Bacterial DnaK reduces the activity of anti-cancer drugs cisplatin and 5FU.

BACKGROUND: Chemotherapy is a primary treatment for cancer, but its efficacy is often limited by cancer-associated bacteria (CAB) that impair tumor suppressor functions. Our previous research found that Mycoplasma fermentans DnaK, a chaperone protein, impairs p53 activities, which are essential for most anti-cancer chemotherapeutic responses. METHODS: To investigate the role of DnaK in chemotherapy, we treated cancer cell lines with M. fermentans DnaK and then with commonly used p53-dependent anti-cancer drugs (cisplatin and 5FU). We evaluated the cells' survival in the presence or absence of a DnaK-binding peptide (ARV-1502). We also validated our findings using primary tumor cells from a novel DnaK knock-in mouse model. To provide a broader context for the clinical significance of these findings, we investigated human primary cancer sequencing datasets from The Cancer Genome Atlas (TCGA). We identified F. nucleatum as a CAB carrying DnaK with an amino acid composition highly similar to M. fermentans DnaK. Therefore, we investigated the effect of F. nucleatum DnaK on the anti-cancer activity of cisplatin and 5FU. RESULTS: Our results show that both M. fermentans and F. nucleatum DnaKs reduce the effectiveness of cisplatin and 5FU. However, the use of ARV-1502 effectively restored the drugs' anti-cancer efficacy. CONCLUSIONS: Our findings offer a practical framework for designing and implementing novel personalized anti-cancer strategies by targeting specific bacterial DnaKs in patients with poor response to chemotherapy, underscoring the potential for microbiome-based personalized cancer therapies.

Microbiological profile of peri-implantitis: Analyses of peri-implant microbiome.

PURPOSE: To characterize the microbiome composition in peri-implant pocket of peri-implantitis and peri-implant sulcus controls using 16S rRNA gene sequencing. MATERIALS AND METHODS: In this controlled clinical cross-sectional study, 23 subjects with control implants (n = 14) and diseased implants (peri-implantitis, n = 21) were included. The peri-implant pocket/sulcus was sampled and used to extract DNA and amplify the 16S rRNA gene using universal primers targeting the V3-V4 regions. The resulting 16S PCR amplicons were sequenced on Illumina MiSeq, and the sequences were processed using DADA2 and the Human Oral Microbiome Database (HOMD) as references. Alpha and Beta diversity, as well as core microbiome and differential abundance analyses, were performed using the MicrobiomeAnalyst workflow. RESULTS: There were no significant differences in microbial diversity between control implants and implants with peri-implantitis (Shannon p = 0.82). Overall bacterial community structure assessed through beta diversity analysis was also not significantly different between the two groups (p = 0.18). However, high levels of Gram-negative bacteria were detected in peri-implant pockets compared to the control sulcus. Abundant species in peri-implantitis were Capnocytophaga leadbetteri, Treponema maltophilum, Peptostreptococcus, Neisseria, P. gingivalis, and Porphyromonas endodontali, Lactococcus lactis and Filifactor alocis (p < 0.05). Gram-positive bacteria such as Streptococcus salivaris, Prevotella melaninogenica, L. wadei, and Actinomyces spp. serve were more abundant in peri-implant control sulcus. CONCLUSIONS: Peri-implant sulcus in control implants harbors predominantly Gram-positive bacteria, whereas pockets of implants with peri-implantitis harbor predominantly Gram-negative bacteria.

Genomic Confirmation of Borrelia garinii, United States.

Lyme disease is a multisystem disorder primarily caused by Borrelia burgdorferi sensu lato. However, B. garinii, which has been identified on islands off the coast of Newfoundland and Labrador, Canada, is a cause of Lyme disease in Eurasia. We report isolation and whole-genome nucleotide sequencing of a B. garinii isolate from a cotton mouse (Peromyscus gossypinus) in South Carolina, USA. We identified a second B. garinii isolate from the same repository. Phylogenetic analysis does not associate these isolates with the previously described isolates of B. garinii from Canada.

Rapid intestinal and systemic metabolic reprogramming in an immunosuppressed environment.

Intrinsic metabolism shapes the immune environment associated with immune suppression and tolerance in settings such as organ transplantation and cancer. However, little is known about the metabolic activities in an immunosuppressive environment. In this study, we employed metagenomic, metabolomic, and immunological approaches to profile the early effects of the immunosuppressant drug tacrolimus, antibiotics, or both in gut lumen and circulation using a murine model. Tacrolimus induced rapid and profound alterations in metabolic activities within two days of treatment, prior to alterations in gut microbiota composition and structure. The metabolic profile and gut microbiome after seven days of treatment was distinct from that after two days of treatment, indicating continuous drug effects on both gut microbial ecosystem and host metabolism. The most affected taxonomic groups are Clostriales and Verrucomicrobiae (i.e., Akkermansia muciniphila), and the most affected metabolic pathways included a group of interconnected amino acids, bile acid conjugation, glucose homeostasis, and energy production. Highly correlated metabolic changes were observed between lumen and serum metabolism, supporting their significant interactions. Despite a small sample size, this study explored the largely uncharacterized microbial and metabolic events in an immunosuppressed environment and demonstrated that early changes in metabolic activities can have significant implications that may serve as antecedent biomarkers of immune activation or quiescence. To understand the intricate relationships among gut microbiome, metabolic activities, and immune cells in an immune suppressed environment is a prerequisite for developing strategies to monitor and optimize alloimmune responses that determine transplant outcomes.

Conventional tobacco products harbor unique and heterogenous microbiomes.

While an increasing number of studies have evaluated tobacco microbiomes, comparative microbiome analyses across diverse tobacco products are non-existent. Moreover, to our knowledge, no previous studies have characterized the metabolically-active (live) fraction of tobacco bacterial communities and compared them across products. To address these knowledge gaps, we compared bacterial communities across four commercial products (cigarettes, little cigars, cigarillos and hookah) and one research cigarette product. After total DNA extraction (n = 414) from all samples, the V3V4 region of the 16S rRNA gene was sequenced on the Illumina HiSeq platform. To identify metabolically-active bacterial communities within these products, we applied a coupled 5-bromo-2'-deoxyuridine labeling and sequencing approach to a subset of samples (n = 56). Each tobacco product was characterized by its signature microbiome, along with a shared microbiome across all tobacco products consisting of Pseudomonas aeruginosa, P. putida, P. alcaligenes, Bacillus subtilis, and Klebsiella pneumoniae. Comparing across products (using Linear discriminant analysis Effect Size (LEfSe)), a significantly higher (p < 0.05) relative abundance of Klebsiella and Acinetobacter was observed in commercial cigarettes, while a higher relative abundance of Pseudomonas and Pantoea was observed in research cigarettes. Methylorubrum and Paenibacillus were higher in hookah, and Brevibacillus, Lactobacillus, Bacillus, Lysinibacillus, and Staphylococcus were higher in little cigars and cigarillos. Across all products, the majority of the metabolically-active bacterial communities belonged to the genus Pseudomonas, followed by several genera within the Firmicutes phylum (Bacillus, Terribacillus, and Oceanobacillus). Identification of some metabolically-active pathogens such as Bacillus cereus and Haemophilus parainfluenzae in commercial products is of concern because of the potential for these microorganisms to be transferred to users' respiratory tracts via mainstream smoke. Future work is warranted to evaluate the potential impact of these tobacco bacterial communities on users' oral and lung microbiomes, which play such an important role on the spectrum from health to disease.

Microbiological profile of peri-implantitis: Analyses of microbiome within dental implants.

PURPOSE: To characterize the microbiome composition within dental implants of peri-implantitis subjects and healthy controls using 16S rRNA gene sequencing. MATERIALS AND METHODS: Twenty-three subjects with healthy (n = 11 implants) and diseased (peri-implantitis, n = 21) implants were included in this controlled clinical cross-sectional study. Samples were obtained from internal surfaces of dental implants using sterile paper points for microbiological analysis. DNA was extracted, and the16S rRNA gene was amplified using universal primers targeting the V3-V4 regions. The resulting 16S polymerize chain reaction amplicons were sequenced on Illumina MiSeq, and the sequences were processed using DADA2 and the Human Oral Microbiome Database (HOMD) as references. Alpha and Beta diversity, as well as core microbiome and differential abundance analyses were then performed using the MicrobiomeAnalyst workflow. RESULTS: A significant increase in microbial diversity was observed in the internal implant surface of healthy implants compared with the internal surfaces of peri-implantitis (Shannon p = 0.02). Bacterial community structure was significantly different among groups (p = 0.012). High levels of Gram-positive bacteria were detected inside implants with peri-implantitis compared to healthy implants, especially Enterococci. CONCLUSIONS: There is a shift in bacterial diversity inside implants with peri-implantitis from the healthy control. The microbial colonization within that space might contribute to the etiology of peri-implant disease.

A novel tick protein supports integrity of gut peritrophic matrix impacting existence of gut microbiome and Lyme disease pathogens.

The peritrophic matrix (PM) is an acellular membrane that covers the gut epithelium in arthropods and physically separates it from the lumen. The structure is thought to play an important role in tick biology. The PM is also known to impact the persistence of tick-borne pathogens like Borrelia burgdorferi, although limited information is available about its molecular constituents or their biological significance. Herein, we characterise a novel PM-associated gut protein in Ixodes scapularis ticks, annotated as Peritrophic Membrane Chitin Binding Protein (PM_CBP), for its role in the integrity and function of the matrix. The PM_CBP displays homology to the chitin deacetylase metalloenzyme, shows upregulation during tick feeding, and is localized at the luminal surface of the gut epithelium. The structural integrity of the PM was impaired both by the knock down of PM_CBP expression via RNA interference and by treatment with anti-PM_CBP antibodies, as revealed by its electron microscopic appearance. Additionally, the duration of tick engorgement on mice and the passage of experimentally-inoculated fluorescent dextran molecules across the PM are affected by the knock down of PM_CBP expression. The transfer of anti-PM_CBP antibodies into the tick gut impacted the overall composition of the resident microbiome, and also influenced B. burgdorferi acquisition in ticks and its transmission to mice. Taken together, these data highlight the biological significance of the Ixodes PM and suggest that the targeting of its molecular constituents may contribute to the development of novel interventions against tick-borne infections.

Variations in Bacterial Communities and Antibiotic Resistance Genes Across Diverse Recycled and Surface Water Irrigation Sources in the Mid-Atlantic and Southwest United States: A CONSERVE Two-Year Field Study.

Reduced availability of agricultural water has spurred increased interest in using recycled irrigation water for U.S. food crop production. However, there are significant knowledge gaps concerning the microbiological quality of these water sources. To address these gaps, we used 16S rRNA gene and metagenomic sequencing to characterize taxonomic and functional variations (e.g., antimicrobial resistance) in bacterial communities across diverse recycled and surface water irrigation sources. We collected 1 L water samples (n = 410) between 2016 and 2018 from the Mid-Atlantic (12 sites) and Southwest (10 sites) U.S. Samples were filtered, and DNA was extracted. The V3-V4 regions of the 16S rRNA gene were then PCR amplified and sequenced. Metagenomic sequencing was also performed to characterize antibiotic, metal, and biocide resistance genes. Bacterial alpha and beta diversities were significantly different (p < 0.001) across water types and seasons. Pathogenic bacteria, such as Salmonella enterica, Staphylococcus aureus, and Aeromonas hydrophilia were observed across sample types. The most common antibiotic resistance genes identified coded against macrolides/lincosamides/streptogramins, aminoglycosides, rifampin and elfamycins, and their read counts fluctuated across seasons. We also observed multi-metal and multi-biocide resistance across all water types. To our knowledge, this is the most comprehensive longitudinal study to date of U.S. recycled water and surface water used for irrigation. Our findings improve understanding of the potential differences in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse irrigation water sources across seasons and U.S. regions.

Viable bacteria abundant in cigarettes are aerosolized in mainstream smoke.

Multiple studies have demonstrated that cigarettes harbor bacterial pathogens. Yet, to our knowledge, there are no published data to date on whether or not these microorganisms can be aerosolized and transmitted to the respiratory tract of users. To address this knowledge gap, we characterized cigarette bacterial communities and evaluated whether or not they could be aerosolized in mainstream smoke. Filtered and unfiltered cigarettes were tested. Non-smoked tobacco leaf, enriched non-smoked tobacco leaf extract and enriched mainstream smoke extract samples (n = 144) were incubated on trypticase soy agar, and resulting bacterial colonies were sequenced. Total DNA was also extracted, followed by PCR amplification of the 16S rRNA gene, sequencing and analysis using UCHIME, QIIME and R packages. The predominant bacterial genera cultured from the mainstream smoke of unfiltered cigarettes were Bacillus, Terribacillus, Paenibacillus and Desulfotomaculum. Culturable bacteria were not recovered from the smoke of filtered products. However, sequencing data demonstrated no significant differences in bacterial community diversity in the smoke of filtered versus unfiltered cigarettes, suggesting that other non-culturable bacteria may be aerosolized in mainstream smoke as well. Our study provides novel evidence that tobacco-associated bacterial communities are viable, can be aerosolized in mainstream smoke, and could potentially be transferred to the oral cavity and respiratory tract of smokers.

Bacterial communities of hookah tobacco products are diverse and differ across brands and flavors.

Young adults are increasingly using non-cigarette products, such as hookahs, since they are perceived as healthier alternatives to cigarette smoking. However, hookah users are exposed to not only carcinogenic compounds but also microorganisms that may play an active role in the development of both infectious and chronic diseases among users. Nevertheless, existing hookah research in this area has focused only on microorganisms that may be transferred to users through the smoking apparatus and not on bacterial communities associated with hookah tobacco. To address this knowledge gap, we conducted time-series experiments on commercially available hookah brands (Al Fakher (flavors: two apple, mint, and watermelon) and Fumari (flavors: white gummy bear, ambrosia, and mint chocolate chill)) stored under three different temperature and relative humidity conditions over 14 days. To characterize bacterial communities, the total DNA was extracted on days 0, 5, 9, and 14, PCR-amplified for the V3V4 region of the bacterial 16S rRNA gene, sequenced on the Illumina HiSeq platform, and analyzed using R. Diversity (alpha and beta) analyses revealed that the microbiotas of Fumari and Al Fakher products differed significantly and that flavor had a significant effect on the hookah microbiota. Overall, Pseudomonas, Bacillus, Sphingomonas, and Methylobacterium were the predominant bacterial taxa across all products. Additionally, we observed compositional differences between hookah brands across the 14-day incubation. These data suggest that the bacterial communities of hookah tobacco are diverse and differ across brands and flavors, which may have critical implications regarding exposures to specific bacteria among hookah users. KEY POINTS: • Commercial hookah products harbor diverse bacterial communities. • Brands and flavors impact the diversity of these communities. • Research on their viability and transmission to users' respiratory tracts is needed.

Comparison of the skin microbiota in acne and rosacea.

Acne and rosacea, despite their similar clinical presentations, follow distinct clinical courses, suggesting that fundamental differences exist in their pathophysiology. We performed a case-control study profiling the skin microbiota in rosacea and acne patients compared to matched controls. Nineteen rosacea and eight acne patients were matched to controls by age ± 5 years, sex and race. DNA was extracted from facial skin swabs. The V3V4 region of the bacterial 16S rRNA gene was sequenced using Illumina MiSeq and analysed using QIIME/Metastats 2.0 software. The mean relative abundance of Cutibacterium acnes in rosacea with inflammatory papules and pustules (20.454% ±16.943%) was more similar to that of acne (19.055% ±15.469%) than that of rosacea without inflammatory papules and pustules (30.419% ±21.862%). C acnes (P = .048) and Serratia marcescens (P = .038) were significantly enriched in individuals with rosacea compared to acne. Investigating the differences between the skin microbiota in acne and rosacea can provide important clues towards understanding the disease progression in each condition.

Nicotine concentration and mentholation affect bacterial community diversity in SPECTRUM research cigarettes.

Previous studies have characterized bacterial communities in menthol versus non-menthol cigarettes. However, these studies evaluated commercial cigarettes, for which levels of chemical constituents are largely unknown, and therefore, could not assess the impact of varying nicotine and menthol concentrations on tobacco bacterial communities. To address this knowledge gap, we performed time-series experiments using SPECTRUM research cigarettes with varying nicotine and menthol levels. Cigarettes were incubated under three storage conditions for 14 days. Cigarette tobacco was then sub-sampled (n = 288), DNA extracted, and subjected to PCR amplification of the V3V4 region of the 16S rRNA gene, followed by Illumina HiSeq sequencing. Sequences were analyzed using QIIME and R. Incubation under varying conditions did not affect bacterial diversity. However, significant differences in bacterial communities were observed across varying nicotine concentrations in menthol and non-menthol products. For example, Pseudomonas spp. was negatively correlated with nicotine concentrations in menthol cigarettes. A significantly higher relative abundance of P. veronii and P. viridiflava was observed in menthols versus non-menthols, while a significantly higher relative abundance of Bacillus foraminis and B. coagulans was found in non-menthols versus menthols. Additional bacteria (e.g., Staphylococcus spp., Jeotgalicoccus psychrophilus, and B. flexus) significantly changed in relative abundance between days 0 and 14. Our findings demonstrate that nicotine and menthol levels have a significant impact on the relative abundance of potential bacterial pathogens present in cigarettes. Future work is needed to demonstrate whether these tobacco-associated bacteria could be transferred to users while smoking, ultimately contributing to adverse respiratory impacts. KEY POINTS: • Varying nicotine levels changes bacterial composition of research cigarettes. • Mentholation affects the tobacco bacterial microbiome. • SPECTRUM research cigarettes are dominated by Pseudomonas and Bacillus.

A roadmap from unknowns to knowns: Advancing our understanding of the microbiomes of commercially available tobacco products.

Tobacco smoking is still the leading cause of preventable diseases and death in the USA and throughout the globe. Under Section 904(a)(3) of the US Federal Food, Drug, and Cosmetic Act, tobacco manufacturing companies need to report on quantities of harmful and potentially harmful constituents (HPHCs) in all tobacco products. While the extensive HPHC list of 2012 includes 93 chemicals, which are categorized as carcinogenic, respiratory, cardiovascular, or reproductive toxicants or addictive compounds, it fails to include microorganisms (bacteria and fungi) that have been shown to contribute to adverse health outcomes among tobacco users. Nevertheless, over the last 50 years, researchers have studied microorganisms in a variety of tobacco products using both culture-based and culture-independent techniques. In this mini-review, we provide an overview of this body of research, detailing the bacterial and fungal microbiomes residing in commercial tobacco products. Overall, studies have characterized over 89 unique bacterial genera and 19 fungal genera in cigarettes, cigars, cigarillos, hookah, and smokeless tobacco. The most predominant bacterial genera are Bacillus, Pseudomonas, and Staphylococcus. Fungal genera identified have included Aspergillus, Penicillium, Mucor, Alternaria, Cladosporium, Streptomyces, and Candida, to name a few. While some of the identified microorganisms are known human pathogens, others are potential opportunistic pathogens. Given the vast array of microorganisms that are present across diverse types of tobacco products, future research should be focused on the viability of these microorganisms, as well as their ability to transfer to the user's respiratory tract, potentially contributing to adverse health outcomes. KEY POINTS: • Commercial tobacco products harbor diverse bacterial and fungal communities. • Some of these microorganisms are known or opportunistic human pathogens. • Research on their viability and transmission to users' respiratory tracts is needed.

Plasticity in early immune evasion strategies of a bacterial pathogen.

Borrelia burgdorferi is one of the few extracellular pathogens capable of establishing persistent infection in mammals. The mechanisms that sustain long-term survival of this bacterium are largely unknown. Here we report a unique innate immune evasion strategy of B. burgdorferi, orchestrated by a surface protein annotated as BBA57, through its modulation of multiple spirochete virulent determinants. BBA57 function is critical for early infection but largely redundant for later stages of spirochetal persistence, either in mammals or in ticks. The protein influences host IFN responses as well as suppresses multiple host microbicidal activities involving serum complement, neutrophils, and antimicrobial peptides. We also discovered a remarkable plasticity in BBA57-mediated spirochete immune evasion strategy because its loss, although resulting in near clearance of pathogens at the inoculum site, triggers nonheritable adaptive changes that exclude detectable nucleotide alterations in the genome but incorporate transcriptional reprograming events. Understanding the malleability in spirochetal immune evasion mechanisms that ensures their host persistence is critical for the development of novel therapeutic and preventive approaches to combat long-term infections like Lyme borreliosis.

Chronic rejection as a persisting phantom menace in organ transplantation: a new hope in the microbiota?

PURPOSE OF REVIEW: The microbiota plays an important role in health and disease. During organ transplantation, perturbations in microbiota influence transplant outcome. We review recent advances in characterizing microbiota and studies on regulation of intestinal epithelial barrier function and mucosal and systemic immunity by microbiota and their metabolites. We discuss implications of these interactions on transplant outcomes. RECENT FINDINGS: Metagenomic approaches have helped the research community identify beneficial and harmful organisms. Microbiota regulates intestinal epithelial functions. Signals released by epithelial cells or microbiota trigger pro-inflammatory or anti-inflammatory effects on innate and adaptive immune cells, influencing the structure and function of the immune system. Assessment and manipulation of microbiota can be used for biomarkers for diagnosis, prognosis, and therapy. SUMMARY: The bidirectional dialogue between the microbiota and immune system is a major influence on immunity. It can be targeted for biomarkers or therapy. Recent studies highlight a close association of transplant outcomes with microbiota, suggesting exciting potential avenues for management of host physiology and organ transplantation.

Microbiological Profile and Human Immune Response Associated with Peri-Implantitis: A Systematic Review.

PURPOSE: To evaluate and synthesize the existing evidence on the microbiological and human immune response associated with peri-implantitis in comparison to healthy implants. MATERIALS AND METHODS: Three electronic databases (MEDLINE, Embase, and Cochrane Library) were searched in October 2019 to identify clinical studies evaluating the microbiota and the immune response associated with peri-implantitis. Two reviewers independently screened the studies and used the full text to extract the data. A qualitative synthesis was performed on the extracted data and summary tables were prepared. Due to clinical and methodological heterogeneity among included studies, no meta-analysis was performed. RESULTS: Forty studies were included in this review. Of these, 20 studies compared the microbiological profile of peri-implantitis with healthy implants. Nineteen studies focused on the immune response associated with peri-implantitis in comparison to healthy implants. Three studies focus on gene polymorphism associated with peri-implantitis. The most commonly reported bacteria associated with peri-implantitis were obligate anaerobe Gram-negative bacteria (OAGNB), asaccharolytic anaerobic Gram-positive rods (AAGPRs), and other Gram-positive species. In regard to immune response, the most frequently reported pro-inflammatory mediators associated with peri-implantitis were IL-1ß, IL-6, IL-17, TNF-α. Osteolytic mediator, e.g., RANK, RANKL, Wnt5a and proteinase enzymes, MMP-2, MMP-9, and Cathepsin-K were also expressed at higher level in peri-implantitis sites compared to control. CONCLUSIONS: Peri-implantitis is associated with complex and different microbiota than healthy implants including bacteria, archaea, fungi, and virus. This difference in the microbiota could provoke higher inflammatory response and osteolytic activity. All of this could contribute to the physiopathology of peri-implantitis.

Unique and specific Proteobacteria diversity in urinary microbiota of tolerant kidney transplanted recipients.

Host-microbiota interactions can modulate the immune system both at local and systemic levels, with potential consequences for organ transplantation outcomes. In this study, we hypothesized that differences in the urinary microbiome following kidney transplantation would be associated with posttransplantation status: stable, minimally immunosuppressed, or tolerant. One hundred thirteen urine samples from stable (n = 51), minimally immunosuppressed (n = 19), and spontaneously tolerant (n = 16) patients, paired with age-matched controls (n = 27) were profiled and compared to each other at a taxonomic level with special interest in the immunosuppressive regimen. All comparisons and correlations were adjusted on sex and time posttransplantation. Our results highlighted a unique and specific urinary microbiota associated with spontaneous tolerance characterized by a high diversity and a clear Proteobacteria profile. Finally, we report that this profile is (1) impacted by gender, (2) inversely correlated with immunosuppressive drugs (calcineurin inhibitors and mammalian target of rapamycin inhibitors), and (3) stable in time.

Antibiotic-resistant Escherichia coli and Klebsiella spp. in greywater reuse systems and pond water used for agricultural irrigation in the West Bank, Palestinian Territories.

Treating and reusing greywater for agricultural irrigation is becoming increasingly prevalent in water-scarce regions such as the Middle East. However, the potential for antibiotic-resistant bacteria to be introduced into food systems or the environment via greywater reuse is a potential area of concern. It is known that off-grid treated greywater often has elevated levels of bacteria, however, little is known regarding the prevalence of antibiotic-resistant bacteria in this water source. To address this knowledge gap, samples (n = 61) of off-grid, household greywater (influent), treated greywater effluent, and irrigation pond water were collected between October 2017 and June 2018 from four farms in the West Bank, Palestinian Territories. Samples were tested for pH, turbidity, dissolved oxygen, electrical conductivity, and oxidation reduction potential. Standard membrane filtration was used to enumerate presumptive Escherichia coli, and isolates (n = 88) were purified, confirmed using 16S rRNA sequencing, and subjected to antimicrobial susceptibility testing using microbroth dilution. The majority of influent (76.5%) and effluent (70.6%) samples had detectable presumptive E. coli. Interestingly, the majority of the isolates were confirmed as Klebsiella sp. (n = 37), followed by E. coli (n = 32), and the remainder were classified as other Enterobacteriaceae (n = 19). A higher percentage of effluent isolates were fully susceptible to all tested antibiotics when compared to influent isolates (28.6% vs 18.6%). Resistance was most commonly observed against ampicillin (69.3% of all isolates), trimethoprim-sulfamethoxazole (11.4%), tetracycline (9.1%), and cefazolin (7.9%), and 7.9% of isolates were observed to be multidrug-resistant. While most water quality parameters were within Israeli and Palestinian wastewater reuse requirements, E. coli levels in effluent violated available standards. These findings suggest that, despite observed decreases in bacteria and an overall decrease in isolates expressing antibiotic resistance from influent to effluent, off-grid greywater treatment systems are still a potential source of both susceptible and antibiotic-resistant bacteria in the agricultural environment.

Mentholation triggers brand-specific shifts in the bacterial microbiota of commercial cigarette products.

Bacterial communities are integral constituents of tobacco products. They originate from tobacco plants and are acquired during manufacturing processes, where they play a role in the production of tobacco-specific nitrosamines. In addition, tobacco bacterial constituents may play an important role in the development of infectious and chronic diseases among users. Nevertheless, tobacco bacterial communities have been largely unexplored, and the influence of tobacco flavor additives such as menthol (a natural antimicrobial) on tobacco bacterial communities is unclear. To bridge this knowledge gap, time series experiments including 5 mentholated and non-mentholated commercially available cigarettes-Marlboro red (non-menthol), Marlboro menthol, Newport menthol box, Newport menthol gold, and Newport non-menthol-were conducted. Each brand was stored under three different temperature and relative humidity conditions. To characterize bacterial communities, total DNA was extracted on days 0 and 14. Resulting DNA was purified and subjected to PCR of the V3V4 region of the 16S rRNA gene, followed by sequencing on the Illumina HiSeq platform and analysis using the QIIME, phyloseq, metagenomeSeq, and DESeq software packages. Ordination analyses showed that the bacterial community composition of Marlboro cigarettes was different from that of Newport cigarettes. Additionally, bacterial profiles significantly differed between mentholated and non-mentholated Newports. Independently of storage conditions, tobacco brands were dominated by Proteobacteria, with the most dominant bacterial genera being Pseudomonas, unclassified Enterobacteriaceae, Bacillus, Erwinia, Sphingomonas, Acinetobacter, Agrobacterium, Staphylococcus, and Terribacillus. These data suggest that the bacterial communities of tobacco products differ across brands and that mentholation of tobacco can alter bacterial community composition of select brands. KEY POINTS: • Bacterial composition differed between the two brands of cigarettes. • Mentholation impacts cigarette microbiota. • Pseudomonas and Bacillus dominated the commercial cigarettes. Graphical abstract.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.