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1. Niche Preference of Escherichia coli in a Peri-Urban Pond Ecosystem

   https://doi.org/10.3390/life11101020  

   NandaKafle, Gitanjali; Huegen, Taylor; Potgieter, Sarah C.; Steenkamp, Emma; Venter, Stephanus N.; Brözel, Volker S. (October 2021, Life)

   Escherichia coli comprises diverse strains with a large accessory genome, indicating functional diversity and the ability to adapt to a range of niches. Specific strains would display greatest fitness in niches matching their combination of phenotypic traits. Given this hypothesis, we sought to determine whether E. coli in a peri-urban pond and associated cattle pasture display niche preference. Samples were collected from water, sediment, aquatic plants, water snails associated with the pond, as well as bovine feces from cattle in an adjacent pasture. Isolates (120) were obtained after plating on Membrane Lactose Glucuronide Agar (MLGA). We used the uidA and mutS sequences for all isolates to determine phylogeny by maximum likelihood, and population structure through gene flow analysis. PCR was used to allocate isolates to phylogroups and to determine the presence of pathogenicity/virulence genes (stxI, stxII, eaeA, hlyA, ST, and LT). Antimicrobial resistance was determined using a disk diffusion assay for Tetracycline, Gentamicin, Ciprofloxacin, Meropenem, Ceftriaxone, and Azithromycin. Our results showed that isolates from water, sediment, and water plants were similar by phylogroup distribution, virulence gene distribution, and antibiotic resistance while both snail and feces populations were significantly different. Few of the feces isolates were significantly similar to aquatic ones, and most of the snail isolates were also different. Population structure analysis indicated three genetic backgrounds associated with bovine, snail, and aquatic environments. Collectively these data support niche preference of E. coli isolates occurring in this ecosystem. 

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2. Antibiotic resistance trends among Vibrio vulnificus and Vibrio parahaemolyticus isolated from the Chesapeake Bay, Maryland: a longitudinal study

   https://doi.org/10.1128/aem.00539-24  

   Morgado, Michele E; Brumfield, Kyle D; Chattopadhyay, Suhana; Malayil, Leena; Alawode, Taiwo; Amokeodo, Ibiyinka; He, Xin; Huq, Anwar; Colwell, Rita R; Sapkota, Amy R (June 2024, Applied and Environmental Microbiology)

   Elkins, Christopher A (Ed.)

   ABSTRACT Antibiotics are often used to treat severeVibrioinfections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009–2012 and 2019–2022).Vibrio parahaemolyticus(n= 134) andVibrio vulnificus(n= 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%–96%) ofV. parahaemolyticusisolates from both sampling periods were resistant to ampicillin and only 2%–6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistantV. vulnificusisolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%–8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns amongV. parahaemolyticusandV. vulnificusrecovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibriospp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, someVibrioisolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed withVibrio vulnificusinfections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severeVibriospp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion. 

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3. Genotypic and Phenotypic Characterization of Incompatibility Group FIB Positive Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources

   https://doi.org/10.3390/genes11111307  

   Aljahdali, Nesreen H.; Khajanchi, Bijay K.; Weston, Kennedi; Deck, Joanna; Cox, Justin; Singh, Ruby; Gilbert, Jeffrey; Sanad, Yasser M.; Han, Jing; Nayak, Rajesh; et al (November 2020, Genes)

   null (Ed.)

   Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998–2003 were compared to those published from subsequent years (2005–2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species. 

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4. Cephalosporins Interfere With Quorum Sensing and Improve the Ability of Caenorhabditis elegans to Survive Pseudomonas aeruginosa Infection

   https://doi.org/10.3389/fmicb.2021.598498  

   Kumar, Lokender; Brenner, Nathanael; Brice, John; Klein-Seetharaman, Judith; Sarkar, Susanta K. (January 2021, Frontiers in Microbiology)

   null (Ed.)

   Pseudomonas aeruginosa utilizes the quorum sensing (QS) system to strategically coordinate virulence and biofilm formation. Targeting QS pathways may be a potential anti-infective approach to treat P. aeruginosa infections. In the present study, we define cephalosporins’ anti-QS activity using Chromobacterium violaceum CV026 for screening and QS-regulated mutants of P. aeruginosa for validation. We quantified the effects of three cephalosporins, cefepime, ceftazidime, and ceftriaxone, on (1) pyocyanin production using spectrophotometric assay, (2) bacterial motility using agar plate assay, and (3) biofilm formation using scanning electron microscopy. We also studied isogenic QS mutant strains of PAO1 (Δ lasR ,Δ rhlR ,Δ pqsA , and Δ pqsR) to compare and distinguish QS-mediated effects on the motility phenotypes and bacterial growth with and without sub-MIC concentrations of antibiotics. Results showed that cephalosporins have anti-QS activity and reduce bacterial motility, pyocyanin production, and biofilm formation for CV026 and PAO1. Also, sub-MICs of cefepime increased aminoglycosides’ antimicrobial activity against P. aeruginosa PAO1, suggesting the advantage of combined anti-QS and antibacterial treatment. To correlate experimentally observed anti-QS effects with the interactions between cephalosporins and QS receptors, we performed molecular docking with ligand binding sites of quorum sensing receptors using Autodock Vina. Molecular docking predicted cephalosporins’ binding affinities to the ligand-binding pocket of QS receptors (CviR, LasR, and PqsR). To validate our results using an infection model, we quantified the survival rate of C aenorhabditis elegans following P. aeruginosa PAO1 challenge at concentrations less than the minimum inhibitory concentration (MIC) of antibiotics. C. elegans infected with PAO1 without antibiotics showed 0% survivability after 72 h. In contrast, PAO1-infected C. elegans showed 65 ± 5%, 58 ± 4%, and 49 ± 8% survivability after treatment with cefepime, ceftazidime, and ceftriaxone, respectively. We determined the survival rates of C. elegans infected by QS mutant strains Δ lasR (32 ± 11%), Δ rhlR (27 ± 8%), Δ pqsA (27 ± 10%), and Δ pqsR (37 ± 6%), which suggest essential role of QS system in virulence. In summary, cephalosporins at sub-MIC concentrations show anti-QS activity and enhance the antibacterial efficacy of aminoglycosides, a different class of antibiotics. Thus, cephalosporins at sub-MIC concentrations in combination with other antibiotics are potential candidates for developing therapies to combat infections caused by P. aeruginosa. 

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5. Rapid, antibiotic incubation-free determination of tuberculosis drug resistance using machine learning and Raman spectroscopy

   https://doi.org/10.1073/pnas.2315670121  

   Ogunlade, Babatunde; Tadesse, Loza F; Li, Hongquan; Vu, Nhat; Banaei, Niaz; Barczak, Amy K; Saleh, Amr_A E; Prakash, Manu; Dionne, Jennifer A (June 2024, Proceedings of the National Academy of Sciences)

   Tuberculosis (TB) is the world’s deadliest infectious disease, with over 1.5 million deaths and 10 million new cases reported anually. The causative organismMycobacterium tuberculosis(Mtb) can take nearly 40 d to culture, a required step to determine the pathogen’s antibiotic susceptibility. Both rapid identification and rapid antibiotic susceptibility testing of Mtb are essential for effective patient treatment and combating antimicrobial resistance. Here, we demonstrate a rapid, culture-free, and antibiotic incubation-free drug susceptibility test for TB using Raman spectroscopy and machine learning. We collect few-to-single-cell Raman spectra from over 25,000 cells of the Mtb complex strain Bacillus Calmette-Guérin (BCG) resistant to one of the four mainstay anti-TB drugs, isoniazid, rifampicin, moxifloxacin, and amikacin, as well as a pan-susceptible wildtype strain. By training a neural network on this data, we classify the antibiotic resistance profile of each strain, both on dried samples and on patient sputum samples. On dried samples, we achieve >98% resistant versus susceptible classification accuracy across all five BCG strains. In patient sputum samples, we achieve ~79% average classification accuracy. We develop a feature recognition algorithm in order to verify that our machine learning model is using biologically relevant spectral features to assess the resistance profiles of our mycobacterial strains. Finally, we demonstrate how this approach can be deployed in resource-limited settings by developing a low-cost, portable Raman microscope that costs <$5,000. We show how this instrument and our machine learning model enable combined microscopy and spectroscopy for accurate few-to-single-cell drug susceptibility testing of BCG. 

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