Development of antiseptic adaptation and cross-adapatation in selected oral pathogens in vitro

There is evidence that pathogenic bacteria can adapt to antiseptics upon repeated exposure. More alarming is the concomitant increase in antibiotic resistance that has been described for some pathogens. Unfortunately, effects of adaptation and cross-adaptation are hardly known for oral pathogens, which are very frequently exposed to antiseptics. Therefore, this study aimed to determine the in vitro increase in minimum inhibitory concentrations (MICs) in oral pathogens after repeated exposure to chlorhexidine or cetylpyridinium chloride, to examine if (cross-)adaptation to antiseptics/antibiotics occurs, if (cross-)adaptation is reversible and what the potential underlying mechanisms are. When the pathogens were exposed to antiseptics, their MICs significantly increased. This increase was in general at least partially conserved after regrowth without antiseptics. Some of the adapted species also showed cross-adaptation, as shown by increased MICs of antibiotics and the other antiseptic. In most antiseptic-adapted bacteria, cell-surface hydrophobicity was increased and mass-spectrometry analysis revealed changes in expression of proteins involved in a wide range of functional domains. These in vitro data shows the adaptation and cross-adaptation of oral pathogens to antiseptics and antibiotics. This was related to changes in cell surface hydrophobicity and in expression of proteins involved in membrane transport, virulence, oxidative stress protection and metabolism.

1 Bacteria exposed to CHX during 10 passages 2 Bacteria exposed to CHX during 10 passages and regrown in absence of antiseptics during 10 passages MIC: Minimum Inhibitory Concentration Statistically significant (P < 0.05) higher MIC values compared to the wild type control are marked with '*' and shown in bold. Statistically significant (P < 0.05) different MIC values when comparing ' 2 ' to ' 1 ' are marked with ' • ' and shown in bold. N = 3. N.I. = not inhibited.

Important role in glycolysis
Upregulated in antibiotic-resistant bacteria 8 Essential for infection 9 Biosynthetic precursors for cell wall components and nucleic acids 10 Thiol:disulfide interchange protein ° (A0A142G1J9) Rescue of secreted proteins damaged by oxidative stress Oxidative stress protection 11 Defects in this protein increase sensitivity to antibiotics 12 4-hydroxy-tetrahydrodipicolinate reductase ° (A0A142G2X6)

Lysine biosynthesis
Increase in peptidoglycan synthesis 13 Found in bacterial species exposed to sub-lethal antibiotic concentrations 13 Phosphoserine aminotransferase (A0A142FYA8) Amino acid metabolism Upregulated in multi-drug resistant bacteria 14 Na(+)-translocating NADH-quinone reductase subunit A ° (A0A142FZX6) Respiratory chain Oxidation-reduction processes Sodium ion transport Alterations of these protein contribute to antibiotic resistance 15 Involved in the physiology of pathogens 16 Related to elevated efflux pump activity and decreased OM permeability 15 D-3-phosphoglycerate dehydrogenase * (A0A142FZ29)

Amino acid transport and metabolism
Upregulated in presence of cell wall-active antibiotics 17 Bacterial adhesion and invasion 18 Tryptophan--tRNA ligase ** (A0A142G174) Tryptophan metabolism and aminoacyl-tRNA biosynthesis Confers high-level resistance to antibiotics 19 Hydrogenase nickel incorporation protein HypB * (A0A142FXT5)

Incorporation of nickel
Significantly associated with resistance to several antibiotics 20 Urease and hydrogenase activities in pathogenic bacteria 21 Involved in colonization and resistance 21 C4-dicarboxylate ABC transporter * (A0A142G2C4)

Transmembrane transport
Highly upregulated in response to antibiotics 22 Resistance to antibiotics 23

Hydrolysis of N-terminal amino acid residues
Alterations of the encoding gene are related to increased antibiotic resistance 26 Involved in epithelial cell cytotoxicity in vitro and virulence 27 ABC transporter ATP-binding protein ° (A0A142FY59)

Transmembrane transport
Actively pumps the drug out of the bacterial cell 28 Involved in pentose-phosphate pathway Produced by MRSA and antibiotic-resistant species 29,30 Lipopolysaccharide production 31 Protein translocase subunit SecD ° (A0A142FYD1)

Lysine biosynthesis
Increase of peptidoglycan synthesis 13 Found in bacterial species exposed to sub-lethal antibiotic concentrations 13 Hydrogenase 3 large subunit ° (A0A142G2F2)

Amino acid transport and metabolism
Upregulated in presence of cell wall-active antibiotics 17 Bacterial adhesion and invasion 18 Hydrogenase nickel incorporation protein HypB * (A0A142FXT5)

Nickel incorporation
Significantly associated with resistance to several antibiotics 20 Urease and hydrogenase activities in pathogenic bacteria 21 Involved in colonization and resistance 21 C4-dicarboxylate ABC transporter * (A0A142G2C4)

Transmembrane transport
High activity in multi-drug resistant bacteria 37 Important influence on the efflux pumps 38 CHX-adapted Aa: A. actinomycetemcomitans exposed to chlorhexidine during 10 passages; CPC-adapted Aa: A. actinomycetemcomitans exposed to cetylpyridinium chloride during 10 passages. ° Proteins also detected in wild type A. actinomycetemcomitans but significantly upregulated in the adapted species (P < 0.05).

CHXadapted Fn
Possible bacterioferrin ° (A5TUE5) Iron acquisition Absence of iron reduces resistance to antibiotics 1 Protects against antibiotics 1 Upregulated in antibiotic-resistant bacteria 2 Peroxiredoxin ° (A5TS03) Antioxidant effect Increases its activity in presence of antibiotics 3 Upregulated in presence of oxidants and persistence in vivo 4 Influences bacterial virulence 5 Beta-lysine 5,6-aminomutase ° (A5TV47) Participation in lysine degradation Protects against oxidative stress 6 Acetate produced by lysine fermentation increases tolerance to antibiotics 6,7 Acyl-CoA dehydrogenase ° (A5TY59) Fatty acid β-oxidation Cell wall and membranes biosynthesis 8 Active in stress response 9 Involved in bacterial survival and virulence 10 Copper (Cu2+)-exporting ATPase * (A5TT66) Exports copper through the membrane Increases the amount of positive charges, thereby reducing influx and adhesion of antiseptics to bacteria 11,12 Possible cobalamin/iron (Fe3+)siderophore ABC superfamily ATP-binding cassette transporter binding protein * (A5TTC9)

Transport activity
Related to the transport of peptide antibiotics, heme, drugs and siderophores 13 L-serine ammonia-lyase * (A5TUB6) Amino acid transport and metabolism Upregulated in response to DNA-damaging agents 14 Uncharacterized protein * (A5TY36) Not identified -Uncharacterized protein ** (5TVL7) Possible ABC transport Possible relation with drug transport 13

Transport activity
Related to the transport of peptide antibiotics, heme, drugs and siderophores 13 L-serine ammonia-lyase * (A5TUB6) Amino acid transport and metabolism Upregulated in response to DNA-damaging agents 14 Uncharacterized protein ** (A5TVD9) Possible lipoprotein -Uncharacterized protein ** (A5TSH6) Possible outer membrane protein Possible influence in permeability and antibiotic resistance 21 CHX-adapted Fn: F. nucleatum exposed to chlorhexidine during 10 passages; CPC-adapted Fn: F. nucleatum exposed to cetylpyridinium chloride during 10 passages. ° Proteins also detected in wild type F. nucleatum but significantly upregulated in the adapted species (P < 0.05). * Proteins uniquely present in both CHX-adapted and CPC-adapted F. nucleatum compared to the wild type species. ** Proteins uniquely present in either CHX-or CPC-adapted F. nucleatum compared to the wild type species.

Adapted species Protein name (accession number) Protein function Native
Associated with resistance/virulence

RNA synthesis
Mutations of the encoding gene increase bacterial antibiotic and biocide resistance 12,13 Thiazole biosynthesis protein ° (A0A1R4DY74)

Detection and response to environmental changes
In response to extracellular glycopeptide antibiotics 21 Regulation of many antibiotic resistance determinants and efflux pumps 21 Arginine-specific cysteine protease RgpA * (A0A1R4ACS4)

RNA synthesis
Mutations of the encoding gene increase bacterial antibiotic and biocide resistance 12,13 UDP-glucose 4-epimerase ° (A0A1R4AFM2) Galactose metabolism and glycoprotein or glycolipid synthesis Absence of the encoding gene reduces bacterial antibiotic resistance 36 Participation in LPS synthesis 36 Acyl-CoA dehydrogenase ° (A0A1R4DX65) Fatty acid β-oxidation Cell wall and membranes biosynthesis 37 Active in stress response 38 Involved in bacterial survival and virulence 37,38 67kDa fimbrillin ° (O83017) Structural protein of the fimbriae Cell adherence and invasion 39,40 Induction of immune response 39,40 Biofilm formation 39,40  Inhibits other bacteria, thereby increasing survival rates with respect to other bacteria 54 Virulence activity 54 Uridylate kinase ** (A0A134DMY9) Participation in pyrimidine metabolism Expressed during the in vivo infection process 55 CHX-adapted Pg: P. gingivalis exposed to chlorhexidine during 10 passages; CPC-adapted Pg: P. gingivalis exposed to cetylpyridinium chloride during 10 passages. ° Proteins also detected in wild type P. gingivalis but significantly upregulated in the adapted species (P < 0.05). * Proteins uniquely present in both CHX-adapted and CPC-adapted P. gingivalis compared to the wild type species. ** Proteins uniquely present in either CHX-or CPC-adapted P. gingivalis compared to the wild type species.