Relevant Bacteria Research Articles

Detection of clinically relevant antibiotic-resistant bacteria in shared fomites, waste water and municipal solid wastes disposed near residential areas of a Nigerian city.

Studies investigating environmental hotspots of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in Nigeria are limited. This study was designed to assess various environmental sources and commonly touched surfaces as potential carriers of ARB and ARGs with implications for public health. A total of 392 samples, including sewage (36), sludge (36), diapers (20), plastics (20), water sachet polythene bags (20), food wastes (20), soil beneath dump sites (20), and frequently touched surfaces such as restroom floors (80), corridors (24), door handles (56), and room floors and walls (60), were collected and screened for the presence of resistant bacteria carrying genes such as bla KPC, bla NDM-1, bla CMY-2, bla IMP, bla OXA66 and MecA. Additionally, we employed standard techniques to detect methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii . We also evaluated the effectiveness of routine disinfection procedures in eliminating ARB from restroom floors. Our findings revealed that sewage, sludge, diapers, food wastes and restroom floors are frequently contaminated with highly and moderately resistant strains of E. coli, K. pneumoniae, P. aeruginosa and MRSA. Notably, we identified two variants of the bla OXA51-like gene (bla OXA-66 and bla OXA-180) in A. baumannii isolated from these environmental sources. Furthermore, we detected seven ESBL- K. pneumoniae , five ESBL- A. baumannii , two ESBL- E. coli and one ESBL- P. aeruginosa , all carrying one or more ARGs (bla KPC, bla NDM-1, bla CMY-2), in isolates recovered from sewage, sludge, restroom floors and plastics. It is of note that ARB persisted on restroom floors even after disinfection procedures. In conclusion, this study highlights that environmental wastes indiscriminately discarded in residential areas and shared surfaces among individuals are heavily colonized by ARB carrying ARGs of significant public health importance.

Coagulase-Negative Staphylococci as an Etiologic Agent of Ovine Mastitis, with a Focus on Subclinical Forms.

The objective of this review is to investigate the distribution and the characteristics of coagulase-negative Staphylococci (CoNS) implicated in ovine mastitis, and especially in subclinical cases, in order to provide a global perspective of the current research data and analyze specific critical aspects of the issue. PRISMA guidelines were implemented in the search of the last 20 years of the related literature in two databases. In total, 139 studies were included in this review. Relevant data were tracked down, assembled, and compared. Regarding the geographical distribution, most studies originated from Europe (68), followed by South America (33). Lacaune was the most examined breed, while S. epidermidis was the predominantly identified species, representing approximately 39% of the obtained isolates. Antibiotic resistance in the relevant bacteria was documented mostly for Penicillin (32.8%) and Amoxicillin (32.1%), while biofilm- and toxin-associated genes were encountered in variable rates because significant inequalities were observed between different articles. Significantly higher rates of antimicrobial resistance were detected in Asia and South America compared to Europe. Finally, the diagnostic procedures carried out in the respective studies were evaluated. Conventional culture and biochemical tests were mostly performed for simple strain identification; therefore, further molecular investigation of isolates should be pursued in future studies, as this will provide important data regarding specific aspects of the implication of CoNS in ovine mastitis.

Antibiotic prophylaxis and hospitalization of horses subjected to median laparotomy: gut microbiota trajectories and abundance increase of Escherichia.

Horse clinics are hotspots for the accumulation and spread of clinically relevant and zoonotic multidrug-resistant bacteria, including extended-spectrum β-lactamase producing (ESBL) Enterobacterales. Although median laparotomy in cases of acute equine colic is a frequently performed surgical intervention, knowledge about the effects of peri-operative antibiotic prophylaxis (PAP) based on a combination of penicillin and gentamicin on the gut microbiota is limited. We collected fecal samples of horses from a non-hospitalized control group (CG) and from horses receiving either a pre-surgical single-shot (SSG) or a peri-operative 5-day (5DG) course of PAP. To assess differences between the two PAP regimens and the CG, all samples obtained at hospital admission (t0), on days three (t1) and 10 (t2) after surgery, were screened for ESBL-producing Enterobacterales and subjected to 16S rRNA V1-V2 gene sequencing. We included 48 samples in the SSG (n = 16 horses), 45 in the 5DG (n = 15), and 20 in the CG (for t0 and t1, n = 10). Two samples of equine patients receiving antibiotic prophylaxis (6.5%) were positive for ESBL-producing Enterobacterales at t0, while this rate increased to 67% at t1 and decreased only slightly at t2 (61%). Shannon diversity index (SDI) was used to evaluate alpha-diversity changes, revealing there was no significant difference between horses suffering from acute colic (5DG, SDImean of 5.90, SSG, SDImean of 6.17) when compared to the CG (SDImean of 6.53) at t0. Alpha-diversity decreased significantly in both PAP groups at t1, while at t2 the onset of microbiome recovery was noticed. Although we did not identify a significant SDImean difference with respect to PAP duration, the community structure (beta-diversity) was considerably restricted in samples of the 5DG at t1, most likely due to the ongoing administration of antibiotics. An increased abundance of Enterobacteriaceae, especially Escherichia, was noted for both study groups at t1. Colic surgery and PAP drive the equine gut microbiome towards dysbiosis and reduced biodiversity that is accompanied by an increase of samples positive for ESBL-producing Enterobacterales. Further studies are needed to reveal important factors promoting the increase and residency of ESBL-producing Enterobacterales among hospitalized horses.

The chitin-binding domain of Bacillus thuringiensis ChiA74 inhibits gram-negative bacterial and fungal pathogens of humans and plants

The chitinase ChiA74 is synthesized by Bacillus thuringiensis and possesses a modular organization composed of four domains. In the C-terminal of the enzyme is located the chitin-binding domain (CBD), which has not been isolated as a single unit or characterized. Here, we aimed to isolate the ChiA74's CBD as a single unit, determine the binding properties, and evaluate its antimicrobial and hemolytic activities. We cloned the ChiA74's CBD and expressed it in Escherichia coli BL21. The single domain was purified, analyzed by SDS-PAGE, and characterized. The recombinant CBD (rCBD) showed a molecular mass of ∼14 kDa and binds strongly to α-chitin, with Kd and Bmax of ∼4.7 ± 0.9 μM and 1.5 ± 0.1 μmoles/g chitin, respectively. Besides, the binding potential (Bmax/Kd) was stronger for α-chitin (∼0.31) than microcrystalline cellulose (∼0.19). It was also shown that the purified rCBD inhibited the growth of the clinically relevant Gram-negative bacteria (GNB) Vibrio cholerae, and V. parahemolyticus CVP2 with minimum inhibitory concentrations (MICs) of 121 ± 9.9 and 138 ± 3.2 μg/mL, respectively, and of one of the most common GNB plant pathogens, Pseudomonas syringae with a MIC of 230 ± 13.8 μg/mL. In addition, the rCBD possessed antifungal activity inhibiting the conidia germination of Fusarium oxysporum (MIC = 192 ± 37.5 μg/mL) and lacked hemolytic and agglutination activities against human erythrocytes. The significance of this work lies in the fact that data provided here show for the first time that ChiA74's CBD from B. thuringiensis has antimicrobial activity, suggesting its potential use against significant pathogenic microorganisms. Future works will be focused on testing the inhibitory effect against other pathogenic microorganisms and elucidating the mechanism of action.

Monitoring the battleground: exploring antimicrobial resistance and virulence factors in wound bacterial isolates.

Antibiotic resistance poses a grave global public health threat, exacerbated by widespread and often inappropriate antibiotic usage. Vigilant surveillance of antibiotic utilization and emergence of antimicrobial resistance (AMR) is essential. Of particular concern in the era of AMR is the persistent issue of chronic wound infections. To address this, we conducted a comprehensive evaluation of wound isolates from chronic wounds at Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH) in Kenya, to identify relevant bacteria and assess their drug resistance patterns.Wound samples were collected and processed using standard microbiological methods. Bacterial isolates were identified and assessed for their susceptibility to a panel of antibiotics using the Kirby-Bauer disk diffusion method. A total of 103 bacterial isolates were obtained from the wound samples, with a higher prevalence in male patients (59%). Staphylococcus aureus (20.7 %) emerged as the most predominant pathogen, followed by Klebsiella spp. (14.8 %), Pseudomonas aeruginosa spp. (14.8 %) and Escherichia coli (4.4 %) in wound samples. High levels of antibiotic resistance were observed among the isolates, with the highest resistance rates reported for cotrimoxazole (48.1 %), clindamycin (25.9 %) and erythromycin (25.9 %). Furthermore, among the isolates, 75 % produced haemolysin and protease, while 50 % produced lipase and phospholipase, factors that enhance virulence and survival. The findings of this study highlight the alarmingly high prevalence of antibiotic resistance among bacterial pathogens isolated from chronic wounds in Kenya. This poses a major challenge to the effective management of chronic wound infections. There is an urgent need to implement effective antimicrobial stewardship programs and develop new antibiotics to combat the growing threat of antibiotic resistance.

Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism

Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. 13C and 2H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of 2H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to 13C-tracers. Furthermore, isotope tracer studies in industrially relevant bacteria that metabolize complex substrates such as cellulose, hemicellulose, or lignocellulosic biomass, are challenging given the difficulty in obtaining these as isotopically labeled substrates.In this study, we examine the potential of deuterated water (2H2O) as an affordable, substrate-neutral isotope tracer for studying central carbon metabolism. We apply 2H2O labeling to investigate the reversibility of glycolytic reactions across three industrially relevant bacterial species -C. thermocellum, Z. mobilis, and E. coli-harboring distinct glycolytic pathways with unique thermodynamics. We demonstrate that 2H2O labeling recapitulates previous reversibility and thermodynamic findings obtained with established 13C and 2H labeled nutrient substrates. Furthermore, we exemplify the utility of this 2H2O labeling approach by applying it to high-substrate C. thermocellum fermentations -a setting in which the use of conventional tracers is impractical-thereby identifying the glycolytic enzyme phosphofructokinase as a major bottleneck during high-substrate fermentations and unveiling critical insights that will steer future engineering efforts to enhance ethanol production in this cellulolytic organism.This study demonstrates the utility of deuterated water as a substrate-agnostic isotope tracer for examining flux and reversibility of central carbon metabolic reactions, which yields biological insights comparable to those obtained using costly 2H-labeled nutrient substrates.

Biocide Tolerance and Impact of Sanitizer Concentrations on the Antibiotic Resistance of Bacteria Originating from Cheese.

In this study, we determined and identified the bacterial diversity of different types of artisanal and industrially produced cheese. The antibiotic (erythromycin, chloramphenicol, kanamycin, ampicillin, clindamycin, streptomycin, tetracycline, and gentamicin) and biocide (peracetic acid, sodium hypochlorite, and benzalkonium chloride) resistance of clinically relevant bacteria was determined as follows: Staphylococcus aureus, Macrococcus caseolyticus, Bacillus sp., Kocuria varians, Escherichia coli, Enterococcus faecalis, Citrobacter freundii, Citrobacter pasteurii, Klebsiella oxytoca, Klebsiella michiganensis, Enterobacter sp., Enterobacter cloacae, Enterobacter sichuanensis, Raoultella ornithinolytica, Shigella flexneri, and Salmonella enterica. Also, the effect of the sub-inhibitory concentration of three biocides on antibiotic resistance was determined. The microbiota of evaluated dairy products comprise diverse and heterogeneous groups of bacteria with respect to antibiotic and disinfectant tolerance. The results indicated that resistance was common in the case of ampicillin, chloramphenicol, erythromycin, and streptomycin. Bacillus sp. SCSSZT2/3, Enterococcus faecalis SRGT/1, E. coli SAT/1, Raoultella ornithinolytica MTT/5, and S. aureus SIJ/2 showed resistance to most antibiotics. The tested bacteria showed sensitivity to peracetic acid and a different level of tolerance to benzalkonium chloride and sodium hypochlorite. The inhibition zone diameter of antibiotics against Enterococcus faecalis SZT/2, S. aureus JS11, E. coli CSKO2, and Kocuria varians GRT/10 was affected only by the sub-inhibitory concentration of peracetic acid.

An Orphan VrgG Auxiliary Module Related to the Type VI Secretion Systems from Pseudomonas ogarae F113 Mediates Bacterial Killing.

The model rhizobacterium Pseudomonas ogarae F113, a relevant plant growth-promoting bacterium, encodes three different Type VI secretion systems (T6SS) in its genome. In silico analysis of its genome revealed the presence of a genetic auxiliary module containing a gene encoding an orphan VgrG protein (VgrG5a) that is not genetically linked to any T6SS structural cluster, but is associated with genes encoding putative T6SS-related proteins: a possible adaptor Tap protein, followed by a putative effector, Tfe8, and its putative cognate immunity protein, Tfi8. The bioinformatic analysis of the VgrG5a auxiliary module has revealed that this cluster is only present in several subgroups of the P. fluorescens complex of species. An analysis of the mutants affecting the vgrG5a and tfe8 genes has shown that the module is involved in bacterial killing. To test whether Tfe8/Tfi8 constitute an effector-immunity pair, the genes encoding Tfe8 and Tfi8 were cloned and expressed in E. coli, showing that the ectopic expression of tfe8 affected growth. The growth defect was suppressed by tfi8 ectopic expression. These results indicate that Tfe8 is a bacterial killing effector, while Tfi8 is its cognate immunity protein. The Tfe8 protein sequence presents homology to the proteins of the MATE family involved in drug extrusion. The Tfe8 effector is a membrane protein with 10 to 12 transmembrane domains that could destabilize the membranes of target cells by the formation of pores, revealing the importance of these effectors for bacterial interaction. Tfe8 represents a novel type of a T6SS effector present in pseudomonads.

Evaluating a robust and easy-to-use biological-activity-based method to assess the presence of potentially adverse bacteria at two riverbank filtration sites along the Danube river: A case study

The Laboratory Biological Activity Reaction Test (LAB-BART) is an easy-to-use assay that utilizes metabolic capabilities to process an array of substrates to semi-quantitatively assess the presence of potentially adverse bacteria in a groundwater sample. Here, we evaluated LAB-BART for the assessment of groundwater samples obtained under real-life conditions from two riverbank filtration (RBF) sites in Austria. Samples were taken monthly for an overall experimental period of six months and analyzed following the manufacturer’s recommendations for measuring iron-related, sulfate-reducing, slime-forming and denitrifying bacteria. Additional measurements were done for analyzing chemical water composition, as well as bacterial community structure to evaluate the suitability of LAB-BART by identifying relevant bacteria. Results imply that while LAB-BART could not give detailed information on bacterial concentrations, it might be able to indicate hydrologically induced changes in biogeochemical processes in a subsurface system, thus allowing operators to determine an adequate response to a potential influx of undesired bacteria. Despite its limitations, LAB-BART might therefore be a valuable tool for monitoring purposes due to its ease of use, but more research is necessary to determine its accuracy in measuring bacterial activity.

The microbiology and pathogenesis of nonfermenting Gram-negative infections.

This review provides an overview of most recent evidence about pathogenesis traits and virulence factors contributing to successful colonization or infection by P. aeruginosa , A. baumannii , S. maltophilia and B. cepacia complex, among the most clinically relevant nonfermenting Gram-negative bacteria (NFGNB). The growing clinical importance of NFGNB as important opportunistic pathogens causing difficult-to-treat infections in a fragile patients' population in stressed by numerous studies. Identification of novel virulence factors and deciphering of their mechanisms of action have greatly furthered our understanding of NFGNB pathogenesis, revealing that each pathogen-specific armamentarium of virulence factors (adhesins, motility, capsule, biofilm, lipopolysaccharide, exotoxins, exoenzymes, secretion systems, siderophores) can be likely responsible for the difference in the pathophysiology even in the context of a similar infection site. Emerging evidence of the immunomodulatory effect of some virulence factors is also acknowledged. NFGNB continue to be a serious global problem as cause of life-threatening opportunistic infections, owing to a highly heterogeneous content of virulence factors and their extensive number of intrinsic resistance mechanisms. Further efforts in development of novel effective antimicrobials and of alternative strategies targeting key virulence factors are warranted.

T9GPred: A Comprehensive Computational Tool for the Prediction of Type 9 Secretion System, Gliding Motility, and the Associated Secreted Proteins.

Type 9 secretion system (T9SS) is one of the least characterized secretion systems exclusively found in the Bacteroidetes phylum, which comprises various environmental and economically relevant bacteria. While T9SS plays a central role in bacterial movement termed gliding motility, survival, and pathogenicity, there is an unmet need for a comprehensive tool that predicts T9SS, gliding motility, and proteins secreted via T9SS. In this study, we develop such a computational tool, Type 9 secretion system and Gliding motility Prediction (T9GPred). To build this tool, we manually curated published experimental evidence and identified mandatory components for T9SS and gliding motility prediction. We also compiled experimentally characterized proteins secreted via T9SS and determined the presence of three unique types of C-terminal domain signals, and these insights were leveraged to predict proteins secreted via T9SS. Notably, using recently published experimental evidence, we show that T9GPred has high predictive power. Thus, we used T9GPred to predict the presence of T9SS, gliding motility, and associated secreted proteins across 693 completely sequenced Bacteroidetes strains. T9GPred predicted 402 strains to have T9SS, of which 327 strains are also predicted to exhibit gliding motility. Further, T9GPred also predicted putative secreted proteins for the 402 strains. In a nutshell, T9GPred is a novel computational tool for systems-level prediction of T9SS and streamlining future experimentation. The source code of the computational tool is available in our GitHub repository: https://github.com/asamallab/T9GPred. The tool and its predicted results are compiled in a web server available at: https://cb.imsc.res.in/t9gpred/.

A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum

BackgroundMicrobial production of aromatic chemicals is an attractive method for obtaining high-performance materials from biomass resources. A non-proteinogenic amino acid, 4-amino-3-hydroxybenzoic acid (4,3-AHBA), is expected to be a precursor of highly functional polybenzoxazole polymers; however, methods for its microbial production have not been reported. In this study, we attempted to produce 4,3-AHBA from glucose by introducing 3-hydroxylation of 4-aminobenzoic acid (4-ABA) into the metabolic pathway of an industrially relevant bacterium, Corynebacterium glutamicum.ResultsSix different 4-hydroxybenzoate 3-hydroxylases (PHBHs) were heterologously expressed in C. glutamicum strains, which were then screened for the production of 4,3-AHBA by culturing with glucose as a carbon source. The highest concentration of 4,3-AHBA was detected in the strain expressing PHBH from Caulobacter vibrioides (CvPHBH). A combination of site-directed mutagenesis in the active site and random mutagenesis via laccase-mediated colorimetric assay allowed us to obtain CvPHBH mutants that enhanced 4,3-AHBA productivity under deep-well plate culture conditions. The recombinant C. glutamicum strain expressing CvPHBHM106A/T294S and having an enhanced 4-ABA biosynthetic pathway produced 13.5 g/L (88 mM) 4,3-AHBA and 0.059 g/L (0.43 mM) precursor 4-ABA in fed-batch culture using a nutrient-rich medium. The culture of this strain in the chemically defined CGXII medium yielded 9.8 C-mol% of 4,3-AHBA from glucose, corresponding to 12.8% of the theoretical maximum yield (76.8 C-mol%) calculated using a genome-scale metabolic model of C. glutamicum.ConclusionsIdentification of PHBH mutants that could efficiently catalyze the 3-hydroxylation of 4-ABA in C. glutamicum allowed us to construct an artificial biosynthetic pathway capable of producing 4,3-AHBA on a gram-scale using glucose as the carbon source. These findings will contribute to a better understanding of enzyme-catalyzed regioselective hydroxylation of aromatic chemicals and to the diversification of biomass-derived precursors for high-performance materials.

A Rapidly Responsive Sensor for Wireless Detection of Early and Mature Microbial Biofilms

AbstractBiofilm‐associated infections, which are able to resist antibiotics, pose a significant challenge in clinical treatments. Such infections have been linked to various medical conditions, including chronic wounds and implant‐associated infections, making them a major public‐health concern. Early‐detection of biofilm formation offers significant advantages in mitigating adverse effects caused by biofilms. In this work, we aim to explore the feasibility of employing a novel wireless sensor for tracking both early‐stage and matured‐biofilms formed by the medically relevant bacteria Staphylococcus aureus and Pseudomonas aeruginosa. The sensor utilizes electrochemical reduction of an AgCl layer bridging two silver legs made by inkjet‐printing, forming a part of near‐field‐communication tag antenna. The antenna is interfaced with a carbon cloth designed to promote the growth of microorganisms, thereby serving as an electron source for reduction of the resistive AgCl into a highly‐conductive Ag bridge. The AgCl−Ag transformation significantly alters the impedance of the antenna, facilitating wireless identification of an endpoint caused by microbial growth. To the best of our knowledge, this study for the first time presents the evidence showcasing that electrons released through the actions of bacteria can be harnessed to convert AgCl to Ag, thus enabling the wireless, battery‐less, and chip‐less early‐detection of biofilm formation.

Antimicrobial resistance- and pathogen patterns in the fecal microbiota of sows and their offspring in German commercial pig farms.

Reducing antibiotic use is one of the biggest challenges in pig farming, as antibiotics have been used for years to control typical problems such as newborn or post-weaning diarrhea. The pressure a one health approach has created on animal production regarding antimicrobial resistance is an opportunity to find other strategies against enterobacterial pathogens in suckling and weaned piglets. A farm-specific approach could have a good success due to the individual farm structures in Germany and other countries. In this study, non-metric multidimensional scaling, hierarchical clustering, and latent class analysis were used to determine the impact of antibiotic use on antibiotic resistance patterns and pathogen prevalence in 20 German pig farms. This may help to develop individualized health strategies. 802 fresh fecal samples were collected from sows and piglets from 20 piglet production and rearing farms at different production times (sows antepartum and postpartum, suckling piglets, weaned piglets). In addition, the use of antibiotics was recorded. DNA extracts were subjected to quantitative real-time qPCR with primers specific for antibiotic resistance genes (int1, sul1-3, dfrA1, mcr-1, blaCTX-M), and virulence factors of relevant bacteria (C. difficile, C. perfringens, Salmonella, Escherichia/Shigella/Hafnia, E. coli). Linear and logistic regression models were used to analyze the relationship between different antibiotics and the major genes contributing to the clustering of observations for the different animal groups. Clustering revealed different farm clusters for sows, suckling piglets, and weaned piglets, with the most remarkable diversity in antibiotic use among weaned piglets. Amoxicillin, lincomycin, and enrofloxacin were identified as the most probable cause of increased odds of the presence of relevant antibiotic resistance genes (mcr1, dfrA1, blaCTX-M). Still, direct effects of a specific antibiotic on its associated resistance gene were rare. Enrofloxacin and florfenicol favored the occurrence of C. difficile in sows. The E. coli fimbriae genes were less affected by antibiotic use in sows and piglets, but the F4 fimbriae gene could be associated with the integrase 1 gene in piglets. The results confirm that multidrug-resistant enterobacteria are widespread in German pig farms and give awareness of the impact of current antibiotic use while searching for alternative health strategies.

Gut microbiology of UK care home residents: a cross-sectional analysis from a randomised controlled trial

ObjectiveTo describe the prevalence of potentially clinically relevant gut pathogens and associations with the carriage of resistant organisms in UK care home residents. MethodsStool samples were collected pre-randomisation from care home residents participating in a randomised placebo-controlled trial. Cultivable clinically relevant bacteria were analysed. Antimicrobial susceptibility testing was performed by agar dilution (amoxicillin, co-amoxiclav, gentamicin, trimethoprim, nitrofurantoin, and ciprofloxacin). We also aimed to detect resistance to third-generation cephalosporins, carbapenems, and vancomycin. ResultsStool samples were available for 159/310 residents participating in the trial (51%) from 23 care homes between 2016 and 2018. In total, 402 bacterial isolates were cultured from 158 stool samples and 29 different species were cultured. The five most common species were Escherichia coli (155/158, 98%), Pseudomonas aeruginosa (40/158, 25%), Enterococcus faecalis (35/158, 22%), Enterococcus faecium (30/158, 19%), and Proteus mirabilis (25/158, 16%). Enterobacterales isolates were cultured from 157 samples (99%), and resistance to at least one of the tested antimicrobials was found in 119 of these (76%). There were high levels of variation in outcomes by care home. DiscussionWe demonstrated that care home residents harbour significant levels of antimicrobial-resistant organisms in their stool. This work emphasises the importance of both enhanced infection control practices and antimicrobial stewardship programmes to support the appropriate use of antimicrobials in this setting.

Synthesis of polymerizable quaternary ammonium bromides and their efficacy against pathogenic and food spoilage bacteria

Despite rigorous cleaning and sanitization protocols, food manufacturing facilities can harbor persistent microorganisms that can cause post processing contamination leading to food safety and food spoilage issues. Of particular concern are ‘growth niches’, regions within a manufacturing facility more likely to accumulate water and debris because they are outside the zones of routine cleaning and sanitization. Such growth niches present a challenge in controlling post-processing microbial contamination which can affect the safety and shelf life of foods and beverages. In recent years, researchers have explored using antimicrobial and nonfouling coatings to control microbial persistence and cross contamination. A challenge in identifying effective coatings is the limited research on efficacy of antimicrobial monomers (suitable for polymerization into antimicrobial coatings) on microorganisms relevant in food safety and spoilage. In this work, we synthesize and characterize the efficacy of five antimicrobial quaternary ammonium bromide (QAB) monomers against pathogenic and food spoilage organisms. The QAB monomers are end-capped with dihydroxy moieties suitable for subsequent polymerization into antimicrobial polyurethanes and polyesters. N,N-bis(2-hydroxyethyl)-N-methyl-N-R-1-ammonium bromides (R= -octan {C8QAB}, -decan {C10QAB}, -dodecan {C12QAB}, -hexadecan {C16QAB}, -octadecan {C18QAB}) were synthesized and characterized using spectral techniques (Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy). Minimum inhibitory concentrations were determined against relevant food safety and food spoilage bacteria Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli, and Pseudomonas poae. Sixteen and eighteen carbon length QAB monomers inhibited growth of L. monocytogenes and P. poae at a concentration of 3 ppm, while sixteen carbon length QAB monomerinhibited growth of Salmonella Typhimurium at 26 ppm and against Escherichia coli at 13 ppm. Quaternary ammonium compounds capped with polymerizable end groups such as those reported here offer an opportunity to reduce persistence and cross-contamination of food pathogens and food spoilage bacteria with particular relevance in growth niches outside of routine cleaning and sanitization regimens.

Sediment-driven plastisphere community assembly on plastic debris in tropical coastal and marine environments

Coastal habitats have been suggested to serve as a sink for unaccounted plastic debris, i.e., “missing plastic” in the sea, and hence, a hotspot of plastic pollution in the marine and coastal environments. Although the accumulation of plastic debris may pose significant threats to coastal ecosystems, we know little about the fate of these plastic debris and their ecological impacts due to the lack of studies on plastic-microbe interactions in coastal habitats, especially for the tropical marine and coastal environments. In this study, we collected plastic debris from 14 sites consisting of various coastal ecosystems (seagrass meadows, mangrove forests, and beaches), and marine ecosystem (coral reef) around Singapore and characterized the prokaryotic and eukaryotic microbial communities colonized on them. Our results showed that the composition of plastisphere communities in these intertidal ecosystems was predominantly influenced by the sediment than by the plastic materials. Compared with surrounding sediment and seawater, the plastic debris enriched potential plastic degraders, such as Muricauda, Halomonas, and Brevundimonas. The plastic debris was also found to host taxa that play significant roles in biogeochemical cycles (e.g., cyanobacteria, Erythrobacter), hygienically relevant bacteria (e.g., Chryseobacterium, Brevundimonas), and potential pathogens that may negatively impact the health of coastal ecosystems (e.g., Thraustochytriaceae, Labyrinthulaceae, Flavobacterium). Taken together, our study provides valuable insights into the plastic-microbe interactions in tropical coastal and marine ecosystems, highlighting the urgent need for plastisphere studies to understand the fate and ecological impacts of plastic debris accumulated in coastal habitats.

In Vitro Activity of Two Novel Antimicrobial Compounds on MDR-Resistant Clinical Isolates.

The development of novel antibiotics is mandatory to curb the growing antibiotic resistance problem resulting in difficult-to-treat bacterial infections. Here, we have determined the spectrum of activity of cystobactamids and chelocardins, two novel and promising classes of molecules with different modes of action. A panel of 297 clinically relevant Gram-negative and Gram-positive isolates with different antibiotic susceptibility profiles, going from wild type to multi- or even extremely drug resistant (MDR, XDR) and including carbapenem-resistant isolates, were tested using broth microdilution assays to determine the minimal inhibitory concentrations (MICs), MIC50s and MIC90s of two cystobactamids derivatives (CN-861-2 and CN-DM-861) and two chelocardin derivatives (CHD and CDCHD). Cystobactamids revealed potent activities on the majority of tested Enterobacterales (MIC50s ranging from 0.25 to 4 µg/mL), except for Klebsiella pneumoniae isolates (MIC50s is 128 µg/mL). Pseudomonas aeruginosa and Acinetobacter baumannii showed slightly higher MIC50s (4 µg/mL and 8 µg/mL, respectively) for cystobactamids. Chelocardins inhibited the growth of Enterobacterales and Stenotrophomas maltophilia at low to moderate MICs (0.25-16 µg/mL) and the chemically modified CDCHD was active at lower MICs. A. baumannii and P. aeruginosa were less susceptible to these molecules with MICs ranging from 0.5 to 32 µg/mL. These molecules show also interesting in vitro efficacies on clinically relevant Gram-positive bacteria with MICs of 0.125-8 µg/mL for cystobactamids and 0.5-8 µg/mL for chelocardins. Taken together, the cystobactamid CN-DM-861 and chelocardin CDCHD showed interesting antibiotic activities on MDR or XDR bacteria, without cross-resistance to clinically relevant antibiotics such as carbapenems, fluoroquinolones, and colistin.

From accurate genome sequence to biotechnological application: The thermophile Mycolicibacterium hassiacum as experimental model.

Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc2 155 being the most common one. In this work, we focus on the use of a thermophilic mycobacterium, Mycolicibacterium hassiacum, which grows optimally above 50°C, as an emerging experimental model valid to extend our general knowledge of mycobacterial biology as well as for application purposes. To that end, accurate genomic sequences are key for gene mining, the study of pathogenicity or lack thereof and the potential for gene transfer. The combination of long- and short-massive sequencing technologies is strictly necessary to remove biases caused by errors specific to long-reads technology. By doing so in M. hassiacum, we obtained from the curated genome clues regarding the genetic manipulation potential of this microorganism from the presence of insertion sequences, CRISPR-Cas, type VII ESX secretion systems, as well as lack of plasmids. Finally, as a proof of concept of the applicability of M. hassiacum as a laboratory and industrial model, we used this high-quality genome of M. hassiacum to successfully knockout a gene involved in the use of phytosterols as source of carbon and energy, using an improved gene cassette for thermostable selection and a transformation protocol at high temperature.

Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations.

Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated.