Detection of anaerobic and aerobic bacteria from commercial tattoo and permanent makeup inks.

Tattooing and use of permanent makeup (PMU) have dramatically increased over the last decade, with a concomitant increase in ink-related infections. Studies have shown evidence that commercial tattoo and PMU inks are frequently contaminated with pathogenic microorganisms. Considering that tattoo inks are placed into the dermal layer of the skin where anaerobic bacteria can thrive and cause infections in low-oxygen environments, the prevalence of anaerobic and aerobic bacteria should be assessed in tattoo and PMU inks. In this study, we tested 75 tattoo and PMU inks using the analytical methods described in the FDA Bacteriological Analytical Manual Chapter 23 for the detection of both aerobic and anaerobic bacterial contamination, followed by 16S rRNA gene sequencing for microbial identification. Of 75 ink samples, we found 26 contaminated samples with 34 bacterial isolates taxonomically classified into 14 genera and 22 species. Among the 34 bacterial isolates, 19 were identified as possibly pathogenic bacterial strains. Two species, namely Cutibacterium acnes (four strains) and Staphylococcus epidermidis (two strains) were isolated under anaerobic conditions. Two possibly pathogenic bacterial strains, Staphylococcus saprophyticus and C. acnes, were isolated together from the same ink samples (n = 2), indicating that tattoo and PMU inks can contain both aerobic (S. saprophyticus) and anaerobic bacteria (C. acnes). No significant association was found between sterility claims on the ink label and the absence of bacterial contamination. The results indicate that tattoo and PMU inks can also contain anaerobic bacteria. IMPORTANCE: The rising popularity of tattooing and permanent makeup (PMU) has led to increased reports of ink-related infections. This study is the first to investigate the presence of both aerobic and anaerobic bacteria in commercial tattoo and PMU inks under aerobic and anaerobic conditions. Our findings reveal that unopened and sealed tattoo inks can harbor anaerobic bacteria, known to thrive in low-oxygen environments, such as the dermal layer of the skin, alongside aerobic bacteria. This suggests that contaminated tattoo inks could be a source of infection from both types of bacteria. The results emphasize the importance of monitoring these products for both aerobic and anaerobic bacteria, including possibly pathogenic microorganisms.

Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

A naturally occurring point mutation in the hyaluronidase gene (hysA1) of Staphylococcus aureus UAMS-1 results in reduced enzymatic activity.

Hyaluronic acid is a high-molecular-weight polysaccharide that is widely distributed in animal tissues. Bacterial hyaluronidases degrade hyaluronic acid as secreted enzymes and have been shown to contribute to infection. Staphylococcus aureus UAMS-1 is a clinical isolate that codes for two hyaluronidases (hysA1 and hysA2). Previous research has shown the presence of a full-length HysA1 protein from the S. aureus UAMS-1 strain with no evidence of enzymatic activity. In this study, the coding and upstream promoter regions of hysA1 from the S. aureus UAMS-1 strain were cloned, sequenced, and compared to the hysA1 gene from the S. aureus Sanger 252 strain. A single base change resulting in an E480G amino acid change was identified in the hysA1 gene from the S. aureus UAMS-1 strain when compared to the hysA1 gene from S. aureus Sanger 252. A plasmid copy of hysA1 from S. aureus Sanger 252 transduced into an S. aureus UAMS-1 hysA2 deletion mutant strain restored near wild-type levels of enzymatic activity. Homology modeling of the HysA1 hyaluronidase was performed with SWISS-MODEL using hyaluronidase from Streptococcus pneumoniae as the template, followed by a series of structural analyses using PyMOL, PLIP, PDBsum, and HOPE servers. This glutamic acid is highly conserved among hyaluronidases from Staphylococcus and other gram-positive bacteria. A series of structural analyses suggested that Glu-480 in HysA1 is critically responsible for maintaining the structural and functional ensemble of the catalytic and tunnel-forming residues, which are essential for enzyme activity. The missense mutation of Glu-480 to Gly introduces a loss of side chain hydrogen bond interactions with key residues Arg-360 and Arg-364, which are responsible for the tunnel topology, resulting in displacement of the substrate from an ideal position for catalysis through a localized conformational change of the active site. There is a high degree of relatedness among several gram-positive bacterial hyaluronidases; the loss of enzymatic activity of HysA1 in the S. aureus UAMS-1 strain is most likely caused by the mutation identified in our study. The role of hyaluronidase in staphylococcal infection and the redundancy of this gene are yet to be determined.

Phylogenetically diverse bacteria isolated from tattoo inks, an azo dye-rich environment, decolorize a wide range of azo dyes.

PURPOSE: There has been an interest in the microbial azo dye degradation as an optional method for the treatment of azo dye-containing wastes. Tattoo ink is an extremely unique azo dye-rich environment, which have never been explored in terms of microorganisms capable of degrading azo dyes. Previously, we isolated 81 phylogenetically diverse bacteria, belonging to 18 genera and 52 species, contaminated in tattoo inks. In this study, we investigated if these bacteria, which can survive in the azo dye-rich environment, have an ability to degrade azo dyes. METHODS: We conducted a two-step azo dye degradation (or decolorization) assay. In step 1, a high-throughput degradability assay was done for 79 bacterial isolates using Methyl Red and Orange II. In step 2, a further degradation assay was done for 10 selected bacteria with a representative of 11 azo dyes, including 3 commercial tattoo ink azo dyes. Degradation of azo dyes were calculated from measuring optical absorbance of soluble dyes at specific wavelengths. RESULTS: The initial high-throughput azo dye assay (step 1) showed that 79 isolates had a complete or partial degradation of azo dyes; > 90% of Methyl Red and Orange II were degraded within 24 h, by 74 and 20 isolates, respectively. A further evaluation of azo dye degradability for 10 selected isolates in step 2 showed that the isolates, belonging to Bacillus, Brevibacillus, Paenibacillus, and Pseudomonas, exhibited an excellent decolorization ability for a wide range of azo dyes. CONCLUSIONS: This study showed that phylogenetically diverse bacteria, isolated from azo dye-rich tattoo inks, is able to degrade a diverse range of azo dyes, including 3 azo dyes used in commercial tattoo inks. Some of the strains would be good candidates for future studies to provide a systematic understanding of azo dye degradation mechanisms.

CYPminer: an automated cytochrome P450 identification, classification, and data analysis tool for genome data sets across kingdoms.

BACKGROUND: Cytochrome P450 monooxygenases (termed CYPs or P450s) are hemoproteins ubiquitously found across all kingdoms, playing a central role in intracellular metabolism, especially in metabolism of drugs and xenobiotics. The explosive growth of genome sequencing brings a new set of challenges and issues for researchers, such as a systematic investigation of CYPs across all kingdoms in terms of identification, classification, and pan-CYPome analyses. Such investigation requires an automated tool that can handle an enormous amount of sequencing data in a timely manner. RESULTS: CYPminer was developed in the Python language to facilitate rapid, comprehensive analysis of CYPs from genomes of all kingdoms. CYPminer consists of two procedures i) to generate the Genome-CYP Matrix (GCM) that lists all occurrences of CYPs across the genomes, and ii) to perform analyses and visualization of the GCM, including pan-CYPomes (pan- and core-CYPome), CYP co-occurrence networks, CYP clouds, and genome clustering data. The performance of CYPminer was evaluated with three datasets from fungal and bacterial genome sequences. CONCLUSIONS: CYPminer completes CYP analyses for large-scale genomes from all kingdoms, which allows systematic genome annotation and comparative insights for CYPs. CYPminer also can be extended and adapted easily for broader usage.

Comparative functional pan-genome analyses to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon metabolism in the genus Mycobacterium.

BACKGROUND: The bacterial genus Mycobacterium is of great interest in the medical and biotechnological fields. Despite a flood of genome sequencing and functional genomics data, significant gaps in knowledge between genome and phenome seriously hinder efforts toward the treatment of mycobacterial diseases and practical biotechnological applications. In this study, we propose the use of systematic, comparative functional pan-genomic analysis to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon (PAH) metabolism in the genus Mycobacterium. RESULTS: Phylogenetic, phenotypic, and genomic information for 27 completely genome-sequenced mycobacteria was systematically integrated to reconstruct a mycobacterial phenotype network (MPN) with a pan-genomic concept at a network level. In the MPN, mycobacterial phenotypes show typical scale-free relationships. PAH degradation is an isolated phenotype with the lowest connection degree, consistent with phylogenetic and environmental isolation of PAH degraders. A series of functional pan-genomic analyses provide conserved and unique types of genomic evidence for strong epistatic and pleiotropic impacts on evolutionary trajectories of the PAH-degrading phenotype. Under strong natural selection, the detailed gene gain/loss patterns from horizontal gene transfer (HGT)/deletion events hypothesize a plausible evolutionary path, an epistasis-based birth and pleiotropy-dependent death, for PAH metabolism in the genus Mycobacterium. This study generated a practical mycobacterial compendium of phenotypic and genomic changes, focusing on the PAH-degrading phenotype, with a pan-genomic perspective of the evolutionary events and the environmental challenges. CONCLUSIONS: Our findings suggest that when selection acts on PAH metabolism, only a small fraction of possible trajectories is likely to be observed, owing mainly to a combination of the ambiguous phenotypic effects of PAHs and the corresponding pleiotropy- and epistasis-dependent evolutionary adaptation. Evolutionary constraints on the selection of trajectories, like those seen in PAH-degrading phenotypes, are likely to apply to the evolution of other phenotypes in the genus Mycobacterium.

Dynamic Response of Mycobacterium vanbaalenii PYR-1 to BP Deepwater Horizon Crude Oil.

We investigated the response of the hydrocarbon-degrading Mycobacterium vanbaalenii PYR-1 to crude oil from the BP Deepwater Horizon (DWH) spill, using substrate depletion, genomic, and proteome analyses. M. vanbaalenii PYR-1 cultures were incubated with BP DWH crude oil, and proteomes and degradation of alkanes and polycyclic aromatic hydrocarbons (PAHs) were analyzed at four time points over 30 days. Gas chromatography-mass spectrometry (GC-MS) analysis showed a chain length-dependent pattern of alkane degradation, with C12 and C13 being degraded at the highest rate, although alkanes up to C28 were degraded. Whereas phenanthrene and pyrene were completely degraded, a significantly smaller amount of fluoranthene was degraded. Proteome analysis identified 3,948 proteins, with 876 and 1,859 proteins up- and downregulated, respectively. We observed dynamic changes in protein expression during BP crude oil incubation, including transcriptional factors and transporters potentially involved in adaptation to crude oil. The proteome also provided a molecular basis for the metabolism of the aliphatic and aromatic hydrocarbon components in the BP DWH crude oil, which included upregulation of AlkB alkane hydroxylase and an expression pattern of PAH-metabolizing enzymes different from those in previous proteome expression studies of strain PYR-1 incubated with pure or mixed PAHs, particularly the ring-hydroxylating oxygenase (RHO) responsible for the initial oxidation of aromatic hydrocarbons. Based on these results, a comprehensive cellular response of M. vanbaalenii PYR-1 to BP crude oil was proposed. This study increases our fundamental understanding of the impact of crude oil on the cellular response of bacteria and provides data needed for development of practical bioremediation applications.

Differential gene expression in Staphylococcus aureus exposed to Orange II and Sudan III azo dyes.

We previously demonstrated the effects of azo dyes and their reduction metabolites on bacterial cell growth and cell viability. In this report, the effects of Orange II and Sudan III on gene expression profiling in Staphylococcus aureus ATCC BAA 1556 were analyzed using microarray and quantitative RT-PCR technology. Upon exposure to 6 µg/ml Orange II for 18 h, 21 genes were found to be differently expressed. Among them, 8 and 13 genes were up- and down-regulated, respectively. Most proteins encoded by these differentially expressed genes involve stress response caused by drug metabolism, oxidation, and alkaline shock indicating that S. aureus could adapt to Orange II exposure through a balance between up and down regulated gene expression. Whereas, after exposure to 6 µg/ml Sudan III for 18 h, 57 genes were differentially expressed. In which, 51 genes were up-regulated and 6 were down-regulated. Most proteins encoded by these differentially expressed genes involve in cell wall/membrane biogenesis and biosynthesis, nutrient uptake, transport and metabolite, and stress response, suggesting that Sudan III damages the bacterial cell wall or/and membrane due to binding of the dye. Further analysis indicated that all differentially expressed genes encoded membrane proteins were up-regulated and most of them serve as transporters. The result suggested that these genes might contribute to survival, persistence and growth in the presence of Sudan III. Only one gene msrA, which plays an important role in oxidative stress resistance, was found to be down-regulated after exposure to both Orange II and Sudan III. The present results suggested that both these two azo dyes can cause stress in S. aureus and the response of the bacterium to the stress is mainly related to characteristics of the azo dyes.

Pleiotropic and epistatic behavior of a ring-hydroxylating oxygenase system in the polycyclic aromatic hydrocarbon metabolic network from Mycobacterium vanbaalenii PYR-1.

Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and its pleiotropic and epistatic behavior(s) responsible for low-molecular-weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparisons using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity directed toward three-ring PAHs. Structure analysis and PAH substrate docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taking the findings together, the Pdo system is an RHO system with the pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also crucial for the metabolic quality and quantity of the PAH-MN.

Unraveling the molecular dynamics of Pseudomonas aeruginosa biofilms at the air-liquid interface.

Aim: The aim of this study was to probe the dynamics of Pseudomonas aeruginosa PA14 air-liquid interface (ALI) biofilms over time through global proteomic analysis. Materials & methods: P. aeruginosa PA14 ALI biofilm samples, collected over 48-144 h, underwent differential expression analysis to identify varying proteins at each time point. Results: A consistent set of 778 proteins was identified, with variable expression over time. Upregulated proteins were mainly linked to 'amino acid transport and metabolism'. Biofilm-related pathways, including cAMP/Vfr and QS, underwent significant changes. Flagella were more influential than pili, especially in early biofilm development. Proteins associated with virulence, transporters and iron showed differential expression throughout. Conclusion: The findings enhance our understanding of ALI biofilm development.

This study looks at how the bacteria Pseudomonas aeruginosa forms a community called a biofilm at the air­liquid interface (ALI), an important environment for bacterial growth. Biofilms at the ALI are resistant to external forces and contribute to antibiotic resistance. Over 48­144 h, we observed a noticeable increase in biofilm thickness. Our data suggested that the flagella, a sort of propeller of the bacterium, plays a crucial role, especially in the initial stages of ALI biofilm formation. Proteins associated with virulence, transporters and iron also showed their significance in ALI biofilms. These findings offer valuable insights into the protein changes and functions involved in P. aeruginosa ALI biofilms, improving our understanding of biofilm development.

Chicken Juice Enhances C. jejuni NCTC 11168 Biofilm Formation with Distinct Morphological Features and Altered Protein Expression.

Campylobacter jejuni is the foodborne pathogen causing most gastrointestinal infections. Understanding its ability to form biofilms is crucial for devising effective control strategies in food processing environments. In this study, we investigated the growth dynamics and biofilm formation of C. jejuni NCTC 11168 in various culture media, including chicken juice (CJ), brain heart infusion (BHI), and Mueller Hinton (MH) broth. Our results demonstrated that C. jejuni exhibited a higher growth rate and enhanced biofilm formation in CJ and in 1:1 mixtures of CJ with BHI or MH broth compared to these measures in BHI or MH broth alone. Electron microscopy unveiled distinct morphological attributes of late-stage biofilm cells in CJ, including the presence of elongated spiral-shaped cells, thinner stretched structures compared to regular cells, and extended thread-like structures within the biofilms. Proteomic analysis identified significant alterations in protein expression profiles in C. jejuni biofilms, with a predominance of downregulated proteins associated with vital functions like metabolism, energy production, and amino acid and protein biosynthesis. Additionally, a significant proportion of proteins linked to biofilm formation, virulence, and iron uptake were suppressed. This shift toward a predominantly coccoid morphology echoed the reduced energy demands of these biofilm communities. Our study unlocks valuable insights into C. jejuni's biofilm in CJ, demonstrating its adaptation and survival.

Identification of the enzyme responsible for N-acetylation of norfloxacin by Microbacterium sp. Strain 4N2-2.

Microbacterium sp. 4N2-2, isolated from a wastewater treatment plant, converts the antibacterial fluoroquinolone norfloxacin to N-acetylnorfloxacin and three other metabolites. Because N-acetylation results in loss of antibacterial activity, identification of the enzyme responsible is important for understanding fluoroquinolone resistance. The enzyme was identified as glutamine synthetase (GS); N-acetylnorfloxacin was produced only under conditions associated with GS expression. The GS gene (glnA) was cloned, and the protein (53 kDa) was heterologously expressed and isolated. Optimal conditions and biochemical properties (K(m) and V(max)) of purified GS were characterized; the purified enzyme was inhibited by Mn(2+), Mg(2+), ATP, and ADP. The contribution of GS to norfloxacin resistance was shown by using a norfloxacin-sensitive Escherichia coli strain carrying glnA derived from Microbacterium sp. 4N2-2. The GS of Microbacterium sp. 4N2-2 was shown to act as an N-acetyltransferase for norfloxacin, which produced low-level norfloxacin resistance. Structural and docking analysis identified potential binding sites for norfloxacin at the ADP binding site and for acetyl coenzyme A (acetyl-CoA) at a cleft in GS. The results suggest that environmental bacteria whose enzymes modify fluoroquinolones may be able to survive in the presence of low fluoroquinolone concentrations.

Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions.

Both sterile and non-sterile pharmaceutical products, which include antiseptics, have been recalled due to Burkholderia cepacia complex (BCC) contamination. Therefore, minimizing the frequency of outbreaks may be conducive to the development of a quick and sensitive approach that can distinguish between live and dead loads of BCC. We have assessed an exo probe-based recombinase polymerase amplification (RPA) with 10 µM propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in various concentrations of antiseptics (i.e., chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions) after 24 h. The optimized assay conducted using a set of primer-probes targeting gbpT was performed at 40 °C for 20 min and shows a detection limit of 10 pg/µL of genomic DNA from B. cenocepacia J2315, equivalent to 104 colony-forming units (CFU/mL). The specificity of a newly designed primer and probe was 80% (20 negatives out of 25). The readings for total cells (i.e., without PMAxx) from 200 µg/mL CHX using PMAxx-RPA exo assay was 310 relative fluorescence units (RFU), compared to 129 RFU with PMAxx (i.e., live cells). Furthermore, in 50-500 µg/mL BZK-treated cells, a difference in the detection rate was observed between the PMAxx-RPA exo assay in live cells (130.4-459.3 RFU) and total cells (207.82-684.5 RFU). This study shows that the PMAxx-RPA exo assay appears to be a valid tool for the simple, rapid and presumptive detection of live BCC cells in antiseptics, thereby ensuring the quality and safety of pharmaceutical products.

Detection, Genophenotypic Characterization, and Antimicrobial Resistance of Microbial Contaminants.

Microbial contamination is the inadvertent presence of microbes or their byproducts in materials or environments [...].

Recalls of tattoo and permanent makeup inks in the United States and a follow-up microbiological survey of inks with a previous recall history.

In this study, we collected voluntary recall records of tattoo and permanent makeup ink from the U.S. Food and Drug Administration (US FDA) Enforcement Report Database. The recall records contain information, such as recall date, manufacturer, ink color, reason for recall, and the microorganisms detected from the ink samples. Between 2003 and 2021, a total of 15 voluntary tattoo ink recalls occurred in the U.S. market, involving over 200 tattoo inks marketed by 13 manufacturers and one distributor. Fourteen recalls were due to microbial contamination, and one recall was due to allergic reaction. As follow-up, a microbiological survey of 28 tattoo inks of new batches from seven manufacturers having products that were previously recalled was conducted. Aerobic plate count (APC) and enrichment culture methods based on the FDA's Bacteriological Analytical Manual (BAM) were used to detect microbial contamination. The results revealed that six out of 28 tattoo inks were contaminated with bacteria and were produced by two manufacturers. The level of microbial contamination was less than 250 CFU/g in three of the tattoo inks and between 1 × 103 and 1 × 105 CFU/g in the other three inks. Eleven bacterial isolates were identified, including spore-forming Bacillus-related species and potentially pathogenic species. Overall, this study shows that some tattoo ink products produced by manufacturers with a recall history continue to be contaminated with microorganisms. This highlights the need for ongoing monitoring and quality control of such products.

CPGminer: An Interactive Dashboard to Explore the Genomic Features and Taxonomy of Complete Prokaryotic Genomes.

Prokaryotes, the earliest forms of life on Earth, play crucial roles in global biogeochemical processes in virtually all ecosystems. The ever-increasing amount of prokaryotic genome sequencing data provides a wealth of information to examine fundamental and applied questions through systematic genome comparison. Genomic features, such as genome size and GC content, and taxonomy-centric genomic features of complete prokaryotic genomes (CPGs) are crucial for various fields of microbial research and education, yet they are often overlooked. Additionally, creating systematically curated datasets that align with research concerns is an essential yet challenging task for wet-lab researchers. In this study, we introduce CPGminer, a user-friendly tool that allows researchers to quickly and easily examine the genomic features and taxonomy of CPGs and curate genome datasets. We also provide several examples to demonstrate its practical utility in addressing descriptive questions.

Functional robustness of a polycyclic aromatic hydrocarbon metabolic network examined in a nidA aromatic ring-hydroxylating oxygenase mutant of Mycobacterium vanbaalenii PYR-1.

In this study, we obtained over 4,000 transposon mutants of Mycobacterium vanbaalenii PYR-1 and analyzed one of the mutants, 8F7, which appeared to lose its ability to degrade pyrene while still being able to degrade fluoranthene. This mutant was identified to be defective in nidA, encoding an aromatic ring-hydroxylating oxygenase (RHO), known to be involved in the initial oxidation step of pyrene degradation. When cultured with pyrene as a sole source of polycyclic aromatic hydrocarbon (PAH), high-pressure liquid chromatography analysis revealed that the nidA mutant showed a significant decrease in the rate of pyrene degradation compared to the wild-type PYR-1, although pyrene was still being degraded. However, when incubated with PAH mixtures including pyrene, phenanthrene, and fluoranthene, the pyrene degradation rate of the mutant was higher than that of the mutant previously incubated with pyrene as a sole source of PAH. There was no significant difference between wild-type PYR-1 and the mutant in the rates of phenanthrene and fluoranthene degradation. From the whole-cell proteome analysis of mutant 8F7 induced by pyrene, we identified expression of a number of RHO enzymes which are suspected to be responsible for pyrene degradation in the nidA mutant, which had no expression of NidA. Taken together, results in this study provide direct evidence for the in vivo functional role of nidA in pyrene degradation at the level of the ring-cleavage-process (RCP) functional module but also for the robustness of the PAH metabolic network (MN) to such a genetic perturbation.

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH interacted in a unique binding mode in which the benzoic acid moiety of Methyl Red and the nicotinamide ring of NADH were not parallel to the flavin isoalloxazine ring, but lay against it at angles of ∼45° and ∼35°, respectively. The adenine ribose moiety of NADH was surrounded by loop ℓ2 on chain B and α3 on chain A in a typical Rossmann fold. There were 12 and 19 amino acid residues that could participate in the binding of Methyl Red and NADH, respectively, especially the residues Tyr-129 and Asp-184. The functional perturbation effects of 13 residues, including Tyr-129 and Asp-184, were mapped to reconstruct the mutation network, which confirmed the proposed binding modes and also provided insights into the interaction among NADH, FMN and Methyl Red.

Concentration-Dependent Global Quantitative Proteome Response of Staphylococcus epidermidis RP62A Biofilms to Subinhibitory Tigecycline.

Staphylococcus epidermidis is a leading cause of biofilm-associated infections on implanted medical devices. During the treatment of an infection, bacterial cells inside biofilms may be exposed to sublethal concentrations of the antimicrobial agents. In the present study, the effect of subinhibitory concentrations of tigecycline (TC) on biofilms formed by S. epidermidis strain RP62A was investigated using a quantitative global proteomic technique. Sublethal concentrations of TC [1/8 (T1) and 1/4 minimum inhibitory concentration (MIC) (T2)] promoted biofilm production in strain RP62A, but 1/2 MIC TC (T3) significantly inhibited biofilm production. Overall, 413, 429, and 518 proteins were differentially expressed in biofilms grown with 1/8 (T1), 1/4 (T2), and 1/2 (T3) MIC of TC, respectively. As the TC concentration increased, the number of induced proteins in each Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway increased. The TC concentration dependence of the proteome response highlights the diverse mechanisms of adaptive responses in strain RP62A biofilms. In both COG and KEGG functional analyses, most upregulated proteins belong to the metabolism pathway, suggesting that it may play an important role in the defense of strain RP62A biofilm cells against TC stress. Sub-MIC TC treatment of strain RP62A biofilms led to significant changes of protein expression related to biofilm formation, antimicrobial resistance, virulence, quorum sensing, ABC transporters, protein export, purine/pyrimidine biosynthesis, ribosomes, and essential proteins. Interestingly, in addition to tetracycline resistance, proteins involved in resistance of various antibiotics, including aminoglycosides, antimicrobial peptides, ß-lactams, erythromycin, fluoroquinolones, fusidic acid, glycopeptides, lipopeptides, mupirocin, rifampicin and trimethoprim were differentially expressed. Our study demonstrates that global protein expression profiling of biofilm cells to antibiotic pressure may improve our understanding of the mechanisms of antibiotic resistance in biofilms.

A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics.

Pharmaceutical products contaminated with Burkholderia cepacia complex (BCC) strains constitute a serious health issue for susceptible individuals. New detection methods to distinguish DNA from viable cells are required to ensure pharmaceutical product quality and safety. In this study, we have assessed a droplet digital PCR (ddPCR) with a variant propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in autoclaved nuclease-free water after 365 days, in 0.001% chlorhexidine gluconate (CHX), and in 0.005% benzalkonium chloride (BZK) solutions after 184 days. Using 10 µM PMAxx and 5 min light exposure, a proportion of dead BCC was quantified by ddPCR. The detection limit of culture-based method was 104 CFU/mL, equivalent to 9.7 pg/µL for B. cenocepacia J2315, while that of ddPCR was 9.7 fg/µL. The true positive rate from nuclease-free water and CHX using PMAxx-ddPCR assay was 60.0% and 38.3%, respectively, compared to 85.0% and 74.6% without PMAxx (p < 0.05), respectively. However, in BZK-treated cells, no difference in the detection rate was observed between the ddPCR assay on samples treated with PMAxx (67.1%) and without PMAxx (63.3%). This study shows that the PMAxx-ddPCR assay provides a better tool for selective detection of live BCC cells in non-sterile pharmaceutical products.