Environmental Bacterial Pathogens Research Articles

Recovering high-quality bacterial genomes from cross-contaminated cultures: a case study of marine Vibrio campbellii

BackgroundEnvironmental monitoring of bacterial pathogens is critical for disease control in coastal marine ecosystems to maintain animal welfare and ecosystem function and to prevent significant economic losses. This requires accurate taxonomic identification of environmental bacterial pathogens, which often cannot be achieved by commonly used genetic markers (e.g., 16S rRNA gene), and an understanding of their pathogenic potential based on the information encoded in their genomes. The decreasing costs of whole genome sequencing (WGS), combined with newly developed bioinformatics tools, now make it possible to unravel the full potential of environmental pathogens, beyond traditional microbiological approaches. However, obtaining a high-quality bacterial genome, requires initial cultivation in an axenic culture, which is a bottleneck in environmental microbiology due to cross-contamination in the laboratory or isolation of non-axenic strains.ResultsWe applied WGS to determine the pathogenic potential of two Vibrio isolates from coastal seawater. During the analysis, we identified cross-contamination of one of the isolates and decided to use this dataset to evaluate the possibility of bioinformatic contaminant removal and recovery of bacterial genomes from a contaminated culture. Despite the contamination, using an appropriate bioinformatics workflow, we were able to obtain high quality and highly identical genomes (Average Nucleotide Identity value 99.98%) of one of the Vibrio isolates from both the axenic and the contaminated culture. Using the assembled genome, we were able to determine that this isolate belongs to a sub-lineage of Vibrio campbellii associated with several diseases in marine organisms. We also found that the genome of the isolate contains a novel Vibrio plasmid associated with bacterial defense mechanisms and horizontal gene transfer, which may offer a competitive advantage to this putative pathogen.ConclusionsOur study shows that, using state-of-the-art bioinformatics tools and a sufficient sequencing effort, it is possible to obtain high quality genomes of the bacteria of interest and perform in-depth genomic analyses even in the case of a contaminated culture. With the new isolate and its complete genome, we are providing new insights into the genomic characteristics and functional potential of this sub-lineage of V. campbellii. The approach described here also highlights the possibility of recovering complete bacterial genomes in the case of non-axenic cultures or obligatory co-cultures.

Molecular and Genomic Epidemiology of Non-O1/Non-O139 and O1/O139 Vibrio cholerae: A Molecular typo-genomics strategy for studying Emerging Potential Environmental Bacterial Pathogens

Recent happening of epidemic, sporadic and pandemic associated with bacteria pathogens around the globe continue to arouse fear amongst the populace. Specific examples amongst such pathogens are the non-O1/non-O139 and O1/O139 Vibrio cholerae which are implicated in acute watery diarrhea (AWD) and cholera cases. It was reported that more than ten Southern/Eastern African countries have suffered the scourge since the beginning of 2018. Considering the highlights on the rapid transmission or spread of AWD/Cholera and the relative impact on public/environmental health, molecular epidemiology of transmitting agents has shown promising relevance in diverse field of interest. This study reviewed researches on some published articles on Cholera/AWD within the Southern African Development Communities (SADC), other East and West African countries and elsewhere. The role of molecular epidemiological tools in understanding the dynamics of Non-O1/Non-O139 and O1/O139 Vibrio cholerae transmission/spread, source tracking of outbreaks and possible management/control in probable and confirmed cases of vibriosis was discussed. It also emphasized on the current updated nature/burden of the pathogen in endemic, epidemic and pandemic area in Southern African countries as the problem of safe-water and hygienic-food for-all continues to resurface. The need for application of molecular and gene based epidemiological approach in profiling bacterial strains cannot be overemphasize especially amongst emerging or reemerging strains of Non-O1/Non-O139 Vibrio cholarae and O1/O139 Vibrio cholerae.

Two Permeases Associated with the Multifunctional CtaP Cysteine Transport System in Listeria monocytogenes Play Distinct Roles in Pathogenesis.

The soil-dwelling bacterium Listeria monocytogenes survives a multitude of conditions when residing in the outside environment and as a pathogen within host cells. Key to survival within the infected mammalian host is the expression of bacterial gene products necessary for nutrient acquisition. Similar to many bacteria, L. monocytogenes uses peptide import to acquire amino acids. Peptide transport systems play an important role in nutrient uptake as well as in additional functions that include bacterial quorum sensing and signal transduction, recycling of peptidoglycan fragments, adherence to eukaryotic cells, and alterations in antibiotic susceptibility. It has been previously described that CtaP, encoded by lmo0135, is a multifunctional protein associated with activities that include cysteine transport, resistance to acid, membrane integrity, and bacterial adherence to host cells. ctaP is located next to two genes predicted to encode membrane-bound permeases lmo0136 and lmo0137, termed CtpP1 and CtpP2, respectively. Here, we show that CtpP1 and CtpP2 are required for bacterial growth in the presence of low concentrations of cysteine and for virulence in mouse infection models. Taken together, the data identify distinct nonoverlapping roles for two related permeases that are important for the growth and survival of L. monocytogenes within host cells. IMPORTANCE Bacterial peptide transport systems are important for nutrient uptake and may additionally function in a variety of other roles, including bacterial communication, signal transduction, and bacterial adherence to eukaryotic cells. Peptide transport systems often consist of a substrate-binding protein associated with a membrane-spanning permease. The environmental bacterial pathogen Listeria monocytogenes uses the substrate-binding protein CtaP not only for cysteine transport but also for resistance to acid, maintenance of membrane integrity, and bacterial adherence to host cells. In this study, we demonstrate complementary yet distinct functional roles for two membrane permeases, CtpP1 and CtpP2, that are encoded by genes linked to ctaP and that contribute to bacterial growth, invasion, and pathogenicity.

Unlocking the molecular realm: advanced approaches for identifying clinically and environmentally relevant bacteria.

Rapid, effective, and specific identification of clinical and environmental bacterial pathogens is of major importance for their control. Traditionally, bacteria have been identified by phenotypic methods based on morphological, biochemical, and metabolic properties. While these methods are very useful in clinical practice, they have limitations including a poor ability to differentiate within and between species and time-consuming workflows. Newly developed molecular methods can greatly improve the accuracy of taxonomic characterization, identifying specific strains of medical or environmental importance. However, due to high costs and the need for trained professionals, these methods are not yet routine in diagnostic laboratories. Thus, disseminating knowledge on advances in molecular identification techniques is pivotal to make these methodologies accessible. The objective of this work was to review and discuss current molecular techniques for bacteria identification aiming to track and monitor microbial agents in clinical and environmental samples.

Black phosphorus-based CuS nanoplatform: Near-infrared-responsive and reactive oxygen species-generating agent against environmental bacterial pathogens

Over the last few years, the two-dimensional (2D) material black phosphorus (BP) has been highlighted for its potential in various biomedical applications, owing to its excellent physical and chemical properties. Nevertheless, the synergistic photothermal–antibacterial effects of BP when combined with other semiconducting materials against pathogenic bacteria have rarely been explored. In this study, the synergistic photothermal–antibacterial activity of a BP-based CuS (CB) nanoplatform against environmental bacterial pathogens was evaluated. A low-temperature solution synthesis method was utilized to prepare CuS nanoparticles that were immobilized onto BP nanosheets. The resulting CB nanocomposite was characterized using X-ray diffraction, transmission electron microscopy, and X-ray photoelectron spectroscopy. The antibacterial activity of the samples was evaluated by determining their minimum inhibitory concentration and bactericidal activity following near-infrared irradiation. The results revealed that CB nanocomposites showed higher antibacterial activity than other samples owing to the high photothermal conversion efficiency. In addition, the plausible antibacterial mechanisms, such as the formation of reactive oxygen species and bacterial membrane disruption, were evaluated. The prepared 2D semiconductor exhibiting synergistic photothermal–antibacterial activity will serve as a good nanoplatform against multidrug-resistant pathogens shed in the environment.

Isolation and Identification of Major Pathogenic Bacteria from Clinical Mastitic Cows in Asella Town, Ethiopia.

Mastitis is a multietiological and complex disease causing inflammation of the parenchyma of mammary glands and is a problem in many dairy cows. The objective of this study was to isolate and identify the pathogenic bacteria that cause bovine clinical mastitis. A cross-sectional study was undertaken between November 2018 to April 2019 on a small scale and government dairy farms in Asella town. Cow's udder and teats were physically examined to detect clinical mastitis. A total of 83 milk samples were collected from 46 cows that show clinical sign of mastitis from a total of 12 farms. Isolation and identification of major bacterial species were carried out by culturing different media and using primary and secondary biochemical tests. Out of the 83 samples collected and examined, all (100%) were positive for the cultural isolation of bacterial species. The bacteria were identified to genus and species level. Among the 83 isolates, 32 (38.6%), 24 (28.9%), and 6 (7.2%) were Staphylococcus aureus, Staphylococcus intermedius, and Staphylococcus hyicus, respectively. Other bacteria like Escherichia coli 12 (14.5%) and Streptococcus species 2 (2.4%) were also isolated. Bacillus species 2 (2.4%), Proteus species 2 (2.4%), and 3 (3.6%) of them were mixed bacterial infections. The present study revealed that both contagious and environmental bacterial pathogens were responsible for the occurrence of clinical mastitis. Proper milking practices and farm husbandry practices and future detailed studies up to the species level and on antibiotic profiles of the pathogens are needed.

Melioidosis in Africa: Time to Uncover the True Disease Load.

Melioidosis is an often fatal infectious disease with a protean clinical spectrum, caused by the environmental bacterial pathogen Burkholderia pseudomallei. Although the disease has been reported from some African countries in the past, the present epidemiology of melioidosis in Africa is almost entirely unknown. Therefore, the common view that melioidosis is rare in Africa is not evidence-based. A recent study concludes that large parts of Africa are environmentally suitable for B. pseudomallei. Twenty-four African countries and three countries in the Middle East were predicted to be endemic, but no cases of melioidosis have been reported yet. In this study, we summarize the present fragmentary knowledge on human and animal melioidosis and environmental B. pseudomallei in Africa and the Middle East. We propose that systematic serological studies in man and animals together with environmental investigations on potential B. pseudomallei habitats are needed to identify risk areas for melioidosis. This information can subsequently be used to target raising clinical awareness and the implementation of simple laboratory algorithms for the isolation of B. pseudomallei from clinical specimens. B. pseudomallei was most likely transferred from Asia to the Americas via Africa, which is shown by phylogenetic analyses. More data on the virulence and genomic characteristics of African B. pseudomallei isolates will contribute to a better understanding of the global evolution of the pathogen and will also help to assess potential differences in disease prevalence and outcome.

Potential human pathogenic bacteria in five hot springs in Eritrea revealed by next generation sequencing.

Human pathogens can survive and grow in hot springs. For water quality assessment, Escherichia coli or Enterococci are the main thermotolerant enteric bacteria commonly used to estimate the load of pathogenic bacteria in water. However, most of the environmental bacteria are unculturable thus culture methods may cause bias in detection of most pathogens. Illumina sequencing can provide a more comprehensive and accurate insight into environmental bacterial pathogens, which can be used to develop better risk assessment methods and promote public health awareness. In this study, high-throughput Illumina sequencing was used to identify bacterial pathogens from five hot springs; Maiwooi, Akwar, Garbanabra, Elegedi and Gelti, in Eritrea. Water samples were collected from the five hot springs. Total community DNA was extracted from samples using the phenol-chloroform method. The 16S rRNA gene variable region (V4—V7) of the extracted DNA was amplified and library construction done according to Illumina sequencing protocol. The sequence reads (length >200 bp) from Illumina sequencing libraries ranged from 22,091 sequences in the wet sediment sample from Garbanabra to 155,789 sequences in the mat sample from Elegedi. Taxonomy was assigned to each OTU using BLASTn against a curated database derived from GreenGenes, RDPII, SILVA SSU Reference 119 and NCBI. The proportion of potential pathogens from the water samples was highest in Maiwooi (17.8%), followed by Gelti (16.7%), Akwar (13.6%) and Garbanabra (10.9%). Although the numbers of DNA sequence reads from Illumina sequencing were very high for the Elegedi (104,328), corresponding proportion of potential pathogens very low (3.6%). Most of the potential pathogenic bacterial sequences identified were from Proteobacteria and Firmicutes. Legionella and Clostridium were the most common detected genera with different species. Most of the potential pathogens were detected from the water samples. However, sequences belonging to Clostridium were observed more abundantly from the mat samples. This study employed high-throughput sequencing technologies to determine the presence of pathogenic bacteria in the five hot springs in Eritrea.

Immune Control of Burkholderia pseudomallei--Common, High-Frequency T-Cell Responses to a Broad Repertoire of Immunoprevalent Epitopes.

Burkholderia pseudomallei (Bp) is an environmental bacterial pathogen that causes potentially lethal sepsis in susceptible individuals and is considered a Category B, Tier-1 biothreat agent. As such, it is crucial to gain an improved understanding of protective immunity and potential vaccine candidates. The nature of immune correlates dictating why most exposed individuals in endemic regions undergo asymptomatic seroconversion while others succumb to life-threatening sepsis is largely uncharted. Bp seroreactive, immunogenic proteins have previously been identified by antigen microarray. We here set out to conduct an analysis of T-cell recognition of the Bp immunome using serodominant antigens represented in the original antigen microarray, examining immune correlates of disease in healthy seropositive individuals and those with acute disease or in convalescence. By screening a library of 739 overlapping peptides representing the sequences of 20 different Bp antigens, we aimed to define immune correlates of protection at the level of immunoprevalent T-cell epitopes. Responses to a large number of epitopes were common in healthy seropositive individuals: we found remarkably broad responsiveness to Bp epitopes, with 235 of 739 peptides recognized by ≥80% of all tested donors. The cumulative response to Bp epitopes in healthy, seropositive, donors from this endemic region were of the order of thousands of spot forming cells per million cells, making Bp recognition a significant component of the T-cell repertoire. Noteworthy among our findings, analysis revealed 10 highly immunoprevalent T-cell epitopes, able to induce Bp-specific IFNγ responses that were high in responding T-cell frequency within the repertoire, and also common across individuals with different human leukocyte antigen types. Acute melioidosis patients showed poor T-cell responses to the immunoprevalent epitopes, but acquired responsiveness following recovery from infection. Our findings suggest that a large repertoire of CD4 T cells, high in frequency and with broad coverage of antigens and epitopes, is important in controlling Bp infection. This offers an attractive potential strategy for subunit or epitope-based vaccines.

Prevalence of bovine subclinical mastitis and antibiotic susceptibility patterns of major mastitis pathogens isolated in Unguja island of Zanzibar, Tanzania.

A cross-sectional study was conducted between January and July 2014 in Unguja island of Zanzibar to establish prevalence of subclinical mastitis (SCM) in smallholder dairy cows and patterns of antibacterial susceptibility of major mastitis pathogens isolated. A total of 416 dairy cows from 201 farmers were randomly selected from three districts of Unguja Island to participate in the study. Questionnaire interview, field observation, individual cow examination, California Mastitis Test (CMT) and bacteriological examination were carried out. Kirby-Bauer disc diffusion technique was used to test drug sensitivity for common bacteria isolated. Based on CMT results, the overall prevalence of SCM was 28.6, 48.8 and 64.7% at quarter, cow and farm level, respectively. Prevalence of bacterial infection was recorded at 42.9, 70.9 and 78.6% at quarter, cow and farm examined, respectively. The common bacteria isolated included Staphylococcus aureus (36.8%), Pseudomonas aeruginosa (17.8%), Staphylococcus epidermidis (16.1%), Klebsiella spp. (9.5%), Micrococcus spp. (6.3%) and Escherichia coli (4.9%). In conclusion, findings of this study demonstrated high level of subclinical mastitis at farms, cows and quarters levels with both contagious and environmental bacterial pathogen involved. Therefore, efforts should be directed to the decreased subclinical mastitis by improving sanitary measures and proper milking practice.

Listeria monocytogenes adapts to long-term stationary phase survival without compromising bacterial virulence

Bacteria withstand starvation during long-term stationary phase through the acquisition of mutations that increase bacterial fitness. The evolution of the growth advantage in stationary phase (GASP) phenotype results in the ability of bacteria from an aged culture to outcompete bacteria from a younger culture when the two are mixed together. The GASP phenotype was first described for Escherichia coli, but has not been examined for an environmental bacterial pathogen, which must balance long-term survival strategies that promote fitness in the outside environment with those that promote fitness within the host. Listeria monocytogenes is an environmental bacterium that lives as a saprophyte in soil, but is capable of replicating within the cytosol of mammalian cells. Herein, we demonstrate the ability of L. monocytogenes to express GASP via the acquisition of mutations during long-term stationary growth. Listeria monocytogenes GASP occurred through mechanisms that were both dependent and independent of the stress-responsive alternative sigma factor SigB. Constitutive activation of the central virulence transcriptional regulator PrfA interfered with the development of GASP; however, L. monocytogenes GASP cultures retained full virulence in mice. These results indicate that L. monocytogenes can accrue mutations that optimize fitness during long-term stationary growth without negatively impacting virulence.

Relationship Between Milk Lactoferrin and Etiological Agent in the Mastitic Bovine Mammary Gland

Bovine mastitis is one of the most deleterious diseases for dairy herds and is mainly caused by contagious and environmental bacterial pathogens. Among contagious bacteria, Staphylococcus aureus is the most prevalent, whereas the main environmental mastitis pathogens are Streptococcus uberis and Escherichia coli. Bovine lactoferrin (bLF) is an approximately 80-kDa glycoprotein present in milk that participates in the innate response of the mammary gland against bacterial infection. The objectives of the current study were to analyze potential changes in bLF milk concentration, which would constitute a response of the mammary gland toward mastitis induced by different etiologic agents, and to evaluate a possible relation between this response and pathogen susceptibility to bLF. Microbiology analysis and bLF quantification in milk from different bovine mammary gland quarters were performed. Infected quarters presented greater concentrations of bLF compared with those from microbiologically negative quarters. Analysis of individual pathogen contributions showed that most of this increase was attributable to Strep. uberis intra-mammary infection. The ability of mammary gland cells to synthesize bLF in response to Strep. uberis challenge was demonstrated by immunodetection of the protein in in vitro infection experiments. Susceptibility of Strep. uberis, E. coli, and Staph. aureus to the antimicrobial activity of bLF was determined by growth inhibition assays conducted with 4 different isolates of each species. Whereas Staph. aureus and E. coli were shown to be susceptible to this protein, Strep. uberis appeared to be resistant to the antimicrobial activity of bLF. Molecular typing of the 4 Strep. uberis isolates used throughout this study showed that this result was representative of the species and not exclusive of a particular strain. Results presented herein suggest that different bacteria species may elicit different mammary gland responses mediated by bLF secretion and that Strep. uberis has probably adapted to this immune reaction by developing resistance to bLF inhibitory action.