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Objective:Diabetic foot ulcers (DFUs) are characterised by the presence of many microbes, some of which may not be identified by traditional culture techniques. Total contact casting (TCC) remains the gold-standard for offloading, yet little is known about the microbiome of wounds that progress from hard-to-heal to closed within a TCC.Method:A patient with a DFU underwent weekly treatment with TCC to closure. Samples for next-generation sequencing (NGS) and bioinformatics analysis of tissue samples were collected during each visit. Detection, identification, characterisation of the microbial community and abundance of microbes in each sample were compared.Results:Abundance of microbes, identified by species and strain, changed with each treatment visit. By the final week of treatment, species diversity of the wound microbiome had decreased significantly, highlighted by an observed decrease in the number of total microorganisms present. Resistance genes for tetracyclines were detected in the first sample, but not in subsequent samples.Conclusion:The results of this study suggest dynamic microbiological changes associated with DFUs as they progress to healing within a TCC. As NGS becomes more readily available, further studies will be helpful to gain an improved understanding of the significance of the wound microbiome in patients with DFUs.

The last 12 months have provided further evidence of the potential for cascading ecological and socio-political crises that were warned of 12 months ago. Then a consensus statement from the Regional Action on Climate Change Symposium warned: “the Earth’s climatic, ecological, and human systems are converging towards a crisis that threatens to engulf global civilization within the lifetimes of children now living.” Since then, the consequences of a broad set of extreme climate events (notably droughts, floods, and fires) have been compounded by interaction with impacts from multiple pandemics (including COVID-19 and cholera) and the Russia–Ukraine war. As a result, new connections are becoming visible between climate change and human health, large vulnerable populations are experiencing food crises, climate refugees are on the move, and the risks of water, food, and climate disruption have been visibly converging and compounding. Many vulnerable populations now face serious challenges to adapt. In light of these trends, this year, RACC identifies a range of measures to be taken at global and regional levels to bolster the resilience of these populations in the face of such emerging crises. In particular, at all scales, there is a need for globally available local data, reliable analytic techniques, community capacity to plan adaptation strategies, and the resources (scientific, technical, cultural, and economic) to implement them. To date, the rate of growth of the support for climate change resilience lags behind the rapid growth of cascading and converging risks. As an urgent message to COP27, it is proposed that the time is now right to devote much greater emphasis, global funding, and support to the increasing adaptation needs of vulnerable populations.

Next-generation sequencing (NGS) and metagenomics were used to identify microbial communities in biofilms of microfiltration (MF) and reverse osmosis (RO) membranes in an advanced water purification facility (AWPF) that treats municipal wastewater to produce potable quality water. Secondary treated wastewater effluent is the source of influent to the AWPF treatment train and was also characterized by NGS. Results show low bacterial diversity in biofilms obtained from the feed-side surfaces of MF and RO membranes. Microbial communities and antibiotic resistance genes (ARGs) in the RO biofilm were compared with those of the MF and influent, revealing lower abundance of bacterial species and ARGs in the RO biofilm than in the other samples. Opportunistic pathogens were detected in all samples; however, indicator bacteria, viruses, and bacteriophages were not detected in the RO biofilm. It is concluded that NGS has great potential for improved detection and characterization of microbial communities in biofilms that form on AWPF MF and RO membranes.

ABSTRACTAn occurrence of Vibrio choleraenon-O1/O139 gastroenteritis in the U.S. Gulf Coast is reported here. Genomic analysis revealed that the isolate lacked known virulence factors associated with the clinical outcome of a V. choleraeinfection but did contain putative genomic islands and other accessory virulence factors. Many of these factors are widespread among environmental strains of V. cholerae, suggesting that there might be additional virulence factors in non-O1/O139 V. choleraeyet to be determined. Phylogenetic analysis revealed that the isolate belonged to a phyletic lineage of environmental V. choleraeisolates associated with sporadic cases of gastroenteritis in the Western Hemisphere, suggesting a need to monitor non-O1/O139 V. choleraein the interest of public health.

Recent molecular advances in microbiology have greatly improved the detection of bacterial pathogens in the environment. These improvements and a downward trend in the cost of molecular detection methods have contributed to increased frequency of detection of pathogenic microorganisms where traditional culture‐based detection methods have failed. Culture methods also have been greatly improved, and the confluence of the two suites of methods provides a powerful tool for detection, isolation, and characterization of pathogens. While molecular detection provides data on the presence and type of pathogens, culturing methods allow a researcher to preserve the organism of interest for “‐omics” studies, such as genomic, metabolomic, secretomic, and transcriptomic analysis, which are rapidly becoming more affordable. This has yielded a clearer understanding of the ecology and epidemiology of microorganisms that cause disease. In this unit, we present commonly accepted methods for isolation, detection, and characterization of V. cholerae, providing more extensive knowledge of the ecology and epidemiology of this organism. This unit has been fully revised and updated from the earlier version with the latest knowledge and additional information not previously included. Curr. Protoc. Microbiol. 26:6A.5.1‐6A.5.51. © 2012 by John Wiley & Sons, Inc.

ABSTRACTVibrio choleraeO1 classical (CL) biotype caused the fifth and sixth pandemics, and probably the earlier cholera pandemics, before the El Tor (ET) biotype initiated the seventh pandemic in Asia in the 1970s by completely displacing the CL biotype. Although the CL biotype was thought to be extinct in Asia and although it had never been reported from Latin America, V. choleraeCL and ET biotypes, including a hybrid ET, were found associated with areas of cholera endemicity in Mexico between 1991 and 1997. In this study, CL biotype strains isolated from areas of cholera endemicity in Mexico between 1983 and 1997 were characterized in terms of major phenotypic and genetic traits and compared with CL biotype strains isolated in Bangladesh between 1962 and 1989. According to sero- and biotyping data, all V. choleraestrains tested had the major phenotypic and genotypic characteristics specific for the CL biotype. Antibiograms revealed the majority of the Bangladeshi strains to be resistant to trimethoprim-sulfamethoxazole, furazolidone, ampicillin, and gentamicin, while the Mexican strains were sensitive to all of these drugs, as well as to ciprofloxacin, erythromycin, and tetracycline. Pulsed-field gel electrophoresis (PFGE) of NotI-digested genomic DNA revealed characteristic banding patterns for all of the CL biotype strains although the Mexican strains differed from the Bangladeshi strains in 1 to 2 DNA bands. The difference was subtle but consistent, as confirmed by the subclustering patterns in the PFGE-based dendrogram, and can serve as a regional signature, suggesting the pre-1991 existence and evolution of the CL biotype strains in the Americas, independent from Asia.

ABSTRACTVibrio parahaemolyticusis an inhabitant of estuarine and marine environments that causes seafood-borne gastroenteritis worldwide. Recently, a type 3 secretion system (T3SS2) able to secrete and translocate virulence factors into the eukaryotic cell has been identified in a pathogenicity island (VP-PAI) located on the smaller chromosome. These virulence-related genes have previously been detected only in clinical strains. Classical virulence genes for this species (tdh, trh) are rarely detected in environmental strains, which are usually considered to lack virulence potential. However, during screening of a collection of environmental V. parahaemolyticusisolates obtained in the North Adriatic Sea in Italy, a number of marine strains carrying virulence-related genes, including genes involved in the T3SS2, were detected. In this study, we investigated the pathogenic potential of these marine V. parahaemolyticusstrains by studying their adherence ability, their cytotoxicity, their effect on zonula occludin protein 1 (ZO-1) of the tight junctions, and their effect on transepithelial resistance (TER) in infected Caco-2 cells. By performing a reverse transcription-PCR, we also tested the expression of the T3SS2 genes vopTand vopB2, encoding an effector and a translocon protein, respectively. Our results indicate that, similarly to clinical strains, marine V. parahaemolyticusstrains carrying vopTand vopB2and that other genes included in the VP-PAI are capable of adhering to human cells and of causing cytoskeletal disruption and loss of membrane integrity in infected cells. On the basis of data presented here, environmental V. parahaemolyticusstrains should be included in coastal water surveillance plans, as they may represent a risk for human health.

Vibrio parahaemolyticus is an inhabitant of estuarine and marine environments that causes seafood-borne gastroenteritis worldwide. Recently, a type 3 secretion system (T3SS2) able to secrete and translocate virulence factors into the eukaryotic cell has been identified in a pathogenicity island (VP-PAI) located on the smaller chromosome. These virulence-related genes have previously been detected only in clinical strains. Classical virulence genes for this species (tdh, trh) are rarely detected in environmental strains, which are usually considered to lack virulence potential. However, during screening of a collection of environmental V. parahaemolyticus isolates obtained in the North Adriatic Sea in Italy, a number of marine strains carrying virulence-related genes, including genes involved in the T3SS2, were detected. In this study, we investigated the pathogenic potential of these marine V. parahaemolyticus strains by studying their adherence ability, their cytotoxicity, their effect on zonula occludin protein 1 (ZO-1) of the tight junctions, and their effect on transepithelial resistance (TER) in infected Caco-2 cells. By performing a reverse transcription-PCR, we also tested the expression of the T3SS2 genes vopT and vopB2, encoding an effector and a translocon protein, respectively. Our results indicate that, similarly to clinical strains, marine V. parahaemolyticus strains carrying vopT and vopB2 and that other genes included in the VP-PAI are capable of adhering to human cells and of causing cytoskeletal disruption and loss of membrane integrity in infected cells. On the basis of data presented here, environmental V. parahaemolyticus strains should be included in coastal water surveillance plans, as they may represent a risk for human health.

Microbiological techniques for sampling the aquatic realm have become increasingly sophisticated, especially with advances in molecular biology. These techniques have been used to detect microorganisms that cannot be cultured by conventional bacteriological methods. This has resulted in a deeper and a clearer understanding of the ecology and epidemiology of microorganisms. Important advances have been made in isolation, detection, and identification of Vibrio choleraeover the past decade. The understanding that V. cholerae, like several other pathogenic bacteria, can enter into a state known as “viable but nonculturable” (VBNC) provided important clues on the epidemiology of the pathogen and its ability to cause sudden explosive epidemics at multiple places almost simultaneously. The advances in techniques have also allowed investigators to discern the intricate aspects of the ecology of this pathogen in the aquatic world. In this unit, we present the most accepted methods for the isolation and detection of V. cholerae.

Presents a speech by Rita R. Colwell, distinguished professor at the University of Maryland College Park, during the Commencement ceremony at the Joint Military Intelligence College in Washington, D.C. in August 2005. Factors which changed the field of intelligence during the recent years; Impact of globalization on the meaning of national security; Role of social science in the field.

The diversity of toxigenic V. choleraeO1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. choleraeO1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed‐field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. choleraeO1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin (CT), which is encoded by a lysogenic bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor which is also the receptor for CTXPhi. The genes for the biosynthesis of TCP are part of a larger genetic element known as the TCP pathogenicity island. To assess their pathogenic potential, we analyzed environmental strains of V. cholerae carrying genetic variants of the TCP pathogenicity island for colonization of infant mice, susceptibility to CTXPhi, and diarrheagenicity in adult rabbits. Analysis of 14 environmental strains, including 3 strains carrying a new allele of the tcpA gene, 9 strains carrying a new allele of the toxT gene, and 2 strains carrying conventional tcpA and toxT genes, showed that all strains colonized infant mice with various efficiencies in competition with a control El Tor biotype strain of V. cholerae O1. Five of the 14 strains were susceptible to CTXPhi, and these transductants produced CT and caused diarrhea in adult rabbits. These results suggested that the new alleles of the tcpA and toxT genes found in environmental strains of V. cholerae encode biologically active gene products. Detection of functional homologs of the TCP island genes in environmental strains may have implications for understanding the origin and evolution of virulence genes of V. cholerae.

ABSTRACTThe major virulence factors of toxigenic Vibrio choleraeare cholera toxin (CT), which is encoded by a lysogenic bacteriophage (CTXΦ), and toxin-coregulated pilus (TCP), an essential colonization factor which is also the receptor for CTXΦ. The genes for the biosynthesis of TCP are part of a larger genetic element known as the TCP pathogenicity island. To assess their pathogenic potential, we analyzed environmental strains of V. choleraecarrying genetic variants of the TCP pathogenicity island for colonization of infant mice, susceptibility to CTXΦ, and diarrheagenicity in adult rabbits. Analysis of 14 environmental strains, including 3 strains carrying a new allele of the tcpAgene, 9 strains carrying a new allele of the toxTgene, and 2 strains carrying conventional tcpAand toxTgenes, showed that all strains colonized infant mice with various efficiencies in competition with a control El Tor biotype strain of V. choleraeO1. Five of the 14 strains were susceptible to CTXΦ, and these transductants produced CT and caused diarrhea in adult rabbits. These results suggested that the new alleles of the tcpAand toxTgenes found in environmental strains of V. choleraeencode biologically active gene products. Detection of functional homologs of the TCP island genes in environmental strains may have implications for understanding the origin and evolution of virulence genes of V. cholerae.

SUMMARYRecently, the role of the environment and climate in disease dynamics has become a subject of increasing interest to microbiologists, clinicians, epidemiologists, and ecologists. Much of the interest has been stimulated by the growing problems of antibiotic resistance among pathogens, emergence and/or reemergence of infectious diseases worldwide, the potential of bioterrorism, and the debate concerning climate change. Cholera, caused by Vibrio cholerae, lends itself to analyses of the role of climate in infectious disease, coupled to population dynamics of pathogenic microorganisms, for several reasons. First, the disease has a historical context linking it to specific seasons and biogeographical zones. In addition, the population dynamics of V. choleraein the environment are strongly controlled by environmental factors, such as water temperature, salinity, and the presence of copepods, which are, in turn, controlled by larger-scale climate variability. In this review, the association between plankton and V. choleraethat has been documented over the last 20 years is discussed in support of the hypothesis that cholera shares properties of a vector-borne disease. In addition, a model for environmental transmission of cholera to humans in the context of climate variability is presented. The cholera model provides a template for future research on climate-sensitive diseases, allowing definition of critical parameters and offering a means of developing more sophisticated methods for prediction of disease outbreaks.

Serratia marcescensbiovar A2/A6, isolated from an Indonesian freshwater source, was identified based on extensive morphological, biochemical and genetic characterization. Formation of pigment was found to be strongly influenced by environmental conditions. Placket-Burman design was used to analyze the effect of carbon and nitrogen sources. Based on results of physiological and biochemical studies, the optimum conditions for growth and pigment formation were incubation 30°C in a neutral to slightly alkaline medium containing lactic acid and beef extract.