Intrinsic tet(L) sub-class in Bacillus velezensis and Bacillus amyloliquefaciens is associated with a reduced susceptibility toward tetracycline.

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes.

The Tetracycline Resistance Gene, tet(W) in Bifidobacterium animalis subsp. lactis Follows Phylogeny and Differs From tet(W) in Other Species.

The tetracycline resistance gene tet(W) encodes a ribosomal protection protein that confers a low level of tetracycline resistance in the probiotic bacterium Bifidobacterium animalis subsp. lactis. With the aim of assessing its phylogenetic origin and potential mobility, we have performed phylogenetic and in silico genome analysis of tet(W) and its flanking genes. tet(W) was found in 41 out of 44 examined B. animalis subsp. lactis strains. In 38 strains, tet(W) was flanked by an IS5-like element and an open reading frame encoding a hypothetical protein, which exhibited a similar GC content (51-53%). These genes were positioned in the same genomic context within the examined genomes. Phylogenetically, the B. animalis subsp. lactis tet(W) cluster in a clade separate from tet(W) of other species and genera. This is not the case for tet(W) encoded by other bifidobacteria and other species where tet(W) is often found in association with transferable elements or in different genomic regions. An IS5-like element identical to the one flanking the B. animalis subsp. lactis tet(W) has been found in a human gut related bacterium, but it was not associated with any tet(W) genes. This suggests that the IS5-like element is not associated with genetic mobility. tet(W) and the IS5 element have previously been shown to be co-transcribed, indicating that co-localization may be associated with tet(W) expression. Here, we present a method where phylogenetic and in silico genome analysis can be used to determine whether antibiotic resistance genes should be considered innate (intrinsic) or acquired. We find that B. animalis subsp. lactis encoded tet(W) is part of the ancient resistome and thereby possess a negligible risk of transfer.

The Novel Membrane-Associated Auxiliary Factors AuxA and AuxB Modulate ß-lactam Resistance in MRSA by stabilizing Lipoteichoic Acids.

A major determinant of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is the drug insensitive transpeptidase, PBP2a, encoded by mecA. Full expression of the resistance phenotype requires auxiliary factors. Two such factors, auxiliary factor A (auxA, SAUSA300_0980) and B (auxB, SAUSA300_1003), were identified in a screen against mutants with increased susceptibility to ß-lactams in the MRSA strain, JE2. auxA and auxB encode transmembrane proteins, with AuxA predicted to be a transporter. Inactivation of auxA or auxB enhanced ß-lactam susceptibility in community-, hospital- and livestock-associated MRSA strains without affecting PBP2a expression, peptidoglycan cross-linking or wall teichoic acid synthesis. Both mutants displayed increased susceptibility to inhibitors of lipoteichoic acid (LTA) synthesis and alanylation pathways and released LTA even in the absence of ß-lactams. The ß-lactam susceptibility of the aux mutants was suppressed by mutations inactivating gdpP, which was previously found to allow growth of mutants lacking the lipoteichoic synthase enzyme, LtaS. Using the Galleria mellonella infection model, enhanced survival of larvae inoculated with either auxA or auxB mutants was observed compared with the wild-type strain following treatment with amoxicillin. These results indicate that AuxA and AuxB are central for LTA stability and potential inhibitors can be tools to re-sensitize MRSA strains to ß-lactams and combat MRSA infections.

Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides.

Antimicrobial peptides (AMPs) are an important part of the human innate immune system for protection against bacterial infections, however the AMPs display varying degrees of activity against Staphylococcus aureus. Previously, we showed that inactivation of the ATP synthase sensitizes S. aureus towards the AMP antibiotic class of polymyxins. Here we wondered if the ATP synthase similarly is needed for tolerance towards various human AMPs, including human ß-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the WT, even when the oxidative burst is inhibited.These results show that the staphylococcal ATP synthase enhance tolerance of S. aureus towards some human AMPs and this indicates that inhibition of the ATP synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMPs of the innate immune system.

Resveratrol enhances the efficacy of aminoglycosides against Staphylococcus aureus.

Development of new antibiotics is costly and time-consuming, and therefore increasing the efficacy of conventional antibiotics is extremely attractive. For the human pathogen, Staphylococcus aureus, inactivation of the ATP synthase increases its susceptibility to gentamicin (an aminoglycoside) 16-fold. Aminoglycosides are rarely used as monotherapy against S. aureus due to the risk of development of resistance and toxic effects. This study explored the possibility of enhancing the efficacy of aminoglycosides against S. aureus and other Gram-positive pathogens by inhibiting the ATP synthase with resveratrol, a polyphenolic ATP synthase inhibitor that is commonly used as a dietary supplement. Co-administration of subinhibitory concentrations of resveratrol increased the activity of aminoglycosides, including gentamicin, kanamycin, neomycin, streptomycin and tobramycin, up to 32-fold against S. aureus, while the effect was lower (2-4-fold reduction in minimum inhibitory concentration) for other Gram-positive pathogens (i.e. Staphylococcus epidermidis, Enterococcus faecium and Enterococcus faecalis). The mechanism by which resveratrol increases the efficacy of aminoglycosides appears to be unrelated to membrane hyperpolarization and disruption of membrane integrity, which have been associated previously with increased aminoglycoside susceptibility. These results demonstrate that inhibition of the ATP synthase increases the efficacy of aminoglycosides against important Gram-positive pathogens, and the ATP synthase should be explored further as a target that may extend the clinical applicability of aminoglycosides.

Multiple pathways towards reduced membrane potential and concomitant reduction in aminoglycoside susceptibility in Staphylococcus aureus.

Staphylococcus aureus is responsible for life-threatening and difficult-to-treat infections worldwide and antimicrobial resistance is an increasing concern. Whilst acquired resistance has been widely studied, little is known of the contributions from chromosomal determinants that upon inactivation may reduce the susceptibility of S. aureus to antibiotics. The aim of this study was to identify genetic determinants that upon inactivation reduce aminoglycoside susceptibility in S. aureus. The Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2 was screened for reduced susceptibility to gentamicin. Nine mutants were confirmed by Etest to display between 2- and 16-fold reduced susceptibility to this antibiotic. All of the identified genes were associated with the electron transport chain and energy metabolism. Four mutant strains (menD, hemB, aroC and SAUSA300_0355) conferred the largest decrease in gentamicin susceptibility and three exhibited a small colony variant phenotype, whereas the remaining mutants (qoxA, qoxB, qoxC, ndh and hemX) displayed colony morphology similar to the wild-type. All of the mutants, except hemX, displayed reduced membrane potential suggesting that reduced uptake of gentamicin is the predominant mechanism leading to reduced susceptibility. The results of this study demonstrate that S. aureus possesses multiple genes that upon inactivation by mutagenesis reduce the membrane potential and thereby reduce the lethal activity of gentamicin.

Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins.

Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened the Nebraska Transposon Mutant Library established in S. aureus strain JE2 for increased susceptibility to polymyxin B. Nineteen mutants displayed at least 2-fold reductions in MIC, while the greatest reductions (8-fold) were observed for mutants with inactivation of either graS, graR, vraF, or vraG or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards polymyxins and that inhibition of the ATP synthase sensitizes S. aureus to this group of compounds. These findings show that by modulation of bacterial metabolism, new classes of antibiotics may show efficacy against pathogens towards which they were previously considered inapplicable. In light of the need for new treatment options for infections with serious pathogens like S. aureus, this approach may pave the way for novel applications of existing antibiotics.IMPORTANCE Bacterial pathogens that cause disease in humans remain a serious threat to public health, and antibiotics are still our primary weapon in treating bacterial diseases. The ability to eradicate bacterial infections is critically challenged by development of resistance to all clinically available antibiotics. Polymyxins constitute an important class of antibiotics for treatment of infections caused by Gram-negative pathogens, whereas Gram-positive bacteria remain largely insusceptible towards class of antibiotics. Here we performed a whole-genome screen among nonessential genes for polymyxin intrinsic resistance determinants in Staphylococcus aureus We found that the ATP synthase is important for polymyxin susceptibility and that inhibition of the ATP synthase sensitizes S. aureus towards polymyxins. Our study provides novel insights into the mechanisms that limit polymyxin activity against S. aureus and provides valuable targets for inhibitors to potentially enable the use of polymyxins against S. aureus and other Gram-positive pathogens.