Introduction of augmented reality to the remote wound care nursing consultation system.

OBJECTIVE: We implemented augmented reality (AR) in remote consultations for enhanced pressure injury (PI) care in homecare nursing and improved the efficiency of on-site technical education for homecare nurses. The study aimed to depict expert techniques using AR technology to improve PI healing time in a male patient. METHOD: We developed and implemented a new system that combines a transparent hand with an image and gives an output as a video image in the existing remote consultation software. The system was used to support remote care of PIs by nurses. RESULTS: We succeeded in superimposing the expert nurse's nonverbal hand gestures onto the patient's wound in real time. The visiting nurse's understanding of the system had improved and there was an improvement in the patient's PI healing. CONCLUSION: These results suggest that remote consultation using AR is effective to observe precise wound care demonstrations of the steps of the PI treatment and provide effective treatment.

Relationship between healing status and microbial dissimilarity in wound and peri-wound skin in pressure injuries.

AIM: Wound infection is the most serious cause of delayed healing for patients with pressure injuries. The wound microbiota, which plays a crucial role in delayed healing, forms by bacterial dissemination from the peri-wound skin. To manage the bioburden, wound and peri-wound skin care has been implemented; however, how the microbiota at these sites contribute to delayed healing is unclear. Therefore, we investigated the relationship between healing status and microbial dissimilarity in wound and peri-wound skin. METHODS: A prospective cohort study was conducted at a long-term care hospital. The outcome was healing status assessed using the DESIGN-R® tool, a wound assessment tool to monitor the wound healing process. Bacterial DNA was extracted from the wound and peri-wound swabs, and microbiota composition was analyzed using 16S rRNA gene analysis. To evaluate microbial similarity, the weighted UniFrac dissimilarity index between wound and peri-wound microbiota was calculated. RESULTS: Twenty-two pressure injuries (7 deep and 15 superficial wounds) were included in the study. For deep wounds, the predominant bacteria in wound and peri-wound skin were the same in the healing wounds, whereas they were different in all cases of hard-to-heal wounds. Analysis based on the weighted UniFrac dissimilarity index, there was no significant difference for healing wounds (p = 0.639), while a significant difference was found for hard-to-heal wounds (p = 0.047). CONCLUSIONS: Delayed healing is possibly associated with formation of wound microbiota that is different in composition from that of the skin commensal microbiota. This study provides a new perspective for assessing wound bioburden.

Expression levels of NPPB, ITGB6, CPNE4, EML5, and ITSN1 in fresh exudates swabbed from critically colonised and infected full-thickness wounds in rats.

Pressure injury management requires reliable identification of critical colonisation due to lack of infection signs. Our research group previously proposed the mRNAs natriuretic peptide B (Nppb), integrin subunit beta 6 (Itgb6), copine 4 (Cpne4), echinoderm microtubule-associated protein like 5, and intersectin 1 as candidate markers in pooled exudates of critically colonised wounds. However, it is unclear whether mRNAs or proteins of the candidate genes would be suitable as biomarkers in fresh exudate. Therefore, this study aimed to evaluate the validity of the mRNAs and proteins as fresh exudate markers for critical colonisation. Three wound models of normal healing, critical colonisation, and infection were created in rats. Fresh swab-collected exudates were collected, and mRNA and protein expression levels were measured. In the fresh wound exudates, the detection frequency of Itgb6 tended to decrease in the critically colonised and infected wounds (P = .067), and those of Cpne4 and Nppb tended to be lower in the infected wounds than in the normal healing and critically colonised wounds (P = .006 and .067, respectively). In contrast, there was no difference in protein expression in the exudates. This study suggests that Itgb6 mRNA in fresh exudates is a promising biomarker for critical colonisation.

Nationwide time-series surveys of pressure ulcer prevalence in Japan.

OBJECTIVE: The Japanese Society of Pressure Ulcers (JSPU) has two purposes: first, to improve knowledge and skills among health professionals related to preventing and managing pressure ulcers (PUs); and second, to represent those in the field managing PUs, including with government and health authorities. Since 2006, JSPU has conducted fact-finding surveys about every four years to identify PU prevalence in Japan (2006, 2010, 2013 and 2016). Based on the prevalence identified by these surveys, an attempt was made to validate the achievements of JSPU's activities. METHOD: Information from one-day surveys of hospitals, long-term care health facilities, long-term care welfare facilities, and home visit nursing care stations was analysed. We used generalised estimating equations to estimate the proportions of PUs and their 95% confidence intervals (CIs) for each survey. RESULTS: A total of 662,419 patients in 2631 facilities participated in the surveys. The estimated proportions for all facilities (95% CI) in chronological order, from the first to the fourth survey, were: 2.67% (2.52-2.83); 2.61% (2.43-2.80); 1.99% (1.83-2.17); and 1.79% (1.65-1.94), respectively. In all facility types, the proportion of PUs was lower in the fourth survey than the first survey. CONCLUSION: The proportion of PUs showed a decreasing trend and was low according to global standards, demonstrating the efficacy of JSPU's activities.

Concurrent validity of biofilm detection by wound blotting on hard-to-heal wounds.

OBJECTIVE: Wound biofilms delay healing of hard-to-heal wounds. Convenient biofilm identification tools for clinical settings are currently not available, hindering biofilm-based wound management. Wound blotting with biofilm staining is a potential tool for biofilm detection, owing to its convenience. Although predictive validity of wound blotting has been established, it is necessary to confirm its concurrent validity. Furthermore, current staining systems employing ruthenium red have some disadvantages for clinical use. This study aimed to evaluate the usability of alcian blue as a substitute for ruthenium red. METHOD: Both in vitro and in vivo clinical samples were used to investigate validity and usability. RESULTS: The in vitro study showed that proteins and extracellular DNA in biofilms did not affect staining ability of ruthenium red and alcian blue in the detection of biofilms. In the in vivo study, using a wound biofilm model with Pseudomonas aeruginosa, the staining sensitivity of ruthenium red was 88.9% and 100% for alcian blue, with correlation coefficients of signal intensities with native polyacrylamide gel electrophoresis (PAGE) of r=0.67 (p=0.035) and r=0.67 (p=0.036) for ruthenium red and alcian blue, respectively. Results from clinical samples were r=0.75 (p=0.001) for ruthenium red and r=0.77 (p<0.001) for alcian blue. The sensitivities of wound blotting staining by ruthenium red and alcian blue were very high and had a good correlation with native PAGE analysis. CONCLUSION: Because the alcian blue procedure is more convenient than the ruthenium red procedure, wound blotting with alcian blue staining would be a promising tool to guide clinicians in delivering biofilm-based wound management.

Ultrasound assessment of deep tissue on the wound bed and periwound skin: A classification system using ultrasound images.

AIMS: Given the utility of ultrasonography in assessing pressure injury, some ultrasonographic findings have already been used as indicators of deep tissue pressure injury. Despite reports showing that a cloud-like ultrasonographic pattern reflected the presence of deep tissue necrosis, identifying cloud-like patterns was difficult given the presence of similar findings, such as a cobblestone-like pattern. This case series reports patients with pressure injuries who presented with a cloud-like (five cases) and cobblestone-like (four cases) pattern during ultrasonography. METHODS: This study was conducted at a Japanese university hospital. Participants included patients who underwent routine examination by an interdisciplinary pressure injury team. Pressure injury severity was assessed using the DESIGN-R® scoring system and the wound size were measured using ImageJ software based on the wound photograph. RESULTS: Among the five cases showing a cloud-like pattern upon ultrasonography, all exhibited an increase in the total DESIGN-R® score, while three exhibited an increase in wound size. On the other hand, all four cases showing a cobblestone-like pattern displayed no increase in the total DESIGN-R® score and a decrease in wound size. CONCLUSION: This study suggested that distinguishing between cloud-like and cobblestone-like ultrasonography patterns is necessary for determining the presence or absence of deep tissue pressure injury. In order to comprehensively assess pressure injuries with ultrasonography, future studies should be conducted in a large number of participants.

Skin tear classification using machine learning from digital RGB image.

AIM: Skin tears are traumatic wounds characterised by separation of the skin layers. Severity evaluation is important in the management of skin tears. To support the assessment and management of skin tears, this study aimed to develop an algorithm to estimate a category of the Skin Tear Audit Research classification system (STAR classification) using digital images via machine learning. This was achieved by introducing shape features representing complicated shape of the skin tears. METHODS: A skin tear image was separated into small segments, and features of each segment were estimated. The segments were then classified into different classes by machine learning algorithms, namely support vector machine and random forest. Their performance in classifying wound segments and STAR categories was evaluated with 31 images using the leave-one-out cross validation. RESULTS: Support vector machine showed an accuracy of 74% and 69% in classifying wound segments and STAR categories, respectively. The corresponding accuracy using random forest were 71% and 63%. CONCLUSION: Machine learning algorithms revealed capable of classifying categories of skin tears. This could offer the potential to aid nurses in their management of skin tears, even if they are not specialised in wound care.

Validity of skin blot examination for albumin and nerve growth factor ß to detect itching of the skin in Indonesian older adults.

AIM: Itching, a common skin disorder, impacts the quality of life of individuals. Itchy skin occurs more with increasing age and the prediction of itchy skin prognosis is necessary to provide good skincare. This study validated biomarkers in skin blotting to identify and measure itching sensation as well as conventional methods to measure skin barrier function. MATERIALS AND METHODS: From a cross-sectional study conducted in Long-term Care (LTC) facilities in Indonesia itching symptoms were obtained through a questionnaire. Skin conditions were assessed using photographs, stratum corneum (SC) hydration, skin pH, and skin blotting for biomarkers: albumin, interleukin 2 (IL2), nerve growth factor ß (NGFß), and thymic stromal lymphopoietin (TSLP). Association of skin measurements with the presence of skin blotting and trends analysis were conducted. RESULTS: Altogether, 564 LTC residents (average age, 70 years) participated. The SC hydration, skin pH, albumin, and NGFß were associated with the presence of itch (p value= <0.001, <0.001, <0.001, and <0.001, respectively). The signal levels of skin blotting biomarkers were higher in itch group than in the non-itch group. Additionally, the higher quantile of SC hydration was significantly associated with a lower intensity level of NGFß and TSLP (p value = 0.005, 0.003, respectively). The lower quantile of skin pH (better skin condition) was significantly associated with lower albumin, NGFß, and TSLP (p value = 0.048, 0.035, and <0.001, respectively). CONCLUSION: The albumin, NGFß, and TSLP could be a candidate for measurement of itchy skin among older adult with disrupted skin barrier function and local skin inflammation.

Combination of urease inhibitor and antiseptic inhibits urea decomposition-induced ammonia production by Proteus mirabilis.

The decomposition of urea into ammonia by urease-producing bacterium shows an elevation in the pH level, which can lead to incontinence-associated dermatitis (IAD). This study aimed to examine the efficacy of a combination of antiseptic and urease inhibitor in inhibiting the decomposition of urea by the urease-producing bacterium Proteus mirabilis. We performed in vitro assays to compare the effects of a combination of antiseptic and urease inhibitor, antiseptic only, urease inhibitor only, and an untreated control with the effects of a urea-containing solution. Cultured P. mirabilis was mixed with urea-containing solution, followed by the addition of antiseptic and/or urease inhibitor. The main outcome used to assess the efficacy of the different treatments was ammonia concentration at 4-hours post-treatment initiation, and multiple comparison analysis was performed using Dunnett's test to compare the results between groups. Ammonia concentrations in samples treated with either antiseptic or urease inhibitor were lower than those in the untreated control, while the combination of antiseptic and urease inhibitor resulted in decreased ammonia concentrations compared with either treatment alone. Therefore, the application of both urease inhibitor and antiseptic is more effective for the inhibition of urea decomposition by urease-producing bacteria. Novel preventive strategies using these reagents may be effective for preventing IAD.

Rapid detection of biofilm by wound blotting following sharp debridement of chronic pressure ulcers predicts wound healing: A preliminary study.

For optimal wound bed preparation, wound debridement is essential to eliminate bacterial biofilms. However, it is challenging for clinicians to determine whether the biofilm is completely removed. A newly developed biofilm detection method based on wound blotting technology may be useful. Thus, we aimed to investigate the effect of biofilm elimination on wound area decrease in pressure ulcers, as confirmed using the wound blotting method. In this retrospective observational study, we enrolled patients with pressure ulcers who underwent sharp debridement with pre- and post-debridement wound blotting. Biofilm was detected on the nitrocellulose membrane using ruthenium red or alcian blue staining. Patients were included if the test was positive for biofilm before wound debridement. Percent decrease in wound area after 1 week was calculated as an outcome measure. We classified the wounds into a biofilm-eliminated group and a biofilm-remaining group based on the post-debridement wound blotting result. Sixteen wound blotting samples from nine pressure ulcers were collected. The percent decrease in wound area was significantly higher in the biofilm-eliminated group (median: 14.4%, interquartile range: 4.6%-20.1%) than in the biofilm-remaining group (median: -14.5%, interquartile range: -25.3%-9.6%; P = .040). The presence of remaining biofilms was an independent predictor for reduced percent decrease in wound area (coefficient = -22.84, P = .040). Biofilm-based wound care guided by wound blotting is a promising measure to help clinicians eliminate bacterial bioburden more effectively for wound area reduction.

Effectiveness of biofilm-based wound care system on wound healing in chronic wounds.

A biofilm plays a crucial role in delaying wound healing. Sharp debridement, a possible effective method for eliminating biofilms, can only be applied to the wound with visible necrotic tissue; thus, no option has been available for eliminating biofilms that are not accompanied by necrotic tissue. Wound blotting was recently developed to visualize biofilm noninvasively and quickly, and ultrasonic debridement is available for biofilm removal. Therefore, the purpose of this study was to investigate the efficacy of "biofilm-based wound care system (BWCS)," a combination of wound blotting as a point-of-care testing and ultrasonic debridement, for promoting wound healing. Firstly, the cross-sectional study was conducted to examine the proportion of biofilm removal by ultrasonic debridement in pressure ulcers [Study 1]. Subsequently, the retrospective cohort study was conducted to examine the effectiveness of BWCS for healing of chronic wounds [Study 2]. The proportions of wound healing between wounds treated with BWCS and those with standard care in the home-visiting clinic were compared by Kaplan-Meier curve, and the Cox proportional hazard modeling was used to assess the effect of BWCS on wound healing. In Study 1, the median of biofilm removal proportion was 38.9% (interquartile range, 12.9-68.0%) for pressure ulcers treated with standard care and 65.2% (41.1-78.8%) for those treated with ultrasonic debridement (p = 0.009). In Study 2, the proportion of wound healing within 90 days was significantly higher in wounds treated with BWCS than in those treated with standard care (p = 0.001). The adjusted hazard ratio of BWCS for wound healing was 4.5 (95% confidence interval, 1.3-15.0; p = 0.015). In conclusion, we demonstrated that our novel approach, BWCS, can be a promising therapeutic strategy for visualizing biofilms that are not accompanied by necrotic tissue and promoting healing in chronic wounds.

Changes of tissue images visualised by ultrasonography in the process of pressure ulcer occurrence.

OBJECTIVE: Ultrasonography is suitable for assessing pressure ulcers, and several features of ultrasonographic images that indicate abnormalities have been reported. However, no study has compared ultrasonographic images between normal and pressure-loaded skin and subcutaneous tissue from the same patients. This study aimed to assess lateral thoracic tissue using ultrasonography for both pre- and postoperative conditions and investigate changes in the tissue caused by loading. Surgeries were performed with patients in the park-bench position. METHOD: A nursing researcher obtained ultrasonographic images of the skin and subcutaneous tissue of the lower thoracic region in areas in contact with the surgical table one or two days before and after surgery. This study focused on three groups of two patients who had a category I pressure ulcer (PU), blanchable erythema, or normal skin on their lateral thoracic region. RESULTS: A total of six patients participated. Postoperatively, muscle layers became thinner and less clear compared with pre-operative conditions in patients with the Category I pressure ulcers. These patients complained of significant pain in the areas of their pressure ulcers. CONCLUSION: Thickness of muscle layers could be an early sign of deep tissue injury.

Assessing subclinical inflammation by peroxidase detection in patients with pressure ulcers.

OBJECTIVE: This study aimed to examine the superiority of peroxidase detection of macroscopic observations using rat wounds, and to test the external validity of the peroxidase analysis in pressure ulcers (PU) in humans. METHOD: In the animal study, rat wounds were analysed. A cross-sectional study analysed, by wound blotting, exudate samples from full-thickness PUs. Peroxidase activity was divided into two groups (ring and non-ring signals). Scores in the 'inflammation/infection' and 'necrotic tissue' components of DESIGN, a classification tool of PUs, were compared between the groups. RESULTS: In the animal study, 20 rat wounds were assessed and in the clinical study, 62 samples were collected from 26 full-thickness PUs of 21 patients aged ≥ 65 years. In the animal study, five of six wounds with clinical inflammation signs showed ring signal (defined as a signal on the wound edge and no signal on the wound bed). While the tissue sections of three wounds with a ring signal showed inflammatory features, they showed no clinical signs of 'inflammation/infection'. In the clinical study, which analysed 630 ring and 32 non-ring signals, 13 samples in the ring signal group and five in the non-ring signal group had 'inflammation/infection; scores of ≥1 (p=0.016). Despite having no clinical signs, 17 samples showed the ring signal. CONCLUSION: This study revealed the external validity of the wound blotting analysis of peroxidase and demonstrated its use to detect subclinical inflammation.

Biofilm detection by wound blotting can predict slough development in pressure ulcers: A prospective observational study.

Bacteria have been found to form multicellular aggregates which have collectively been termed "biofilms." It is hypothesized that biofilm formation is a means to protect bacterial cells including protection form the immune response of humans. This protective mechanism is believed to explain persistent chronic wound infections. At times, the biofilms are abundant enough to see, and remove by simple wiping. However, recent evidence has shown that the removal of these visible portions are not sufficient, and that biofilms can continue to form even with daily wiping. In this work, we tested an approach to detect the biofilms which are present after clinically wiping or sharp wound debridement. Our method is based on a variation of impression cytology in which a nitrocellulose membrane was used to collect surface biofilm components, which were then differentially stained. In this prospective study, members of an interdisciplinary pressure ulcer team at a university hospital tested our method's ability to predict the generation of wound slough in the week that followed each blotting. A total of 70 blots collected from 23 pressure ulcers produced 27 wounds negative for staining and 43 positive. In the negative blots 55.6% were found to have decreased wound slough, while 81.4% with positive staining had either increase or unchanged wound slough generation. These results lead to an odds ratio of positive blotting cases of 9.37 (95% confidence intervals: 2.47-35.5, p = 0.001) for slough formation; suggesting that the changes in wound slough formation can be predicted clinically using a non-invasive wound blotting method.

Lower temperature at the wound edge detected by thermography predicts undermining development in pressure ulcers: a pilot study.

Undermined pressure ulcers (PUs) are troublesome complications that are likely to delay wound healing. Early skin incision and debridement can prevent the deterioration of undermined PUs, thus it is necessary to identify devitalised tissue areas to determine the appropriate timing for such interventions. This retrospective cohort study evaluated whether a lower temperature at the wound edge than the wound bed and periwound skin, detected by thermography, can predict undermining development in PUs 1 week after the assessment. Twenty-two participants with category III, IV, or unstageable PUs who were examined by interdisciplinary PU team and were followed up for at least two consecutive weeks were analysed. We found 9/11 PUs without a lower temperature at the wound edge did not develop undermining development, whereas 8/11 PUs with the lower temperature did develop undermining. The relative risk of undermining development after 1 week in PUs with the lower temperature was 4·00 (95% confidence intervals: 1·08-14·7). The sensitivity, specificity, positive predictive value and negative predictive value were 0·80, 0·75, 0·73 and 0·81, respectively. A thermal imaging assessment focusing on a lower temperature pattern at the wound edge may provide sufficient information to predict undermining development.

Exploring the prevalence of skin tears and skin properties related to skin tears in elderly patients at a long-term medical facility in Japan.

The identification of appropriate skin tear prevention guidelines for the elderly requires clinicians to focus on local risk factors such as structural alterations of the epidermis and dermis related to skin tears. The aim of this cross-sectional study is to explore the prevalence of skin tears and to explore skin properties related to skin tears in elderly Japanese patients at a long-term medical facility. After doing the prevalence study, 18 participants with skin tears and 18 without were recruited and an evaluation of their skin properties using 20-MHz ultrasonography, skin blotting and also Corneometer CM-825, Skin-pH-meterPH905, VapoMeter, Moisture Meter-D and CutometerMPA580 was undertaken. A total of 410 patients were examined, the median age was 87 years and 73·2% were women. The prevalence of skin tears was 3·9%, and 50% of skin tears occurred on the dorsal forearm. The changes in skin properties associated with skin tears included increased low-echogenic pixels (LEP) by 20-MHz ultrasonography, decreased type IV collagen and matrix metalloproteinase-2, and increased tumour necrosis factor-α by skin blotting. In conclusion, this study suggests that increased dermal LEP, including solar elastosis, may represent a risk factor for skin tears; this indicates that skin tear risk factors might not only represent chronological ageing but also photoageing.

Prediction of healing progress of pressure ulcers by distribution analysis of protein markers on necrotic tissue: A retrospective cohort study.

Predicting the short-term healing progress of pressure ulcers is important for providing timely and appropriate intervention. Although there are some prediction methods available, these are unsuitable for ulcers with abundant necrotic tissue. We aimed to elucidate the relationship between necrotic tissue alteration and protein distributions on ulcers to establish a new prediction method. Thirty-eight pressure ulcers were retrospectively analyzed. Protein distributions on necrotic tissue were evaluated by the wound blotting at three levels: marker protein positivity, signal patterns (speckled, heterogeneous, or homogeneous), and the occupation of heterogeneous pattern. Peroxidase, alkaline phosphatase, tumor necrosis factor α, and matrix metalloproteinase-2 were used as marker proteins. One-week necrotic tissue alteration was classified as liquefaction or nonliquefaction, and associations with protein distributions were analyzed. The peroxidase positivity was significantly higher in the liquefaction than in the nonliquefaction (p = 0.031). In peroxidase-positive samples, the proportion of nonliquefaction samples was significantly higher in the heterogeneous pattern (p = 0.029). In the heterogeneous-patterned samples, the proportion of samples with an occupation values greater than the median value tended to be higher in the nonliquefaction (p = 0.087). There was no significant relationship between liquefaction and other markers. Peroxidase positivity predicts 1-week liquefaction of necrotic tissue, while a heterogeneous pattern indicates nonliquefaction.

Hydrocellular foam dressings promote wound healing associated with decrease in inflammation in rat periwound skin and granulation tissue, compared with hydrocolloid dressings.

The effects of modern dressings on inflammation, which represent the earliest phase of wound healing, are poorly understood. We investigated the effects of modern hydrocellular foam dressings (HCFs) on wound healing and on the gene expression levels of the inflammatory markers--interleukin (IL)-1ß, IL-6, and IL-10--in rat periwound skin and granulation tissue by quantitative reverse transcription-polymerase chain reaction. HCF absorbed significantly higher volume of water than hydrocolloid dressing (HCD) and increased the contraction of wounds. Polymorphonuclear neutrophils were massively infiltrated to the wound edge and boarded between granulation and dermis in the HCD group. IL-1ß, IL-6, and IL-10 mRNA levels were significantly decreased in the periwound skin around the wounds and granulation tissue covered with HCF. These findings suggest that HCF may promote wound healing along with decrease in inflammation by reducing gene expression levels of IL-1ß, IL-6, and IL-10.

Fulminant adenovirus hepatitis after hematopoietic stem cell transplant: Retrospective real-time PCR analysis for adenovirus DNA in two cases.

BACKGROUND: Viral infection is one of the major causes of mortality in patients undergoing hematopoietic stem cell transplant (HSCT). Systemic infection of adenovirus (AdV) has emerged as a not uncommon viral infection with significant morbidity and mortality as with cytomegalovirus and Epstein-Barr virus infection. Routine surveillance for these viruses has become a clinical practice and subsequent preemptive therapy improves patients' outcomes; however, the effectiveness of preemptive therapy for AdV has not been fully investigated in patients with a lethal form of AdV infection. METHODS: Sequential AdV loads were retrospectively analyzed in children with fulminant AdV hepatitis after HSCT. RESULTS: The AdV DNA became detectable (1 × 10(4) copies/mL) as early as 2 weeks after HSCT. These levels reached >1 × 10(8) copies/mL at the onset of fulminant hepatitis. However, we determined that γ-glutamyltransferase levels were elevated to >100 IU/L at least 2 weeks before the diagnosis of hepatitis. CONCLUSIONS: Our observation raises the possibility that elevated γ-glutamyltransferase could be a sentinel marker for AdV hepatitis, which prompts elaborated monitoring of AdV load and targeted treatment.

Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes.

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA(Arg)ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA(Arg)CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA(Arg)CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA(Arg) and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA(Arg)ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA(Arg)ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA(Arg)CCG and tadA, and then acquired a new tRNA(Arg)UCG. This permitted further tRNA(Arg)ACG mutations with tRNA(Arg)GCG or its disappearance, leaving a single tRNA(Arg)UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.