Comparative transcriptomics of multidrug-resistant Acinetobacter baumannii in response to antibiotic treatments

Abstract Multidrug-resistant Acinetobacter baumannii, a major hospital-acquired pathogen, is a serious health threat and poses a great challenge to healthcare providers. Although there have been many genomic studies on the evolution and antibiotic resistance of this species, there have been very limited transcriptome studies on its responses to antibiotics. We conducted a comparative transcriptomic study on 12 strains with different growth rates and antibiotic resistance profiles, including 3 fast-growing pan-drug-resistant strains, under separate treatment with 3 antibiotics, namely amikacin, imipenem, and meropenem. We performed deep sequencing using a strand-specific RNA-sequencing protocol, and used de novo transcriptome assembly to analyze gene expression in the form of polycistronic transcripts. Our results indicated that genes associated with transposable elements generally showed higher levels of expression under antibiotic-treated conditions, and many of these transposon-associated genes have previously been linked to drug resistance. Using co-expressed transposon genes as markers, we further identified and experimentally validated two novel genes of which overexpression conferred significant increases in amikacin resistance. To the best of our knowledge, this study represents the first comparative transcriptomic analysis of multidrug-resistant A. baumannii under different antibiotic treatments, and revealed a new relationship between transposons and antibiotic resistance

Arginine deiminase pathway is far more important than urease for acid resistance and intracellular survival in Laribacter hongkongensis: a possible result of arc gene cassette duplication

BACKGROUND: Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community-acquired gastroenteritis and traveler's diarrhea. Its mechanisms of adaptation to various environmental niches and host defense evasion are largely unknown. During the process of analyzing the L. hongkongensis genome, a complete urease cassette and two adjacent arc gene cassettes were found. We hypothesize that the urease cassette and/or the arc gene cassettes are important for L. hongkongensis to survive in acidic environment and macrophages. In this study, we tested this hypothesis by constructing single, double and triple non-polar deletion mutants of the urease and two arc gene cassettes of L. hongkongensis using the conjugation-mediated gene deletion system and examining their effects in acidic environment in vitro, in macrophages and in a mouse model. RESULTS: HLHK9ureA, HLHK9ureC, HLHK9ureD and HLHK9ureE all exhibited no urease activity. HLHK9arcA1 and HLHK9arcA2 both exhibited arginine deiminase (ADI) activities, but HLHK9arcA1/arcA2 double deletion mutant exhibited no ADI activity. At pH 2 and 3, survival of HLHK9arcA1/arcA2 and HLHK9ureA/arcA1/arcA2 were markedly decreased (p < 0.001) but that of HLHK9ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. Survival of HLHK9ureA/arcA1/arcA2 and HLHK9arcA1/arcA2 in macrophages were also markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9ureA was slightly decreased (p < 0.05), compared to HLHK9, although expression of arcA1, arcA2 and ureA genes were all upregulated. Using a mouse model, HLHK9ureA exhibited similar survival compared to HLHK9 after passing through the murine stomach, but survival of HLHK9arcA1/arcA2 and HLHK9ureA/arcA1/arcA2 were markedly reduced (p < 0.01). CONCLUSIONS: In contrast to other important gastrointestinal tract pathogens, ADI pathway is far more important than urease for acid resistance and intracellular survival in L. hongkongensis. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis.published_or_final_versio

Pharmacokinetic/Pharmacodynamic Correlation of Cefquinome Against Experimental Catheter-Associated Biofilm Infection Due to Staphylococcus aureus.

Biofilm formations play an important role in Staphylococcus aureus pathogenesis and contribute to antibiotic treatment failures in biofilm-associated infections. The aim of this study was to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) profiles of cefquinome against an experimental catheter-related biofilm model due to S. aureus, including three clinical isolates and one non-clinical isolate. The minimal inhibitory concentration (MIC), minimal biofilm inhibitory concentration (MBIC), biofilm bactericidal concentration (BBC), minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) and in vitro time-kill curves of cefquinome were studied in both planktonic and biofilm cells of study S. aureus strains. The in vivo post-antibiotic effects (PAEs), PK profiles and efficacy of cefquinome were performed in the catheter-related biofilm infection model in murine. A sigmoid E max model was utilized to determine the PK/PD index that best described the dose-response profiles in the model. The MICs and MBICs of cefquinome for the four S. aureus strains were 0.5 and 16 μg/mL, respectively. The BBCs (32-64 μg/mL) and MBECs (64-256 μg/mL) of these study strains were much higher than their corresponding BPC values (1-2 μg/mL). Cefquinome showed time-dependent killing both on planktonic and biofilm cells, but produced much shorter PAEs in biofilm infections. The best-correlated PK/PD parameters of cefquinome for planktonic and biofilm cells were the duration of time that the free drug level exceeded the MIC (fT &gt; MIC, R (2) = 96.2%) and the MBIC (fT &gt; MBIC, R (2) = 94.7%), respectively. In addition, the AUC24h/MBIC of cefquinome also significantly correlated with the anti-biofilm outcome in this model (R (2) = 93.1%). The values of AUC24h/MBIC for biofilm-static and 1-log10-unit biofilm-cidal activity were 22.8 and 35.6 h; respectively. These results indicate that the PK/PD profiles of cefquinome could be used as valuable guidance for effective dosing regimens treating S. aureus biofilm-related infections

Human streptococcus agalactiae strains in aquatic mammals and fish

&lt;p&gt;Background: In humans, Streptococcus agalactiae or group B streptococcus (GBS) is a frequent coloniser of the rectovaginal tract, a major cause of neonatal infectious disease and an emerging cause of disease in non-pregnant adults. In addition, Streptococcus agalactiae causes invasive disease in fish, compromising food security and posing a zoonotic hazard. We studied the molecular epidemiology of S. agalactiae in fish and other aquatic species to assess potential for pathogen transmission between aquatic species and humans.&lt;/p&gt; &lt;p&gt;Methods: Isolates from fish (n = 26), seals (n = 6), a dolphin and a frog were characterized by pulsed-field gel electrophoresis, multilocus sequence typing and standardized 3-set genotyping, i.e. molecular serotyping and profiling of surface protein genes and mobile genetic elements.&lt;/p&gt; &lt;p&gt;Results: Four subpopulations of S. agalactiae were identified among aquatic isolates. Sequence type (ST) 283 serotype III-4 and its novel single locus variant ST491 were detected in fish from Southeast Asia and shared a 3-set genotype identical to that of an emerging ST283 clone associated with invasive disease of adult humans in Asia. The human pathogenic strain ST7 serotype Ia was also detected in fish from Asia. ST23 serotype Ia, a subpopulation that is normally associated with human carriage, was found in all grey seals, suggesting that human effluent may contribute to microbial pollution of surface water and exposure of sea mammals to human pathogens. The final subpopulation consisted of non-haemolytic ST260 and ST261 serotype Ib isolates, which belong to a fish-associated clonal complex that has never been reported from humans.&lt;/p&gt; &lt;p&gt;Conclusions: The apparent association of the four subpopulations of S. agalactiae with specific groups of host species suggests that some strains of aquatic S. agalactiae may present a zoonotic or anthroponotic hazard. Furthermore, it provides a rational framework for exploration of pathogenesis and host-associated genome content of S. agalactiae strains.&lt;/p&gt

Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression

As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.published_or_final_versio

Nanoparticle-encapsulated chlorhexidine against oral bacterial biofilms

Background: Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms. Methodology/Principal Findings: The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50-200 mu g/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h. Conclusions/Significance: This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.published_or_final_versio

Human oropharynx as natural reservoir of Streptobacillus hongkongensis

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On complex oscillation theory, quasi-exact solvability and Fredholm Integral Equations

Biconfluent Heun equation (BHE) is a confluent case of the general Heun equation which has one more regular singular points than the Gauss hypergeometric equation on the Riemann sphere $\hat{\mathbb{C}}$. Motivated by a Nevanlinna theory (complex oscillation theory) approach, we have established a theory of \textit{periodic} BHE (PBHE) in parallel with the Lam\'e equation verses the Heun equation, and the Mathieu equation verses the confluent Heun equation. We have established condition that lead to explicit construction of eigen-solutions of PBHE, and their single and double orthogonality, and a related first-order Fredholm-type integral equation for which the corresponding eigen-solutions must satisfy. We have also established a Bessel polynomials analogue at the BHE level which is based on the observation that both the Bessel equation and the BHE have a regular singular point at the origin and an irregular singular point at infinity on the Riemann sphere $\hat{\mathbb{C}}$, and that the former equation has orthogonal polynomial solutions with respect to a complex weight. Finally, we relate our results to an equation considered by Turbiner, Bender and Dunne, etc concerning a quasi-exact solvable Schr\"odinger equation generated by first order operators such that the second order operators possess a finite-dimensional invariant subspace in a Lie algebra of $SL_2(\mathbb{C})$Comment: This paper has been withdrawn by the authors due to a new version with different title "Galoisian approach to complex oscillation theory of Hill equations" and many contents change

Transcriptomic Analysis of Laribacter hongkongensis Reveals Adaptive Response Coupled with Temperature

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Self-cleaning and antibiofouling enamel surface by slippery liquid-infused technique.

We aimed to create a slippery liquid-infused enamel surface with antibiofouling property to prevent dental biofilm/plaque formation. First, a micro/nanoporous enamel surface was obtained by 37% phosphoric acid etching. The surface was then functionalized by hydrophobic low-surface energy heptadecafluoro-1,1,2,2-tetra- hydrodecyltrichlorosilane. Subsequent infusion of fluorocarbon lubricants (Fluorinert FC-70) into the polyfluoroalkyl-silanized rough surface resulted in an enamel surface with slippery liquid-infused porous surface (SLIPS). The results of water contact angle measurement, diffuse-reflectance Fourier transform infrared spectroscopy, and atomic force microscope confirmed that the SLIPS was successfully constructed on the enamel surface. The antibiofouling property of the SLIPS was evaluated by the adsorption of salivary protein of mucin and Streptococcus mutans in vitro, as well as dental biofilm formation using a rabbit model in vivo. The results showed that the SLIPS on the enamel surface significantly inhibited mucin adhesion and S. mutans biofilm formation in vitro, and inhibited dental plaque formation in vivo.published_or_final_versio