericidal concentrations, MIC ratios, and TDtest results identified many strains with cefoxitin tolerance. Sequencing of pbp1, pbp2, pbp3, pbp4, gdpP, and yjbH, and the promoter of pbp4 revealed mutations in BORSA isolates and derivatives, despite their absence in parent isolates, suggesting that mutations in PBPs confer OXA/cefoxitin tolerance in BORSA strains.
Get full access to this article
View all access options for this article.
References
1.
LakhundiS., and ZhangK.. 2018. Methicillin-resistant Staphylococcus aureus: molecular characterization, evolution, and epidemiology. Clin. Microbiol. Rev. 31:e00020-18.
2.
ChoS.Y., and ChungD.R.. 2017. Infection prevention strategy in hospitals in the era of community-associated methicillin-resistant Staphylococcus aureus in the Asia-pacific region: a review. Clin. Infect. Dis. 64:S82–S90.
3.
FeltenA., GrandryB., LagrangeP.H, and CasinI.. 2002. Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen latex agglutination test. J. Clin. Microbiol. 40:2766–2771.
4.
BeckerK., BallhausenB., KöckR., and KriegeskorteA.. 2014. Methicillin resistance in Staphylococcus isolates: the “mec alphabet” with specific consideration of mecC, a mec homolog associated with zoonotic S. aureus lineages. Int. J. Med. Microbiol. 304:794–804.
5.
ArgudínM.A., RoisinS., NienhausL., DodémontM., de MendonçaR., NonhoffC., DeplanoA., and DenisO.. 2018. Genetic diversity among Staphylococcus aureus isolates showing oxacillin and/or cefoxitin resistance not linked to the presence of mec genes. Antimicrob. Agents Chemother. 62:e00091-18.
6.
BanerjeeR., GretesM., HarlemC., BasuinoL., and ChambersH.F.. 2010. A mecA-negative strain of methicillin-resistant Staphylococcus aureus with high-level β-lactam resistance contains mutations in three genes. Antimicrob. Agents Chemother. 54:4900–4902.
7.
BaX., HarrisonE.M, EdwardsG.F, HoldenM.T, LarsenA.R, PetersenA., SkovR.L, PeacockS.J, ParkhillJ., PatersonG.K, and HolmesM.A.. 2014. Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene. J. Antimicrob. Chemother. 69:594–597.
8.
HryniewiczM.M., and GarbaczK.. 2017. Borderline oxacillin-resistant Staphylococcus aureus (BORSA)—a more common problem than expected? J. Med. Microbiol. 66:1367–1373.
9.
LeahyT.R., YauY.C, AtenafuE., CoreyM., RatjenF., and WatersV.. 2011. Epidemiology of borderline oxacillin-resistant Staphylococcus aureus in pediatric cystic fibrosis. Pediatr. Pulmonol. 46:489–496.
10.
Varaldo, P.E. 1993. The ‘borderline methicillin-susceptible’ Staphylococcus aureus. J. Antimicrob. Chemother. 31:1–4.
11.
JonasD., SpeckM., DaschnerF.D, and GrundmannH.. 2002. Rapid PCR-based identification of methicillin-resistant Staphylococcus aureus from screening swabs. J. Clin. Microbiol. 40:1821–1823.
12.
SteggerM., AndersenP.S, KearnsA., PichonB., HolmesM.A, EdwardsG., LaurentF., TealeC., SkovR., and LarsenA.R.. 2012. Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251. Clin. Microbiol. Infect. 18:395–400.
13.
BeckerK., LarsenA.R, SkovR.L, PatersonG.K, HolmesM.A, SabatA.J, FriedrichA.W, KöckR., PetersG., and KriegeskorteA.. 2013. Evaluation of a modular multiplex-PCR methicillin-resistant Staphylococcus aureus detection assay adapted for mecC detection. J. Clin. Microbiol. 51:1917–1919.
14.
García-ÁlvarezL., HoldenM.T, LindsayH., WebbC.R, BrownD.F, CurranM.D, WalpoleE., BrooksK., PickardD.J, TealeC., ParkhillJ., BentleyS.D, EdwardsG.F, GirvanE.K, KearnsA.M, PichonB., HillR.L, LarsenA.R, SkovR.L, PeacockS.J, MaskellD.J, and HolmesM.A.. 2011. Methicillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect. Dis. 11:595–603.
15.
ShopsinB., GomezM., MontgomeryS.O, SmithD.H, WaddingtonM., DodgeD.E, BostD.A, RiehmanM., NaidichS., and KreiswirthB.N.. 1999. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J. Clin. Microbiol. 37:3556–3563.
16.
HarmsenD., ClausH., WitteW., RothgängerJ., ClausH., TurnwaldD., and VogeletU.. 2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol. 41:5442–5448.
17.
GefenO., ChekolB., StrahilevitzJ., and BalabanN.Q.. 2017. TDtest: Easy detection of bacterial tolerance and persistence in clinical isolates by a modified disk-diffusion assay. Sci. Rep. 7:41284.
18.
KotkováH., CabrnochováM., LicháI., TkadlecJ., FilaL., BartošováJ., and MelterO.. 2019. Evaluation of TD test for analysis of persistence or tolerance in clinical isolates of Staphylococcus aureus. J. Microbiol. Methods, 167:105705.
19.
FullerE., ElmerC., NattressF., EllisR., HorneG., CookP., and FawcettT.. 2005. Beta-lactam resistance in Staphylococcus aureus cells that do not require a cell wall for integrity. Antimicrob. Agents Chemother. 49:5075–5080.
20.
PozziC., WatersE.M, RudkinJ.K, SchaefferC.R, LohanA.J, TongP., LoftusB.J, PierG.B, FeyP.D, MasseyR.C, and O'GaraJ.P.. 2012. Methicillin resistance alters the biofilm phenotype and attenuates virulence in Staphylococcus aureus device-associated infections. PLoS Pathog. 8:e1002626.
21.
ArgudínM.A., DodémontM., TaguemountM., RoisinS., de MendonçaR., DeplanoA., NonhoffC., and DenisO.. 2017. In vitro activity of ceftaroline against clinical Staphylococcus aureus isolates collected during a national survey conducted in Belgian hospitals. J. Antimicrob. Chemother. 72:56–59.
22.
MorroniG., BrencianiA., BresciniL., FioritiS., SimoniS., PocognoliA., MingoiaM., GiovanettiE., BarchiesiF., GiacomettiA., and CirioniO.. 2018. High rate of ceftobiprole resistance among clinical methicillin-resistant Staphylococcus aureus isolates from a hospital in central Italy. Antimicrob. Agents Chemother. 62:e01663-18.
23.
BaX., KalmarL., HadjirinN.F, KerschnerH., ApfalterP., MorganF.J, PatersonG.K, GirvanS.L, ZhouR., HarrisonE.M, and HolmesM.A.. 2019. Truncation of GdpP mediates β-lactam resistance in clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 74:1182–1191.
24.
KuehlR., MorataL., MeylanS., MensaJ., and SorianoA.. 2020. When antibiotics fail: a clinical and microbiological perspective on antibiotic tolerance and persistence of Staphylococcus aureus. J. Antimicrob. Chemother. 75:1071–1086.
25.
BraunerA., FridmanO., GefenO., and BalabanN.Q.. 2016. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat. Rev. Microbiol. 14:320–330.
26.
BalabanN.Q., MerrinJ., ChaitR., KowalikL., and LeiblerS.. 2004. Bacterial persistence as a phenotypic switch. Science. 305:1622–1625.
ConlonB.P., RoweS.E, GandtA.B, NuxollA.S, DoneganN.P, ZalisE.A, ClairG., AdkinsJ.N, CheungA.L, and LewisK.. 2016. Persister formation in Staphylococcus aureus is associated with ATP depletion. Nat. Microbiol. 1:16051.
29.
FridmanO., GoldbergA., RoninI., ShoreshN., and BalabanN.Q.. 2014. Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations. Nature. 513:418–421.
30.
SpringerM.T., SinghV.K, CheungA.L, DoneganN.P, and ChamberlainN.R.. 2016. Effect of clpP and clpC deletion on persister cell number in Staphylococcus aureus. J. Med. Microbiol. 65:848–857.
31.
FreesD., ChastanetA., QaziS., SørensenK., HillP., MsadekT., and IngmerH.. 2004. Clp ATPases are required for stress tolerance, intracellular replication and biofilm formation in Staphylococcus aureus. Mol. Microbiol. 54:1445–1462.
32.
MascioC.T., AlderJ.D, and SilvermanJ.A.. 2007. Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells. Antimicrob. Agents Chemother. 51:4255–4260.
33.
ZalisE.A., NuxollA.S, ManuseS., ClairG., RadlinskiL.C, ConlonB.P, AdkinsJ., and LewisK.. 2019. Stochastic variation in expression of the tricarboxylic acid cycle produces persister cells. mBio, 10:e01930-19.
34.
RegisA., VilletQ.C, WangY., EstabrooksZ., MedeirosH., and HooperD.C.. 2014. Regulation of expression of abcA and its response to environmental conditions. J. Bacteriol. 196:1532–1539.
35.
DomanskiT.L., de JongeB.L.M, and BaylesK.W.. 1997. Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4. J. Bacteriol. 179:2651–2657.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
