Microbial contamination of tattoo and permanent makeup inks marketed in the US: a follow-up study.

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.

Microbiological survey of commercial tattoo and permanent makeup inks available in the United States.

AIMS: Tattooing and use of permanent makeup (PMU) has dramatically increased over the last decade, with a concomitant increase in ink-related infections. The aim of this study was to determine whether micro-organisms are present, and if so, the number and their identification in the commercial tattoo and PMU inks available in the United States. METHODS AND RESULTS: We surveyed 85 unopened tattoo and PMU inks, purchased from 13 companies. We incubated 100 µl of ink samples on trypticase soy agar plates for bacterial growth, 7H10 Middlebrook medium for mycobacterial growth, and Sabouraud dextrose medium for fungal growth. In total, 42 inks were contaminated with micro-organisms (49%). Thirty-three inks were contaminated with bacteria, 2 inks with fungi, and 7 inks had both bacterial and fungal growth. Mycobacteria were not detected in any of the examined tattoo and PMU inks. In 26 inks, microbial concentrations ranged between 101 and 103 CFU per ml, but higher counts (>103 CFU per ml) were recorded in 16 inks. We identified 83 bacteria by their 16S rDNA sequences, including 20 genera and 49 species. Strains of Bacillus spp. (53%) were dominant, followed by Lysinibacillus fusiformis (7%) and Pseudomonas aeruginosa (5%). Thirty-four (41%) possibly clinically relevant strains were identified, including P. aeruginosa, Dermacoccus barathri and Roseomonas mucosa, some of which have been previously reported to be associated with human skin infections. CONCLUSIONS: The results indicate that commercial tattoo and PMU inks on the US market surveyed in this study contain a wide range of micro-organisms, including pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial contaminants in tattoo and PMU inks are an emerging safety concern for public health. This study provides evidence that microbial contamination of tattoo and PMU inks available in the United States is more common than previously thought and highlights the importance of monitoring these products for potentially pathogenic micro-organisms.

Workshop report: the 2012 antimicrobial agents in veterinary medicine: exploring the consequences of antimicrobial drug use: a 3-D approach.

Antimicrobial resistance is a global challenge that impacts both human and veterinary health care. The resilience of microbes is reflected in their ability to adapt and survive in spite of our best efforts to constrain their infectious capabilities. As science advances, many of the mechanisms for microbial survival and resistance element transfer have been identified. During the 2012 meeting of Antimicrobial Agents in Veterinary Medicine (AAVM), experts provided insights on such issues as use vs. resistance, the available tools for supporting appropriate drug use, the importance of meeting the therapeutic needs within the domestic animal health care, and the requirements associated with food safety and food security. This report aims to provide a summary of the presentations and discussions occurring during the 2012 AAVM with the goal of stimulating future discussions and enhancing the opportunity to establish creative and sustainable solutions that will guarantee the availability of an effective therapeutic arsenal for veterinary species.

Evidence for significantly enhancing reduction of Azo dyes in Escherichia coli by expressed cytoplasmic Azoreductase (AzoA) of Enterococcus faecalis.

Although cytoplasmic azoreductases have been purified and characterized from various bacteria, little evidence demonstrating that these azoreductases are directly involved in azo dye reduction in vivo is known. In order to evaluate the contribution of the enzyme to azo dye reduction in vivo, experiments were conducted to determine the effect of a recombinant cytoplasmic azoreductase (AzoA) from Enterococcus faecalis expressed in Escherichia coli on the rate of metabolism of Methyl Red, Ponceau BS and Orange II. The intact cells that contained IPTG induced AzoA had a higher rate of dye reduction with increases of 2 (Methyl Red), 4 (Ponceau BS) and 2.6 (Orange II)-fold compared to noninduced cells, respectively. Metabolites of Methyl Red isolated from induced cultures were identified as N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid through liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analyses. In conclusion, our data demonstrate that AzoA from Ent. faecalis is capable of increasing the reduction of azo dyes in intact E. coli cells and that cytoplasmic azoreductase is involved in bacterial dye degradation in vivo.

Microarray analysis of antimicrobial resistance genes in Salmonella enterica from preharvest poultry environment.

AIMS: To detect antimicrobial resistance genes in Salmonella isolates from turkey flocks using the microarray technology. METHODS AND RESULTS: A 775 gene probe oligonucleotide microarray was used to detect antimicrobial resistance genes in 34 isolates. All tetracycline-resistant Salmonella harboured tet(A), tet(C) or tet(R), with the exception of one Salmonella serotype Heidelberg isolate. The sul1 gene was detected in 11 of 16 sulfisoxazole-resistant isolates. The aadA, aadA1, aadA2, strA or strB genes were found in aminoglycoside-resistant isolates of Salm. Heidelberg, Salmonella serotype Senftenberg and untypeable Salmonella. The prevalence of mobile genetic elements, such as class I integron and transposon genes, in drug-resistant Salmonella isolates suggested that these elements may contribute to the dissemination of antimicrobial resistance genes in the preharvest poultry environment. Hierarchical clustering analysis demonstrated a close relationship between drug-resistant phenotypes and the corresponding antimicrobial resistance gene profiles. CONCLUSIONS: Salmonella serotypes isolated from the poultry environment carry multiple genes that can render them resistant to several antimicrobials used in poultry and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple antimicrobial resistance genes in environmental Salmonella isolates could be identified efficiently by microarray analysis. Hierarchical clustering analysis of the data was also found to be a useful tool for analysing emerging patterns of drug resistance.

Echinacea purpurea supplementation stimulates select groups of human gastrointestinal tract microbiota.

BACKGROUND AND OBJECTIVE: The objective of this research was to determine the effects of the dietary supplement Echinacea purpurea on aerobic and anaerobic bacteria common to the human gastrointestinal (GI) tract. Botanical extracts have shown in vitro antimicrobial effects against certain pathogenic bacteria. It is uncertain if medicinal herbs have any effect against pathogenic bacteria or on the native GI microbiota. METHODS: Fifteen human subjects consumed 1000 mg of standardized E. purpurea for 10 days. Faecal samples were collected at baseline, 10 days and 17-18 days following supplementation. Samples were tested for select aerobic and anaerobic bacteria using plate culture microbiological methods. RESULTS AND DISCUSSION: Significant increases were found for total aerobic bacteria, Bacteroides group and Bacteroides fragilis after E. purpurea exposure. Supplementation did not significantly alter the number of enteric bacteria, enterococci, lactobacilli, bifidobacteria or total anaerobic bacteria. CONCLUSION: Echinacea supplementation has altered the GI microbiota. The health consequences associated with this change are unknown but previous research has shown increased Bacteroides concentrations associated with diarrhoea, inflammatory bowel disease and increased risk of colon cancer. Additional research should delineate the role of Echinacea in the stimulation of Bacteroides and describe the effects of other botanical supplements to the GI microbiota.

Approaches in the safety evaluations of veterinary antimicrobial agents in food to determine the effects on the human intestinal microflora.

The administration of antimicrobial agents to livestock creates potential for antibiotic residues to enter the food supply and be consumed by humans. Therefore, as a process of food animal drug registration, national regulatory agencies and international committees evaluate data regarding the chemical, microbiologic, pharmacokinetic, pharmacodynamic, pharmacologic, toxicologic, and antimicrobial properties of veterinary drugs to assess the safety of ingested antimicrobial residues to consumers. Currently, European, Australian and United States guidelines for veterinary drug registration require a safety assessment of microbiologic hazards from consumption of antimicrobial residues taking into account the potentially adverse effects on human intestinal microflora. The main concerns addressed are selection of resistant bacteria in the gastrointestinal tract and disruption of the colonization barrier of the resident intestinal microflora. Current requirements differ among national agencies. Efforts are ongoing internationally to review and harmonize approaches and test methods and protocols for application to these microbiologic safety evaluations of antimicrobial drug residues in food. This review describes the background to current regulatory approaches used in applying in vitro and in vivo methods to set a microbiologic acceptable daily intake for residues in food derived from animals treated with an antimicrobial agent. This paper also examines the current research needs to support these evaluations.

Genetic diversity and virulence gene determinants of antibiotic-resistant Salmonella isolated from preharvest turkey production sources.

This study evaluated the molecular diversity of 29 Salmonella serotypes isolated from turkey ceca and the production environment. Isolates were resistant to bacitracin (100%), erythromycin (100%), novobiocin (100%), rifampin (100%), streptomycin (62%), gentamicin (52%), spectinomycin (48%), tetracycline (31%), sulfamethoxazole/trimethoprim (SXT) (3%) and tobramycin (3%). The minimum inhibitory concentration (MIC) values ranged from 32 to >/=1024 microg/ml. The pulsed-field gel electrophoresis (PFGE) and ribotyping patterns were identical within each of the serotypes Heidelberg, Worthington and Muenster. The plasmid profiles were identical within each of the Salmonella serotypes. Two different clones of Salmonella anatum were differentiated by PFGE typing but not by ribotyping. Heidelberg isolates from nine turkey ceca and three drinker samples had identical antibiotic resistance, PFGE, ribotype and plasmid patterns, suggesting that transmission of this particular clone may have occurred between the birds and the drinkers. Identical PFGE, ribotype and plasmid patterns were observed in one Salmonella worthington isolate from turkey ceca in one flock and two S. worthington isolates from feeder contents and drinkers from a subsequent flock, suggesting transmission of this pathogen between flocks. Individual and multiple polymerase chain reaction (PCR) analyses revealed the presence of the virulence genes invA, aceK and sopB and the absence of the h-1i gene in all isolates. A combination of genotypic and phenotypic markers can be useful in studying genetic variation among natural salmonellae populations in turkey production and delineating possible transmission pathways.

Phenotypic and genotypic characterization of competitive exclusion products for use in poultry.

AIMS: Phenotypic and genotypic bacteria identification methods were compared for their efficacy in determining the composition of competitive exclusion (CE) products. METHODS AND RESULTS: Phenotypic methods used for bacterial identification were fatty acid methyl ester profiles, biochemical assays and carbohydrate utilization profiles. Genotypic methods were MicroSeq16S rRNA sequence analysis and BLAST searches of the GenBank sequence database. Agreement between phenotypic and genotypic methods for identification of bacteria isolated from the Preempt CE product was 20%. A defined test mixture of bacteria was identified to the species level 100% by BLAST analysis, 64% by MicroSeq and 36% by phenotypic techniques. CONCLUSIONS: The wide range of facultative and obligate anaerobic bacteria present in a CE product are more accurately identified with 16S rRNA sequence analyses than with phenotypic identification techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will provide guidelines for manufacturers of CE products to submit more reliable product information for market approval by regulatory agencies.

Degradation of phenanthrene and naphthalene by a Burkholderia species strain.

Burkholderia sp. TNFYE-5 was isolated from soil for the ability to grow on phenanthrene as sole carbon and energy source. Unlike most other phenanthrene-degrading bacteria, TNFYE-5 was unable to grow on naphthalene. Growth substrate range experiments coupled with the ring-cleavage enzyme assay data suggest that TNFYE-5 initially metabolizes phenanthrene to 1-hydroxy-2-naphthoate with subsequent degradation through the phthalate and protocatechuate and beta-ketoadipate pathway. A metabolite in the degradation of naphthalene by TNFYE-5 was isolated by high-pressure liquid chromatography (HPLC) and was identified as salicylate by UV-visible spectral and gas chromatography-mass spectrometry analyses. Thus, the inability to degrade salicylate is apparently one major reason for the incapability of TNFYE-5 to grow on naphthalene.

Development of a membrane-array method for the detection of human intestinal bacteria in fecal samples.

A membrane-array method was developed for the detection of human intestinal bacteria in fecal samples without using the expensive microarray-arrayer and laser-scanner. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to nitrocellulose membranes. Digoxigenin (DIG)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or pure cultured bacteria using two universal primers, and were hybridized to the membrane-array. Hybridization signals were read by NBT/BCIP color development. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, B. vulgatus, B. fragilis, B. distasonis, Clostridium clostridiiforme, C. leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, R. bromii, R. callidus, R. albus, Bifidobacterium longum, B. adolescentis, B. infantis, Eubacterium biforme, E. aerofaciens, Lactobacillus acidophilus,Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the membrane-array method is a reliable technique for the detection of predominant human intestinal bacteria in the fecal samples. The result was also confirmed by using specific PCR methods for these bacteria.

Degradation of biphenyl by Mycobacterium sp. strain PYR-1.

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.

Utilization of mixtures of polycyclic aromatic hydrocarbons by bacteria isolated from contaminated sediment.

Abstract The ability of sediment bacteria to utilize polycyclic aromatic hydrocarbons (PAHs) when present as components of mixtures was investigated. One strain, identified as Mycobacterium flavescens, could utilize fluoranthene in the presence of pyrene, although utilization of pyrene was slower in the presence of fluoranthene than in its absence. The second strain, a Rhodococcus species, could utilize fluoranthene in the presence of anthracene, although the presence of fluoranthene slowed the rate of utilization of anthracene. Cometabolism of fluoranthene in these strains was confirmed by the isolation of metabolites of fluoranthene and by kinetic analysis of the rate of utilization of the growth substrate in the presence of fluoranthene. In both strains, metabolism of fluoranthene occurred on the fused ring of the fluoranthene molecule, producing 9-fluorenone-1-carboxylic acid. In the Rhodococcus sp., a second metabolite, a-(carboxymethylene)fluorene-1-carboxylic acid, was identified, indicating that this strain has the capacity to metabolize fluoranthene via ortho as well as meta cleavage. The presence of PAHs in a mixture produces interactive effects which can either increase or decrease the rate of utilization of individual PAHs, results which need to be taken into account when estimating rates of degradation in contaminated environments.

Metabolism of anthracene by a Rhodococcus species.

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans.

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides.

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.

Biotransformation of malachite green by the fungus Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 micromol x h(-1) (mg of cells)(-1). Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.

Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1.

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5' to the 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large alpha subunit did not cluster with most of the known alpha-subunit sequences but rather with three newly described alpha subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.

A rapid method for determining the tuberculocidal activity of liquid chemical germicides.

A rapid, quantitative method has been developed for determining the tuberculocidal activity of liquid chemical germicides. In this method, a test strain of Mycobacterium bovis that carries the firefly luciferase gene is exposed to a germicide, and the surviving bacteria are detected by bioluminescence. The tuberculocidal activities of five commercially available glutaraldehyde-based disinfectants were tested, and all reduced the number of surviving mycobacteria by greater than five orders of magnitude. In contrast, a phenol-based disinfectant with tuberculocidal claims gave less than one order of magnitude reduction of the test organism. With this method for determining tuberculocidal activity, results can be obtained in less than one day, compared with weeks or months for the standard tuberculocidal assays.

Validation of a polymerase chain reaction method for the detection of rendered bovine-derived materials in feedstuffs.

This study validated a polymerase chain reaction-based method for the detection of a specific bovine mitochondrial gene derived from rendered bovine tissues and admixed with complete animal feed. Four laboratories participated in this effort: one state laboratory and three Food and Drug Administration (FDA) laboratories, including one FDA field laboratory. The protocol used a statistical approach of 90% probability, with a 95% confidence interval for determining acceptable rates of false-positive and false-negative samples. Each participating laboratory analyzed 30 samples of feed each containing 0, 0.125, and 2.0% bovine meat and bone meal (BMBM), for a total of 90 feed samples. The samples were randomized such that the analysts were unaware of the true identity of the test samples. The results demonstrated that all laboratories met the acceptance criteria established for this protocol. The overall rates of false-negative results were 0.83% (1/120) at the level of 0.125% BMBM and 1.67% (2/120) at the level of 2% BMBM. The overall rate of false-negative results for all levels of BMBM was 1.25% (3/240). The rate for false-positive results was 0.83%.