Drinking water quality of various sources in Peshawar, Mardan, Kohat and Swat districts of Khyber Pakhtunkhwa province, Pakistan / Qualidade de água potável de váris fontes dos distritos de Peshawar, Mardan, Kohat e Swat da província de Khyber Pakhtunkhwa, Paquistão

Abstract The present study involves the chemical and bacteriological analysis of water from different sources i.e., bore, wells, bottle, and tap, from Peshawar, Mardan, Swat and Kohat districts of Khyber Pakhtunkhwa (KP) province, Pakistan. From each district, 50 water samples (10 samples from each source), regardless of urban and rural status, were collected from these sources and analysed for sulphates, nitrates, nitrites, chlorides, total soluble solids and coliforms (E. coli). Results indicated that majority of the water sources had unacceptable E. coli count i.e.> 34 CFU/100mL. E. coli positive samples were high in Mardan District, followed by Kohat, Swat and Peshawar district. Besides this, the some water sources were also chemically contaminated by different inorganic fertilizers (nitrates/nitrites of sodium, potassium) but under safe levels whereas agricultural and industrial wastes (chloride and sulphate compounds) were in unsafe range. Among all districts, the water quality was found comparatively more deteriorated in Kohat and Mardan districts than Peshawar and Swat districts. Such chemically and bacteriologically unfit water sources for drinking and can cause human health problems.

Resumo O presente estudo envolve a análise química e bacteriológica de água de diferentes fontes, ou seja, furo, poços, garrafa e torneira, dos distritos de Peshawar, Mardan, Swat e Kohat da província de Khyber Pakhtunkhwa (KP), Paquistão. De cada distrito, 50 amostras de água (10 amostras de cada fonte), independentemente do status urbano e rural, foram coletadas dessas fontes e analisadas para sulfatos, nitratos, nitritos, cloretos, sólidos solúveis totais e coliformes (E. coli). Os resultados indicaram que a maioria das fontes de água tinha uma contagem inaceitável de E. coli, ou seja, > 34 UFC / 100 mL. As amostras positivas para E. coli foram elevadas no distrito de Mardan, seguido por Kohat, Swat e distrito de Peshawar. Além disso, algumas fontes de água também foram contaminadas quimicamente por diferentes fertilizantes inorgânicos (nitratos/nitritos de sódio, potássio), mas em níveis seguros, enquanto os resíduos agrícolas e industriais (compostos de cloreto e sulfato) estavam em níveis inseguros. Entre todos os distritos, a qualidade da água foi considerada comparativamente mais deteriorada nos distritos de Kohat e Mardan do que nos distritos de Peshawar e Swat. Essas fontes de água química e bacteriologicamente impróprias para beber podem causar problemas à saúde humana.

Antibacterial effect of Asphodelus fistulosus aqueous and ethanolic crude extracts on gram positive and gram negative bacteria / Efeito antibacteriano de extratos brutos aquosos e etanólicos de Asphodelus fistulosus sobre bactérias gram-positivas e gram-negativas

Asphodelus fistulosus (A. fistulosus) is a wild plant grows in Jordan. Traditionally, it is used to treat different medical conditions and diseases such as respiratory ailments, against burns and dermatomucosal infections.This study aims to find out the effects of A. fistulosus aqueous and ethanolic crude extracts on Staphylococcus aureus(S. aureus) as gram positive bacteria and Escherichia coli (E. coli) as gram negative bacteria and to investigate which one will be affected either by aqueous and/or ethanolic crude extracts of A. fistulosus shooting parts that were collected from Jerash in the north of Jordan. Agar well diffusion method was used to evaluate the antibacterial activity of the crude extracts. In addition, MIC (minimum inhibitory concentration) as well as MBC (minimum bactericidal concentration) were determined against both types of bacteria. The results showed that flower aqueous extract of A. fistulosus was very effective against E. coli (20.0 ± 0.50) mm and caused a (14.0 ± 0.50) mm inhibition to S. aureus. The ethanolic extract of stem was very effective cauesed a (19.0 ± 0.50) mm inhibition in both bacterial species. Respectively, both S. aureus and E. coli were inhibited by ethanolic and aqueous extracts (mixture1 and mixture2) (15.0 ± 0.00 mm and 10.5 ± 0.50 mm). The highest antimbacterial activity was observed for the leaves aqueous extract against E.coli (0.06120 mg/mL). The obtained MIC values from A. fistulosus parts extracts demonstrated antibacterial activity ranged between 7.606 and 0.06120 mg/mL. The highest antimicrobial activity was recorded in the leaves aqueous extract against E. coli.The MBC value of stem aqueous extract was 5.00 mg/mL against both S. aureus and E. coli. On the other hand, ethanolic and aqueous extracts of the leaves gave MBC values 5.00 mg/mL, and 0.156 mg/mL, respectively, against E. coli.Based on the results of this study, it can be concluded that there is good inhibitory effect of aqueous and ethanolic of A. fistulosus shooting parts extracts on growth of E. coli and S. aureus. Adding to that, stem ethanolic extract has the most effective against S. aureus while aqueous extract of flower has the most effective against E. coli.So, it is recommended to have further future studies on the A. fistulosus shooting parts crude extract bioactive components and the mechanism of how these constituents affect these types of bacteria.

Asphodelus fistulosus (A. fistulosus) é uma planta selvagem que cresce na Jordânia. Tradicionalmente, é usada para tratar diferentes condições médicas e doenças, como doenças respiratórias, contra queimaduras e infecções dermatomucosas. Bactérias positivas e Escherichia coli (E. coli) como bactérias gram-negativas e investigar qual delas será afetada por extratos brutos aquosos e/ou etanólicos de partes de tiro de A. fistulosus que foram coletadas em Jerash no norte da Jordânia. O método de difusão em poço de ágar foi utilizado para avaliar a atividade antibacteriana dos extratos brutos. Além disso, MIC (concentração inibitória mínima) e MBC (concentração bactericida mínima) foram determinados contra ambos os tipos de bactérias. Os resultados mostraram que o extrato aquoso de flores de A. fistulosus foi muito eficaz contra E. coli (20,0 + 0,50 mm) e causou uma inibição (14,0 + 0,50 mm) para S. aureus. O extrato etanólico do caule foi muito eficaz, causando inibição (19,0 + 0,50 mm) em ambas as espécies bacterianas. Respectivamente, tanto S. aureus quanto E. coli foram inibidos pelos extratos etanólico e aquoso (mistura 1 e mistura 2) (15,0 + 0,00 mm e 10,5 + 0,50 mm). A maior atividade antibacteriana foi observada para o extrato aquoso das folhas contra E. coli (0,06120 mg/mL). Os valores de CIM obtidos dos extratos de partes de A. fistulosus demonstraram atividade antibacteriana variando entre 7,606 e 0,06120 mg/mL. A maior atividade antimicrobiana foi registrada no extrato aquoso das folhas contra E. coli. O valor de CBM do extrato aquoso do caule foi de 5,00 mg/mL contra S. aureus e E. coli. Por outro lado, os extratos etanólico e aquoso das folhas apresentaram valores de CBM de 5,00 mg/mL e 0,156 mg/mL, respectivamente, contra E. coli, efeito de extratos aquosos e etanólicos de partes de tiro de A. fistulosus no crescimento de E. coli e S. aureus. Somando-se a isso, o extrato etanólico do caule é o mais eficaz contra S. aureus enquanto o extrato aquoso da flor é o mais eficaz contra E. coli. Assim, recomenda-se a realização de estudos futuros sobre os componentes bioativos do extrato bruto de partes fotografais de A. fistulosus e o mecanismo de como esses constituintes afetam esses tipos de bactérias.

Antibiotic resistance pattern and pathological features of avian pathogenic Escherichia coli O78: K80 in chickens / Padrão de resistência a antibióticos e características patológicas de Escherichia coli patogênica aviária O78: K80 em galinhas

Avian pathogenic Escherichia coli (APEC) induces colibacillosis, an acute and systemic disease, resulting in substantial economic losses in the poultry sector. This study aimed to investigate the antibiotic resistance pattern associated with frequent virulence gene distribution in APEC O78:K80 that may cause pathological alterations in chickens. The antibiogram profile showed high resistance to erythromycin, chloramphenicol, tetracycline, ampicillin, and co-trimoxazole, followed by intermediate resistance to ciprofloxacin, levofloxacin, enrofloxacin, norfloxacin, nitrofurantoin, and doxycycline hydrochloride, and sensitive to amikacin, streptomycin, gentamicin, and colistin. Virulence gene distribution identifies eight (irp-2, iutA, ompT, iss, iucD, astA, hlyF, iroN) genes through a conventional polymerase chain reaction. APEC O78:K80 caused significantly high liver enzyme concentrations, serum interleukin-6 and tumor necrosis factor-alpha levels in experimental birds. Also, infected birds have hypoproteinemia, hypoalbuminemia, and hyperglobulinemia. Necropsy examination revealed fibrinous perihepatitis and pericarditis, congested lungs, intestinal ecchymotic hemorrhages and necrotizing granulomatosis of the spleen. Histopathological examination depicted hepatocellular degeneration, myocardial necrosis, interstitial nephritis, intestinal hemorrhages and lymphopenia in the spleen. This study is the first evidence to assess the antibiotic resistance profile linked with virulence genes and clinicopathological potential of APEC O78:K80 in chickens in Pakistan, which could be a useful and rapid approach to prevent and control the disease by developing the control strategies.

A Escherichia coli patogênica aviária (APEC) induz a colibacilose, uma doença aguda e sistêmica, resultando em perdas econômicas substanciais no setor avícola. Este estudo teve como objetivo investigar o padrão de resistência a antibióticos associado à frequente distribuição de genes de virulência em APEC O78:K80 que podem causar alterações patológicas em galinhas. O perfil do antibiograma mostrou alta resistência à eritromicina, cloranfenicol, tetraciclina, ampicilina e cotrimoxazol; resistência intermediária à ciprofloxacina, levofloxacina, enrofloxacina, norfloxacina, nitrofurantoína e cloridrato de doxiciclina; e sensível à amicacina, estreptomicina, gentamicina e colistina . A distribuição de genes de virulência identificou oito genes (irp-2, iutA, ompT, iss, iucD, astA, hlyF e iroN) por meio de uma reação em cadeia da polimerase convencional. A APEC O78:K80 causou concentrações significativamente altas de enzimas hepáticas, níveis séricos de interleucina-6 e fator de necrose tumoral alfa em aves experimentais. Além disso, aves infectadas apresentaram hipoproteinemia, hipoalbuminemia e hiperglobulinemia. O exame de necropsia revelou peri-hepatite e pericardite fibrinosa, pulmões congestos, hemorragias equimóticas do intestino e granulomatose necrosante do baço. O exame histopatológico mostrou degeneração hepatocelular, necrose miocárdica, nefrite intersticial, hemorragias intestinais e linfopenia no baço. Este estudo é a primeira evidência para avaliar o perfil de resistência a antibióticos associado a genes de virulência e potencial clínico-patológico de APEC O78:K80 em galinhas no Paquistão, o que pode ser uma abordagem útil e rápida para prevenir e controlar a doença por meio do desenvolvimento de estratégias de controle.

Screening, biochemical characterization and antibiotics resistance/susceptibility of bacteria isolated from native soil and water samples / Triagem, caracterização bioquímica e resistência/suscetibilidade a antibióticos de bactérias isoladas de amostras nativas de solo e água

The present study was conducted to isolate and characterize bacteria from water and soil sample taken from the Lahore Canal at different sites i.e. Mall Road, Mohlanwal and Khera site. Isolated bacterial strains were identified on the basis of morphological and biochemical tests. Identification was confirmed by culturing bacteria on selective media. Antibiotic resistance test was also performed to observe the resistance of bacteria against different antibiotics. Blood agar test was performed for identification of different pathogenic bacteria. The result revealed that water and soil samples of Lahore Canal Lahore from different sites were contaminated with Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. and Staphylococcus spp. Due to presence of these pathogens, this water is not suitable for any domestic and irrigation use. Study also revealed that water of the Lahore Canal is harmful for human health as it is contaminated with bacteria that can cause severe disease e.g., Escherichia coli can cause gastroenteritis, Bacillus spp. can cause nausea and vomiting, Enterococcus may infect urinary tract, Salmonella sp. is responsible for Bacteremia, Staphylococcus spp. can cause mild fever and Vibrio sp. can be the reason of cholera. Thus it is rendered unfit for any kind of human use even other than drinking like swimming, bathing, washing etc., until and unless some remedial measures are employed to eradicate pathogenic microorganisms by WASA and LWMS according to standards of WHO. Similarly, it is quite harmful, when and where ever it is used for irrigation without proper treatment.

O presente estudo foi realizado para isolar e caracterizar bactérias de amostras de água e solo retiradas do Canal Lahore, em Lahore, em diferentes locais, ou seja, Mall Road, Mohlanwal e Khera. As cepas bacterianas isoladas foram identificadas com base em testes morfológicos e bioquímicos. A identificação foi confirmada por cultura de bactérias em testes de meios seletivos. O teste de resistência aos antibióticos também foi realizado para observar a resistência das bactérias a diferentes antibióticos. Foi realizado o teste de ágar sangue para identificar diferentes bactérias patogênicas. O resultado revelou que amostras de água e solo do Canal Lahore, Lahore, de diferentes localidades estavam contaminadas com Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. e Staphylococcus spp. Por causa da presença desses patógenos, essa água não é adequada para qualquer uso doméstico e de irrigação. O estudo revelou que a água do Canal Lahore é prejudicial à saúde humana, pois está contaminada com bactérias que podem causar doenças graves, por exemplo: Escherichia coli pode ocasionar gastroenterite; Bacillus spp. pode causar náuseas e vômitos; Enterococcus sp. pode infectar o trato urinário; Salmonella sp. é responsável pela bacteremia; Staphylococcus spp. pode causar febre leve; e Vibrio sp. pode ser a razão da cólera. Assim, torna-se imprópria para uso humano, como natação, banho, lavagem etc., até que algumas medidas corretivas sejam empregadas para erradicar microrganismos patogênicos por WASA e LWMS de acordo com os padrões da OMS. Da mesma forma, é bastante prejudicial, quando usada para irrigação sem tratamento adequado.

New Genetic Markers Differentiating IPEC and ExPEC Pathotypes-A New Approach to Genome-Wide Analysis Using a New Bioinformatics Tool.

The increasingly expanding genomic databases generate the need for new tools for their processing and further use. In the paper, a bioinformatics tool, which is a search engine of microsatellite elements-trinucleotide repeat sequences (TRS) in files of FASTA type-is presented. An innovative approach was applied in the tool, which consists of connecting-within one search engine-both mapping of TRS motifs and extracting sequences that are found between the mapped TRS motifs. Accordingly, we present hereby the tool called TRS-omix, which comprises a new engine for searching information on genomes and enables generation of sets of sequences and their number, providing the basis for making comparisons between genomes. In our paper, we showed one of the possibilities of using the software. Using TRS-omix and other IT tools, we showed that we were able to extract sets of DNA sequences that can be assigned only to the genomes of the extraintestinal pathogenic Escherichia coli strains or to the genomes of the intestinal pathogenic Escherichia coli strains, as well as providing the basis for differentiation of the genomes/strains belonging to each of these clinically essential pathotypes.

Metal-Responsive Transcription Factors Co-Regulate Anti-Sigma Factor (Rsd) and Ribosome Dimerization Factor Expression.

Bacteria exposed to stress survive by regulating the expression of several genes at the transcriptional and translational levels. For instance, in Escherichia coli, when growth is arrested in response to stress, such as nutrient starvation, the anti-sigma factor Rsd is expressed to inactivate the global regulator RpoD and activate the sigma factor RpoS. However, ribosome modulation factor (RMF) expressed in response to growth arrest binds to 70S ribosomes to form inactive 100S ribosomes and inhibit translational activity. Moreover, stress due to fluctuations in the concentration of metal ions essential for various intracellular pathways is regulated by a homeostatic mechanism involving metal-responsive transcription factors (TFs). Therefore, in this study, we examined the binding of a few metal-responsive TFs to the promoter regions of rsd and rmf through promoter-specific TF screening and studied the effects of these TFs on the expression of rsd and rmf in each TF gene-deficient E. coli strain through quantitative PCR, Western blot imaging, and 100S ribosome formation analysis. Our results suggest that several metal-responsive TFs (CueR, Fur, KdpE, MntR, NhaR, PhoP, ZntR, and ZraR) and metal ions (Cu2+, Fe2+, K+, Mn2+, Na+, Mg2+, and Zn2+) influence rsd and rmf gene expression while regulating transcriptional and translational activities.

Essentiality of the Escherichia coli YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme.

Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylates some tRNAs. The growth of cells lacking YgfZ is compromised especially at low temperature. The RimO enzyme is homologous to MiaB and thiomethylates a conserved aspartic acid in ribosomal protein S12. To quantitate thiomethylation by RimO, we developed a bottom-up LC-MS2 analysis of total cell extracts. We show here that the in vivo activity of RimO is very low in the absence of YgfZ and independent of growth temperature. We discuss these results in relation to the hypotheses relating to the role of the auxiliary 4Fe-4S cluster in the Radical SAM enzymes that make Carbon-Sulfur bonds.

Bioinspired Synthesis of Magnetic Nanoparticles Based on Iron Oxides Using Orange Waste and Their Application as Photo-Activated Antibacterial Agents.

Magnetic nanoparticles based on iron oxides (MNPs-Fe) have been proposed as photothermal agents (PTAs) within antibacterial photothermal therapy (PTT), aiming to counteract the vast health problem of multidrug-resistant bacterial infections. We present a quick and easy green synthesis (GS) to prepare MNPs-Fe harnessing waste. Orange peel extract (organic compounds) was used as a reducing, capping, and stabilizing agent in the GS, which employed microwave (MW) irradiation to reduce the synthesis time. The produced weight, physical-chemical features and magnetic features of the MNPs-Fe were studied. Moreover, their cytotoxicity was assessed in animal cell line ATCC RAW 264.7, as well as their antibacterial activity against Staphylococcus aureus and Escherichia coli. We found that the 50GS-MNPs-Fe sample (prepared by GS, with 50% v/v of NH4OH and 50% v/v of orange peel extract) had an excellent mass yield. Its particle size was ~50 nm with the presence of an organic coating (terpenes or aldehydes). We believe that this coating improved the cell viability in extended periods (8 days) of cell culture with concentrations lower than 250 µg·mL-1, with respect to the MNPs-Fe obtained by CO and single MW, but it did not influence the antibacterial effect. The bacteria inhibition was attributed to the plasmonic of 50GS-MNPs-Fe (photothermal effect) by irradiation with red light (630 nm, 65.5 mW·cm-2, 30 min). We highlight the superparamagnetism of the 50GS-MNPs-Fe over 60 K in a broader temperature range than the MNPs-Fe obtained by CO (160.09 K) and MW (211.1 K). Therefore, 50GS-MNPs-Fe could be excellent candidates as broad-spectrum PTAs in antibacterial PTT. Furthermore, they might be employed in magnetic hyperthermia, magnetic resonance imaging, oncological treatments, and so on.

Evaluation of L-Alanine Metabolism in Bacteria and Whole-Body Distribution with Bacterial Infection Model Mice.

The World Health Organization has cautioned that antimicrobial resistance (AMR) will be responsible for an estimated 10 million deaths annually by 2050. To facilitate prompt and accurate diagnosis and treatment of infectious disease, we investigated the potential of amino acids for use as indicators of bacterial growth activity by clarifying which amino acids are taken up by bacteria during the various growth phases. In addition, we examined the amino acid transport mechanisms that are employed by bacteria based on the accumulation of labeled amino acids, Na+ dependence, and inhibitory effects using a specific inhibitor of system A. We found that 3H-L-Ala accurately reflects the proliferative activity of Escherichia coli K-12 and pathogenic EC-14 in vitro. This accumulation in E. coli could be attributed to the amino acid transport systems being different from those found in human tumor cells. Moreover, biological distribution assessed in infection model mice with EC-14 using 3H-L-Ala showed that the ratio of 3H-L-Ala accumulated in infected muscle to that in control muscle was 1.20. By detecting the growth activity of bacteria in the body that occurs during the early stages of infection by nuclear imaging, such detection methods may result in expeditious diagnostic treatments for infectious diseases.

Impact of Negative Feedbacks on De Novo Pyrimidines Biosynthesis in Escherichia coli.

Earlier studies aimed at investigating the metabolism of endogenous nucleoside triphosphates in synchronous cultures of E. coli cells revealed an auto-oscillatory mode of functioning of the pyrimidine and purine nucleotide biosynthesis system, which the authors associated with the dynamics of cell division. Theoretically, this system has an intrinsic oscillatory potential, since the dynamics of its functioning are controlled through feedback mechanisms. The question of whether the nucleotide biosynthesis system has its own oscillatory circuit is still open. To address this issue, an integral mathematical model of pyrimidine biosynthesis was developed, taking into account all experimentally verified negative feedback in the regulation of enzymatic reactions, the data of which were obtained under in vitro conditions. Analysis of the dynamic modes of the model functioning has shown that in the pyrimidine biosynthesis system, both the steady-state and oscillatory functioning modes can be realized under certain sets of kinetic parameters that fit in the physiological boundaries of the investigated metabolic system. It has been demonstrated that the occurrence of the oscillatory nature of metabolite synthesis depended on the ratio of two parameters: the Hill coefficient, hUMP1-the nonlinearity of the UMP effect on the activity of carbamoyl-phosphate synthetase, and the parameter r characterizing the contribution of the noncompetitive mechanism of UTP inhibition to the regulation of the enzymatic reaction of UMP phosphorylation. Thus, it has been theoretically shown that the E. coli pyrimidine biosynthesis system possesses its own oscillatory circuit whose oscillatory potential depends to a significant degree on the mechanism of regulation of UMP kinase activity.

Antimicrobial resistance from a One Health perspective in Zambia: a systematic review.

BACKGROUND: Antimicrobial resistance (AMR) is widely acknowledged as a global health problem, yet its extent is not well evaluated, especially in low-middle income countries. It is challenging to promote policies without focusing on healthcare systems at a local level, therefore a baseline assessment of the AMR occurrence is a priority. This study aimed to look at published papers relating to the availability of AMR data in Zambia as a means of establishing an overview of the situation, to help inform future decisions. METHODS: PubMed, Cochrane Libraries, Medical Journal of Zambia and African Journals Online databases were searched from inception to April 2021 for articles published in English in accordance with the PRISMA guidelines. Retrieval and screening of article was done using a structured search protocol with strict inclusion/exclusion criteria. RESULTS: A total of 716 articles were retrieved, of which 25 articles met inclusion criteria for final analysis. AMR data was not available for six of the ten provinces of Zambia. Twenty-one different isolates from the human health, animal health and environmental health sectors were tested against 36 antimicrobial agents, across 13 classes of antibiotics. All the studies showed a degree of resistance to more than one class of antimicrobials. Majority of the studies focused on antibiotics, with only three studies (12%) highlighting antiretroviral resistance. Antitubercular drugs were addressed in only five studies (20%). No studies focused on antifungals. The most common organisms tested, across all three sectors, were Staphylococcus aureus, with a diverse range of resistance patterns found; followed by Escherichia coli with a high resistance rate found to cephalosporins (24-100%) and fluoroquinolones (20-100%). CONCLUSIONS: This review highlights three important findings. Firstly, AMR is understudied in Zambia. Secondly, the level of resistance to commonly prescribed antibiotics is significant across the human, animal, and environmental sectors. Thirdly, this review suggests that improved standardization of antimicrobial susceptibility testing in Zambia could help to better delineate AMR patterns, allow comparisons across different locations and tracking of AMR evolution over time.

Inhibitory effect of 405 nm laser light on bacterial biofilm in urethral stent.

The clinical use of urethral stents is usually complicated by various adverse effects, including dysuria, fever, and urinary tract infection (UTI). Biofilms (formed by bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) adhering to the stent cause UTIs in stented patients (approximately 11%). The undesirable consequences of antibiotics use include bacterial resistance, weight gain, and type 1 diabetes, which occur when antibiotics are used for a long time. We aimed to assess the efficacy of a new optical treatment with a 405 nm laser to inhibit bacterial growth in a urethral stent in vitro. The urethral stent was grown in S. aureus broth media for three days to induce biofilm formation under dynamic conditions. Various irradiation times with the 405 nm laser light were tested (5, 10, and 15 min). The efficacy of the optical treatment on biofilms was evaluated quantitatively and qualitatively. The production of reactive oxygen species helped eliminate the biofilm over the urethral stent after 405 nm irradiation. The inhibition rate corresponded to a 2.2 log reduction of colony-forming units/mL of bacteria after 0.3 W/cm2 of irradiation for 10 min. The treated stent showed a significant reduction in biofilm formation compared with the untreated stent, as demonstrated by SYTO 9 and propidium iodide staining. MTT assays using the CCD-986sk cell line revealed no toxicity after 10 min of irradiation. We conclude that optical treatment with 405 nm laser light inhibits bacterial growth in urethral stents with no or minimal toxicity.

Size limits the sensitivity of kinetic schemes.

Living things benefit from exquisite molecular sensitivity in many of their key processes, including DNA replication, transcription and translation, chemical sensing, and morphogenesis. At thermodynamic equilibrium, the basic biophysical mechanism for sensitivity is cooperative binding, for which it can be shown that the Hill coefficient, a sensitivity measure, cannot exceed the number of binding sites. Generalizing this fact, we find that for any kinetic scheme, at or away from thermodynamic equilibrium, a very simple structural quantity, the size of the support of a perturbation, always limits the effective Hill coefficient. We show how this bound sheds light on and unifies diverse sensitivity mechanisms, including kinetic proofreading and a nonequilibrium Monod-Wyman-Changeux (MWC) model proposed for the E. coli flagellar motor switch, representing in each case a simple, precise bridge between experimental observations and the models we write down. In pursuit of mechanisms that saturate the support bound, we find a nonequilibrium binding mechanism, nested hysteresis, with sensitivity exponential in the number of binding sites, with implications for our understanding of models of gene regulation and the function of biomolecular condensates.

Molecular surveillance reveals the emergence and dissemination of NDM-5-producing Escherichia coli high-risk clones in Germany, 2013 to 2019.

BackgroundCarbapenemase-producing Enterobacterales (CPE) are rapidly increasing worldwide, also in Europe. Although prevalence of CPE in Germany is comparatively low, the National Reference Centre for Multidrug-resistant Gram-negative Bacteria noted annually increasing numbers of NDM-5-producing Escherichia coli isolates.AimAs part of our ongoing surveillance programme, we characterised NDM-5-producing E. coli isolates received between 2013 and 2019 using whole genome sequencing (WGS).MethodsFrom 329 identified NDM-5-producing E. coli, 224 isolates from known geographical locations were subjected to Illumina WGS. Analyses of 222 sequenced isolates included multilocus sequence typing (MLST), core genome (cg)MLST and single-nucleotide polymorphism (SNP)-based analyses.ResultsResults of cgMLST revealed genetically distinct clusters for many of the 43 detected sequence types (ST), of which ST167, ST410, ST405 and ST361 predominated. The SNP-based phylogenetic analyses combined with geographical information identified sporadic cases of nosocomial transmission on a small spatial scale. However, we identified large clusters corresponding to clonal dissemination of ST167, ST410, ST405 and ST361 strains in consecutive years in different regions in Germany.ConclusionOccurrence of NDM-5-producing E. coli rose in Germany, which was to a large extent due to the increased prevalence of isolates belonging to the international high-risk clones ST167, ST410, ST405 and ST361. Of particular concern is the supra-regional dissemination of these epidemic clones. Available information suggest community spread of NDM-5-producing E. coli in Germany, highlighting the importance of epidemiological investigation and an integrated surveillance system in the One Health framework.

Antibiotic-Lysobacter enzymogenes proteases combination as a novel virulence attenuating therapy.

Minimizing antibiotic resistance is a key motivation strategy in designing and developing new and combination therapy. In this study, a combination of the antibiotics (cefixime, levofloxacin and gentamicin) with Lysobacter enzymogenes (L. enzymogenes) bioactive proteases present in the cell- free supernatant (CFS) have been investigated against the Gram-positive methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) and the Gram-negative Escherichia coli (E. coli O157:H7). Results indicated that L. enzymogenes CFS had maximum proteolytic activity after 11 days of incubation and higher growth inhibitory properties against MSSA and MRSA compared to E. coli (O157:H7). The combination of L. enzymogenes CFS with cefixime, gentamicin and levofloxacin at sub-MIC levels, has potentiated their bacterial inhibition capacity. Interestingly, combining cefixime with L. enzymogenes CFS restored its antibacterial activity against MRSA. The MTT assay revealed that L. enzymogenes CFS has no significant reduction in human normal skin fibroblast (CCD-1064SK) cell viability. In conclusion, L. enzymogenes bioactive proteases are natural potentiators for antimicrobials with different bacterial targets including cefixime, gentamicin and levofloxacin representing the beginning of a modern and efficient era in the battle against multidrug-resistant pathogens.

Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells.

Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.

Antibacterial and anti-biofilm properties of carvacrol alone and in combination with cefixime against Escherichia coli.

BACKGROUND: Plant-derived compounds can be used as antimicrobial agents in medicines and as food preservatives. These compounds can be applied along with other antimicrobial agents to strengthen the effect and/or reduce the required treatment dose. RESULTS: In the present study, the antibacterial, anti-biofilm and quorum sensing inhibitory activity of carvacrol alone and in combination with the antibiotic cefixime against Escherichia coli was investigated. The MIC and MBC values for carvacrol were 250 µg/mL. In the checkerboard test, carvacrol showed a synergistic interaction with cefixime against E. coli (FIC index = 0.5). Carvacrol and cefixime significantly inhibited biofilm formation at MIC/2 (125 and 62.5 µg/mL), MIC/4 (62.5 and 31.25 µg/mL) and MIC/8 (31.25 and 15.625 µg/mL) for carvacrol and cefixime, respectively. The antibacterial and anti-biofilm potential effect of carvacrol confirmed by the scanning electron microscopy. Real-time quantitative reverse transcription PCR revealed significant down-regulation of the luxS and pfs genes following treatment with a MIC/2 (125 µg/mL) concentration of carvacrol alone and of only pfs gene following treatment with MIC/2 of carvacrol in combination with MIC/2 of cefixime (p < 0.05). CONCLUSIONS: Because of the significant antibacterial and anti-biofilm activity of carvacrol, the present study examines this agent as an antibacterial drug of natural origin. The results indicate that in this study the best antibacterial and anti-biofilm properties are for the combined use of cefixime and carvacrol.

Irinotecan-gut microbiota interactions and the capability of probiotics to mitigate Irinotecan-associated toxicity.

BACKGROUND: Irinotecan is a chemotherapeutic agent used to treat a variety of tumors, including colorectal cancer (CRC). In the intestine, it is transformed into SN-38 by gut microbial enzymes, which is responsible for its toxicity during excretion. OBJECTIVE: Our study highlights the impact of Irinotecan on gut microbiota composition and the role of probiotics in limiting Irinotecan-associated diarrhea and suppressing gut bacterial ß-glucuronidase enzymes. MATERIAL AND METHODS: To investigate the effect of Irinotecan on the gut microbiota composition, we applied 16S rRNA gene sequencing in three groups of stool samples from healthy individuals, colon cancer, and Irinotecan treated patients (n = 5/group). Furthermore, three Lactobacillus spp.; Lactiplantibacillus plantarum (L. plantarum), Lactobacillus acidophilus (L. acidophilus), Lacticaseibacillus rhamnosus (L. rhamnosus) were used in a single and mixed form to in-vitro explore the effect of probiotics on the expression of ß-glucuronidase gene from E. coli. Also, probiotics were introduced in single and mixed forms in groups of mice before the administration of Irinotecan, and their protective effects were explored by assessing the level of reactive oxidative species (ROS) as well as studying the concomitant intestinal inflammation and apoptosis. RESULTS: The gut microbiota was disturbed in individuals with colon cancer and after Irinotecan treatment. In the healthy group, Firmicutes were more abundant than Bacteriodetes, which was the opposite in the case of colon-cancer or Irinotecan treated groups. Actinobacteria and Verrucomicrobia were markedly present within the healthy group, while Cyanobacteria were noted in colon-cancer and the Irinotecan-treated groups. Enterobacteriaceae and genus Dialister were more abundant in the colon-cancer group than in other groups. The abundance of Veillonella, Clostridium, Butryicicoccus, and Prevotella were increased in Irinotecan-treated groups compared to other groups. Using Lactobacillus spp. mixture in mice models significantly relieved Irinotecan-induced diarrhea through the reduction of both ß-glucuronidase expression and ROS, in addition to guarding gut epithelium against microbial dysbiosis and proliferative crypt injury. CONCLUSIONS: Irinotecan-based chemotherapy altered intestinal microbiota. The gut microbiota participates greatly in determining both the efficacy and toxicity of chemotherapies, of which the toxicity of Irinotecan is caused by the bacterial ß-glucuronidase enzymes. The gut microbiota can now be aimed and modulated to promote efficacy and decrease the toxicity of chemotherapeutics. The used probiotic regimen in this study lowered mucositis, oxidative stress, cellular inflammation, and apoptotic cascade induction of Irinotecan.

Black glucose-releasing silicon elastomer rings for fed-batch operation allow measurement of the oxygen transfer rate from the top and optical signals from the bottom for each well of a microtiter plate.

BACKGROUND: In industrial microbial biotechnology, fed-batch processes are frequently used to avoid undesirable biological phenomena, such as substrate inhibition or overflow metabolism. For targeted process development, fed-batch options for small scale and high throughput are needed. One commercially available fed-batch fermentation system is the FeedPlate®, a microtiter plate (MTP) with a polymer-based controlled release system. Despite standardisation and easy incorporation into existing MTP handling systems, FeedPlates® cannot be used with online monitoring systems that measure optically through the transparent bottom of the plate. One such system that is broadly used in biotechnological laboratories, is the commercial BioLector. To allow for BioLector measurements, while applying the polymer-based feeding technology, positioning of polymer rings instead of polymer disks at the bottom of the well has been proposed. This strategy has a drawback: measurement requires an adjustment of the software settings of the BioLector device. This adjustment modifies the measuring position relative to the wells, so that the light path is no longer blocked by the polymer ring, but, traverses through the inner hole of the ring. This study aimed at overcoming that obstacle and allowing for measurement of fed-batch cultivations using a commercial BioLector without adjustment of the relative measurement position within each well. RESULTS: Different polymer ring heights, colours and positions in the wells were investigated for their influence on maximum oxygen transfer capacity, mixing time and scattered light measurement. Several configurations of black polymer rings were identified that allow measurement in an unmodified, commercial BioLector, comparable to wells without rings. Fed-batch experiments with black polymer rings with two model organisms, E. coli and H. polymorpha, were conducted. The identified ring configurations allowed for successful cultivations, measuring the oxygen transfer rate and dissolved oxygen tension, pH, scattered light and fluorescence. Using the obtained online data, glucose release rates of 0.36 to 0.44 mg/h could be determined. They are comparable to formerly published data of the polymer matrix. CONCLUSION: The final ring configurations allow for measurements of microbial fed-batch cultivations using a commercial BioLector without requiring adjustments of the instrumental measurement setup. Different ring configurations achieve similar glucose release rates. Measurements from above and below the plate are possible and comparable to measurements of wells without polymer rings. This technology enables the generation of a comprehensive process understanding and target-oriented process development for industrial fed-batch processes.

Prevalence and molecular characteristics of mcr-1-positive Escherichia coli isolated from duck farms and the surrounding environments in coastal China.

The emergence of colistin-resistance is considered a threat to public health and colistin-resistant bacteria have recently been reported in animal, environmental and human sources. Whereas, the epidemic and dissemination of colistin-resistant bacteria in duck farms have not been surveyed, especially the surrounding environmental contamination from duck farms. We investigated the prevalence and molecular characteristics of mcr-1-positive E. coli from duck farms in coastal China. 360 mcr-1-positive E. coli isolates were collected from 1112 samples from duck farms and surrounding environments. The prevalence of mcr-1-positive E. coli in Guangdong province was higher than other two provinces we examined. PFGE analysis indicated clonal spread of mcr-1-positive E. coli between duck farms and surrounding environments, including water and soil. MLST analysis demonstrated that ST10 was more common than ST1011, ST117, and ST48. Phylogenomic analysis also suggested mcr-1-positive E. coli collected from distinct cities were assigned to the same lineage and mcr-1 was primarily located on IncI2 and IncHI2 plasmids. Genomic environment analysis showed mobile gene elements ISApl1 most likely plays a key role in the horizontal transmission of mcr-1. WGS further revealed that mcr-1 was found associated with 27 different ARGs. Our findings emphasize the urgent need for effective colistin resistance surveillance in humans, animals and the environment.