Facultative Pathogenic Bacteria Research Articles

Findaureus: An open-source application for locating Staphylococcus aureus in fluorescence-labelled infected bone tissue slices.

Staphylococcus aureus (S. aureus) is a facultative pathogenic bacterium that can cause infections in various tissue types in humans. Fluorescence imaging techniques have been employed to visualize the bacteria in ex-vivo samples mostly in research, aiding in the understanding of the etiology of the pathogen. However, the multispectral images generated from fluorescence microscopes are complex, making it difficult to locate bacteria across image files, especially in consecutive planes with different imaging depths. To address this issue, we present Findaureus, an open-source application specifically designed to locate and extract critical information about bacteria, especially S. aureus. Due to the limited availability of datasets, we tested the application on a dataset comprising fluorescence-labelled infected mouse bone tissue images, achieving an accuracy of 95%. We compared Findaureus with other state-of-the-art image analysis tools and found that it performed better, given its specificity toward bacteria localization. The proposed approach has the potential to aid in medical research of bone infections and can be extended to other tissue and bacteria types in the future.

Regulation of the Gene for Alanine Racemase Modulates Amino Acid Metabolism with Consequent Alterations in Cell Wall Properties and Adhesive Capability in Brucella spp.

Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp.

Antibiotic resistances from slaughterhouse effluents and enhanced antimicrobial blue light technology for wastewater decontamionation

The frequencies of 6 different facultative pathogenic bacteria of the ESKAPE group (priority list WHO) and a total of 14 antibiotic resistance genes (ARGs) with different priorities for human medicine were quantified in wastewaters of poultry and pig slaughterhouses using molecular biological approaches. Raw sewage from poultry and pig slaughterhouses was found to be contaminated not only with facultative pathogenic bacteria but also with various categories of clinically relevant ARGs, including ARGs against the reserve antibiotics group. The concentration of the different gene targets decreased after on-site conventional biological or advanced oxidative wastewater treatments, but was not eliminated. Hence, the antimicrobial BlueLight (aBL) in combination with a porphyrin photo-sensitizer was studied with ESKAPE bacteria and real slaughterhouse wastewaters. The applied broad LED-based blue light (420–480 nm) resulted in groups of sensitive, intermediate, and non-sensitive ESKAPE bacteria. The killing effect of aBL was increased in the non-sensitive bacteria Klebsiella pneumoniae and Enterococcus faecium due to the addition of porphyrins in concentrations of 10−6 M. Diluted slaughterhouse raw wastewater was treated with broad spectrum aBL and in combination with porphyrin. Here, the presence of the photo-sensitizer enhanced the aBL biocidal impact.

Molecular Docking Simulation-based Pharmacophore Modeling to Design Translation Inhibitors Targeting c-di-GMP Riboswitch of Vibrio cholera

Background: Vibrio cholera is a facultative pathogenic bacterium that causes cholera pandemics, primarily in nations with hot and humid climates and large bodies of water containing a large quantity of organic debris. Consumption of V. cholera contaminated water or food causes acute diarrheal illness, followed by severe dehydration and mortality. Cholera is a highly infectious illness, with over 4 million cases recorded globally each year, and over a hundred thousand deaths. Objective: The only known therapy for cholera infection is oral rehydration solution along with antibiotics. Excessive antibiotic use causes pathogens to acquire antimicrobial drug resistance, resulting in a loss of efficacy. Furthermore, antibiotics are accompanied with a plethora of unfavorable side effects, restricting their usage. Methods: A Riboswitch is a non-homologous proteinaceous therapeutic target that plays a regulatory role in the crucial process of bacterial translation. As a result, the bacterial riboswitch was investigated as a surrogate target for developing a therapeutic medication against V. cholera. Results: In-silico screening with 24407 ligands was performed against the bacterial riboswitch to identify potential lead candidates, followed by pharmacophore modeling and bioisosteric lead modifications to design potential leads having an antagonistic impact on the pathogenic bacterial riboswitch Conclusion: The riboswitch-based innovative therapy was anticipated to be devoid of the issues connected with the development of antimicrobial drug resistance as well as the unwanted side effects associated with antibiotic usage.

Success of different therapy concepts for bacterial endometritis in stud farm practice

Bacterial endometritis is among the major problems in equine reproduction and is usually treated with antibiotics. Data on clinical success rates of antibiotic as well as non-antibiotic treatment is scarce. The study aimed to collect data of mares diagnosed with intrauterine growth of facultative pathogenic bacteria and compare the outcome of different therapies for bacterial endometritis in stud farm practice. Data on mares (different breeds) with positive uterine culture results was collected retrospectively in private veterinary practices (n=5) from 2018 to 2022. Information relating to 30 factors (mare, diagnostics, therapy and pregnancy rate) of cases with bacterial endometritis (Endometritis, n=1005) were recorded and analysed. Possible effects on treatment success (negative control swab or positive pregnancy result in the first heat after treatment) were tested by binomial logistic regression. For comparison of pregnancy rates, data on mares with negative uterine culture and without any treatment (Control; n=446) was also collected and compared with Endometritis group by chi square testing. In group Endometritis, uterine swabbing (technique: 72% double-guarded swab, 28% low-volume lavage) revealed presence of β-hemolytic streptococci in most cases (n=918). Pseudomonas aeruginosa (n=21), Klebsiella pneumoniae (n=15), Staphylococcus aureus (n=13) and Candida sp. (n=10) were observed less frequently. Results positive for E. coli were not included. In group Endometritis, treatment occurred regardless of the cycle stage either with trimethoprim-sulfonamides (n=532), procain-penicillium (n=277),marbofloxacin (n=71), other antibiotics (n=66) or without antibiotics (n=59) after susceptibility testing (performed in 549/1005 cases). Antibiotics were administered rather intrauterine (n=34) than systemically (n=911). Uterine lavage was reported in 46% of mares. Additional local treatment with different substances (e.g., n-acetylcysteine, dimethyl sulfoxide or Coca Cola) was performed in 164 mares. Uterotonic drugs were administered in 39.9% of mares. A control uterine culture was not obtained in most cases (n=677). Different breeding programs in this study included artificial insemination (AI) with chilled semen (n=667), AI with frozen semen (n=169), transfer of fresh (n=112) or cryopreserved (n=27) embryos and natural cover (n=27). In the control group, AI with chilled semen (n=230), AI with frozen semen (n=106), embryo transfer (fresh: n=49; cryopreserved: n=58) and natural cover (n=2) were used. After the first breeding after treatment, pregnancy rate was 47% in group Endometritis and 54% in the control group (p<0.05). End of season pregnancy rate was 72% in treated mares and 76% in the control group (p>0.05). Treatment success was affected significantly by duration of antibiotic treatment, usage of uterotonic drugs, veterinary practice, and mare's age, but neither by the chosen antibiotic drug nor administration route nor covering method. In conclusion, end of season pregnancy rate did not differ among mares treated for bacterial endometritis vs. mares with negative uterine cultures.

Enhancement of photocatalytic-based processes by mono- and bimetallic (CuPd) rutile loaded nanoparticles for antibiotic resistance genes and facultative pathogenic bacteria removal

The aim of the study was the strong reduction of facultative pathogenic bacteria (FPB), and clinically relevant antibiotic resistance genes (ARGs) from secondary effluent. To evaluate the ARGs removal efficiency comparative study ofindividual unit processes and combined AOPs has been performed. The present work investigated: i) removal of selected ARGs, namely blaTEM, ermB, qnrS, tetM, five FPB as well as 16S rDNA and the integrase gene intl1 involved in horizontal gene transfer; ii) bacterial regrowth potential after treatment; (iii) the removal of the total genomic DNA content, by mono- and bimetallic TiO2-CuPd rutile photocatalysts under visible light radiation in real secondary effluent.The nanoparticles were characterized by UV–vis, XRD, HRTEM, Raman and XPS. TiO2-CuPd/VIS inactivation gives the highest (more than 2 log units removal) efficiency for almost all targets (blaTEM, ermB, tetM, intl 1, and 16S rRNA) compared to monometallic catalysts. The highest efficiency toward blaTEM was achieved after ozonation and TiO2-CuPd/O3/VIS (3.22 and 3.23 LRV, respectively). It was found that despite similar FPB inactivation, lower DNA destruction was found after O3 compared to TiO2-CuPd/O3/VIS treatment. The blaTEM and enterococci were completely removed during TiO2-CuPd/O3/Vis and TiO2-Cu/O3/Vis. While, catalytic ozonation leads to only slightly blaTEM removal (0.64 and 0.44 LRV for TiO2-CuPd/O3 and TiO2-Cu/O3, respectively). Photocatalytic ozonation with the application of TiO2-CuPd leads to significant removal of FPB and ARGs due to synergistic effect between Pd and Cu, which leads to acceleration of ozone – catalysts reaction. Photocatalytic ozonation has the greatest potential giving promising possibilities for eliminating AMR.

Facultative pathogenic bacteria and antibiotic resistance genes in swine livestock manure and clinical wastewater: A molecular biology comparison

Manure contains vast amounts of biological contaminants of veterinary origin. Only few studies analyse clinically critical resistance genes against reserve antibiotics in manure. In general, resistances against these high priority antibiotics involve a high potential health risk. Therefore, their spread in the soil as well as the aquatic environment has to be prevented.Manures of 29 different swine livestock were analysed. Abundances of facultative pathogenic bacteria including representatives of the clinically critical ESKAPE-pathogens (P. aeruginosa, K. pneumoniae, A. baumannii, E. faecium) and E. coli were investigated via qPCR. Antibiotic resistance genes against commonly used veterinary antibiotics (ermB, tetM, sul1) as well as various resistance genes against important (mecA, vanA) and reserve antibiotics (blaNDM, blaKPC3, mcr-1), which are identified by the WHO, were also obtained by qPCR analysis. The manures of all swine livestock contained facultative pathogenic bacteria and commonly known resistance genes against antibiotics used in veterinary therapies, but more important also a significant amount of clinically critical resistance genes against reserve antibiotics for human medicine. To illustrate the impact the occurrence of these clinically critical resistance genes, comparative measurements were taken of the total wastewater of a large tertiary care hospital (n = 8). Both manure as well as raw hospital wastewaters were contaminated with significant abundances of gene markers for facultative pathogens and with critical resistance genes of reserve antibiotics associated with genetic mobile elements for horizontal gene transfer. Hence, both compartments bear an exceptional potential risk for the dissemination of facultative pathogens and critical antibiotic resistance genes.

Application of microwave heating in the production of homogeneous paste from the fruits of small-fruited apple trees

The paper presents data on the chemical, physical and microbiological changes during storage of paste from the fruits of small-fruited apple trees processed with microwaves. After six month of storage the content of water soluble solids increased 1.02 times (from 21.50 % to 21.93 %). The sugar-acid index decreased 1.23 times (from 10.25 % to 8.35 %); the titratable acidity increased 1.13 times (from 9.92 g/dm3 to 11.21 g/dm3). The mass fraction of pectin substances decreased 2.51 times (from 2.31 % to 0.92 %) and the content of Vitamin C decreased 1.26 times (from 24.1 mg/100 g to 19.12 mg/100 g). The number of mesophilic aerobic and facultative anaerobic microorganisms did not exceed 200 colony-forming units (CFU) per gram. The number of yeasts did not exceed 260 CFU/g and the number of filamentous fungi did not exceed 620 CFU/g. No pathogenic or facultative pathogenic bacteria were found during all the period of storage.

Platelets, Bacterial Adhesins and the Pneumococcus.

Systemic infections with pathogenic or facultative pathogenic bacteria are associated with activation and aggregation of platelets leading to thrombocytopenia and activation of the clotting system. Bacterial proteins leading to platelet activation and aggregation have been identified, and while platelet receptors are recognized, induced signal transduction cascades are still often unknown. In addition to proteinaceous adhesins, pathogenic bacteria such as Staphylococcus aureus and Streptococcus pneumoniae also produce toxins such as pneumolysin and alpha-hemolysin. They bind to cellular receptors or form pores, which can result in disturbance of physiological functions of platelets. Here, we discuss the bacteria-platelet interplay in the context of adhesin–receptor interactions and platelet-activating bacterial proteins, with a main emphasis on S. aureus and S. pneumoniae. More importantly, we summarize recent findings of how S. aureus toxins and the pore-forming toxin pneumolysin of S. pneumoniae interfere with platelet function. Finally, the relevance of platelet dysfunction due to killing by toxins and potential treatment interventions protecting platelets against cell death are summarized.

Bacterial Toxins from Staphylococcus aureus and Bordetella bronchiseptica Predispose the Horse's Respiratory Tract to Equine Herpesvirus Type 1 Infection.

Respiratory disease in horses is caused by a multifactorial complex of infectious agents and environmental factors. An important pathogen in horses is equine herpesvirus type 1 (EHV-1). During co-evolution with this ancient alphaherpesvirus, the horse’s respiratory tract has developed multiple antiviral barriers. However, these barriers can become compromised by environmental threats. Pollens and mycotoxins enhance mucosal susceptibility to EHV-1 by interrupting cell junctions, allowing the virus to reach its basolateral receptor. Whether bacterial toxins also play a role in this impairment has not been studied yet. Here, we evaluated the role of α-hemolysin (Hla) and adenylate cyclase (ACT), toxins derived from the facultative pathogenic bacterium Staphylococcus aureus (S. aureus) and the primary pathogen Bordetella bronchiseptica (B. bronchiseptica), respectively. Equine respiratory mucosal explants were cultured at an air–liquid interface and pretreated with these toxins, prior to EHV-1 inoculation. Morphological analysis of hematoxylin–eosin (HE)-stained sections of the explants revealed a decreased epithelial thickness upon treatment with both toxins. Additionally, the Hla toxin induced detachment of epithelial cells and a partial loss of cilia. These morphological changes were correlated with increased EHV-1 replication in the epithelium, as assessed by immunofluorescent stainings and confocal microscopy. In view of these results, we argue that the ACT and Hla toxins increase the susceptibility of the epithelium to EHV-1 by disrupting the epithelial barrier function. In conclusion, this study is the first to report that bacterial exotoxins increase the horse’s sensitivity to EHV-1 infection. Therefore, we propose that horses suffering from infection by S. aureus or B. bronchiseptica may be more susceptible to EHV-1 infection.

Environmental Surveillance and Characterization of Antibiotic Resistant Staphylococcus aureus at Coastal Beaches and Rivers on the Island of Hawai'i.

Staphylococcus aureus are human facultative pathogenic bacteria and can be found as contaminants in the environment. The aim of our study was to determine whether methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolated from coastal beach and river waters, anchialine pools, sand, and wastewater on the island of Hawaiʻi, Hawaiʻi, are a potential health risk. Samples were collected from three regions on Hawaiʻi Island from July to December 2020 during the COVID-19 pandemic and were characterized using whole-genome sequencing (WGS). From WGS data, multilocus sequence typing (MLST), SCCmec type, antimicrobial resistance genes, virulence factors, and plasmids were identified. Of the 361 samples, 98.1% were positive for Staphylococcus spp. and 7.2% were S. aureus positive (n = 26); nine MRSA and 27 MSSA strains were characterized; multiple isolates were chosen from the same sample in two sand and seven coastal beach water samples. The nine MRSA isolates were multi-drug resistant (6–9 genes) sequence type (ST) 8, clonal complex (CC) 8, SCCmec type IVa (USA300 clone), and were clonally related (0–16 SNP differences), and carried 16–19 virulence factors. The 27 MSSA isolates were grouped into eight CCs and 12 STs. Seventy-eight percent of the MSSA isolates carried 1–5 different antibiotic resistance genes and carried 5–19 virulence factors. We found S. aureus in coastal beach and river waters, anchialine pools, and sand at locations with limited human activity on the island of Hawaiʻi. This may be a public health hazard.

Codon usage bias and environmental adaptation in microbial organisms

In each genome, synonymous codons are used with different frequencies; this general phenomenon is known as codon usage bias. It has been previously recognised that codon usage bias could affect the cellular fitness and might be associated with the ecology of microbial organisms. In this exploratory study, we investigated the relationship between codon usage bias, lifestyles (thermophiles vs. mesophiles; pathogenic vs. non-pathogenic; halophilic vs. non-halophilic; aerobic vs. anaerobic and facultative) and habitats (aquatic, terrestrial, host-associated, specialised, multiple) of 615 microbial organisms (544 bacteria and 71 archaea). Principal component analysis revealed that species with given phenotypic traits and living in similar environmental conditions have similar codon preferences, as represented by the relative synonymous codon usage (RSCU) index, and similar spectra of tRNA availability, as gauged by the tRNA gene copy number (tGCN). Moreover, by measuring the average tRNA adaptation index (tAI) for each genome, an index that can be associated with translational efficiency, we observed that organisms able to live in multiple habitats, including facultative organisms, mesophiles and pathogenic bacteria, are characterised by a reduced translational efficiency, consistently with their need to adapt to different environments. Our results show that synonymous codon choices might be under strong translational selection, which modulates the choice of the codons to differently match tRNA availability, depending on the organism’s lifestyle needs. To our knowledge, this is the first large-scale study that examines the role of codon bias and translational efficiency in the adaptation of microbial organisms to the environment in which they live.

Determination of antibiotic resistance genes in a WWTP-impacted river in surface water, sediment, and biofilm: Influence of seasonality and water quality

Many pathogenic bacteria are adapted to live in aquatic habitats, which makes rivers possible sources and spread pathways of antibiotic resistance, since they usually receive effluents from wastewater treatment plants (WWTP), possibly containing antibiotic residues and also antibiotic-resistant bacteria. This study investigates different monitoring strategies to identify the occurrence of antibiotic-resistant bacteria in rivers. We analyzed the presence of 13 antibiotic resistance genes (ARGs) and seven gene markers for facultative pathogenic bacteria (FPB) with qPCR in sampling sites upstream and downstream of a small WWTP in Southern Germany. Five sampling campaigns were conducted from February to June 2019. Surface water, sediment, and biofilm samples were analyzed. The biofilm was collected from an artificial sampler placed in the river. blaTEM, ermB, tetM, and sul1 genes were detected in all samples analyzed. The results showed there was a previous background in the river, but the WWTP and the water quality of the river influenced the concentration and occurrence of ARGs and FPB. Genes representing resistance against strong or last-resort antibiotics, such as mecA, blaCMY-2, blaKPC-3, and mcr-1, and multidrug resistance were also detected, mainly in samples collected downstream of the WWTP. Downstream of the WWTP, the occurrence of ARG and FPB correlated with ammoniacal nitrogen, while upstream of the WWTP correlated with turbidity, suspended solids, and seasonal factors such as UVA radiation and the presence of macrophytes. Biofilm samples presented higher abundances of ARGs and FPB. The biofilm sampler was efficient and allowed to collect biofilms from specific periods, which helped to identify seasonal patterns.

Clinically relevant antibiotic-resistant bacteria in aquatic environments – An optimized culture-based approach

The emergence of antibiotic-resistant clinically relevant facultative pathogenic bacteria in the environment has become one of the most important global health challenges. Antibiotic-resistant bacteria (ARB) have been found in surface waters and wastewater treatment plants. Drinking water guidelines and the EU bathing water directive 2006/7/EC include the surveillance of defined microbiological parameters on species level, while the monitoring of ARB is missing in all existing guidelines. However, standardized methods for the detection of ARB exist for clinical investigations of human materials only. They are based on cultivation on selective agar plates. These methods cannot be used directly for environmental samples, because of the high amount and diversity of bacterial background flora which interferes with the detection of human-relevant ARB. The aim of this study was to introduce a proposal for future normative standard operation procedures, with international relevance, for the culture-based detection of clinically-relevant antibiotic resistant bacteria in aquatic environmental samples like wastewater and surface water: gram-negative bacteria resistant against 3rd generation cephalosporins (ESBL) and against carbapenems (CARBA), gram-positive vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). The final adaptation of standardized cultivation methods included increasing the standard incubation temperature from 36 °C to 42 °C, which effectively inhibits the environmental background flora on agar plates while the desired target species survive. This enables the detection of target species in suitable sample volumes. Putative target colonies which belong to the remaining background flora had to be excluded by morphological and physiological differentiation. Therefore, a time and cost optimized testing scheme with good performance was developed, which allows an effective exclusion of non-target isolates in samples. Depending on the target species and sample type, sensitivity of up to 100% is achieved, and specificity ranges from 91.1% to 99.7%, while the positive predictive value, negative predicted value and accuracy rate are always >90%.

Research Note: Tracing pathways of entry and persistence of facultative pathogenic and antibiotic-resistant bacteria in a commercial broiler farm with substantial health problems

On a commercial broiler farm with substantial health problems, shown by a reported loss rate of approx. 10% and regular antibiotic use, samples were taken at different locations in 2 barns, with the aim of analyzing possible entry routes and persistence of pathogens and antibiotic-resistant bacteria as well as revealing weak points in sanitation. Therefore, swab samples for biofilm and water samples from animal drinking water lines and the spray cooling system were taken twice immediately before restocking. In addition, swab samples from drain holes and air samples were collected. At restocking, hatchlings that died during transportation and chick paper were sampled. All samples were analyzed for the occurrence of Pseudomonas aeruginosa, total coliform count, and antibiotic-resistant bacteria, namely, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., Acinetobacter baumannii, P. aeruginosa, and vancomycin-resistant Enterococci (VRE). No MRSA or VRE were detectable. In all samples from drinking water and sprinkler system pipes, P. aeruginosa was detectable; in most cases, antibiotic-resistant P. aeruginosa was also detected, with varying resistance profiles. Samples from the hatchlings and chick paper were contaminated with antibiotic-resistant Enterobacter spp., with resistance to piperacillin, fosfomycin, and the third-generation cephalosporins cefotaxime and ceftazidime. Therefore, the initial entry of antibiotic-resistant Enterobacteriaceae likely occurred via exposure at the hatchery, resulting in colonization of the chicks. Animals on the fattening farm were treated with colistin, amoxicillin, and lincomycin in the 3 production cycles before sampling. Owing to the frequent administration of several antibiotic classes during the fattening period via piped water in both barns, resistance of isolates from water pipes accumulated, showing additional resistance to chloramphenicol and frequently to ciprofloxacin and levofloxacin. To prevent the development of secondary diseases caused by the facultative pathogen P. aeruginosa in chicks with a weak immune status, the hygiene management for drinking water lines and the spray cooling system was changed. These changes resulted in an improvement in water line sanitation, shown by the absence of antibiotic-resistant bacteria and rare detection of P. aeruginosa.

The Serratia grimesii outer membrane vesicles-associated grimelysin triggers bacterial invasion of eukaryotic cells.

Serratia grimesii are facultative pathogenic bacteria that can penetrate a wide range of host cells and cause infection, especially in immunocompromised patients. Previously, we have found that bacterial metalloprotease grimelysin is a potential virulence determinant of S. grimesii invasion (E. S. Bozhokina et al., (2011).Cell Biology International, 35(2), 111-118). Protease is characterized as an actin-hydrolyzing enzyme with a narrow specificity toward other cell proteins. It is not known, however, whether grimelysin is transported into eukaryotic cells. Here, we show, for the first time, that S. grimesii can generate outer membrane vesicles (OMVs) displayed specific proteolytic activity against actin, characteristic of grimelysin. The presence of grimelysin was also confirmed by the Western blot analysis of S. grimesii OMVs lysate. Furthermore, confocal microscopy analysis revealed that the S. grimesii grimelysin-containing OMVs attached to the host cell membrane. Finally, pretreatment of HeLa cells with S. grimesii OMVs before the cells were infected with bacteria increased the bacterial penetration several times. These data strongly suggest that protease grimelysin promotes S. grimesii internalization by modifying bacterial and/or host molecule(s) when it is delivered as a component of OMVs.

Evaluation of antibiotic resistance dissemination by wastewater treatment plant effluents with different catchment areas in Germany

The study quantified the abundances of antibiotic resistance genes (ARGs) and facultative pathogenic bacteria (FPB) as well as one mobile genetic element in genomic DNA via qPCR from 23 different wastewater treatment plant (WWTP) effluents in Germany. 12 clinically relevant ARGs were categorized into frequently, intermediately, and rarely occurring genetic parameters of communal wastewaters. Taxonomic PCR quantifications of five FPB targeting Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and enterococci were performed. The WWTPs differed in their catchment areas being impacted by hospitals, food processing companies, or housing areas only. The total discharges of the analyzed ARGs and FPB were found to cluster independently of the sizes of the WWTPs with a maximum difference of two log units within one cluster. Initially, quantitative data evaluations revealed no significant difference between ARG categories and WWTP catchment areas. More distinct correlations became obvious with a Pearson correlation approach, where each single taxonomic marker is compared to each ARG target. Here, increased correlation of FPB (i.e. E. coli, K. pneumoniae, P. aeruginosa, and enterococci) with clinically relevant ARGs of the category of rarely occurring resistance genes (blaNDM-1, vanA) was found in WWTP effluents being influenced by hospital wastewaters.

Multi-drug resistant facultative pathogenic bacteria colonizing the vagina of pregnant women with premature rupture of membrane, Tanzania

Background: Premature rupture of membrane (PROM) contributes to approximately one-third of premature birth and 10% perinatal mortality worldwide. Here, we report the patterns of facultative pathogenic bacteria colonizing the vagina of pregnant women to guide prophylactic antibiotic treatment in the management of PROM.Methods: This comparative cross-sectional study was conducted between August 2015 and March 2016. High vaginal swabs were collected and processed to detect the presence of facultative pathogenic bacteria. Isolate identification and antibiotic susceptibility testing was conducted using MALDI-TOF MS and VITEK-2 system, respectively. Data were analyzed using STATA version 13.Results: A total of 175 pregnant women with PROM and 175 without PROM were investigated. The median age of the pregnant women with PROM was significantly higher than that of pregnant women without PROM: 27 [21-32] vs. 25[21-29], p=0.026. Pregnant women with PROM were significantly more likely to be colonized with facultative pathogenic bacteria 59/175 (33.7%), 95% CI; 26.7-40.7 than pregnant women without PROM; 27/175 (15.4%), 95% CI; 10.1-20.7, P<0.001. Escherichia coli were significantly more isolated from pregnant women with PROM than those without PROM: 36 (73.5%) vs. 13(26.5%), p<0.001. The proportion of resistance among pathogenic isolates from women with PROM to ampicillin, trimethoprim/sulfamethoxazole and cefotaxime were 100%, 66.7% and 40%, respectively.Conclusions: The vagina of pregnant women with PROM is significantly more colonized by multi-resistant facultative pathogenic bacteria than that of pregnant women without PROM. Further studies should be done to elucidate the impact of these bacteria in relation to PROM and the pregnancy outcome.

Staphylococcus aureus Pneumonia: Preceding Influenza Infection Paves the Way for Low-Virulent Strains.

Staphylococcus aureus is a facultative pathogenic bacterium that colonizes the nasopharyngeal area of healthy individuals, but can also induce severe infection, such as pneumonia. Pneumonia caused by mono- or superinfected S. aureus leads to high mortality rates. To establish an infection, S. aureus disposes of a wide variety of virulence factors, which can vary between clinical isolates. Our study aimed to characterize pneumonia isolates for their virulent capacity. For this, we analyzed isolates from colonization, pneumonia due to S. aureus, and pneumonia due to S. aureus/influenza virus co-infection. A total of 70 strains were analyzed for their virulence genes and the host–pathogen interaction was analyzed through functional assays in cell culture systems. Strains from pneumonia due to S. aureus mono-infection showed enhanced invasion and cytotoxicity against professional phagocytes than colonizing and co-infecting strains. This corresponded to the high presence of cytotoxic components in pneumonia strains. By contrast, strains obtained from co-infection did not exhibit these virulence characteristics and resembled strains from colonization, although they caused the highest mortality rate in patients. Taken together, our results underline the requirement of invasion and toxins to cause pneumonia due to S. aureus mono-infection, whereas in co-infection even low-virulent strains can severely aggravate pneumonia.

Dissemination prevention of antibiotic resistant and facultative pathogenic bacteria by ultrafiltration and ozone treatment at an urban wastewater treatment plant

Conventional wastewater treatment is not sufficient for the removal of hygienically relevant bacteria and achieves only limited reductions. This study focuses on the reduction efficiencies of two semi-industrial ultrafiltration units operating at a large scale municipal wastewater treatment plant. In total, 7 clinically relevant antibiotic resistance genes, together with 3 taxonomic gene markers targeting specific facultative pathogenic bacteria were analysed via qPCR analyses before and after advanced treatment. In parallel with membrane technologies, an ozone treatment (1 g ozone/g DOC) was performed for comparison of the different reduction efficiencies. Both ultrafiltration units showed increased reduction efficiencies for facultative pathogenic bacteria and antibiotic resistance genes of up to 6 log units, resulting mostly in a strong reduction of the bacterial targets. In comparison, the ozone treatment showed some reduction efficiency, but was less effective compared with ultrafiltration due to low ozone dosages frequently used for micro-pollutant removal at municipal wastewater treatment plants. Additionally, metagenome analyses demonstrated the accumulation of facultative pathogenic bacteria, antibiotic resistance genes, virulence factor genes, and metabolic gene targets in the back flush retentate of the membranes, which opens further questions about retentate fluid material handling at urban wastewater treatment plants.