Inside-out, antimicrobial resistance mediated by efflux pumps in clinical strains of Acinetobacter baumannii isolated from burn wound infections.

Acinetobacter baumannii belongs to the ESKAPE group. It is classified as a critical priority group by the World Health Organization and a global concern on account of its capacity to acquire and develop resistance mechanisms to multiple antibiotics. Data from the United States indicates 500 deaths annually. Resistance mechanisms of this bacterium include enzymatic pathways such as ß-lactamases, carbapenemases, and aminoglycoside-modifying enzymes, decreased permeability, and overexpression of efflux pumps. A. baumannii has been demonstrated to possess efflux pumps, which are classified as members of the MATE family, RND and MFS superfamilies, and SMR transporters. The aim of our work was to assess the distribution of efflux pumps and their regulatory gene expression in clinical strains of A. baumannii isolated from burned patients. METHODS: From the Clinical Microbiology Laboratory at the Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra collection in Mexico, 199 strains were selected. Antibiotics susceptibilities were performed by broth microdilutions to determine minimal inhibitory concentrations. Phenotypic assays with efflux pump inhibitors were conducted using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and phenylalanine-arginine ß-naphthylamide (PAßN) in conjunction with amikacin, ceftazidime, imipenem, meropenem and levofloxacin. A search was conducted for structural genes that are linked to efflux pumps, and the relative expression of the adeR, adeS, and adeL genes was analyzed. RESULTS: Among a total of 199 strains, 186 exhibited multidrug resistance (MDR). Fluoroquinolones demonstrated the highest resistance rates, while minocycline and amikacin displayed comparatively reduced resistance rates (1.5 and 28.1, respectively). The efflux activity of fluorquinolones exhibited the highest phenotypic detection (from 85 to 100%), while IMP demonstrated the lowest activity of 27% with PAßN and 43.3% with CCCP. Overexpression was observed in adeS and adeL, with adeR exhibiting overexpression. Concluding that clinical strains of A. baumannii from our institution exhibited efflux pumps as one of the resistance mechanisms.

Comparing antibiotic resistance and virulence profiles of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa from environmental and clinical settings.

Antibiotic resistance and virulence profiles of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa, isolated from water sources collected in informal settlements, were compared to clinical counterparts. Cluster analysis using repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) indicated that, for each respective species, low genetic relatedness was observed between most of the clinical and environmental isolates, with only one clinical P. aeruginosa (PAO1) and one clinical K. pneumoniae (P2) exhibiting high genetic similarity to the environmental strains. Based on the antibiograms, the clinical E. faecium Ef CD1 was extensively drug resistant (XDR); all K. pneumoniae isolates (n = 12) (except K. pneumoniae ATCC 13883) were multidrug resistant (MDR), while the P. aeruginosa (n = 16) isolates exhibited higher susceptibility profiles. The tetM gene (tetracycline resistance) was identified in 47.4 % (n = 6 environmental; n = 3 clinical) of the E. faecium isolates, while the blaKPC gene (carbapenem resistance) was detected in 52.6 % (n = 7 environmental; n = 3 clinical) and 15.4 % (n = 2 environmental) of the E. faecium and K. pneumoniae isolates, respectively. The E. faecium isolates were predominantly poor biofilm formers, the K. pneumoniae isolates were moderate biofilm formers, while the P. aeruginosa isolates were strong biofilm formers. All E. faecium and K. pneumoniae isolates were gamma (γ)-haemolytic, non-gelatinase producing (E. faecium only), and non-hypermucoviscous (K. pneumoniae only), while the P. aeruginosa isolates exhibited beta (ß)-haemolysis and produced gelatinase. The fimH (type 1 fimbriae adhesion) and ugE (uridine diphosphate galacturonate 4-epimerase synthesis) virulence genes were detected in the K. pneumoniae isolates, while the P. aeruginosa isolates possessed the phzM (phenazine production) and algD (alginate biosynthesis) genes. Similarities in antibiotic resistance and virulence profiles of environmental and clinical E. faecium, K. pneumoniae, and P. aeruginosa, thus highlights the potential health risks posed by using environmental water sources for daily water needs in low-and-middle-income countries.

Significant variation of filamentation phenotypes in clinical Candida albicans strains.

Introduction: Candida albicans is an opportunistic human pathogen that typically resides as part of the microbiome in the gastrointestinal and genitourinary tracts of a large portion of the human population. This fungus lacks a true sexual cycle and evolves in a largely clonal pattern. The ability to cause disease is consistent across the species as strains causing systemic infections appear across the known C. albicans intra-species clades. Methods: In this work, strains collected from patients with systemic C. albicans infections isolated at the Nebraska Medicine clinical laboratory were typed by MLST analysis. Since the ability to form filaments has been linked to pathogenesis in C. albicans, these clinical strains, as well as a previously genotyped set of clinical strains, were tested for their ability to filament across a variety of inducing conditions. Results: Genotyping of the clinical strains demonstrated that the strains isolated at one of the major medical centers in our region were as diverse as strains collected across the United States. We demonstrated that clinical strains exhibit a variety of filamentation patterns across differing inducing conditions. The only consistent pattern observed in the entire set of clinical strains tested was an almost universal inability to filament in standard solid inducing conditions used throughout the C. albicans field. A different solid filamentation assay that produces more robust filamentation profiles from clinical strains is proposed in this study, although not all strains expected to filament in vivo were filamentous in this assay. Discussion: Our data supports growing evidence that broad phenotypic diversity exists between the C. albicans type strain and clinical strains, suggesting that the type strain poorly represents filamentation patterns observed in most clinical isolates. These data further highlight the need to use diverse clinical strains in pathogenesis assays.

Genetic and Functional Characterization of Congenital HCMV Clinical Strains in Ex Vivo First Trimester Placental Model.

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, leading to a variety of symptoms in the unborn child that range from asymptomatic to death in utero. Our objective was to better understand the mechanisms of placental infection by HCMV clinical strains, particularly during the first trimester of pregnancy. We thus characterized and compared the replication kinetics of various HCMV clinical strains and laboratory strains by measuring viral loads in an ex vivo model of first trimester villi and decidua, and used NGS and PCA analysis to analyze the genes involved in cell tropism and virulence factors. We observed that first trimester villi and decidua are similarly permissive to laboratory and symptomatic strains, and that asymptomatic strains poorly replicate in decidua tissue. PCA analysis allowed us to segregate our clinical strains based on their clinical characteristics, suggesting a link between gene mutations and symptoms. All these results bring forth elements that can help better understand the mechanisms that induce the appearance of symptoms or in the congenitally infected newborn.

Antibacterial, Cytotoxic and Genotoxic Assessment of New Sulfonamide Derivatives.

In the last few years, the interest in sulfonamides has expanded owing to their broad spectrum of biological activities. Their flexible structure turns them into amazing candidates to replace old drugs or develop modern multi-target agents. In this study, a series of new sulfonamides (sul1-5) was evaluated, in vitro, for the antibacterial, cytotoxic and genotoxic effects. The antibacterial activity was investigated against 12 clinical and 4 reference strains. Cytotoxic activity was carried out by the brine shrimp bioassay and the genotoxicity was assessed in the Ames test. An interesting antibacterial activity was showed especially against Gram negative strains. The inhibition zones varied between 15 and 30 mm, and the Minimum Inhibitory Concentrations (MIC's) values between 0.5 and 256 µg/ml. No antibacterial activity was shown with S. aureus isolates. Only Sul1 and Sul4 were active against P. aeruginosa. Compounds Sul1 and Sul2 showed a significant cytotoxicity with LC50 equal to 18.29 and 18 µg/ml respectively, and a genotoxic effect against TA100 and TA1535 Salmonella strains. Only compounds Sul3, Sul4 and Sul5 with an interesting antibacterial activity, no cytotoxicity and no genotoxic effects, could be exploited against resistant pathogens as new drugs.

Heterogeneity among Clinical Intestinal Escherichia coli Isolates upon Acquired Streptomycin Resistance.

Escherichia coli isolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (StrR) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous StrR strain of the intestinal adherent-invasive E. coli (AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation in rpsL that commonly confers StrR, rpsL-K43N. StrR NC101 exhibited a striking loss of aggregation and significantly increased motility, behaviors that can impact host-microbe interactions. Behavioral changes were associated with reduced transcription of csgA, encoding the biofilm component curli, and increased transcription of fliC, encoding flagellin. Scanning electron microscopy (SEM) detailed morphologic changes consistent with the observed alterations in multicellular behavior. Because intestinal E. coli isolates exhibit remarkable strain-specific differences, we generated spontaneous StrR mutants of 10 clinical E. coli phylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 StrR clinical strains, two had altered colony morphology on Congo red agar (suggesting changes in extracellular products), and three had significant changes in motility. These changes were not associated with a particular rpsL mutation nor with the presence of virulence genes encoding the inflammation-associated E. coli metabolites yersiniabactin or colibactin. We conclude that common mutations in rpsL, which confer StrR, can differentially alter disease-associated phenotypes across intestinal E. coli strains. These findings highlight the heterogeneity among seemingly similar intestinal E. coli strains and reveal the need to carefully study the strain-specific effects of antibiotic resistance mutations, particularly when using these mutations during strain selection studies. IMPORTANCE We demonstrate that StrR, commonly acquired through a single point mutation in rpsL (a gene encoding part of the 30S bacterial ribosome), strikingly alters the morphology and behavior of a key intestinal AIEC strain, NC101. These changes include remarkably diminished aggregation and significantly increased motility, traits that are linked to AIEC-defining features and disease development. Phenotypic changes were heterogeneous among other StrR clinical E. coli strains, underscoring the need to evaluate the strain-specific effects of commonly acquired antibiotic resistance mutations. This is important, as the results of studies using mutant StrR Enterobacteriaceae strains (e.g., for cloning or in vivo selection) may be confounded beyond our demonstrated effects. Long term, these findings can help researchers better distinguish the contribution of specific E. coli traits to functional changes in the microbiota. Evaluating these strain-level differences could provide insight into the diversity of IBD symptoms and lead to improved therapies for microbiota-driven intestinal disorders.

Clinical and Environmental Vibrio cholerae Non-O1, Non-O139 Strains from Australia Have Similar Virulence and Antimicrobial Resistance Gene Profiles.

Cholera caused by pathogenic Vibrio cholerae is still considered one of the major health problems in developing countries including those in Asia and Africa. Australia is known to have unique V. cholerae strains in Queensland waterways, resulting in sporadic cholera-like disease being reported in Queensland each year. We conducted virulence and antimicrobial genetic characterization of O1 and non-O1, non-O139 V. cholerae (NOVC) strains (1983 to 2020) from Queensland with clinical significance and compared these to environmental strains that were collected as part of a V. cholerae monitoring project in 2012 of Queensland waterways. In this study, 87 V. cholerae strains were analyzed where O1 (n = 5) and NOVC (n = 54) strains from Queensland and international travel-associated NOVC (n = 2) (61 in total) strains were sequenced, characterized, and compared with seven previously sequenced O1 strains and 18 other publicly available NOVC strains from Australia and overseas to visualize the genetic context among them. Of the 61 strains, three clinical and environmental NOVC serogroup strains had cholera toxin-producing genes, namely, the CTX phage (identified in previous outbreaks) and the complete Vibrio pathogenicity island 1. Phylogenetic analysis based on core genome analysis showed more than 10 distinct clusters and interrelatedness between clinical and environmental V. cholerae strains from Australia. Moreover, 30 (55%) NOVC strains had the cholix toxin gene (chxA) while only 11 (20%) strains had the mshA gene. In addition, 18 (34%) NOVC strains from Australia had the type three secretion system and discrete expression of type six secretion system genes. Interestingly, four NOVC strains from Australia and one NOVC strain from Indonesia had intSXT, a mobile genetic element. Several strains were found to have beta-lactamase (blaCARB-9) and chloramphenicol acetyltransferase (catB9) genes. Our study suggests that Queensland waterways can harbor highly divergent V. cholerae strains and serve as a reservoir for various V. cholerae-associated virulence genes which could be shared among O1 and NOVC V. cholerae strains via mobile genetic elements or horizontal gene transfer. IMPORTANCE Australia has its own V. cholerae strains, both toxigenic and nontoxigenic, that are associated with cholera disease. This study aimed to characterize a collection of clinical and environmental NOVC strains from Australia to understand their virulence and antimicrobial resistance profile and to place strains from Australia in the genetic context of international strains. The findings from this study suggest the toxigenic V. cholerae strains in the Queensland River water system are of public health concern. Therefore, ongoing monitoring and genomic characterization of V. cholerae strains from the Queensland environment are important and would assist public health departments to track the source of cholera infection early and implement prevention strategies for future outbreaks. Understanding the genomics of V. cholerae could also inform the natural ecology and evolution of this bacterium in natural environments.

Clinical strains of Mycobacterium tuberculosis exhibit differential lipid metabolism-associated transcriptome changes in in vitro cholesterol and infection models.

Many studies have identified host-derived lipids, characterised by the abundance of cholesterol, as a major source of carbon nutrition for Mycobacterium tuberculosis during infection. Members of the Mycobacterium tuberculosis complex are biologically different with regards to degree of disease, host range, pathogenicity and transmission. Therefore, the current study aimed at elucidating transcriptome changes during early infection of pulmonary epithelial cells and on an in vitro cholesterol-rich minimal media, in M. tuberculosis clinical strains F15/LAM4/KZN and Beijing, and the laboratory H37Rv strain. Infection of pulmonary epithelial cells elicited the upregulation of fadD28 and hsaC in both the F15/LAM4/KZN and Beijing strains and the downregulation of several other lipid-associated genes. Growth curve analysis revealed F15/LAM4/KZN and Beijing to be slow growers in 7H9 medium and cholesterol-supplemented media. RNA-seq analysis revealed strain-specific transcriptomic changes, thereby affecting different metabolic processes in an in vitro cholesterol model. The differential expression of these genes suggests that the genetically diverse M. tuberculosis clinical strains exhibit strain-specific behaviour that may influence their ability to metabolise lipids, specifically cholesterol, which may account for phenotypic differences observed during infection.

Isolation and Characterization of Lytic Bacteriophages Active against Clinical Strains of E. coli and Development of a Phage Antimicrobial Cocktail.

Pathogenic E. coli cause urinary tract, soft tissue and central nervous system infections, sepsis, etc. Lytic bacteriophages can be used to combat such infections. We investigated six lytic E. coli bacteriophages isolated from wastewater. Transmission electron microscopy and whole genome sequencing showed that the isolated bacteriophages are tailed phages of the Caudoviricetes class. One-step growth curves revealed that their latent period of reproduction is 20-30 min, and the average value of the burst size is 117-155. During co-cultivation with various E. coli strains, the phages completely suppressed bacterial host culture growth within the first 4 h at MOIs 10-7 to 10-3. The host range lysed by each bacteriophage varied from six to two bacterial strains out of nine used in the study. The cocktail formed from the isolated bacteriophages possessed the ability to completely suppress the growth of all the E. coli strains used in the study within 6 h and maintain its lytic activity for 8 months of storage. All the isolated bacteriophages may be useful in fighting pathogenic E. coli strains and in the development of phage cocktails with a long storage period and high efficiency in the treatment of bacterial infections.

Fist report on fumagillin production by Aspergillus fumigatus sensu stricto gliotoxigenic strains recovered from raw cow milk and clinical samples in Argentina / Primer reporte en Argentina sobre la producción de fumagilina por cepas deAspergillus fumigatus sensu stricto productoras de gliotoxina, aisladas de lechecruda y de casos clínicos

Abstract In Argentina there are no reports on Aspergillus fumigatus fumagillin-producingstrains. In this study we describe the isolation and mycotoxin production capacity of ten A.fumigatus strains isolated from farm and clinical samples. Farm strains were isolated frommilk samples taken from dairy cows in Córdoba province, some of which were associated withsubclinical mastitis. A culture medium was defined to optimize fumagillin production and adetection method was developed by HPLC chromatography. It is known that in addition to thehost immune status, strain virulence is a fundamental characteristic that will determine itspathogenicity and, in this sense, fumagillin is considered to be among the virulence factors. Inthe present work, all the strains tested for the production of fumagillin were able to synthesizeit, highlighting that the strain A. fumigatus RC2243, from a milk sample from a cow with clinicalmastitis, was the most productive. The existence of fumagillin-producing strains represents apotential risk of mycotoxins being transferred to raw milk, constituting a public health risk.

Resumen En Argentina no existen reportes sobre cepas de Aspergillus fumigatus productoras de fumagilina. En este trabajo se describe el aislamiento y la producción de dicha micotoxina clínicaspor 10 cepas, provenientes del medioambiente rural y aisladas de muestras clínicas. Las cepasde origen rural fueron aisladas de vacas lecheras en tambos de la provincia de Córdoba, yalgunas de esas cepas se asociaron a casos de mastitis subclínica. Se definió la composición deun medio de cultivo para optimizar la producción de fumagilina y se desarrolló un método decromatografía HPLC para su determinación. Es conocido que, además del estado inmunitario delhuésped, la virulencia de la cepa es una de las características fundamentales que determinansu potencial patogénico y, en este sentido, la fumagilina es considerada un factor de virulencia. En el presente trabajo todas las cepas estudiadas fueron capaces de sintetizarla y la cepa A.fumigatus RC2243, proveniente de leche de una vaca con mastitis subclínica, se destacó comola cepa más productora. La existencia de cepas productoras de fumagillina representa un riesgopotencial por el pasaje de dicha micotoxina a la leche, lo cual constituye un problema para lasalud pública.

In Vitro Analysis of Extracts of Plant Used in Mexican Traditional Medicine, Which Are Useful to Combat Clostridioides difficile Infection.

Recently, a worrying acceleration of the emergence of antibiotic-resistant bacteria has been reported. The increase in antibiotic-associated diseases, such as Clostridioides difficile infection (CDI), has promoted research on new treatments that could be more effective and less aggressive for CDI patients. This study evaluates eight plants with antimicrobial activity commonly used in Mexican traditional medicine to evaluate their potential against C. difficile. We provide essential information about these plants' activities and action mechanisms against C. difficile and their effect on different bacterial infection activities: motility, adherence, sporulation, and germination. The selected plants are rosemary, estafiate, rue, epazote, mint, toloache, ajenjo, and thyme. We used clinical isolates to test their activity against strains responsible for current outbreaks to provide more information about the clinical impact of these extracts. We found that thyme, ajenjo, and mint were the most effective against the isolates. We identified that the extracts affected protein synthesis. In addition, the extracts affect the strains' motility, and some, such as thyme extract, affect adherence, whereas rue extract affects sporulation. These results led to the identification of new compounds beneficial to CDI treatment.

Heteroaryl-Ethylenes as New Effective Agents for High Priority Gram-Positive and Gram-Negative Bacterial Clinical Isolates.

The World Health Organization has identified antimicrobial resistance as a public health emergency and developed a global priority pathogens list of antibiotic-resistant bacteria that can be summarized in the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales species), reminding us of their ability to escape the effect of antibacterial drugs. We previously tested new heteroaryl-ethylene compounds in order to define their spectrum of activity and antibacterial capability. Now, we focus our attention on PB4, a compound with promising MIC and MBC values in all conditions tested. In the present study, we evaluate the activity of PB4 on selected samples of ESKAPE isolates from nosocomial infections: 14 S. aureus, 6 E. faecalis, 7 E. faecium, 12 E. coli and 14 A. baumannii. Furthermore, an ATCC control strain was selected for all species tested. The MIC tests were performed according to the standard method. The PB4 MIC values were within very low ranges regardless of bacterial species and resistance profiles: from 0.12 to 2 mg/L for S. aureus, E. faecalis, E. faecium and A. baumannii. For E. coli, the MIC values obtained were slightly higher (4-64 mg/L) but still promising. The PB4 heteroaryl-ethylenic compound was able to counteract the bacterial growth of both high-priority Gram-positive and Gram-negative clinical strains. Our study contributes to the search for new molecules that can fight bacterial infections, in particular those caused by MDR bacteria in hospitals. In the future, it would be interesting to evaluate the activity of PB4 in animal models to test for its toxicity.

Fist report on fumagillin production by Aspergillus fumigatus sensu stricto gliotoxigenic strains recovered from raw cow milk and clinical samples in Argentina.

In Argentina there are no reports on Aspergillus fumigatus fumagillin-producing strains. In this study we describe the isolation and mycotoxin production capacity of ten A. fumigatus strains isolated from farm and clinical samples. Farm strains were isolated from milk samples taken from dairy cows in Córdoba province, some of which were associated with subclinical mastitis. A culture medium was defined to optimize fumagillin production and a detection method was developed by HPLC chromatography. It is known that in addition to the host immune status, strain virulence is a fundamental characteristic that will determine its pathogenicity and, in this sense, fumagillin is considered to be among the virulence factors. In the present work, all the strains tested for the production of fumagillin were able to synthesize it, highlighting that the strain A. fumigatus RC2243, from a milk sample from a cow with clinical mastitis, was the most productive. The existence of fumagillin-producing strains represents a potential risk of mycotoxins being transferred to raw milk, constituting a public health risk.

Multiplexed Strain Phenotyping Defines Consequences of Genetic Diversity in Mycobacterium tuberculosis for Infection and Vaccination Outcomes.

There is growing evidence that genetic diversity in Mycobacterium tuberculosis, the causative agent of tuberculosis, contributes to the outcomes of infection and public health interventions, such as vaccination. Epidemiological studies suggest that among the phylogeographic lineages of M. tuberculosis, strains belonging to a sublineage of Lineage 2 (mL2) are associated with concerning clinical features, including hypervirulence, treatment failure, and vaccine escape. The global expansion and increasing prevalence of this sublineage has been attributed to the selective advantage conferred by these characteristics, yet confounding host and environmental factors make it difficult to identify the bacterial determinants driving these associations in human studies. Here, we developed a molecular barcoding strategy to facilitate high-throughput, experimental phenotyping of M. tuberculosis clinical isolates. This approach allowed us to characterize growth dynamics for a panel of genetically diverse M. tuberculosis strains during infection and after vaccination in the mouse model. We found that mL2 strains exhibit distinct growth dynamics in vivo and are resistant to the immune protection conferred by Bacillus Calmette-Guerin (BCG) vaccination. The latter finding corroborates epidemiological observations and demonstrates that mycobacterial features contribute to vaccine efficacy. To investigate the genetic and biological basis of mL2 strains' distinctive phenotypes, we performed variant analysis, transcriptional studies, and genome-wide transposon sequencing. We identified functional genetic changes across multiple stress and host response pathways in a representative mL2 strain that are associated with variants in regulatory genes. These adaptive changes may underlie the distinct clinical characteristics and epidemiological success of this lineage. IMPORTANCE Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, is a remarkably heterogeneous disease, a feature that complicates clinical care and public health interventions. The contributions of pathogen genetic diversity to this heterogeneity are uncertain, in part due to the challenges of experimentally manipulating M. tuberculosis, a slow-growing, biosafety level 3 organism. To overcome these challenges, we applied a molecular barcoding strategy to a panel of M. tuberculosis clinical isolates. This novel application of barcoding permitted the high-throughput characterization of M. tuberculosis strain growth dynamics and vaccine resistance in the mouse model of infection. Integrating these results with genomic analyses, we uncover bacterial pathways that contribute to infection outcomes, suggesting targets for improved therapeutics and vaccines.

Purpureocillium lilacinum an emergent pathogen: antifungal susceptibility of environmental and clinical strains.

Purpureocillium lilacinum is a filamentous and hyaline fungus cosmopolitan, saprophytic, largely used in the biological control of plant-parasitic nematodes and insects, also considered an emerging and opportunistic human pathogen. The standard treatment for hyalohyphomycosis caused by P. lilacinum is not yet defined, since this fungus is resistant to different antifungals, in vitro and in vivo. The aim of this study was to evaluate and compare in vitro antifungal activity against environmental and clinical P. lilacinum isolates and our results demonstrated that these isolates can be resistant to newer generation triazoles, such as voriconazole, and to caspofungin, a drug of the echinocandin class. In summary, we highlight the importance of knowing the different susceptibility profiles of P. lilacinum isolates, and besides that, the emergence of uncommon human and animal opportunistic fungi, such P. lilacinum, especially during COVID-19, highlight the need for antifungal susceptibility testing of isolates since empirical therapy with different treatment schedules failed in great number of patients.

Dual RNA Sequencing of Mycobacterium tuberculosis-Infected Human Splenic Macrophages Reveals a Strain-Dependent Host-Pathogen Response to Infection.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host-pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host-pathogen interaction.

Antibiotic resistance and siderophores production by clinical Escherichia coli strains.

The phenomenon of antibiotic resistance has dramatically increased in the last few decades, especially in enterobacterial pathogens. Different strains of Escherichia coli have been reported to produce a variety of structurally different siderophores. In the present study, 32 E. coli strains were collected from different clinical settings in Cairo, Egypt and subjected to the antibiotic susceptibility test by using 19 antibiotics belonging to 7 classes of chemical groups. The results indicated that 31 strains could be considered as extensively drug-resistant and only one strain as pan drug-resistant. Siderophores production by all the tested E. coli strains was determined qualitatively and quantitatively. Two E. coli strains coded 21 and 49 were found to be the most potent siderophores producers, with 79.9 and 46.62%, respectively. Bacterial colonies with cured plasmids derived from strain 49 showed susceptibility to all the tested antibiotics. Furthermore, E. coli DH5α cells transformed with the plasmid isolated from E. coli strain 21 or E. coli strain 49 were found to be susceptible to ansamycins, quinolones, and sulfonamide groups of antibiotics. In contrast, both plasmid-cured and plasmid-transformed strains did not produce siderophores, indicating that the genes responsible for siderophores production were located on plasmids and regulated by genes located on the chromosome. On the basis of the obtained results, it could be concluded that there is a positive correlation between antibiotic resistance, especially to quinolones and sulfonamide groups, and siderophores production by E. coli strains used in this study.

In Vitro Susceptibility to Miltefosine of Leishmania infantum (syn. L. chagasi) Isolates from Different Geographical Areas in Brazil.

Treatment of visceral leishmaniasis in Brazil still relies on meglumine antimoniate, with less than ideal efficacy and safety, making new therapeutic tools an urgent need. The oral drug miltefosine was assayed in a phase II clinical trial in Brazil with cure rates lower than previously demonstrated in India. The present study investigated the susceptibility to miltefosine in 73 Brazilian strains of Leishmania infantum from different geographic regions, using intracellular amastigote and promastigote assays. The EC50 for miltefosine of 13 of these strains evaluated in intracellular amastigotes varied between 1.41 and 4.57 µM. The EC50 of the 73 strains determined in promastigotes varied between 5.89 and 23.7 µM. No correlation between in vitro miltefosine susceptibility and the presence of the miltefosine sensitive locus was detected among the tested strains. The relatively low heterogeneity in miltefosine susceptibility observed for the 73 strains tested in this study suggests the absence of decreased susceptibility to miltefosine in Brazilian L. infantum and does not exclude future clinical evaluation of miltefosine for VL treatment in Brazil.

Detection of a clinical carbapenem-resistant Citrobacter portucalensis strain and the dissemination of C. portucalensis in clinical settings.

OBJECTIVES: A clinical carbapenem-resistant Citrobacter portucalensis was characterised by whole-genome sequencing (WGS). METHODS: Strain 3839 was identified by VITEK®2.0 and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antimicrobial susceptibility testing was performed by microbroth dilution. WGS followed by bioinformatics analysis was performed. RESULTS: Strain 3839 was initially identified as Citrobacter freundii by VITEK and MALDI-TOF/MS, and was subsequently demonstrated to be C. portucalensis by WGS analysis. Through average nucleotide identity analysis, we detected 55 C. portucalensis genomes misidentified as C. freundii in GenBank, and at least 22 were clinically-associated, suggesting that the occurrence of C. portucalensis in the clinical setting might be underestimated by the conventional method. Strain 3839 was extensively drug-resistant and the presence of multiple resistance determinants was detected by WGS, including blaNDM-1, blaSHV-12, blaCMY-150, blaOXA-1, qnrB9, qnrA1, aac(6')-Ib-cr, aph(6)-Id_1, aph(3'')-Ib, tetA, tet34 and catB3. A total of 45 insertion sequence (IS) elements, 8 phages and 1 integron gene cassette were identified. The blaNDM-1 gene was carried by an IncX3 plasmid that was identical to a plasmid detected in a C. freundii strain. The genetic context of blaNDM-1 was IS30-blaNDM-1-bleMBL-trpF-dsbD-cutA1-groES-groEL-IS91. CONCLUSION: To our knowledge, this is the first report of carbapenem-resistant C. portucalensis isolated from a clinical sample. The blaNDM-1 gene carried by C. portucalensis may transmit among Citrobacter spp. mediated by plasmids. Together with underestimation of its clinical occurrence caused by misidentification, our study warrants the necessity of preventing the dissemination of such emerging drug-resistant species in clinical settings.

Lacticaseicin 30 and Colistin as a Promising Antibiotic Formulation against Gram-Negative ß-Lactamase-Producing Strains and Colistin-Resistant Strains.

Antimicrobial resistance is a global health concern across the world and it is foreseen to swell if no actions are taken now. To help curbing this well announced crisis different strategies are announced, and these include the use of antimicrobial peptides (AMP), which are remarkable molecules known for their killing activities towards pathogenic bacteria. Bacteriocins are ribosomally synthesized AMP produced by almost all prokaryotic lineages. Bacteriocins, unlike antibiotics, offer a set of advantages in terms of cytotoxicity towards eukaryotic cells, their mode of action, cross-resistance and impact of microbiota content. Most known bacteriocins are produced by Gram-positive bacteria, and specifically by lactic acid bacteria (LAB). LAB-bacteriocins were steadily reported and characterized for their activity against genetically related Gram-positive bacteria, and seldom against Gram-negative bacteria. The aim of this study is to show that lacticaseicin 30, which is one of the bacteriocins produced by Lacticaseibacillus paracasei CNCM I-5369, is active against Gram-negative clinical strains (Salmonella enterica Enteritidis H10, S. enterica Typhimurium H97, Enterobacter cloacae H51, Escherichia coli H45, E. coli H51, E. coli H66, Klebsiella oxytoca H40, K. pneumoniae H71, K. variicola H77, K. pneumoniae H79, K. pneumoniae H79), whereas antibiotics failed. In addition, lacticaseicin 30 and colistin enabled synergistic interactions towards the aforementioned target Gram-negative clinical strains. Further, the combinations of lacticaseicin 30 and colistin prompted a drastic downregulation of mcr-1 and mcr-9 genes, which are associated with the colistin resistance phenotypes of these clinical strains. This report shows that lacticaseicin 30 is active against Gram-negative clinical strains carrying a rainbow of mcr genes, and the combination of these antimicrobials constitutes a promising therapeutic option that needs to be further exploited.