Novel gold(III)-dithiocarbamate complex targeting bacterial thioredoxin reductase: antimicrobial activity, synergy, toxicity, and mechanistic insights.

Introduction: Antimicrobial resistance is a pressing global concern that has led to the search for new antibacterial agents with novel targets or non-traditional approaches. Recently, organogold compounds have emerged as a promising class of antibacterial agents. In this study, we present and characterize a (C^S)-cyclometallated Au(III) dithiocarbamate complex as a potential drug candidate. Methods and results: The Au(III) complex was found to be stable in the presence of effective biological reductants, and showed potent antibacterial and antibiofilm activity against a wide range of multidrug-resistant strains, particularly gram-positive strains, and gram-negative strains when used in combination with a permeabilizing antibiotic. No resistant mutants were detected after exposing bacterial cultures to strong selective pressure, indicating that the complex may have a low propensity for resistance development. Mechanistic studies indicate that the Au(III) complex exerts its antibacterial activity through a multimodal mechanism of action. Ultrastructural membrane damage and rapid bacterial uptake suggest direct interactions with the bacterial membrane, while transcriptomic analysis identified altered pathways related to energy metabolism and membrane stability including enzymes of the TCA cycle and fatty acid biosynthesis. Enzymatic studies further revealed a strong reversible inhibition of the bacterial thioredoxin reductase. Importantly, the Au(III) complex demonstrated low cytotoxicity at therapeutic concentrations in mammalian cell lines, and showed no acute in vivo toxicity in mice at the doses tested, with no signs of organ toxicity. Discussion: Overall, these findings highlight the potential of the Au(III)-dithiocarbamate scaffold as a basis for developing novel antimicrobial agents, given its potent antibacterial activity, synergy, redox stability, inability to produce resistant mutants, low toxicity to mammalian cells both in vitro and in vivo, and non-conventional mechanism of action.

Clinical Escherichia coli: From Biofilm Formation to New Antibiofilm Strategies.

Escherichia coli is one of the species most frequently involved in biofilm-related diseases, being especially important in urinary tract infections, causing relapses or chronic infections. Compared to their planktonic analogues, biofilms confer to the bacteria the capacity to be up to 1000-fold more resistant to antibiotics and to evade the action of the host's immune system. For this reason, biofilm-related infections are very difficult to treat. To develop new strategies against biofilms, it is important to know the mechanisms involved in their formation. In this review, the different steps of biofilm formation in E. coli, the mechanisms of tolerance to antimicrobials and new compounds and strategies to combat biofilms are discussed.

Antibiotic Resistance and Virulence Profiles of Klebsiella pneumoniae Strains Isolated From Different Clinical Sources.

Klebsiella pneumoniae is a Gram-negative bacterium capable of colonizing, invading, and causing infections in different anatomical sites of the human body. Its ability to evade the immune system, its increasing antimicrobial resistance and the emergence of hypervirulent pathotypes have become a major challenge in the medical field. In this study, 127 strains from different clinical sources (urine, respiratory tract or blood) were characterized for antimicrobial resistance, the presence of virulence factor genes, serum resistance, hypermucoviscosity and the ability to form biofilms. Specific characteristics of the uropathogenic strains were examined and compared with the other clinical groups. Differences were found between urine and the other groups of strains. Urine strains showed the highest antibiotic resistance (64.91%) compared to blood (63.64%) or respiratory strains (51.35%) as well as the highest extended-spectrum beta-lactamases (ESBL) production. These strains also showed statistically significant high resistance to fosfomycin (24.56%) compared to the other groups (p = 0.008). Regarding virulence, 84.21% of the urine strains presented the uge gene, showing a statistically significant difference (p = 0.03) compared to the other clinical sources, indicating a possible role of this gene in the development of urinary tract infection. In addition, 46% of biofilm-forming strains belonged to the urine sample group (p = 0.043). In conclusion, K. pneumoniae strains isolated from urine samples showed higher antimicrobial resistance, ESBL production, and biofilm-forming ability compared to those isolated from respiratory or blood samples. The rapid spread of clinical strains with these characteristics is of concern, and new therapeutic alternatives are essential to mitigate their harmful effects.

Correlation Between Antimicrobial Resistance, Virulence Determinants and Biofilm Formation Ability Among Extraintestinal Pathogenic Escherichia coli Strains Isolated in Catalonia, Spain.

Escherichia coli is a well-characterized bacterium highly prevalent in the human intestinal tract and the cause of many important infections. The aim of this study was to characterize 376 extraintestinal pathogenic E. coli strains collected from four hospitals in Catalonia (Spain) between 2016 and 2017 in terms of antimicrobial resistance, siderophore production, phylogroup classification, and the presence of selected virulence and antimicrobial resistance genes. In addition, the association between these characteristics and the ability to form biofilms was also analyzed. The strains studied were classified into four groups according to their biofilm formation ability: non-biofilm formers (15.7%), weak (23.1%), moderate (35.6%), and strong biofilm formers (25.6%). The strains were highly resistant to ciprofloxacin (48.7%), trimethoprim-sulfamethoxazole (47.9%), and ampicillin (38%), showing a correlation between higher resistance to ciprofloxacin and lower biofilm production. Seventy-three strains (19.4%) were ESBL-producers. However, no relationship between the presence of ESBL and biofilm formation was found. The virulence factor genes fimH (92%), pgaA (84.6%), and irp1 (77.1%) were the most prevalent in all the studied strains. A statistically significant correlation was found between biofilm formation and the presence of iroN, papA, fimH, sfa, cnf, hlyA, iutA, and colibactin-encoding genes clbA, clbB, clbN, and clbQ. Interestingly, a high prevalence of colibactin-encoding genes (19.9%) was observed. Colibactin is a virulence factor, which interferes with the eukaryotic cell cycle and has been associated with colorectal cancer in humans. Most colibactin-encoding E. coli isolates belonged to phylogroup B2, exhibited low antimicrobial resistance but moderate or high biofilm-forming ability, and were significantly associated with most of the virulence factor genes tested. Additionally, the analysis of their clonal relatedness by PFGE showed 48 different clusters, indicating a high clonal diversity among the colibactin-positive strains. Several studies have correlated the pathogenicity of E. coli and the presence of virulence factor genes; however, colibactin and its relationship to biofilm formation have been scarcely investigated. The increasing prevalence of colibactin in E. coli and other Enterobacteriaceae and the recently described correlation with biofilm formation, makes colibactin a promising therapeutic target to prevent biofilm formation and its associated adverse effects.

Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922.

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

Enterococcus faecalis inhibits Klebsiella pneumoniae growth in polymicrobial biofilms in a glucose-enriched medium.

Catheter-related urinary tract infections are one of the most common biofilm-associated diseases. Within biofilms, bacteria cooperate, compete, or have neutral interactions. This study aimed to investigate the interactions in polymicrobial biofilms of Klebsiella pneumoniae and Enterococcus faecalis, two of the most common uropathogens. Although K. pneumoniae was the most adherent strain, it could not maintain dominance in the polymicrobial biofilm due to the lactic acid produced by E. faecalis in a glucose-enriched medium. This result was supported by the use of E. faecalis V583 ldh-1/ldh-2 double mutant (non-producer of lactic acid), which did not inhibit the growth of K. pneumoniae. Lyophilized cell-free supernatants obtained from E. faecalis biofilms also showed antimicrobial/anti-biofilm activity against K. pneumoniae. Conversely, there were no significant differences in planktonic polymicrobial cultures. In summary, E. faecalis modifies the pH by lactic acid production in polymicrobial biofilms, which impairs the growth of K. pneumoniae.

First report of a Klebsiella pneumoniae ST466 strain causing neonatal sepsis harbouring the blaCTX-M-15 gene in Rabat, Morocco.

Klebsiella pneumoniae is one of the Gram-negative bacilli most commonly found in urine of pregnant women and causing neonatal sepsis. The aim of this study was to analyse in terms of epidemiology and antimicrobial resistance of 23 K. pneumoniae isolates collected from vaginal swabs or urine of pregnant women, from pharyngeal and ear swabs of apparently healthy newborns and from peripheral cultures and hemocultures of newborns with suspected invasive neonatal infection in Rabat, Morocco. The prevalence of K. pneumoniae was 0.6 and 0.9% among pregnant women and neonates, respectively. These strains showed lower antimicrobial resistance levels regarding the developed countries. Thus, only one strain from a neonate presented an ESBL. This is the first report of a K. pneumoniae strain causing neonatal sepsis harbouring the blaCTX-M-15 gene in an IncFII plasmid and belonging to ST466 in this area.