Bacteria produce extracellular enzymes to obtain resources from complex chemical substrates, but this strategy is vulnerable to cheating by cells that take up reaction products without paying the cost of enzyme production. We hypothesized that cheating would suppress enzyme production in co-cultures of cheater and producer bacteria, particularly under well-mixed conditions. To test this hypothesis, we monitored protease expression and frequencies of Pseudomonas fluorescens producer and cheater genotypes over time in mixed liquid cultures and on agar plates. In mixed culture inoculated with equal frequencies of cheaters and producers, enzyme concentration declined to zero after 20 days, consistent with our hypothesis. We observed a similar decline in cultures inoculated with producers only, suggesting that cheater mutants arose de novo and swept the population. DNA sequencing showed that genetic changes most likely occurred outside the protease operon. In one experimental replicate, the population regained the ability to produce protease, likely due to further genetic changes or population dynamics. Under spatially structured conditions on agar plates, cheaters did not sweep the population. Instead, we observed a significant increase in the variation of enzyme activity levels expressed by clones isolated from the population. Together these results suggest that restricted diffusion favors a diversity of enzyme production strategies. In contrast, well-mixed conditions favor population sweeps by cheater strains, consistent with theoretical predictions. Cheater and producer strategies likely coexist in natural environments with the frequency of cheating increasing with diffusion rate.

The elimination of bacterial biofilms remains a major challenge due to their recalcitrant nature. Bacteriophages, viruses that infect bacteria, have been gaining increasing attention as biofilm control agents. However, the development of a successful phage-based strategy requires in-depth analysis of different parameters. It is particularly important to determine the ability of a given phage to diffuse, propagate and remain viable within the complex biofilm structure. Here, we examine some of these properties for two staphylophages, vB_SauM_phiIPLA-RODI and vB_SepM_phiIPLA-C1C. Both Staphylococcus aureus and Staphylococcus epidermidis are important opportunistic pathogens that readily form biofilms on a wide array of biotic and abiotic surfaces. Our results confirmed that both phages could penetrate through biofilms formed by several bacterial strains with varying degrees of susceptibility to the viruses and biofilm-forming abilities. However, phage penetration differed depending on the specific bacterium or combination of bacteria. The data presented here suggest that the factors determining the diffusion rate of phages in biofilms include the amount of attached biomass, susceptibility of the strain, initial phage titer, phage entrapment in the extracellular matrix, and phage inactivation. This information will help to further characterize phage-bacteria interactions within biofilm communities and will be valuable for the development of antistaphylococcal products based on these phages.

The molecular epidemiology of HIV-1 in Italy is becoming increasingly complex, mainly due to the spread of non-B subtypes and the emergence of new recombinant forms. We previously characterized the outbreak of the first Italian circulating recombinant form (CRF60_BC), occurring among young MSM living in Apulia between the years 2009 and 2011. Here we show a 5-year follow-up surveillance to trace the evolution of CRF60_BC and to investigate its further spread in Italy. We collected additional sequences and clinical data from patients harboring CRF60_BC, enrolled at the Infectious Diseases Clinic of the University of Bari. In addition to the 24 previously identified sequences, we retrieved 27 CRF60_BC sequences from patients residing in Apulia, whose epidemiological and clinical features did not differ from those of the initial outbreak, i.e., the Italian origin, young age at HIV diagnosis (median: 24 years; range: 18-37), MSM risk factor (23/25, 92%) and recent infection (from 2008 to 2017). Sequence analysis revealed a growing overall nucleotide diversity, with few nucleotide changes that were fixed over time. Twenty-seven additional sequences were detected across Italy, spanning multiple distant regions. Using a BLAST search, we also identified a CRF60_BC sequence isolated in United Kingdom in 2013. Three patients harbored a unique second generation recombinant form in which CRF60_BC was one of the parental strains. Our data show that CRF60_BC gained epidemic importance, spreading among young MSM in multiple Italian regions and increasing its population size in few years, as the number of sequences identified so far has triplicated since our first report. The observed further divergence of CRF60_BC is likely due to evolutionary bottlenecks and host adaptation during transmission chains. Of note, we detected three second-generation recombinants, further supporting a widespread circulation of CRF60_BC and the increasing complexity of the HIV-1 epidemic in Italy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the coronavirus disease 2019 (COVID-19) pandemic, which started as a severe pneumonia outbreak in Wuhan, China, in December 2019. Italy has been the first European country affected by the pandemic, registering a total of 300,363 cases and 35,741 deaths until September 24, 2020. The geographical distribution of SARS-CoV-2 in Italy during early 2020 has not been homogeneous, including regions severely affected as well as administrative areas being only slightly interested by the infection. Among the latter, Sardinia represents one of the lowest incidence areas likely due to its insular nature.

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (D L ) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that D L is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli. We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host.

In the cation diffusion facilitator (CDF) family, the transported substrates are confined to divalent metal ions, such as Zn2+, Fe2+, and Mn2+. However, this study identifies a novel CDF member designated MceT from the moderate halophile Planococcus dechangensis. MceT functions as a Na+(Li+, K+)/H+ antiporter, together with its capability of facilitated Zn2+ diffusion into cells, which have not been reported in any identified CDF transporters as yet. MceT is proposed to represent a novel CDF group, Na-CDF, which shares significantly distant phylogenetic relationship with three known CDF groups including Mn-CDF, Fe/Zn-CDF, and Zn-CDF. Variation of key function-related residues to "Y44-S48-Q150" in two structural motifs explains a significant discrimination in cation selectivity between Na-CDF group and three major known CDF groups. Functional analysis via site-directed mutagenesis confirms that MceT employs Q150, S158, and D184 for the function of MceT as a Na+(Li+, K+)/H+ antiporter, and retains D41, D154, and D184 for its facilitated Zn2+ diffusion into cells. These presented findings imply that MceT has evolved from its native CDF family function to a Na+/H+ antiporter in an evolutionary strategy of the substitution of key conserved residues to "Q150-S158-D184" motif. More importantly, the discovery of MceT contributes to a typical transporter model of CDF family with the unique structural motifs, which will be utilized to explore the cation-selective mechanisms of secondary transporters.

The majority of environmental microbiomes are not amenable to cultivation under standard laboratory growth conditions and hence remain uncharacterized. For environmental applications, such as bioremediation, it is necessary to isolate microbes performing the desired function, which may not necessarily be the fast growing or the copiotroph microbiota. Toward this end, cultivation and isolation of microbial strains using diffusion chambers (DC) and/or microbial traps (MT) have both been recently demonstrated to be effective strategies because microbial enrichment is facilitated by soil nutrients and not by synthetically defined media, thus simulating their native habitat. In this study, DC/MT chambers were established using soils collected from two US Department of Energy (DOE) sites with long-term history of heavy metal contamination, including mercury (Hg). To characterize the contamination levels and nutrient status, soils were first analyzed for total mercury (THg), methylmercury (MeHg), total carbon (TC), total nitrogen (TN), and total phosphorus (TP). Multivariate statistical analysis on these measurements facilitated binning of soils under high, medium and low levels of contamination. Bacterial and fungal microbiomes that developed within the DC and MT chambers were evaluated using comparative metagenomics, revealing Chthoniobacter, Burkholderia and Bradyrhizobium spp., as the predominant bacteria while Penicillium, Thielavia, and Trichoderma predominated among fungi. Many of these core microbiomes were also retrieved as axenic isolates. Furthermore, canonical correspondence analysis (CCA) of biogeochemical measurements, metal concentrations and bacterial communities revealed a positive correlation of Chthoniobacter/Bradyrhizobium spp., to THg whereas Burkholderia spp., correlated with MeHg. Penicillium spp., correlated with THg whereas Trichoderma spp., and Aspergillus spp., correlated with MeHg, from the MT approach. This is the first metagenomics-based assessment, isolation and characterization of soil-borne bacterial and fungal communities colonizing the diffusion chambers (DC) and microbial traps (MT) established with long-term metal contaminated soils. Overall, this study provides proof-of-concept for the successful application of DC/MT based assessment of mercury resistant (HgR) microbiomes in legacy metal-contaminated soils, having complex contamination issues. Overall, this study brings out the significance of microbial communities and their relevance in context to heavy metal cycling for better stewardship and restoration of such historically contaminated systems.

The effects of environmental stresses on microorganisms have been well-studied, and cellular responses to stresses such as heat, cold, acids, and salts have been extensively discussed. Although high pressure processing (HPP) is becoming more popular as a preservation method in the food industry, the characteristics of the cellular damage caused by high pressure are unclear, and the microbial response to this stress has not yet been well-explored. We exposed the pathogen Listeria monocytogenes to HPP (400 MPa, 8 min, 8°C) and found that the high pressure created plasma membrane pores. Using a common staining technique involving propidium iodide (PI) combined with high-frequency fluorescence microscopy, we monitored the rate of diffusion of PI molecules into hundreds of bacterial cells through these pores on days 0, 1, 2, 3, and 4 after pressurization. We also developed a mathematical dynamic model based on mass transfer and passive diffusion laws, calibrated using our microscopy experiments, to evaluate the response of bacteria to HPP. We found that the rate of diffusion of PI into the cells decreased over the 4 consecutive days after exposure to HPP, indicating repair of the pressure-created membrane pores. The model suggested a temporal change in the size of pores until closure. To the best of our knowledge, this is the first time that pressure-created membrane pores have been quantitatively described and shown to diminish with time. In addition, we found that the membrane repair rate in response to HPP was linear, and growth was temporarily arrested at the population level during the repair period. These results support the existence of a progressive repair process in some of the cells that take up PI, which can therefore be considered as being sub-lethally injured rather than dead. Hence, we showed that a subgroup of bacteria survived HPP and actively repaired their membrane pores.

Flavobacterium psychrophilum is the most important bacterial pathogen for freshwater farmed salmonids in Chile. The aims of this study were to determine the susceptibility to antimicrobials used in fish farming of Chilean isolates and to calculate their epidemiological cut-off (COWT) values. A number of 125 Chilean isolates of F. psychrophilum were isolated from reared salmonids presenting clinical symptoms indicative of flavobacteriosis and their identities were confirmed by 16S rRNA polymerase chain reaction. Susceptibility to antibacterials was tested on diluted Mueller-Hinton by using an agar dilution MIC method and a disk diffusion method. The COWT values calculated by Normalized Resistance Interpretation (NRI) analysis allow isolates to be categorized either as wild-type fully susceptible (WT) or as manifesting reduced susceptibility (NWT). When MIC data was used, NRI analysis calculated a COWT of ≤0.125, ≤2, and ≤0.5 μg mL-1 for amoxicillin, florfenicol, and oxytetracycline, respectively. For the quinolones, the COWT were ≤1, ≤0.5, and ≤0.125 μg mL-1 for oxolinic acid, flumequine, and enrofloxacin, respectively. The disk diffusion data sets obtained in this work were extremely diverse and were spread over a wide range. For the quinolones there was a close agreement between the frequencies of NWT isolates calculated using MIC and disk data. For oxolinic acid, flumequine, and enrofloxacin the frequencies were 45, 39, and 38% using MIC data, and 42, 41, and 44%, when disk data were used. There was less agreement with the other antimicrobials, because NWT frequencies obtained using MIC and disk data, respectively, were 24 and 10% for amoxicillin, 8 and 2% for florfenicol, and 70 and 64% for oxytetracycline. Considering that the MIC data was more precise than the disk diffusion data, MIC determination would be the preferred method for susceptibility testing for this species and the NWT frequencies derived from the MIC data sets should be considered as the more authoritative. Despite the high frequency of isolates showing full susceptibility to florfenicol, the significant frequencies of isolates exhibiting reduced susceptibility to oxytetracycline and quinolones may result in treatment failures when these agents are used.

This study reports the simplified carbapenem inactivation method (sCIM) to detect carbapenemase-producing gram-negative bacilli in a simple and accurate manner. This method is based on the modified carbapenem inactivation method (mCIM) with the improvement of experimental procedures. Instead of incubating the antibiotic disk in the organism culture media, the organism to be tested was smeared directly onto the antibiotic disk in the sCIM. For evaluating the sensitivity and specificity of the method, a total of 196 Enterobacteriaceae, 73 Acinetobacter baumannii, and 158 Pseudomonas aeruginosa isolates were collected. Polymerase chain reaction (PCR) was used to detect the carbapenemase genes. Phenotypic evaluations were performed using both the sCIM and the mCIM. PCR results showed that, of the 196 Enterobacteriaceae strains, 147 expressed the carbapenemase genes blaKPC-2 (58.5%), blaIMP-4 (21.8%), blaIMP-2 (2.0%), blaVIM-1 (6.1%), blaNDM-1 (10.2%), and blaOXA-48 (1.4%). sCIM results had high concordance with PCR results (99.5%) and mCIM results (100%) with the exception of one Klebsiella pneumoniae strain, which had an minimal inhibitory concentration (MIC) for imipenem of 0.25 mg/L. PCR demonstrated that 53 of the 73 A. baumannii isolates expressed the carbapenemase genes blaOXA-23 (98.1%) and blaVIM-2 (1.8%). sCIM and PCR results corresponded but all A. baumannii isolates were carbapenemase negative by the mCIM. PCR demonstrated that 25 of the 158 P. aeruginosa isolates expressed carbapenemase genes blaVIM-1 (52%) , blaVIM-2 (8%) , blaVIM-4 (36%), and blaIMP-4 (4%). sCIM results had high concordance with PCR results (100%) and the mCIM results (99.4%) with the exception of one P. aeruginosa isolate that expressed the blaVIM-4 gene. The sCIM offers specificity and sensitivity comparable to PCR but has the advantage of being more user-friendly. This method is suitable for routine use in most clinical microbiology laboratories for the detection of carbapenemase-producing gram-negative bacilli.

Ciprofloxacin is the choice treatment for infections caused by Salmonella Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn5 transposon mutants of S. Typhi were screened. S. Typhi zxx::EZ-Tn5 mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn5 transposon insertions were sequenced. Null mutants of interrupted genes, as well as inducible genetic constructs, were produced using site-directed mutagenesis, to corroborate phenotypes. SDS-PAGE and Real-time PCR were used to evaluate the expression of proteins and genes, respectively. Five mutants with increased ciprofloxacin susceptibility were found in the screening. The first confirmed mutant was the glutamine synthetase-coding gene glnA. Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the glnA mutant. Expression of ompF increased four times in the glnA null mutant compared to WT strain. To understand the relationship between the expression of glnA and ompF, a strain with the glnA gene under control of the tetracycline-inducible Ptet promoter was created, to modulate glnA expression. Induction of glnA decreased expression of ompF, at the same time that reduced susceptibility to ciprofloxacin. Expression of sRNA MicF, a negative regulator of OmpF was reduced to one-fourth in the glnA mutant, compared to WT strain. In addition, expression of glnL and glnG genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the glnA mutant. Further studies indicate that deletion of glnG decreases susceptibility to CIP, while deletion of micF gene increases susceptibility CIP. Our findings indicate that glnA inactivation promotes ompF expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.

Colisitin-associated resistance in bacteria of food producing animals has gained significant attention with the mcr gene being linked with resistance. Recently, newer variants of mcr have emerged with more than nine variants currently recognized. Reports of mcr associated resistance in Escherichia coli of poultry appear to be relatively limited, but its prevalence requires assessment since poultry is one of the most important and cheapest sources of the world's protein and the emergence of resistance could limit our ability to treat disease outbreaks. Here, 107 E. coli isolates from production poultry were screened for the presence of mcr 1-9. The isolates were collected between April 2015 and June 2016 from broiler chickens and free-range layer hens in Rio de Janeiro, Brazil. All isolates were recovered from the trachea and cloaca of healthy birds and an additional two isolates were recovered from sick birds diagnosed with colibacillosis. All isolates were screened for the presence of mcr-1 to 9 using PCR and Sanger sequencing for confirmation of positive genes. Additionally, pulse field gel electrophoresis (PFGE) analysis, avian fecal E. coli (APEC) virulence associated gene screening, plasmid replicon typing and antimicrobial resistance phenotype and resistance gene screening, were also carried out to further characterize these isolates. The mcr-1 gene was detected in 62 (57.9%) isolates (61 healthy and 1 APEC) and the mcr-5 gene was detected in 3 (2.8%) isolates; mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, and mcr-9 were not detected in any isolate. In addition, mcr 1 and 5 positive isolates were phenotypically resistant to colistin using the agar dilution assay (> 8ug/ml). PFGE analysis found that most of the isolates screened had unique fingerprints suggesting that the emergence of colistin resistance was not the result of clonal dissemination. Plasmid replicon types IncI2, FIB, and B/O were found in 38, 36, and 34% of the mcr positive isolates and were the most prevalent replicon types detected; tetA and tetB (32 and 26%, respectively) were the most prevalent antimicrobial resistance genes detected and iutA, was the most prevalent APEC virulence associated gene, detected in 50% of the isolates. Approximately 32% of the isolates examined could be classified as APEC-like, based on the presence of 3 or more genes of APEC virulence associated path panel (iroN, ompT, hlyF, iss, iutA). This study has identified a high prevalence of mcr-1 in poultry isolates in Brazil, suggesting that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. Emergence of the mcr gene and associated colisitin resistance in production poultry warrants continued monitoring from the animal health and human health perspective.

This study evaluates the potential application of silver nanoparticles (AgNPs) as antimicrobial or nematicidal agents produced by the extremophile Bacillus pumilus, which was isolated from the alkaline Wadi El-Natrun Lake in Egypt. The AgNPs were characterized by ultraviolet-visible absorption spectroscopy, transmission electron microscopy, and energy dispersive x-ray spectroscopy. The size of AgNPs formed ranged from 20.12 to 29.48 nm. Panagrellus redivivus was exposed to different concentrations (0, 50, 100, 150, and 200 μg/mL) of AgNPs in a 5 mL nematode suspension (1 × 103 mL-1). The best result occurred at AgNP concentrations of 150 and 200 μg/mL, with death rates of 80 and 91%, respectively, following 48 h of exposure. AgNPs also exhibited potent antimicrobial properties when using Gram-negative and Gram-positive human pathogens, with MIC and MBC values of 5 and 10 μg/mL, respectively. These laboratory assays prove that biologically synthesized AgNPs are an ecofriendly material that can be used in lieu of solvents or toxic chemicals.

Several environmental bacteria are considered as opportunistic pathogens in cystic fibrosis (CF) and are able to persistently colonize the CF respiratory tract (CFRT). Beside Pseudomonas aeruginosa and Burkholderia cepacia complex, Pandoraea spp. are defined as pathogenic. During chronic colonization, adaptive evolution and diversified population have been demonstrated, notably for P. aeruginosa. However, the persistence of Pandoraea in the CFRT remains largely unexplored. We studied genomic and phenotypic traits of Pandoraea pulmonicola isolates successively recovered from the airways of a single CF patient and relate the results to qualitative and quantitative evolution of other cultivable pathogens and to patient clinical status. A total of 31 isolates recovered from 18 sputum samples over a 7-year period in a single CF patient were studied. Genome dynamics was assessed by pulsed-field gel electrophoresis, ERIC-PCR fingerprinting and 16S rRNA gene PCR-temporal temperature gel electrophoresis. Phenotypic features included antimicrobial susceptibility, motility, biofilm production, and virulence in Caenorhabditis elegans model. Variability was observed for all the characteristics studied leading to highly diversified patterns (24 patterns) for the 31 clonally related isolates. Some of these modifications, mainly genomic events were concomitantly observed with CFRT microbiota composition shifts and with severe exacerbations. The diversity of P. pulmonicola population studied, observed for isolates recovered from successive samples but also within a sample suggested that existence of a diversified population may represent a patho-adaptive strategy for host persistence in the heterogeneous and fluctuating CFRT environment.

Androgenetic alopecia (AGA) is a dihydrotestosterone (DHT)-mediated hair loss disorder characterized by shortened anagen hair cycle. Oligosaccharides derived from seaweeds possess diverse biological functions. However, little is known about their effects on AGA. In this study, algal oligosaccharide (AOS) was characterized for its mitigation effects on key features involved in AGA pathogenesis, such as DHT- mediated cellular signaling and shortened anagen hair cycle. AOS with varying degrees of polymerization (DP), namely, AOS (DP2), AOS (DP4-6), and AOS (DP8-12), were prepared by agar biodegradation with Flammeovirga pacifica WPAGA1, an agarolytic bacterium isolated from deep-sea sediments. In vitro results showed that AOS with varying DPs significantly ameliorated the DHT-induced alterations of regulatory factors in human hair follicle dermal papilla cells in a dose- and DP-dependent manner, as revealed by the normalization of several hair-growth-stimulating or inhibitory factors. In vivo studies showed that AOS (DP2) extended the anagen phase and thereby delayed catagen progression in mice. Furthermore, AOS (DP2) stimulated dorsal hair growth in mice by increasing hair length, density, and thickness. Therefore, our findings indicated that AOS antagonized key factors involved in AGA pathogenesis, suggesting the potential application of AOS in the prevention and the treatment of AGA.

Campylobacter spp. may cause fever, vomiting, and diarrhea in humans. Antibiotic treatment is suggested for patients with severe campylobacteriosis. However, the interpretative criteria for antibiotic susceptibility are inconsistent between Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines. The aim of the study is to investigate the antibiotic susceptibility and prevalence of cytolethal distending toxin genes and to evaluate antibiotic susceptibility testing methods in the clinical laboratories of two tertiary medical centers in Taiwan. In total, 236 bacterial isolates were collected between 2001 and 2014. The disk diffusion and E-test methods were used to evaluate the antibiotic susceptibility, and broth dilution results were used as a reference. The virulence genes cdtA, cdtB, cdtC, and ceuE were detected through polymerase chain reaction. The antimicrobial sensitivity rates for erythromycin, ciprofloxacin, and tetracycline using the broth dilution assay were 80.4, 5.4, and 3.4%, respectively. No significant differences were observed in the antibiotic susceptibility of the isolates obtained from southern and northern Taiwan. However, some differences were observed between species. The susceptibility test for erythromycin (disk diffusion) showed that the isolates with small inhibition zone diameters were all resistant, and five isolates (4.0%) with large IZDs were non-sensitive. The error rate of the disk diffusion method according to the CLSI M45-A3 guideline was 5.4% (8/148). The incompatibility rates between the E-test and broth dilution methods for erythromycin, ciprofloxacin, and tetracycline were 8.0, 5.3, and 1.3%, respectively. The positive rates of the genes cdtA and cdtC were considerably higher in Campylobacter jejuni than in C. coli. Erythromycin is recommended as the first choice of treatment for campylobacteriosis. The disk diffusion method is suitable for prescreening Campylobacter susceptibility by using the CLSI M45-A2 and EUCAST criteria (low error rate of 3.4%). If antibiotic treatment fails or IZDs are between 6 and 20 mm, minimum inhibitory concentration testing by using the E-test method is highly recommended because the results of the E-test and broth dilution methods exhibit high agreement. The error rate of disk diffusion method using CLSI M45-A3 criteria is only slightly higher than B, which is also a suitable criteria.

There have been reports of increasing azole resistance in Candida tropicalis, especially in the Asia-Pacific region. Here we report on the epidemiology and antifungal susceptibility of C. tropicalis causing invasive candidiasis in China, from a 9-year surveillance study.

Twelve strains of Leuconostoc carnosum from meat products were investigated in terms of biochemical, physiological, and functional properties. The spectrum of sugars fermented by L. carnosum strains was limited to few mono- and disaccharides, consistently with the natural habitats of the species, including meat and fermented vegetables. The strains were able to grow from 4 to 37°C with an optimum of approximately 32.5°C. The ability to grow at temperatures compatible with refrigeration and in presence of up to 60 g/L NaCl explains the high loads of L. carnosum frequently described in many meat-based products. Six strains produced exopolysaccharides, causing a ropy phenotype of colonies, according to the potential involvement on L. carnosum in the appearance of slime in packed meat products. On the other side, the study provides evidence of a potential protective role of L. carnosum WC0321 and L. carnosum WC0323 against Listeria monocytogenes, consistently with the presence in these strains of the genes encoding leucocin B. Some meat-based products intended to be consumed without cooking may harbor up to 108 CFU/g of L. carnosum; therefore, we investigated the potential impact of this load on health. No strains survived the treatment with simulated gastric juice. Three selected strains were challenged for the capability to colonize a mouse model and their immunomodulatory properties were investigated. The strains did not colonize the intestine of mice during 10 days of daily dietary administration. Intriguingly, despite the loss of viability during the gastrointestinal transit, the strains exhibited different immunomodulatory effect on the maturation of dendritic cells in vivo, the extent of which correlated to the production of exopolysaccharides. The ability to stimulate the mucosal associated immune system in such probiotic-like manner, the general absence of antibiotic resistance genes, and the lack of the biosynthetic pathways for biogenic amines should reassure on the safety of this species, with potential for exploitation of selected starters.

Quantitative techniques are a critical part of contemporary biology research, but students interested in biology enter college with widely varying quantitative skills and attitudes toward mathematics. Course-based undergraduate research experiences (CUREs) may be an early way to build student competency and positive attitudes. Here we describe the design, implementation, and assessment of an introductory quantitative CURE focused on halophilic microbes. In this CURE, students culture and isolate halophilic microbes from environmental and food samples, perform growth assays, then use mathematical modeling to quantify the growth rate of strains in different salinities. To assess how the course may impact students' future academic plans and attitudes toward the use of math in biology, we used pre- and post-quarter surveys. Students who completed the course showed more positive attitudes toward science learning and an increased interest in pursuing additional quantitative biology experiences. We argue that the classroom application of microbiology methods, combined with mathematical modeling using student-generated data, provides a degree of student ownership, collaboration, iteration, and discovery that makes quantitative learning both relevant and exciting to students.

Purpose: To investigate the susceptibility of carbapenemase-producing Enterobacterales (CPE) to mecillinam based on the recently updated European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for uncomplicated Urinary Tract Infection (uUTI). Methods: The challenge collection consisted of 105 molecularly characterized Enterobacterales [Klebsiella spp. (N = 49), Escherichia coli (N = 30), Enterobacter cloacae (n = 13), Citrobacter freundii (N = 9), Proteus mirabilis (N = 3), and Raoultella ornithinolytica (N = 1)]. Isolates produced OXA-48 (N = 18), OXA-48-like (N = 18), VIM (N = 22), NDM (N = 22), KPC (N = 12), IMI (N = 9), IMP (N = 6), GES (N = 1), OXA-58 (N = 2) or combinations thereof (N = 5). MICs of carbapenems were determined by agar gradient diffusion (AGD). MICs of mecillinam were assessed by agar dilution (reference method) and compared to disk diffusion (DD) and AGD. Results: Overall 23/105 CPE (21.9%) were susceptible to mecillinam. Susceptibility was observed in E. coli (N = 12), E. cloacae (N = 7), and Klebsiella pneumoniae (N = 4) producing IMI, OXA-48, OXA-48-like, and NDM-1 carbapenemases. MIC50 for mecillinam in all isolates was 128 mg/L while MIC50 for meropenem was 8 mg/L. Lower MICs for mecillinam were found in IMI (MIC50 8 mg/L) and OXA-48-like (MIC50 16 mg/L) producers. The comparison of the different susceptibility methods showed very major errors of 12.2% with AGD and 8.5% with disk diffusion when compared to the reference method. Conclusion: Mecillinam susceptibility was restricted to isolates producing IMI-, OXA-48-like, and NDM-1 carbapenemases and was documented despite high carbapenem MICs in some isolates. Mecillinam could be a promising oral antimicrobial in uUTI caused by E. coli and E. cloacae isolates carrying IMI- and OXA-48-like carbapenemases; however, susceptibility testing by AGD and disk diffusion remains problematic.