AcrB-TolC efflux system is essential for macrolide resistance in Helicobacter pylori

The prevalence of Helicobacter pylori strains resistant to macrolide is increasing worldwide. Macrolide molecules can be generally extruded by the AcrB-TolC system in bacteria. The H. pylori 26695 genome was assessed for putative translocases and the outer membrane efflux of AcrB (HP607) and TolC (HP605) proteins. We investigated the role of the AcrB-TolC efflux system in macrolide resistant (M-R) H. pylori. Both acrB- and tolC-mutant M-R strains were constructed from M-R strains by insertional inactivation of the acrB and tolC genes. The minimal inhibition concentrations (MICs) of erythromycin (EM) and clarithromycin (CLR) were determined by an agar dilution assay. To investigate the efflux ability of macrolides, intracellular accumulation of radiolabeled EM in the H. pylori 26695 strain, M-R strain, and acrB- and tolC-mutant M-R strains was measured by a liquid scintillation counter. For Post antibiotic effect (PAE), EM-treated H. pylori was diluted 1000-fold to remove antimicrobial activity. After additional 24 hours incubation, the CFU was measured. The decrease in the levels of resistance to EM and CLR was 32-fold higher for the acrB- and tolC-mutant M-R strains than the M-R strains. The intracellular EM concentration significantly increased in the acrB- and tolC-mutant M-R strains than the H. pylori 26695 and M-R strains. Diluted acrB, and tolC M-R mutant H. pylori after EM treatment was markedly reduced compared to M-R H. pylori. Our result showed that the M-R mechanism of H. pylori is significantly associated with AcrB-TolC efflux system.

Effect of Subinhibitory Concentrations of Antibiotics and Disinfectants on ISAba-Mediated Inactivation of Lipooligosaccharide Biosynthesis Genes in Acinetobacter baumannii

Inactivation of the lipooligosaccharide (LOS) biosynthesis genes lpxA, lpxC and lpxD by ISAba insertion elements results in high-level resistance to colistin in A. baumannii. In the present study, we quantify the rate of spontaneous insertional inactivation of LOS biosynthesis genes by ISAba elements in the ATCC 19606-type strain and two multidrug clinical isolates. Using insertional inactivation of lpxC by ISAba11 in the ATCC 19606 strain as a model, we determine the effect of several subinhibitory concentrations of the antibiotics, namely tetracycline, ciprofloxacin, meropenem, kanamycin and rifampicin, as well as the disinfectants ethanol and chlorhexidine on ISAba11 insertion frequencies. Notably, subinhibitory concentrations of tetracycline significantly increased ISAba11 insertion, and rifampicin completely inhibited the emergence of colistin resistance due to ISAba11 inactivation of lpxC. Sequencing of ISAba11 insertion sites within the lpxC gene demonstrated that insertions clustered between nucleotides 382 and 618 (58.3% of unique insertions detected), indicating that this may be a hotspot for ISAba11 insertion. The alignment of insertion sites revealed a semi-conserved AT-rich consensus sequence upstream of the ISAba11 insertion site, suggesting that ISAba11 insertion sites may be sequence-dependent. This study explores previously uncharacterized aspects regarding the acquisition of colistin resistance through insertional activation in LOS biosynthesis genes in A. baumannii.

Insertional Inactivation of Prevotella intermedia OxyR Results in Reduced Survival with Oxidative Stress and in the Presence of Host Cells

One of the most abundant bacteria in the subgingival pockets of patients with bleeding following mechanical periodontal therapy is Prevotella intermedia. However, despite its abundance, the molecular mechanisms of its contribution to periodontal disease are not well known. This is mainly due to the lack of genetic tools that would allow examination of the role of predicted virulence factors in the pathogenesis of this bacterium. Here, we report on the first mutant in the P. intermedia OMA14 strain. The mutation is an allelic exchange replacement of the sequences coding for a putative OxyR regulator with ermF sequences coding for the macrolide–lincosamide resistance in anaerobic bacteria. The mutant is severely impaired in its ability to grow with eukaryotic cells, indicating that it is an important target for interventional strategies. Further analyses reveal that its ability to grow with oxidative stress species, in the form of hydrogen peroxide and oxygen, is severely affected. Transcriptome analysis reveals that the major deregulated genes code for the alkylhydroperoxide reductase system, AhpCF, mediating protection from peroxide stress. Moreover, genes coding for Dps, CydA and Ftn are downregulated in the mutant strain, as further verified using qRT-PCR analysis. In conclusion, we succeeded in generating the first P. intermedia mutant and show that the OxyR-deficient strain is unable to survive with a variety of host cells as well as with oxidative stress.

Colistin resistance in Acinetobacter baumannii is driven by multiple genomic traits: Evaluating the role of ISAba1-driven eptA overexpression among Indian isolates

Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrAB two component system and overexpression of eptA due to upstream insertion of ISAba1 play a major role. To characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 224 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrAB genes were characterized by Whole Genome Shotgun sequencing. Twenty eight complete genomes were further characterized for insertional inactivation of lpx genes and the association of ISAba1-eptA using hybrid assembly approach. Non-synonymous mutations like M12I in pmrA, A138T and A444V in pmrB and E117K in lpxD were identified. Four of the five colistin resistant A. baumannii isolates had insertion of ISAba1 upstream eptA. No mcr genes were identified. Overall, the present study highlights the diversity of colistin resistance mechanisms in A. baumannii. ISAba1-driven eptA overexpression could be responsible for colistin resistance among Indian isolates of colistin resistant A. baumannii.

CcpN: a moonlighting protein regulating catabolite repression of gluconeogenic genes in Bacillus subtilis also affects cell length and interacts with DivIVA

CcpN is a transcriptional repressor in Bacillus subtilis that binds to the promoter region of gapB and pckA, downregulating their expression in the presence of glucose. CcpN also represses sr1, which encodes a small noncoding regulatory RNA that suppresses the arginine biosynthesis gene cluster. CcpN has homologues in other Gram-positive bacteria, including Enterococcus faecalis. We report the interaction of CcpN with DivIVA of B. subtilis as determined using bacterial two-hybrid and glutathione S-transferase pull-down assays. Insertional inactivation of CcpN leads to cell elongation and formation of straight chains of cells. These findings suggest that CcpN is a moonlighting protein involved in both gluconeogenesis and cell elongation.

Untethering and Degradation of the Polysaccharide Matrix Are Essential Steps in the Dispersion Response of Pseudomonas aeruginosa Biofilms

ABSTRACT Biofilms are multicellular aggregates of bacteria that are encased in an extracellular matrix. The biofilm matrix of Pseudomonas aeruginosa PAO1 is composed of eDNA, proteins, and the polysaccharides Pel and Psl. This matrix is thought to be degraded during dispersion to liberate cells from the biofilms, with dispersion being apparent not only by single cells escaping from the biofilm but also leaving behind eroded or hollowed-out biofilm. However, little is known of the factors involved in matrix degradation. Here, we focused on the glycoside hydrolases PelA and PslG. We demonstrate that induction of pelA but not pslG expression resulted in dispersion. As Psl is tethered to the matrix adhesin CdrA, we furthermore explored the role of CdrA in dispersion. cdrA mutant biofilms were hyperdispersive, while lapG mutant biofilms were impaired in dispersion in response to glutamate and nitric oxide, indicating the presence of the surface-associated matrix protein CdrA impedes the dispersion response. In turn, insertional inactivation of cdrA enabled pslG-induced dispersion. Lowering of the intracellular c-di-GMP level via induction of PA2133 encoding a phosphodiesterase was not sufficient to induce dispersion by wild-type strains and strains overexpressing pslG, indicating that pslG-induced dispersion is independent of c-di-GMP modulation and, likely, LapG. IMPORTANCE Pseudomonas aeruginosa forms multicellular aggregates or biofilms encased in a matrix. We show for the first time here that dispersion by P. aeruginosa requires the endogenous expression of pelA and pslG, leading to the degradation of both Pel and Psl polysaccharides, with PslG-induced dispersion being CdrA dependent. The findings suggested that endogenously induced Psl degradation is a sequential process, initiated by untethering of CdrA-bound Psl or CdrA-dependent cell interactions to enable Psl degradation and ultimately, dispersion. Untethering likely involves CdrA release in a manner independent of c-di-GMP modulation and thus LapG. Our findings not only provide insight into matrix degrading factors contributing to dispersion but also identify key steps in the degradation of structural components of the P. aeruginosa biofilm matrix.

Transposable temperate phages promote the evolution of divergent social strategies in Pseudomonas aeruginosa populations

Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore overproducing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvd genes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signalling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.

Transposable temperate phages promote the evolution of divergent social strategies inPseudomonas aeruginosapopulations

Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Here we show, using experimental evolution ofPseudomonas aeruginosain iron-limited and iron-rich environments causing differential expression of siderophore cooperation, that transposable phages promoted divergence into extreme siderophore production phenotypes in iron-limited populations. Iron-limited populations with transposable phages evolved siderophore over-producing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations inpvdgenes, encoding pyoverdine biosynthesis, andpqsgenes, encoding quinolone signaling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.

MntC-Dependent Manganese Transport Is Essential forStaphylococcus aureusOxidative Stress Resistance and Virulence

ABSTRACTStaphylococcus aureusis a human pathogen that has developed several approaches to evade the immune system, including a strategy to resist oxidative killing by phagocytes. This resistance is mediated by production of superoxide dismutase (SOD) enzymes which use manganese as a cofactor.S. aureusencodes two manganese ion transporters, MntABC and MntH, and a possible Nramp family manganese transporter, exemplified byS. aureusN315 SA1432. Their relative contributions to manganese transport have not been well defined in clinically relevant isolates. For this purpose, insertional inactivation mutations were introduced intomntC,mntH, and SA1432 individually and in combination.mntCwas necessary for full resistance to methyl viologen, a compound that generates intracellular free radicals. In contrast, strains with an intactmntHgene had a minimal increase in resistance that was revealed only inmntCstrains, and no change was observed upon mutation of SA1432 in strains lacking bothmntCandmntH. Similarly, MntC alone was required for high cellular SOD activity. In addition,mntCstrains were attenuated in a murine sepsis model. To further link these observations to manganese transport, anS. aureusMntC protein lacking manganese binding activity was designed, expressed, and purified. While circular dichroism experiments demonstrated that the secondary and tertiary structures of this protein were unaltered, a defect in manganese binding was confirmed by isothermal titration calorimetry. Unlike complementation with wild-typemntC, introduction of the manganese-binding defective allele into the chromosome of anmntCstrain did not restore resistance to oxidative stress or virulence. Collectively, these results underscore the importance of MntC-dependent manganese transport inS. aureusoxidative stress resistance and virulence.IMPORTANCEWork outlined in this report demonstrated that MntC-dependent manganese transport is required forS. aureusvirulence. These study results support the model that MntC-specific antibodies elicited by a vaccine have the potential to disruptS. aureusmanganese transport and thus abrogate to its virulence.

Characterization of Extensively Drug-Resistant or Pandrug-Resistant Sequence Type 147 and 101 OXA-48-Producing Klebsiella pneumoniae Causing Bloodstream Infections in Patients in an Intensive Care Unit

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae causes important health care-associated infections worldwide. An outbreak of sequence type 11 (ST11) OXA-48-producing K. pneumoniae (OXA-48-Kp) isolates occurred in Tzaneio Hospital in 2012 and was contained until 2014, when OXA-48-Kp reemerged. The present study involved 19 bloodstream infection (BSI) OXA-48-Kp isolates recovered from 19 intensive care unit (ICU) patients hospitalized between August 2014 and July 2016. MICs were determined by broth microdilution. Beta-lactamase genes were detected by PCR. All isolates were typed by pulsed-field gel electrophoresis/multilocus sequence typing (PFGE/MLST), and 10 representative isolates were typed by next-generation sequencing (NGS). Of the 19 study patients, 9 had previous hospitalizations, and 10 carried OXA-48-Kp prior to BSI isolation; median time from ICU admission to BSI was 29 days. Four OXA-48-Kp isolates belonged to PFGE profile A (ST147) and were pandrug resistant (PDR), while 15 isolates exhibited PFGE profile B (ST101) and were extensively drug resistant. Genes detected via NGS resistome analysis accounted for most of the resistance phenotypes, except for tigecycline and fosfomycin. Insertional inactivation of mgrB (distinct per clone) conferred colistin resistance in all 19 isolates. NGS single nucleotide polymorphism (SNP) analysis validated the clonal relatedness of the ST147 and ST101 strains and revealed the possible presence of two index ST147 strains and the microevolution of ST101 strains. Distinct, but highly related, IncL OXA-48-encoding plasmid lineages were identified; plasmids of the ST147 strains were identical with the plasmid of ST11 OXA-48-Kp which caused the 2012 outbreak. In conclusion, biclonal circulation of OXA-48-Kp and, alarmingly, emergence of a PDR clone are reported. These observations, along with the challenging phenotypic detection of OXA-48 producers and the high reported transmissibility of blaOXA-48, necessitate intensive efforts to prevent their further spread.