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      Are Semi-Quantitative Clinical Cultures Inadequate? Comparison to Quantitative Analysis of 1053 Bacterial Isolates from 350 Wounds

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          Abstract

          Early awareness and management of bacterial burden and biofilm is essential to wound healing. Semi-quantitative analysis of swab or biopsy samples is a relatively simple method for measuring wound microbial load. The accuracy of semi-quantitative culture analysis was compared to ‘gold standard’ quantitative culture analysis using 428 tissue biopsies from 350 chronic wounds. Semi-quantitative results, obtained by serial dilution of biopsy homogenates streaked onto culture plates divided into 4 quadrants representing occasional, light, moderate, and heavy growth, were compared to total bacterial load quantified as colony-forming units per gram (CFU/g). Light growth, typically considered an insignificant finding, averaged a clinically significant 2.5 × 10 5 CFU/g (SE = 6.3 × 10 4 CFU/g). Occasional growth (range: 10 2–10 6 CFU/g) and light growth (10 3–10 7 CFU/g) corresponded to quantitative values that spanned a 5-log range; moderate and heavy growth corresponded to a range of 4-log and 6-log, respectively, with a high degree of overlap in range of CFU/g per category. Since tissue biopsy and quantitative culture cannot be widely practiced and semi-quantitative analysis is unreliable, other clinically relevant approaches are required to determine wound bioburden and guide best management practices. Fluorescence imaging is a point-of-care technology that offers great potential in this field.

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          Bacterial biofilms: a common cause of persistent infections.

          Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.
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            Recent findings on the viable but nonculturable state in pathogenic bacteria.

            Many bacteria, including a variety of important human pathogens, are known to respond to various environmental stresses by entry into a novel physiological state, where the cells remain viable, but are no longer culturable on standard laboratory media. On resuscitation from this 'viable but nonculturable' (VBNC) state, the cells regain culturability and the renewed ability to cause infection. It is likely that the VBNC state is a survival strategy, although several interesting alternative explanations have been suggested. This review describes the VBNC state, the various chemical and physical factors known to induce cells into this state, the cellular traits and gene expression exhibited by VBNC cells, their antibiotic resistance, retention of virulence and ability to attach and persist in the environment, and factors that have been found to allow resuscitation of VBNC cells. Along with simple reversal of the inducing stresses, a variety of interesting chemical and biological factors have been shown to allow resuscitation, including extracellular resuscitation-promoting proteins, a novel quorum-sensing system (AI-3) and interactions with amoeba. Finally, the central role of catalase in the VBNC response of some bacteria, including its genetic regulation, is described.
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              Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing

              Background Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D), venous leg ulcers (V), and pressure ulcers (P). Results There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods. Conclusion If clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in order to improve the outcomes of patients. This survey will also foster the pioneering and development of new molecular diagnostic tools, which can be used to identify the community compositions of chronic wound pathogenic biofilms and other medical biofilm infections.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Diagnostics (Basel)
                Diagnostics (Basel)
                diagnostics
                Diagnostics
                MDPI
                2075-4418
                12 July 2021
                July 2021
                : 11
                : 7
                : 1239
                Affiliations
                [1 ]SerenaGroup Research Foundation, Cambridge, MA 02140, USA
                [2 ]Phil Bowler Consulting Ltd., Warrington WA1 1RG, UK; philbowler.consulting@ 123456gmail.com
                [3 ]Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL 32610, USA; schultzg@ 123456ufl.edu
                [4 ]MolecuLight Inc., Toronto, ON M5G 1T6, Canada; adsouza@ 123456moleculight.com (A.D.); mrennie@ 123456moleculight.com (M.Y.R.)
                Author notes
                [* ]Correspondence: serena@ 123456serenagroups.com ; Tel.: +1-81-(46)-884000
                Author information
                https://orcid.org/0000-0003-1032-3578
                https://orcid.org/0000-0003-2919-2221
                Article
                diagnostics-11-01239
                10.3390/diagnostics11071239
                8303231
                34359322
                0a9b15d2-43df-467c-b29e-476133a5fc15
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 17 June 2021
                : 05 July 2021
                Categories
                Article

                semi-quantitative culture,quantitative culture,wound biopsy,chronic wounds,wound microbiology,fluorescence imaging

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