Actionable data on antimicrobial use is important when planning strategic interventions such as antimicrobial stewardship to address the challenge of drug resistance, particularly in resource-constrained settings.To assess the prevalence of antibiotic use, the pattern of commonly used antibiotics and patient factors that may be associated with the increased use of antibiotics in the study hospitals.This was a cross-sectional study conducted using the WHO Methodology for Point Prevalence Surveys in hospitals. Chi-squared analysis, Fisher’s exact test and logistic regression were employed to analyse statistically the data obtained.P = 0.04] and increasing age  were associated with lower risk of antibiotic use.The overall prevalence of antibiotic use in the hospitals was 60.5%. The commonest indications for antibiotic recommendations were community-acquired infections (36.5%), surgical prophylaxis (26.1%) and hospital-acquired infections (15.7%), among others. Very few (2.7%) of the patients had their samples taken for culture and susceptibility testing to guide therapy. Penicillins (48.7%), cephalosporins (23.5%) and fluoroquinolones (17.4%) were the most commonly prescribed antibiotics. Concurrent malaria infection [adjusted OR (AOR) 0.33, 95% CI 0.11–0.94, AWaRe classification for the selection of antibiotics and increased use of culture and susceptibility data to guide infectious disease therapy.The prevalence of antibiotic consumption in the hospitals was lower than that reported in similar studies in Ghana, but high relative to some reports from high-income countries. Most antibiotic therapy was empirical and not guided by culture and susceptibility testing. There is the need for application of the WHO  Antimicrobial resistance (AMR) is an important subject in healthcare and public health as it is associated with increased mortality and morbidity among patients. The risk of AMR is high as every individual is susceptible to developing drug-resistant infections.AWaRe Classification database, first developed in 2017, which has 180 antibiotics classified as Access, Watch or Reserve (AWaRe), based on pharmacological class, anatomical therapeutic chemical (ATC) codes and their WHO Essential Medicines List status. This classification provides a useful means to monitor antibiotic use across the classes as an indicator of optimal antibiotic use. Antibiotics in the Access group are those that show a lower potential for resistance than the other groups with a good range of activity against many common pathogens. Watch antibiotics are those with much higher resistance potential and as such require key targets for stewardship interventions to reduce their inappropriate use. The Reserve group are those used for treating infections due to multidrug-resistant pathogens and so should be used as a last line, after all alternatives have been explored, in order to protect them against resistance.Data and information on the use of antimicrobials in relevant sectors are indispensable in policy-making to tackle AMR. Information-driven solutions make the most sustainable impact with minimal resources and wastage. Real world data is needed to implement locally tailored antimicrobial stewardship programmes (ASPs) to optimize antibiotic use. WHO develops various tools and strategies to support stewardship activities in countries.Several studies have investigated antibiotic use in various settings in Ghana and across the world with varied prevalence of antibiotic use and patient parameters affecting their use.We employed the WHO Methodology for Point Prevalence survey (PPS) on antibiotic use in hospitals, version 1.1, which has been described elsewhere.The facilities were classified according to the criteria set forth in the WHO methodology for PPSUHS provides services to more than 200 000 Ghanaians annually. The hospital provides a wide range of services ranging from ambulatory to in-patient care. The hospital has a functioning infection prevention and control programme as well as a drug and therapeutics committee supporting optimization of patient care.APH, the secondary hospital, is a 250 bed capacity hospital which caters to patients from all over Ghana and neighbouring countries, especially for ophthalmological care. It serves a municipal catchment area of over 170 822 persons. It is designated as Collaborating Centre for the University of Ghana School of Public Health, Training Centre for Buruli Ulcer Treatment, Ministry of Health (MOH)/WHO designated centre for training in the surgical management of buruli ulcer and one of two sites in Ghana and eight sites in Africa for Malaria Vaccine Trial.EGH is a primary hospital facility under the Ghana Health service in a municipality of over 143 762 persons.The PPS was a cross-sectional survey conducted on 26 and 27 November 2019 at a tertiary hospital (UHS) and primary level facility (EGH), respectively and on 10 December 2019 at a secondary level hospital (APH). The different dates were necessary to ensure that all in-patients at each facility were surveyed with the resources available.Each patient present on the ward at the time of the survey was targeted for inclusion in the study unless the patient did not meet inclusion criteria. Generally, any form with more than 10% missing data would be excluded from the analysis. TableAnonymized data was collected from the hospital records of in-patients who met the inclusion criteria. The data was collected by the research team with help from select final year Doctor of Pharmacy students from KNUST. A training programme was organized by the research team led by O.K.O.A. and N.K.A.B. on the 25 November 2019 for UHS and EGH as well as on 9 December 2019 for APH, to ensure accuracy and minimize error during data collection. This training involved simulations on obtaining information from patient records, and managing situations and records that strayed from the norm. A mock data collection exercise was conducted to improve accuracy during data collection. A total of 16 persons collected patient data at UHS and EGH and 14 persons at APH. All data was collected on hard copies of forms used in the protocol. No patient sampling was done.® database and exported into Stata™ 14 for analyses.,P value of <0.05 was considered statistically significant.Data collected after surveying each hospital was sorted and organized to prevent mix-up during data entry. All the data collected in the study was entered into a REDCapEthics approval was obtained from the KNUST Committee on Human Research, Publications and Ethics after getting approval from each facility. (CHRPE/AP/654/19).190 out of 211 inpatients met the inclusion criteria for the study: 44 out of 49 from UHS, 45 of 49 from EGH and 101 of 113 from APH.The overall prevalence of antibiotic use was 60.5% Table. APH, thTableThe commonly prescribed antibiotics were penicillins (48.7%) cephalosporins (23.5%) and fluoroquinolones (17.4%) as shown in FigureJAC Online).According to the univariate analysis, surgery, urinary catheter and peripheral vascular catheter use all showed statistically significant association with antibiotic use Table. ResultsAccording to the multivariate analysis, the associations between surgery, peripheral vascular catheter and urinary catheter use with antibiotic use were not statistically significant on adjusting for other variables. Increasing age and malaria were associated with decreased adjusted odds of being on antibiotics Table.Watch category in the WHO AWaRe classification.,,,,,,,,According to this study, the prevalence of antibiotic use is high relative to other studies, with many patients using two or more antibiotics, and antibiotics in The majority of patients on antibiotics were on two antibiotics at the time of the study, which may be a key target for improvement. An example is a case observed in the study where a patient was on ceftriaxone, gentamicin and azithromycin for clinical sepsis. Such a combination is usually aimed at preventing or surmounting possible resistance. The conventional mantra in infectious disease management is to ‘hit hard and to hit early’, which may often include combination of antimicrobial agents for synergy.However, only a small fraction of patients had a sample taken for culture and drug susceptibility analyses before antibiotic therapy was started. This is an important finding as directed therapy is essential in optimizing therapy with antibiotics compared with empirical therapy. Local antibiograms and antimicrobial guidelines are needed as interventions in the hospitals to direct empirical therapy in the face of limited resources. Amoxicillin, ciprofloxacin, ceftriaxone, cefuroxime and ampicillin were the most used antibiotics overall. This is comparable to the top three antibiotics prescribed worldwide.Reserve category of the WHO AWaRe system, similar to another study in Ghana,Watch group with three of them among the most used agents overall, which was higher than findings in Finland (23%) but lower than in Iran (77.3%). Use of Access agents could be increased in Ghana (47%) when compared with that in Singapore (100%) for neonates.,Watch category as much could be potentially problematic. This can be addressed effectively and inexpensively with hospital-specific formularies with the AWaRe classification taken into consideration. Another intervention could be to increase use of Access agents and restrict the use of Reserve agents to require preauthorization from qualified persons to maintain their low use in the health facilities. Prescriber training and information posters could also be provided to support appropriate prescribing and compliance to rational antibiotic use strategies. Increasing age was associated with slightly decreased adjusted odds of being on an antibiotic. This may be attributed to paediatric patients having immature immune systems and so are at an understandably higher risk of infections requiring antibiotics compared with older individuals. Older persons have more developed immune systems which have evolved to provide protection through exposure to various foreign agents and pathogens from childhood to adulthood.None of the facilities at the time of the study had patients on antibiotics classified in the A concurrent diagnosis of malaria was protective from receiving antibiotics in patients. Malaria is a febrile illness, endemic to Ghana,in situ and being on an antibiotic in univariate analyses. This finding may be due to their invasive nature, but could be confounded by the severity of illness in these patients requiring such devices or procedures. Catheter-related urinary tract infections are one of the most common nosocomial infections and a common complication of urinary catheters.,There were statistically significant associations between having surgery on admission, having a urinary catheter, a peripheral vascular catheter (PVC) Antibiotic consumption data are essential in the fight against AMR as they provide an indication of prescribers’ behaviours and are an important metric for improving antibiotic use. This study represents the successful use of the WHO toolWatch category. Increasing age and patients diagnosed with malaria had lower odds of being prescribed antibiotics. There is the need for pragmatic stewardship initiatives in these hospitals to control the prescribing and consumption of antimicrobials. An example may be the application of the WHO AWaRe classification for the selection of antibiotics, as well as increased use of culture and antimicrobial susceptibility data to guide infectious disease therapy.The prevalence of antibiotic consumption in the hospitals was low relative to reports from some studies in Ghana, but high relative to similar studies from some high-income countries. Most of the antibiotic therapy was empirical and not guided by culture and drug susceptibility testing. The most commonly prescribed antibiotics were penicillins, cephalosporin and fluoroquinolones, with a great proportion being those in the WHO dlab008_Supplementary_DataClick here for additional data file.


Selenium is an essential trace element. Although this chalcogen forms a wide variety of compounds, there are surprisingly few small-molecule organic selenium compounds (OSeCs) in biology. Besides its more prominent relative selenocysteine (SeCys), the amino acid selenomethionine (SeMet) is one example. SeMet is synthesized in plants and some fungi and, via nutrition, finds its way into mammalian cells. In contrast to its sulfur analog methionine (Met), SeMet is extraordinarily redox active under physiological conditions and via its catalytic selenide (RSeR’)/selenoxide (RSe(O)R’) couple provides protection against reactive oxygen species (ROS) and other possibly harmful oxidants. In contrast to SeCys, which is incorporated via an eloquent ribosomal mechanism, SeMet can enter such biomolecules by simply replacing proteinogenic Met. Interestingly, eukaryotes, such as yeast and mammals, also metabolize SeMet to a small family of reactive selenium species (RSeS). Together, SeMet, proteins containing SeMet and metabolites of SeMet form a powerful triad of redox-active metabolites with a plethora of biological implications. In any case, SeMet and its family of natural RSeS provide plenty of opportunities for studies in the fields of nutrition, aging, health and redox biology. The few notable exceptions found Nature include compounds such as the histidine derivative and ergothionine analog selenoneine present in tuna fish and inorganic selenocyanate (SeCN−) in the green freshwater algae Chlorella vulgaris [Selenium is an essential trace element in humans . Since ivulgaris ,5.γ-glutamylselenocysteine and Se-adenosylselenohomocysteine. In humans, SeMet is absorbed via intestinal transport channels and subsequently enters the methionine pool, where it is stored and consequently recruited from to become integrated into proteins [trans-selenation pathways to produce S-adenosyl SeMet, adenosyl selenohomocysteine, homocysteine, selenocystathionine and SeCys, as shown in Indeed, SeCys dominates most discussions of selenium in proteins and enzymes, which is hardly surprising, as most of the roughly 25 selenium proteins identified in humans to date contain one or more SeCys residues . In factproteins . Once inAs part of this minireview, we shall therefore provide some essential information about this fascinating and in many aspects unique proteinogenic amino acid, from its biosynthesis in plants and metabolism in yeast to its ability to replace Met in proteins and its role as a food supplement. From the onset, we would like to emphasize that we have focused on some of the more recent and exciting discoveries in this field and therefore shall refer to the existing literature for more basic information ,11,12,13Saccharomyces cerevisiae) and a few edible mushrooms, such as the Shiitake (Lentinula edodes) and King Bolete (Boletus edulis) mushrooms [32− and SeO42− from the soil and convert these inorganic salts to different organic forms of selenium following a sequence of biochemical events, as shown in 42− and SeO32− in plants is controlled tightly and involves certain transporters, such as sulfate (SO42−) and phosphate (PO43−) transporters in rice and wheat, respectively [42− interacts with ATP in the presence of ATP sulfurylase (ATPS) to produce adenosylphosphoselenate, which is subsequently reduced by adenosine phosphosulfate reductase to SeO32− consuming the reduced form of glutathione (GSH) as the electron donor [32−, either absorbed via PO43− transporters or reduced from SeO42−, interacts spontaneously with GSH to produce selenodiglutathione (GSSeSG), which is subsequently reduced by GSH reductase (GSR) to generate glutathioneselenol (GSeH), which is again reduced by GSR in the presence of GSH to form hydrogen selenide (H2Se) [2Se with O-acetyl serine in the presence of cysteine synthase results in the formation of SeCys [Higher organisms, such as mammals and humans, are unable to synthesize SeMet. This task is left to plants and fungi, including yeast  . The intof SeCys .Met responsible for the incorporation of Met does not distinguish between Met and SeMet, an issue of the blatant hijacking of a sulfur pathway by selenium we shall discuss on several occasions during this review. Indeed, the production of SeMet in plants is not linked to any specific stimuli or requirements and is mainly running side by side to the synthesis of Met [SeMet is synthesized from SeCys in several steps. The α-amino acid homoserine interacts with SeCys to produce a selenocystathionine adduct, which is subsequently hydrolyzed to selenohomocysteine, pyruvate and ammonia, as illustrated in s of Met . Whether32− and synthesize SeMet following the footsteps of plants, although it also produces other selenium species, including elemental red selenium Se±0, often in form of nanoparticles, a metabolic detoxification pathway actually also common in many bacteria and fungi, including Lactobacillus plantarum, Escherichia coli and S. cerevisiae [32− into GSSeSG, H2Se and SeCys, yet do not process SeCys to SeMet [Yeast as a eukaryote can reduce SeOrevisiae . Compareto SeMet .42− or SeO32− follows the one in plants [S. cerevisiae) is very low, i.e., around 0.75%, based on dry weight [The nonspecific and accidental biosynthesis of SeMet in yeast from SeOn plants . Interesn plants ,28. It iy weight . The exty weight ,31.Furthermore, in such fungi, SeMet can requisite the machinery responsible for changing Met into many other natural sulfur compounds, and this results in the production of a plethora of interesting selenium compounds, some of which are shown in S. cerevisiae treated with SeO32−, SeO42− and SeMet [32− or SeO42−. Treatment of yeast with SeMet significantly enhances the intracellular level of SeMet in yeast in less than 15 min, confirming a surprisingly fast and comprehensive uptake of SeMet by these cells. SeO32− and SeO42− are also taken up and processed in yeast to organic selenides, such as selenohomocysteine (SeHcy), gamma-glutamylselenocysteine, selenoglutathione (GSeH), 5′-methylselenoadenosine, Se-adenosylhomocysteine (SeAHcy) and Se-adenosylmethionine (SeAM), in addition to elemental selenium as mentioned before.Liquid chromatography coupled with orbitrap mass spectrometry, for example, has been utilized to hunt for such OSeCs in S. cerevisiae. Popular notions that selenium-enriched yeast contains mostly SeMet therefore are not entirely correct. Among the various selenium compounds found in this fungus, the rather exotic SeAHcy shown in S-adenosylmethionine (SAM), which acts as a methyl donor in various eukaryotic cells. SeAM serves, for example, as a precursor in the synthesis of SeAHcy and is also involved in the methylation of lipids, proteins, nucleic acids and various secondary metabolites. Furthermore, methyltransferases are able to transfer methyl groups from SeAM to different nucleic acids, i.e., ribosomal and transfer RNAs and even DNA [Notably, while yeast can synthesize SeMet following the sulfur pathway, it is not the preferred selenium compound produced in even DNA .S-adenosylhomocysteine hydrolase to adenosine and SeHcy, as shown in S-adenosylhomocysteine hydrolase actually demonstrates a slight discrimination, as its hydrolase activity against SeAHcy is relatively low as compared to its activity against S-adenosylhomocysteine. This also explains why SeHcy is found in yeast in relatively small amounts of around 8.5% of selenium compounds, despite the presence of excessive amounts of SeAHcy, for instance in yeast cells treated with SeMet. The presence of 5′-methylselenoadenosine, a rather exceptional selenide shown in S. cerevisiae, with more exotic substances such as SeAM and chiefly SeAHcy dominating the field.SeAHcy is hydrolyzed by −. Nonetheless, the literature is rich in examples underlining the biological redox activity of SeMet.As mentioned already, many organisms are unable to distinguish between SeMet and Met and therefore tend to accept SeMet in many physiological processes in place of Met. This has notable consequences, as SeMet tends to be more reactive than Met and often acts as a redox cycler and catalyst. This strong antioxidant potential associated with selenium is found for SeMet as a free amino acid, for SeMet in proteins and enzymes and, notably, also for a range of other SeMet precursors and follow-on products containing the same selenide RSeR’ motif . Then agk ~2.4 × 103 M−1s−1, which is almost tenfold higher than the one for Met at just 3.64 × 102 M−1s−1. Not surprisingly, SeMet therefore competes significantly with cellular targets at similar concentrations [2O2, the enzymatic monooxygenase system, amino acid-, peptide- and protein-bound hydroperoxides, hypothiocyanous acid HOSCN and hypochlorite HOCl-derived chloramines [2O2 [In simple assays, SeMet protects dihydrorhodamine 123 and supercoiled plasmid DNA from oxidation mediated by peroxynitrous acid (ONOOH) ,37. Thistrations ,39. The oramines ,41,42. Mnes [2O2 . SeMet iEqually notably, SeMet itself is not only a small-molecule antioxidant, and its ability to become part of proteins and enzymes in the position of Met enables SeMet to be present when and where such oxidative damage may occur. In sharp contrast, the oxygen analog of SeMet and Met, the amino acid methoxinine, is unable to hijack pathways designated for Met, possibly because the oxygen atom is smaller than sulfur and selenium, albeit smaller atoms usually are less demanding and more flexible in such situations. Regardless of the deeper philosophical questions of why Se, and not O, may disguise itself as sulfur, the implications of this Trojan Horse issue include the more or less random replacement of Met by SeMet in proteins, such as human membrane protein B7-1. The glycoprotein B7-1 comprises six Met residues out of a total of 201 amino acids and SeMet has been reported to replace 60% of these Met residues. In another human protein, angiopoietin-2 (Ang2), which contains four Met residues out of a total of 215 amino acids, SeMet has been reported to replace 93% of Met. A similar trend has been reported for other human proteins, including Tie2, netrin-1, plexinB3, ADAM10 and EphA3, where SeMet replaces more than 85% of the Met residues ,45,46.2O, as depicted in 2k ranging from 103 to 104 M−1s−1. Apart from a spontaneous reduction of SeMetO in the presence of GSH, it also looks as if thioredoxin reductase (TR) has the ability to accept SeMetO as substrate, reducing SeMetO to SeMet [This replacement does not go unnoticed, as the biological behavior of SeMet differs from the one of Met. Although both chalcogens are redox active and may be oxidized to sulfoxide (RS(O)R’) and selenoxide (RSe(O)R’), respectively, selenides and selenoxides are usually more (re-)active. The selenide, for instance, is the far better reducing agent and antioxidant compared to the sulfide, hence providing stronger antioxidant protection for the protein containing SeMet in place of Met and also a direct protection for the environment this protein is placed in, as shown in to SeMet .It is therefore not surprising that proteins and enzymes loaded with SeMet exhibit somewhat different and de facto additional properties compared to their unsubstituted analogs. In general, these proteins and enzymes are now also good and often catalytic antioxidants, albeit not as strong as enzymes based on SeCys, such as GPx or human TR.Taken together, the selenide/selenoxide redox pair therefore is a spontaneous redox cycler and catalyst, while the sulfoxide itself is more or less inert and requires the assistance of enzymes such as sulfoxide reductases MsrA or MsrB to get reduced to sulfide .Please note that not every SeMet found in a plant, fungal or mammalian cell is incorporated into proteins. In humans, dietary SeMet intake corresponds to the total Se content in the blood. Here, SeMet is incorporated primarily by erythrocytes into hemoglobin. In plasma, SeMet is found mostly in the albumin fraction. In humans, SeMet is often associated with albumin. For example, SeMet concentrations of 28.3 and 53.4 ng/g have been reported in individuals residing in a selenium-deficient region of China. This concentration of SeMet corresponds to around 20% of the total selenium content in albumin. In contrast, individuals residing in selenium-rich areas of China exhibited elevated levels of SeMet, which constituted around 47.5% of the total selenium content in albumin .The high redox activity of SeMet has major physiological consequences, which can be classified as the result of (a) SeMet as an antioxidant, (b) SeMet as a GPx-like redox catalyst, (c) SeMet as a provider of selenium, (d) SeMet as an intermediate for a family of RSeS and (e) SeMet as a substitute for Met in proteins and enzymes. In most instances, a medley of these different aspects unique to this selenium amino acid is responsible for the physiological activities recorded in practice. Here, the scope of biological activities of SeMet is extremely broad, including simple selenium supplementation as a nutraceutical, antioxidant, anti-inflammatory and preventive activities. As there are numerous reports on such possible activities in the literature a few selected and rather interesting highlights of such activities need to suffice as a part of this review, with relevant literature provided for further information.The population above the age of 65 is more prone to suffer from cardiovascular diseases which are occasionally associated with elevated levels of ROS . The maiAging also interferes with atherogenesis via numerous pathways, and it is highly unlikely that one specific factor dominates the overall pathophysiological mechanism. . Aging iDietary selenium supplementation has been reported to significantly enhance the host immunity against viruses . The topCancer is not only related to OS, it is also to some extent linked to aging . The pre3SeH, which in turn causes the oxidation of thiols and generation of superoxide radical anions (O2●−). Together, these pro-oxidant actions affect the cellular thiolstat and, especially under the conditions of sufficient OS, may also cause apoptosis.Because SeMet is able to counteract each of these changes, it may be beneficial in the fight against cancer in several ways and via several avenues. As discussed already, SeMet is a good antioxidant and may therefore prevent DNA damage. SeMet and its follow-on products are also able to kill cancer cells. Indeed, SeMet is cleaved by the γ—lyase methioninase and therefore produces CH32− supplementation in these studies has triggered extensive metastasis [Not surprisingly, SeMet has been reported to inhibit different cancer cell lines, including colorectal (SW-480), prostate (DU-145), breast (MCF-7/S) and melanoma (UACC-375) cell lines ,70. In mtastasis .Notably, a variety of cancer cells depend on a sufficient supply of the amino acid Met, a feature often referred to as the Hoffmann effect . As SeMeOur brief and necessarily incomplete overview of the biochemistry and possible preventive and therapeutic roles of the second selenium amino acid SeMet has shown that this amino acid is as colorful as its more popular relative SeCys. SeMet is produced in plants and certain mushrooms and may also be taken up and processed in mammals and humans. SeMet is not only an excellent supplier of the trace element selenium; it is also redox active and may act as redox catalyst inside cells. Notably, SeMet is very similar to its sulfur analog Met and often uses the cellular pathways of Met, a red herring to allow SeMet to enter a range of sulfur proteins. Evidence for the health benefits of SeMet, for instance, as a selenium-containing food supplement, is therefore mounting, and there are early reports on possible roles in age-related human diseases, such as cardiovascular diseases and atherosclerosis and also during viral infections. The roles of SeMet as the 22nd proteogenic human amino acid are therefore definitely worth more research.


Institutional academic productivity remains an influential factor in an applicant's selection of fellowship training. This study aimed to determine the quality and quantity of research in the United States orthopaedic sports medicine fellowship programs and identify those with highest productivity.The Arthroscopy Association of North America Fellowship Directory was used to evaluate 88 fellowships in the United States. Publication data and Hirschberg indices (h-index) were collected from the Scopus database. Subanalysis was performed based on the number of publications and mean h-index.P = 0.05). The five programs with the highest number of publications included Hospital for Special Surgery, Rush University, University of Pittsburgh Medical Center, Mayo Clinic in Rochester, and Boston Children's Hospital.Total number of publications per faculty member ranged from 0 to 866, with a median of 20. The median h-index per faculty member was 9. The number of fellows was correlated with a higher mean average h-index of faculty members (Most academic productivity in sports medicine is produced by a relatively small number of fellowship programs in the United States. Of interest, the number of fellows or faculty does not affect significantly the quality or quantity of research productivity at top institutions. However, increased work-hour restrictions and supervision requirements have altered the clinical experience of orthopaedic surgery residency.6 Publicly available rankings for orthopedic hospitals exist through agencies such as US News & World Report, but these rankings include a combination of objective measures  and subjective measures of reputation score based on physician survey results.7 These measures may not apply specifically to fellowship programs that may have vastly different desirability.Deciding on where to complete fellowship training involves factors including surgical experience, academic productivity, location, program reputation, and potential career opportunities. Although financial effects of completing fellowship training have been well documented within the orthopaedic literature, many of these factors are not readily quantifiable.8-10Academic experience available to fellows in training can be measured using several metrics. Aside from the total number of publications, Hirschberg indices (h-index) is widely used and validated to capture both the number and impact of publications produced by authors and being predictive for future scientific achievement in different medical specialties.This study aimed to use both publication data and h-index to determine the quality and quantity of research among faculty members within orthopaedic sports medicine fellowship programs in the United States. Quantifying this academic achievement and impact allows for applicants to decide which programs align with their overall career goals.11 Programs were included if they had an active website with listed faculty members. Programs were excluded if they no longer had an active fellowship based on their program website or if the faculty members were not listed. The program websites were screened by three authors  to assure the program was an active fellowship and to determine sports medicine faculty and the number of fellows accepted each year. We further evaluated the eligible websites to identify full-time sports medicine faculty and exclude pediatric and shoulder/elbow surgeons from the final analysis.The Arthroscopy Association of North America (AANA) Fellowship Directory was searched  to identify orthopaedic sports medicine fellowships available in the United States.12 To ensure accurate count of publications for each department, duplicate publications counted for multiple faculty members within a single department were excluded from the overall total for each department. Subanalysis was performed on the top 10 programs based on the number of publications and mean h-index. Additional analysis was performed to assess the effect of total number of publications within each department by grouping based on total publications, including <50, 50 to 149, 150 to 299, and >300 publications. Authors with exceptionally high volumes of publication, >100 and >200 individual publications, were separately investigated to determine the effects of high-volume publication on h-index.The Scopus database (Elsevier BV), which includes research published from 1995 and, later, was subsequently queried to assess faculty-specific publication data from January 1, 1995, to May 15, 2020. The Scopus database maintains the largest available repository of citation tracking for the peer-reviewed articles. The Scopus was further used to obtain the cumulative h-index for each author over the study period. The h-index is a metric used to evaluate the impact of an author and is typically calculated based the highest number of publications for which an author has an equal or greater number of citations by other authors, with scores >20 considered to be great.t-tests (significant set at P = 0.05). Multivariate logistic regressions were performed to evaluate the complete set of programs and the top 10 programs based on number of total publications to identify the effect of faculty size and number of fellows on total number of publications and mean h-index. Further multivariate logistic regression was performed to identify the effect of high-volume publications by authors on h-index.Data were pooled and assessed using Microsoft Excel 2016 (Microsoft Corporation). Statistical analyses were performed using Stata IC 16 (StataCorp LLC) with continuous data evaluated using two-tailed two-sample unequal variances A total of 94 programs were identified on the AANA Fellowship Directory. After screening of program websites, six programs were excluded from analysis because they were no longer active fellowship programs. Eighty-eight total sports medicine fellowships were evaluated with 689 associated faculty members. Within the period available on the Scopus database (1995 to 2020), a total of 30,210 publications were identified after removal of duplicates within each individual department. The total number of publications per faculty member over the 25-year period ranged from 0 to 866, with a median of 20. The median h-index of faculty members was 9 (range 0 to 106).Within the 88 fellowship programs examined, a total of 222 fellowship positions were identified. The average number of fellows per program was 3 range 1 to 9), with an average faculty-to-fellow ratio of 3.5. The top 10 programs based on total number of publications and h-index were identified, and the results are listed in Table  to 9, wiP = 0.42 and 0.36) and h-index (P = 0.86 and P = 0.06) of these top 10 performing programs. However, when analyzing across all programs, including those outside of the top 10, both the number of fellows and faculty members had a positive correlation with total number of publications (P < 0.001 and P < 0.001). Increased number of fellows was correlated with a higher mean average h-index (P = 0.05). The higher number of faculty was not associated with a change in the average h-index (P = 0.606).Among the top 10 programs, the number of fellows and faculty per program had no effect on both publication (P = 0.012). No significant difference was found in the number of fellows per year or faculty to fellow ratio in programs within high-volume publication cohorts . The mean h-index of these cohorts increased as total number of publications of the program increased with a mean of 5 (range 1 to 9), 10 (range 3 to 20), 15 (range 6 to 23), and 21 (range 5 to 40) respectively, suggesting higher volume of publications is sufficient to increase h-index (P = 0.0048). When analyzing faculty with exceptionally high volumes of publications, including >100 and >200 individual articles, a strongly positive correlation was associated with increasing number of publications and h-index (P < 0.0001).Programs were grouped based on total publications including <50, 50 to 149, 150 to 299, and >300 publications. The total number of sports medicine faculty present at each institution increased in each consecutive group from a mean of 5 (range 1 to 11), 5.5 (range 1 to 11), 8 (range 4 to 16), and 10 (range 4 to 42), respectively . This finding was further supported by our regression analysis of faculty with significantly high-volume publication numbers demonstrating a positive correlation with h-index. Despite these findings, h-index remains a widely used key metric within orthopaedics and across medicine to evaluate academic productivity.Although total publications capture academic productivity, it does not capture the effect that these publications have. In this context, h-indices have become an increasingly relied measure of academic productivity.18 found a mean h-index of 12.8. Similarly, academic spine surgeons had a mean h-index of 13.6 as determined by Schoelfeld et al. In a 2015 study by Lopez et al, fellowship-associated academic hand surgeons had a mean h-index of 10.2.19 Finally, in foot and ankle fellowship programs, Sherman et al20 found the mean h-index of faculty to be 11.9. The results of these studies suggest that the mean h-index among academic sports medicine fellowship faculty is higher on average than other orthopaedic subspecialties.In our study, the mean h-index among all sports medicine faculties was 15.02. However, this number was significantly higher when calculated across the top 10 programs, with a mean h-index of 24.13. Our findings indicate that although the academic productivity of orthopaedic sports medicine fellowship faculty members varies widely, the overall h-index compares favorably with other surgical subspecialties within orthopaedic surgery. Amongst adult reconstruction surgeons, Khan et al22 In 2016, Cvetanovich et al21 investigated the factors associated with academic productivity and institutional academic rank among orthopaedic sports medicine fellowships. Higher h-index and years of total practice were positively correlated with academic rank within each institution. In addition, Cvetanovich et al21 identified regional differences in overall academic productivity of faculty. However, the authors did not rank any programs by name and instead focused their efforts on analyzing the various factors of programs and faculty members who were associated with higher h-index.21 Another study by Clark et al23 surveyed fellowship program directors in sports medicine based on potential variables that may influence academic productivity including amount of protected research time, number of publications per fellow, requirement to do research, and the presence of dedicated research staff to assist with projects. The authors concluded that the presence of dedicated research staff and >25 publications annually were likely associated with higher fellowship productivity; however, specific programs were not ranked or named, and the overall survey response rate was noted to be only 33% of all programs, potentially limiting the generalizability of the conclusions drawn.23Few previous studies have created objective lists of sports medicine fellowship program's academic productivity.13 These rankings include a combination of objective measures alongside subjective measures of reputation based on physician survey results. Furthermore, these rankings are nonspecific to fellowship and, instead, based on academic medical center orthopaedic departments. The results of our study provide sports medicine fellowship applicants with an objective list of programs based on academic metrics to help better inform them in their fellowship selection.In our study, there were a select few programs which ranked among the top 10 in total number of publications and h-index, including Rush University, Hospital for Special Surgery, Mayo Clinic in Rochester, and the University of Virginia. To our knowledge, no previous studies ranked fellowship programs by name to allow applicants to identify the most productive academic centers. Although the abovementioned sources (Doximity and US News and World Report) have created widely popularized rankings, they have several limitations regarding orthopaedic fellowship applicants.24 Although this study did not examine the reputation of each sports medicine fellowship program as whole, we provided an updated rank list of academic productivity  and basic program characteristics to facilitate the program selection process by applicants and potentially stimulate the programs to improve their academic records. Future studies should focus on evaluating the sports medicine fellowship programs based on balanced measures , to provide an accurate program profile to the academic community.Although academic productivity is commonly included in the criteria for selection of sports medicine fellowship program by the applicants, other factors play a role toward this decision. The surgical experience and opportunity for surgical autonomy is extremely important to applicants. Other factors taken into account include sport team coverage, program location and accessibility, family-related matters such as spouse employment opportunities and childcare, social support, opportunity to attend scientific events, financial compensation, and others. A previous study surveying orthopedic residents applying to a single sports medicine fellowship program noted that the variety and complexity of surgical exposure, autonomy, and faculty reputation were the top criteria for residents in choosing where to apply for sports fellowship programs; however, it is important to note that quality or quantity of research was not included as an option in this survey.Our study has several limitations. The identification of programs and faculty members was based on website information, which can be incomplete or imperfect. This was evidenced by the fact that six programs listed in our initial search of the AANA registry no longer had active fellowship training programs. In addition, identifying those orthopaedic sports medicine faculty who are directly involved in training of fellows while excluding both pediatric and shoulder and elbow faculty relied on website information. We attempted to mitigate this limitation by manual screening of each individual website and faculty member profile. Although we eliminated repeat publications within departments, we did not consider author position or number of authors. This could result in repeat publications for different departments if faculty members had changed academic institutions. Furthermore, faculty listed on program websites may not accurately reflect who fellows work with in practice. A website may list more faculty than fellows work with, and consequently, this may have reduced our calculated research productivity.25-27 However, addition of further metrics of productivity was considered to be unnecessarily complex without providing fellowship applicants with helpful information.This study did not account for other measures of academic productivity such as grant funding, national presentations, lectures, or visiting professorship and, instead, relied on number of publications and h-index. Although h-index is a widely used and validated measure of academic productivity, there are inherent limitations to the h-index as a completely accurate measure. As evidenced by our findings, attendings with increasing publishing volume demonstrated an associated increase in h-index on regression analysis, suggesting that high publication volume alone is sufficient to increase h-index. Furthermore, h-index is subject to false elevation in instances of self-citation, which cannot be accounted for but is believed to play a minor role in orthopaedics and its subspecialties.Most academic productivity in sports medicine is attributed to a relatively small number of fellowship programs in the United States. Of interest, the number of fellows or faculty does not affect significantly the quality or quantity of this research productivity at the top programs. Finally, only a few numbers of select programs ranked among the top 10 for both total publications and mean h-index. These results allow for applicants to identify mentors and institutions that align with their career goals to optimize academic productivity in fellowship.


In this study, the impact of steel fibres and Silica Fume (SF) on the mechanical properties of recycled aggregate concretes made of two different types of Recycled Coarse Aggregates (RCA) sourced from both low- and high-strength concretes were evaluated through conducting 60 compressive strength tests. The RCAs were used as replacement levels of 50% and 100% of Natural Coarse Aggregates (NCA). Hook-end steel fibres and SF were also used in the mixtures at the optimised replacement levels of 1% and 8%, respectively. The results showed that the addition of both types of RCA adversely affected the compressive strength of concrete. However, the incorporation of SF led to compressive strength development in both types of concretes. The most significant improvement in terms of comparable concrete strength and peak strain with ordinary concrete at 28 days was observed in the case of using a combination of steel fibres and SF in both recycled aggregate concretes, especially with RCA sourced from high strength concrete. Although using SF slightly increased the elastic modulus of both recycled aggregate concretes, a substantial improvement in strength was observed due to the reinforcement with steel fibre and the coexistence of steel fibre and SF. Moreover, existing models to predict the elastic modulus of both non-fibrous and fibrous concretes are found to underestimate the elastic modulus values. The incorporation of SF changed the compressive stress-strain curves for both types of RCA. The addition of steel fibre and SF remarkably improved the post-peak ductility of recycled aggregates concretes of both types, with the most significant improvement observed in the case of RCA sourced from a low-strength parent concrete. The existing model to estimate the compressive stress-strain curve for steel fibre-reinforced concrete with natural aggregates was found to reasonably predict the compressive stress-strain behaviour for steel fibres-reinforced concrete with recycled aggregate. Concrete is considered the most widely used construction material in the world ,11,12,13The properties of concrete made by recycled aggregates have been analysed in many research studies ,28. TherLike concrete-containing natural aggregates, recycled aggregate concrete also exhibits brittle behaviour in tension and flexure . TherefoTo mitigate the weaker performance of RCA and make it more comparable to conventional concrete, multiple approaches have been utilized in previous research, including the addition of supplementary cementitious materials (SCMs) such as fly ash, electric arc furnace slag, ground granulated blast furnace slag, and SF . Such SCIn addition to the desirable impact of the mentioned materials, previous research studies indicate the superb performance of SF in the enhancement of mechanical and durability properties of recycled concrete . The addCarneiro et al.  measuredMeesala  studied A better understanding of the compressive stress-strain behaviour and elastic modulus of recycled aggregate concrete containing steel fibre, SF, and their combination needs to be accepted by many designers, contractors, and policymakers as a sustainable alternative to conventional concrete. Therefore, the current study aims to evaluate the impacts of steel fibre and SF and their combination on the compressive stress-strain behaviour and elastic modulus of different recycled aggregate concretes. In published research, it is proven that the replacement levels of up to 30% of NCA by RCA does not significantly jeopardise the mechanical properties of concrete. A recent study reported a 5.0–9.3% reduction in compressive strength when different amounts of RCA were utilized . However, respectively. The attached mortar was obtained according to the thermal method, as recommended by other researchers [mi and mf are the initial and final masses of the sample, respectively.The cementitious materials used in this study were ordinary Portland cement (OPC), equivalent to ASTM Type I, and SF. Their chemical compositions and physical properties are summarised in Table 2earchers ,71. In tThe mix design of the concrete samples is shown in A constant water-to-binder ratio equal to 0.4 was used in all the mixtures. Two different RCA contents included the partial replacement of NCA (50% by mass) and full replacement (100% by mass). It should be noted that the replacements were made by mass because the RCA featured a different density compared with the NCA.The trial-and-error method was used to find the suitable mixing procedure. First, the fine aggregates and binders were mixed using a Hobart mixer for one minute until a homogenous mixture was obtained. Next, half of the mixing water and super-plasticiser were added to the mix of binder and aggregates and were mixed for two minutes. The coarse aggregates and the other half of the water were then added, and the mixing process was resumed for five minutes. Finally, the fibres were added, and the mixing was resumed for five minutes.The freshly mixed concrete was poured into cylindrical moulds with a 100 mm diameter and 200 mm in height to undergo the compressive strength tests and determine the stress-strain curves ,72. NextThe slump values of all the mix designs adopted in this study were set to be between 50 mm and 75 mm, which is a reasonable value for practical applications. Moreover, the workability of the samples was slightly reduced while replacing the natural aggregates with both types of recycled concrete aggregates, or by using higher amounts of silica fume. Detailed information regarding the sample preparation, compression tests, and the used standards can be found in recent research studies conducted by the authors ,74. The The compressive stress-strain behaviours of the non-fibrous concretes containing RCA types A and B are shown in The typical failure patterns of all the concrete samples are shown in The calculated elastic modulus values of all the concretes are presented in 2 equal to 0.87. The measured elastic modulus values were compared with those predicted by existing models for both the non-fibrous and steel fibre-reinforced concretes to examine the feasibility of using existing models. In the case of the non-fibrous concrete, the models proposed by Warner et al. [A correlation between the compressive strength and the modulus of elasticity of the non-fibrous recycled aggregates concretes was established, as shown in r et al.  and Thomr et al.  for steeThe effect of SF addition on the toughness of both types of steel fibre-reinforced recycled aggregates concretes was calculated from the area under the compressive stress-strain curve in each concrete. The results are summarised in The consequences of adding SF on the peak compressive strain and compressive strength of both non-fibrous and fibrous recycled aggregates concretes are shown in As shown in coε is the strain corresponding to the compressive strength , and the combined use of steel fibres and SF on the mechanical properties of recycled aggregate concretes containing 50% and 100% recycled coarse types aggregates (RCA), sourced from both low- and high-strength concretes, were investigated. The following main conclusions were drawn based on the experimental and prediction studies:For future research studies, it is recommended to explore the effects of different water/cement ratios on the same mix designs. The investigation of the impact of using other types of fibres on the engineering properties of the mix designs adopted in this research is also suggested.


Porphyromonas gingivalis in human chronic infections may be an important virulence factor and have severe implications for therapy. In this review, we intend to i) define and summarize the significance of the VBNC state in general and ii) discuss the VBNC state of oral bacteria with regard to P. gingivalis. Future studies focused on this phenomenon of intraoral VBNC would provide novel molecular insights on the virulence and persistence of oral pathogens during chronic infections and identify potential novel therapies.Bacteria are exposed to stresses during their growth and multiplication in their ecological systems to which they respond in multiple ways as expert survivalists. One such response mechanism is to convert to a viable but not culturable (VBNC) state. As the name indicates, bacteria in the VBNC state have lost their ability to grow on routine growth medium. A large number of bacteria including many pathogenic species have been reported to be able to enter a VBNC state. VBNC differs from culturable cells in various physiological properties which may result in changes in chemical resistance, adhesion, cellular morphology, metabolism, gene expression, membrane and cell wall composition and/or virulence. The ability of VBNC bacteria to return to the culturable state or resuscitate, when the stressor is removed poses a considerable threat to public health. There have been few publications that overtly describe the ability of oral pathogenic species to enter the VBNC state. However, the presence of VBNCs among oral pathogens such as  Saccharomyces cerevisiae , wine spoilage yeast Brettanomyces  as well as in fungi . At least one bacterial species has been reported to express a virulence factor during the VBNC state [Bacterial species have evolved multiple mechanisms to survive ecological, nutritional and chemical stresses. Among these is to enter a viable but not culturable (VBNC) state . As the Vibrio vulnificus [Citrobacter freundii [The stressful conditions that induce bacterial cells to enter the VBNC state are known for numerous species and have been found to vary not only among species but also strains. Some of the more common stressors described to date include starvation, antibiotic treatment and temperature changes . Most imlnificus  or as lofreundii .Mycobacterium tuberculosis has been demonstrated to exist in a VBNC state and latent TB reactivation may be a attributed to resuscitation from the VBNC state [Enterococcus faecalis, Escherichia coli, Haemophilus influenza, Helicobacter pylori and Mycobacterium smegmatis was significantly associated with increased resistance to antibiotics.Pathogenic VBNC bacteria initially gained attention due to their ability to exist at non-detectable thresholds by traditional water and food safety techniques. Several pathogens in this state may contribute initially to non-apparent infections, which are later followed by a manifestation of the disease. For example, NC state . ImportaNC state , the VBNE. coli and Vibrio cholerae cell [The VBNC state was first discovered in 1982 in The VBNC state of bacteria was traditionally referred to as a dormant bacterial state. However, it is now clear that there exists a difference between VBNC and dormant stages based on their metabolic activity. The VBNC state is characterized by a metabolic activity that is always measurable, whereas during the dormant state, this activity declines to levels that are lower than the level of detection . Thus thCampylobacter spp. change from the characteristic spiral shape during normal exponential phase growth to a coccoid shape during transition to the VBNC state [Burkholderia pseudomallei and V. cholerae cells were also observed to change morphology from their characteristic rod to cocci shape during transition to the VBNC [Cellular morphology is a critical aspect of the phenotype of a cell. Since the cell wall/peptidoglycan determines the shape of the cell, as it enables the tensile strength and diffusion barriers that are necessary to attain a particular shape, any differences in the cell wall composition may affect the cellular morphology. This occurs in some VBNC bacterial cells as many have shown differences in cellular morphology such as cell dwarfing and rounding ,12. It iNC state . Similarthe VBNC ,18. Howethe VBNC .E. coli and Salmonella typhimurium include rpoS and oxyR. rpoS is a sigma factor that is essential for survival in stationary phase as well as during stress responses [oxyR is a lysR-type transcriptional regulator which has a characteristic N-terminal DNA-binding domain that is known to regulate oxidative stress-related genes and plays a crucial role in the induction of the VBNC state [Bacterial cells undergo a change in their physiological state to VBNCs in response to various conditions including but not limited to environmentally unfavorable conditions , starvatesponses ,27. oxyRNC state ,29.E.coli VBNCs including the levels of outer membrane proteins (Omp), the 43 β-subunit antigen, TolC, and OmpT while there was a shift in 106 proteins in VBNCs resulting from exposure to natural seawater and light [V. vulnificus cells to the VBNC state, the levels and composition of unsaturated fatty acids increased with a major shift towards fatty acids with less than 16 carbons and increases in hexadecanoic, hexadecenoic, and octadecanoic acids [E. faecalis VBNCs compared to culturable cells [The lower metabolic rate of VBNCs results in changes in proteins, fatty acids and peptidoglycan in their cell walls and membranes. For example, there was an observed shift in the outer membrane subproteome in nd light . Signifind light . During ic acids . Furtherle cells .V. cholerae [tuf, rpoS, and relA genes that are responsible for protein synthesis and stress responses [VBNC cells also have a variable gene expression profile compared to their culturable counterparts. For example, 58 genes related to regulatory functions, cellular processes, energy metabolism as well as transport and binding were all induced by more than 5-fold in cholerae , while aesponses .V. vulnificus were observed to be resistant to mechanical destruction by sonication [M. smegmatis were found to be more resistant to high temperatures [Vibrio parahaemolyticus [Campylobacter jejuni [E. faecalis [These regulatory and physiological changes produce VBNCs that exhibit strong resistance to various physical, chemical and temperature stress conditions. For example, VBNC cells of nication , while teratures . VBNC ceolyticus , Campylor jejuni  and E. ffaecalis , were alAn array of viability markers have been used to differentiate viable and VBNC cells. The most common method is differential staining combined with direct microscopic enumeration, the LIVE/DEAD® Bac Light™ assay. This assay employs two fluorescent dyes, Syto 9 and propidium iodide, which have variable cell permeability characteristics that can differentiate cells with different membrane integrities .The p-iodonitrotetrazolium violet (INT) assay, is another method and is based on the activity of an electron transport system . As onlyUntargeted metabolomics is being developed as a new method to identify the VBNC state ,43. WithSalmonella enteritidis cells with the subsequent addition of HI broth. Two decades later, this phenomenon was again reported by Baffone et al. [The term ‘resuscitation’ was first used to describe the recovery of non-culturable e et al. , who defPseudomonas aeruginosa and M. tuberculosis [M. tuberculosis existing in a VBNC state.Another common threat, especially in the case of several host-associated human pathogens, occurs when an antibiotic is prescribed to treat an infection. The presence of an antibiotic can induce a bacterium to enter the VBNC state. In some cases, when the antibiotic is discontinued, the VBNC bacteria resuscitate, allowing the infection to reoccur. There is recent evidence that suggests some intracellular pathogens can enter the VBNC state inside their host cells, thus ‘hiding’ from the cell’s antibacterial mechanisms and allowing them to persist. This ability of a bacterial species to enter a state of VBNC has been linked to the survival and pathogenicity of multiple host-associated species including rculosis ,46. For The signaling mechanisms of resuscitation are not well understood because there are no readily available methods/techniques that can distinguish between culturable cells and those that arise from resuscitation. Thus far documented resuscitation has been reported only in approximately half of the human pathogens that are known to exist in the VBNC state. Thus mechanisms that govern resuscitation of pathogenic VBNC cells are mostly unknown which complicates more fully understanding of host-bacterial interactions and outcomes.V. cholerae and E. faecalis [The issue becomes further complicated because of the ‘resuscitation window’, as first proposed by Pinto et al.  and defifaecalis ,47.Various factors have been tested as a stimulus for resuscitation including a shift in temperature , the useThe VBNC and persistence states were independently described decades apart but the phenotypic similarities between them require definitions that distinguish VBNC cells from persistent cells. Persistence occurs when the majority of a population of the cells are susceptible to an antibiotic, while a subpopulation of the cells are tolerant to the antibiotic. Thus persistent cells can be considered as phenotypic variants in a population . SimilarBamford and colleagues, using a single-cell imaging technique, proposed a new identification and isolation system for persisters and VBNC cells which is based on their differential promoter activity expression . This meP. gingivalis or any other oral species to enter the VBNC state, with the exception of E. fecalis for which the oral cavity is a secondary habitat. However, we propose such a state not only occurs but is key to the survival of P. gingivalis in vivo, thereby contributing to chronic infections.The oral cavity can be defined as a mixture of distinct dynamic ecological habitats that support growth of a specific microbial community because of their characteristic biological features. To date, there are no publications that overtly describe the ability of P. gingivalis in diseased tissues [P. gingivalis within the ECV-304 cells using species-specific fluorescently tagged antibodies combined with deconvolution microscopy. This determination was crucial, as dead P. gingivalis cannot invade these nonphagocytic cells. Thus, blood agar did not contain the signal for growth and division but host cells did. The intriguing question then became, what was it about P. gingivalis in vivo that was different from P. gingivalis cultured in vitro?There are few publications that provide support for this hypothesis. First, laboratories worldwide attempted for many years to culture periodontal bacteria from diseased atherosclerotic vessels without success. This was partially in response to the criticism that studies that identified genomic DNA of oral pathogens in these diseased tissues did not prove the presence of viable pathogens, only the presence of the DNA, which could have been transported to the diseased site by macrophages. Then, in 2005, our laboratory published the first proof of the presence of viable  tissues . This waP. gingivalis strain W83 in either endothelial or smooth muscle cells could be enumerated by colony counting, for up to 48 hours of co-culture. However after 48 hours, the numbers of colonies detected on plates decreased to essentially zero. This occurred at the same time numerous intact P. gingivalis were visualized inside the cells by microscopy. Furthermore, when the infected cells were lysed and added to uninfected cells, there was a significant number of colonies culturable on blood agar plates. These results suggested that the intracellular P. gingivalis, although not culturable after 48 hours, were nevertheless viable and the addition of fresh uninfected host cells provided a signal for transformation into the culturable state.A second important publication, by Li et al. , demonstP. gingivalis from atheroma plaques using the co-culture approach . We have spent the ensuing years contemplating the state of P. gingivalis in human atheromatous tissues that prevents them from growing on normally permissive medium. Our hypothesis is that they become VBNCs while residing intracellularly in vivo.Finally, Kozarov and coworkers were subsequently able to culture and isolate additional strains of P. gingivalis has been linked to a large number of other systemic chronic diseases, some of which include cardiovascular diseases, rheumatoid arthritis, preterm birth, Alzheimer’s disease, and several types of cancers. In a recent observation by Ursula H et al., 2020, P. gingivalis was observed to exist in a VBNC state in neuronal cultures in vitro. We propose that the chronicity of P. gingivalis associated infections, both oral and systemic, is due to the ability of P. gingivalis to enter the VBNC state and subsequently periodically resuscitate within cells of the oral cavity or elsewhere within the body.Listeria monocytogenes changes from the cytoplasmic motile lifestyle, in which it uses host cell actin to transit from one host cell to another, to a state in which the bacteria lose their ability to interact with actin and instead localize within LAMP1 positive lysosomal-like vacuoles [L. monocytogenes can ‘revert’ to its normal state of replication upon co-culturing with the infected host cells. The authors suggest that VBNC represents a state of persistence that can then result in the asymptomatic carriage of this bacterium by human hosts, thereby lengthening the incubation period of listeriosis and promoting L. monocytogenes survival/resistance.Most investigations of the VBNC state to date have studied bacteria in freeliving conditions, without the complication of the bacterial cells being intracellular. However, it was recently reported that intracellular vacuoles . Surprisgingivalis entering into a VBNC state. Our first experiments were limited to in vitro conditions. We reasoned that oxidative stress would be one condition to investigate, since P. gingivalis encounters oxidative stress in vivo. To this end, oxidative stress was induced by the addition of 10 mM hydrogen peroxide to stationary P. gingivalis cultures. After 30 min of exposure, the cultures were plated to determine colony-forming units (CFUs) and viability was assayed using the LIVE/DEAD BacLight Bacterial Viability Kit. As can be seen in P. gingivalis strain W83 had indeed entered the VBNC state after exposure to oxidative stress.Based on the studies cited above, we began an investigation of the possibility of P. 2O2 by the addition of 0.6 mM sodium pyruvate for 60 min. The rationale behind the selection of sodium pyruvate was due to the cytoprotection ability of it against oxidative stress. These cultures were then plated and viability was determined. As can be seen in We next attempted resuscitation of the VBNC cells that were exposed to HP. gingivalis strain W83 could convert to the VBNC state. Using our human Coronary Artery Endothelial Cell (HCAEC) model, we observed that after 72 hours of infection, no viable P. gingivalis could be detected by plate counting . Howevecounting ). TogethE. coli and E. faecalis. RpoD in PgW83 is a putative homologue of rpoS, an RNA polymerase sigma factor, required for E. coli to enter the VBNC state. PgW83 has a comparable two-component signaling system (Pg1797 and Pg1089), which has been proposed to be a sensor to initiate the VBNC state in E. coli. PgW83 also has homologues to two Vibrio genes required for the VBNC state. Finally, we have identified possible PgW83 homologues to resuscitation genes characterized in Rhodococcus, Mycobacterium, and Vibrio.Furthermore, we have identified a number of putative PgW83 homologues of gene products that have been used as VBNC markers or required for the VBNC or resuscitation states in other bacteria . PorR, PP. gingivalis strain W83 and likely other strains of P. gingivalis as well. We now are aware that P. gingivalis strain W83 appears to have the necessary genes for the VBNC state and resuscitation and is capable of entering a VBNC state and can resuscitate, once an appropriate environment/signal is present, to a culturable state. These observations are significant not only in terms of the pathobiology of P. gingivalis strain W83 but also call into question the conclusions of previously published studies of trafficking of P. gingivalis within host cells. The previously published investigations should be reevaluated and reinterpreted, since the vast majority of such studies used only CFU enumeration to determine live bacteria within the host cells.These findings offer new insights into the lifecycle of Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguinus were also reported to display phenotypes that closely resemble the description of VBNC states. E. fecalis for which the oral cavity is a secondary habitat is observed to adopt the VBNC state in response to environmental conditions. As additional studies are done, we expect that a variety of oral bacteria will also demonstrate that the VBNC state is part of their lifestyle. Thus investigation of the VBNC state in oral bacterial pathogens is vital to more completely define and characterize all phases of their lifestyle, thereby more fully understanding their virulence/survival mechanisms. This understanding of a previously unknown lifestyle should result in new and novel preventative approaches and treatments for oral and other diseases. For example, the identification of the molecular determinants required for the transition of P. gingivalis to the VBNC state and/or resuscitation during infection could lead to the development of therapeutics that target the chronicity of infections by this bacterium. In addition to antimicrobials, disinfectants also have been shown to trigger entry into the VBNC state. It is thus entirely possible that disinfecting agents used in oral care, e.g. in endodontics may induce the associated oral bacteria to enter the VBNC state. The chronic presence of VBNC of oral bacteria may have severe implications for therapy for infections of the oral cavity as well as infections caused by these pathogens at various and multiple sites within the body.It is almost certain that other oral bacterial species can enter and resuscitate from a VBNC state. P. gingivalis but other pathogens as well. More importantly, future studies of this previously unknown and unappreciated stage of the life cycle of oral pathogens will define mechanisms that can be targeted for novel antimicrobial agents designed to interfere with the VBNC and/or resuscitation states. The identification of such drug targets would have the potential to prevent the persistence and chronicity of not only P. gingivalis infections, but those of other bacterial species involved in chronic diseases.The precise role of the VBNC state in bacterial pathogenesis is yet to be elucidated. It is possible that its role and significance differ from bacterium to bacterium. It will be crucial to understand the role of VBNCs in evasion of host immune detection. By this publication, we seek to convey to the community studying the oral microbial populations that there is a need to initiate studies to further investigate the significance, role and molecular signaling mechanisms of the VBNC and resuscitation states not only in