Background: Rieske non-heme iron aromatic ring-hydroxylating oxygenases (RHOs) are multi-component enzyme systems that are remarkably diverse in bacteria isolated from diverse habitats. Since the first classification in 1990, there has been a need to devise a new classification scheme for these enzymes because many RHOs have been discovered, which do not belong to any group in the previous classification. Here, we present a scheme for classification of RHOs reflecting new sequence information and interactions between RHO enzyme components. Result: We have analyzed a total of 130 RHO enzymes in which 25 well-characterized RHO enzymes were used as standards to test our hypothesis for the proposed classification system. From the sequence analysis of electron transport chain (ETC) components of the standard RHOs, we extracted classification keys that reflect not only the phylogenetic affiliation within each component but also relationship among components. Oxygenase components of standard RHOs were phylogenetically classified into 10 groups with the classification keys derived from ETC components. This phylogenetic classification scheme was converted to a new systematic classification consisting of 5 distinct types. The new classification system was statistically examined to justify its stability. Type I represents two-component RHO systems that consist of an oxygenase and an FNR_C-type reductase. Type II contains other two-component RHO systems that consist of an oxygenase and an FNR_N-type reductase. Type III represents a group of three-component RHO systems that consist of an oxygenase, a [2Fe-2S]-type ferredoxin and an FNR_N-type reductase. Type IV represents another three-component systems that consist of oxygenase, [2Fe-2S]-type ferredoxin and GR-type reductase. Type V represents another different three-component systems that consist of an oxygenase, a [3Fe-4S]-type ferredoxin and a GR-type reductase. Conclusion: The new classification system provides the following features. First, the new classification system analyzes RHO enzymes as a whole. RwithSecond, the new classification system is not static but responds dynamically to the growing pool of RHO enzymes. Third, our classification can be applied reliably to the classification of incomplete RHOs. Fourth, the classification has direct applicability to experimental work. Fifth, the system provides new insights into the evolution of RHO systems based on enzyme interaction.

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. 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. 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.

Pseudomonas aeruginosa is the most common Gram-negative pathogen causing nosocomial multidrug resistant infections. It is a good biofilm producer and has the potential for contaminating medical devices. Despite the widespread use of antibacterial-impregnated catheters, little is known about the impacts of antibacterial coating on the pathogenesis of P. aeruginosa. In this study, we investigated the adaptive resistance potential of P. aeruginosa strain PAO1 in response to continuous antibiotic exposure from clindamycin/rifampicin-impregnated catheters (CR-IC). During exposure for 144 h to clindamycin and rifampicin released from CR-IC, strain PAO1 formed biofilms featuring elongated and swollen cells. There were 545 and 372 differentially expressed proteins (DEPs) identified in the planktonic and biofilm cells, respectively, by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Both Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the planktonic cells responded to the released antibiotics more actively than the biofilm cells, with metabolism and ribosomal biosynthesis-associated proteins being significantly over-expressed. Exposure to CR-IC increased the invasion capability of P. aeruginosa for Hela cells and upregulated the expression of certain groups of virulence proteins in both planktonic and biofilm cells, including the outer membrane associated (flagella, type IV pili and type III secretion system) and extracellular (pyoverdine) virulence proteins. Continuous exposure of P. aeruginosa to CR-IC also induced the overexpression of antibiotic resistance proteins, including porins, efflux pumps, translation and transcription proteins. However, these upregulations did not change phenotypic minimum inhibitory concentration (MIC) during the experimental timeframe. The concerning association between CR-IC and overexpression of virulence factors in P. aeruginosa suggests the need for additional investigation to determine if it results in adverse clinical outcomes.

In a 2018 survey, U.S. Food and Drug Administration (FDA) identified microbial contamination in 42 (49%) of 85 unopened tattoo and permanent makeup (PMU) inks purchased from 13 manufacturers in the US between November 2015 and April 2016. To confirm the results of our previous survey, we evaluated the level of microbial contamination in an additional 27 samples from 10 manufacturers from September 2017 to December 2017, including 21 unopened tattoo and PMU inks which were selected based on our previous survey results and 6 ink diluents that were not previously analysed. Aerobic plate count and enrichment culture methods from the FDA’s Bacteriological Analytical Manual revealed 11 (52%) out of 21 inks, from six manufacturers, were contaminated with micro-organisms, with contamination levels up to 3·6 × 10⁸ CFU per gram, consistent with our previous survey results. We identified 25 bacterial strains belonging to nine genera and 19 species. Strains of Bacillus sp. (11 strains, 44%) were dominant, followed by Paenibacillus sp. (5 strains, 20%). Clinically relevant strains, such as Kocuria rhizophila and Oligella ureolytica, were also identified, as similar to the findings in our previous survey. No microbial contamination was detected in any of the six ink diluents.

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.

Despite increased awareness and public health initiatives, the incidence of microbial infections related to tattoos has increased since 2000. Building on the first paper in this two-part Series, which detailed the microbiological aspects of tattoo-related infections over the past two centuries from 1820 to 2023, this second paper describes the patterns, causes, and other related epidemiological factors of these infections. Since 2000, bacterial outbreaks, particularly those caused by non-tuberculous mycobacteria, have increased, prompting a re-evaluation of tattoos as a serious public health risk. Insufficient hygiene practices have been the primary cause of microbial infections, with contaminated tattoo inks also contributing substantially, leading to 11 outbreaks and subsequent ink recalls. Although rare, the tattooing process can occasionally lead to life-threatening infections and fatalities. Tattoos by both professional and non-professional artists were associated with infections, suggesting that regulated environments do not necessarily eliminate risk. Additionally, individuals with compromised immune systems, especially those with HIV, were particularly vulnerable to infections such as Leishmania. Although permanent make-up is often perceived as safer than conventional tattoos, infections still occur, with 11 cases reported since 2010. Furthermore, polymicrobial infections involving multiple pathogens have posed challenges for diagnosis and treatment. Overall, these insights highlight the historical and emerging patterns of tattoo-related infections and can inform the development of more effective public health guidelines, enhance preventive measures, and guide future research on reducing the risks associated with tattoos.