Unique physiological and genetic features of ofloxacin-resistant Streptomyces mutants

New antimicrobial agents are urgently needed to combat the emergence and spread of multidrug-resistant bacteria. Activating the cryptic biosynthetic gene clusters for actinomycete secondary metabolites can provide essential clues for research into new antimicrobial agents. An effective method for this purpose is based on drug resistance selection. This report describes interesting results for drug resistance selection using antibiotics that target DNA replication can effectively potentiate secondary metabolite production by actinomycetes. Ofloxacin-resistant mutants were isolated from five different streptomycetes. Ofloxacin is an antibiotic that binds to DNA complexes and type II topoisomerase, causing double-stranded breaks in bacterial chromosomes. Physiological and genetic characterization of the mutants revealed that the development of ofloxacin resistance in streptomycetes leads to the emergence of various types of secondary metabolite-overproducing strains. In Streptomyces coelicolor A3(2), ofloxacin-resistant mutants that overproduced actinorhodin, undecylprodigiosin, or carotenoid were identified. Also, an ofloxacin-resistant mutant that overproduces methylenomycin A, whose biosynthetic gene cluster is located on the endogenous plasmid, SCP1, was isolated. These observations indicate that ofloxacin resistance might activate biosynthetic genes on both chromosomes and on endogenous plasmids. We also identified the mutations that are probably involved in the phenotype of ofloxacin resistance and secondary metabolite overproduction in S. coelicolor A3(2). Furthermore, we observed an interesting phenomenon in which several ofloxacin-resistant mutants overproduced antibiotics in the presence of ofloxacin. Based on these results, we present the unique physiological and genetic characteristics of ofloxacin-resistant Streptomyces mutants and discuss the importance and potential development of the new findings. IMPORTANCE The abuse or overuse of antibacterial agents for therapy and animal husbandry has caused an increased population of antimicrobial-resistant bacteria in the environment. Consequently, there are now fewer effective antimicrobials available. Due to the depleted antibiotic pipeline, pandemic outbreaks caused by antimicrobial-resistant bacteria are deeply concerned, and the development of new antibiotics is now an urgent issue. Promising sources of antimicrobial agents include cryptic biosynthetic gene clusters for secondary metabolites in streptomycetes and rare actinomycetes. This study’s significance is an unprecedented activation method to accelerate drug discovery research on a global scale. The technique developed in this study could allow for simultaneous drug discovery in different countries, maximizing the world’s microbial resources.

Candida glabrata MSH2 deletion increases antifungal tolerance in vitro and during intra-abdominal candidiasis (IAC), it does not impact pathogenesis of peritonitis or intra-abdominal abscesses

Background: IAC is the second most common type of invasive Candidiasis, but its pathogenesis is poorly understood. We have shown that Candida albicans DNA damage response genes are strongly induced within intra-abdominal abscesses. Deletion of C. glabrata MSH2, A DNA mismatch repair (MMR) gene, results in a mutator phenotype that facilitates multidrug resistance in vitro and in mouse gastrointestinal tracts. Our goal was to determine if CGMSH2 Contributed to pathogenesis or resistance to the new antifungal rezafungin during IAC. Methods: We createdΔMSH2 in BG2 using SAT-Flipper, and tested virulence and rezafungin responses in a mouse model of IAC. Results: ΔMSH2 displayed no growth defects at 30°C in liquid (YPD, Ypglycerol) or solid media (YPD+0.02% MMS, 1MM H2O2, 1M NACL, 20 UG/ML CW, 250 UG/ML OR 0.02% SDS). ΔMSH2 longevity in YPD was comparable to BG2. Caspofungin-, Rezafungin- and Fluconazole-resistant mutants arose 24-, 16- and 3-fold more often, respectively, for ΔMSH2 than BG2 (108-106 CFU overnight in YPD, selected on 8XMIC-Containing plates). However, respective minimum inhibitory concentrations (MICS) were not different, nor were rezafungin time-kills.ΔMSH2 was comparable to BG2 in peritonitis and abscess burdens in mouse IAC.ΔMSH2 demonstrated significantly greater caspofungin- and fluconazole-tolerance than BG2 in abscesses. Rezafungin reduced peritonitis and abscess burdens ofΔMSH2,BG2 ANDFKS mutant strains to similar extents. Conclusions: CgMSH2 deletionincreased the frequency of spontaneously-arising echinocandin- and fluconazole-resistant colonies in vitro and tolerance in intra-abdominal abscesses, but it did not attenuate virulence or rezafungin responses during IAC.

Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput

Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.

Mcc1229, an Stx2a-amplifying microcin, is produced in vivo and requires CirA for activity

Enterohemorrhagic E. coli (EHEC) strains, including the foodborne pathogen E. coli O157:H7, are responsible for thousands of hospitalizations each year. Various environmental triggers can modulate pathogenicity in EHEC by inducing expression of Shiga toxin (Stx), which is encoded on a lambdoid prophage and transcribed together with phage late genes. Cell-free supernatants of the sequence type (ST) 73 E. coli strain 0.1229 are potent inducers of Stx2a production in EHEC, suggesting that 0.1229 secretes a factor that activates the SOS response and leads to phage lysis. We previously demonstrated that this factor, designated microcin (Mcc) 1229, was proteinaceous and plasmid-encoded. To further characterize Mcc1229 and support its classification as a microcin, we investigated its regulation, determined its receptor, and identified loci providing immunity. Production of Mcc1229 was increased upon iron limitation, as determined by ELISA, lacZ fusions, and qRT-PCR. Spontaneous Mcc1229-resistant mutants and targeted gene deletion revealed that CirA was the Mcc1229 receptor. TonB, which interacts with CirA in the periplasm, was also essential for Mcc1229 import. Subcloning of the Mcc1229 plasmid indicated that Mcc activity was neutralized by two ORFs, each predicted to encode a domain of unknown function (DUF)-containing protein. In a germfree mouse model of infection, colonization with 0.1229 suppressed subsequent colonization of EHEC. Although Mcc1229 was produced in vivo , it was dispensable for colonization suppression. The regulation, import, and immunity determinants identified here are consistent with features of other Mccs, suggesting that Mcc1229 be included in this class of small molecules.

New Delhi metallo-beta-lactamase facilitates the emergence of cefiderocol resistance in Enterobacter cloacae .

Cefiderocol is a promising novel siderophore cephalosporin for the treatment of multi-drug resistant Gram-negative bacilli and with stability against degradation by metallo-β-lactamases. Nonetheless, the emergence of cefiderocol in metallo-β-lactamase-producing Enterobacterales during therapy has been reported on more than one occasion. To understand the underlying mechanisms and factors facilitating the resistance development, we conducted an in vitro evolution experiment using clinical E. cloacae isolates via serial passaging under cefiderocol pressure. In this study, we show that the presence of the New-Delhi metallo-β-lactamase (NDM) facilitates the emergence of resistance via non-synonymous mutations of the CirA catecholate siderophore receptor. Inhibition of metallo-β-lactamase activity using dipicolinic acid prevented the emergence of cefiderocol-resistant mutants successfully. This finding implies that caution should be taken, when using cefiderocol for the treatment of infections caused by metallo-β-lactamase- producing bacteria.

Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfcrt and pfmdr1 genes in isolates from Honduras and Nicaragua, 2018–2021

Background Central America and the island of Hispaniola have set out to eliminate malaria by 2030. However, since 2014 a notable upturn in the number of cases has been reported in the Mosquitia region shared by Nicaragua and Honduras. In addition, the proportion of Plasmodium falciparum malaria cases has increased significantly relative to vivax malaria. Chloroquine continues to be the first-line drug to treat uncomplicated malaria in the region. The objective of this study was to evaluate the emergence of chloroquine resistant strains of P. falciparum using a genetic approach. Plasmodium vivax populations are not analysed in this study. Methods 205 blood samples from patients infected with P. falciparum between 2018 and 2021 were analysed. The pfcrt gene fragment encompassing codons 72–76 was analysed. Likewise, three fragments of the pfmdr1 gene were analysed in 51 samples by nested PCR and sequencing. Results All samples revealed the CVMNK wild phenotype for the pfcrt gene and the N86, Y184F, S1034C, N1042D, D1246 phenotype for the pfmdr1 gene. Conclusions The increase in falciparum malaria cases in Nicaragua and Honduras cannot be attributed to the emergence of chloroquine-resistant mutants. Other possibilities should be investigated further. This is the first study to report the genotype of pfmdr1 for five loci of interest in Central America.

Selection for phage resistance reduces virulence of Shigella flexneri .

There is increasing interest in phage therapy as an alternative to antibiotics for treating bacterial infections, especially using phages that select for evolutionary trade-offs between increased phage resistance and decreased fitness traits such as virulence in target bacteria. A vast repertoire of virulence factors allows the opportunistic bacterial pathogen, Shigella flexneri , to invade human gut epithelial cells, replicate intracellularly, and evade host immunity through intercellular spread. It is previously shown that OmpA is necessary for intercellular spread of S. flexneri . We hypothesized that a phage which uses OmpA as a receptor to infect S. flexneri , should select for phage-resistant mutants with attenuated intercellular spread. Here we show that phage A1-1, requires OmpA as a receptor and selects for reduced virulence in S. flexneri . We characterized five phage-resistant mutants by measuring phenotypic changes in various traits: cell-membrane permeability, total lipopolysaccharide (LPS), sensitivity to antibiotics, and susceptibility to other phages. Results separated the mutants into two groups: R1 and R2 phenotypically resembled ompA knockouts, whereas R3, R4 and R5 were similar to LPS-deficient strains. Whole genome sequencing confirmed that R1 and R2 had mutations in ompA , while R3, R4 and R5 showed mutations in LPS inner-core biosynthesis genes gmhA and gmhC . Bacterial plaque assays confirmed that all phage-resistant mutants were incapable of intercellular spread. We concluded that selection for S. flexneri resistance to phage A1-1 generally reduced virulence (i.e. intercellular spread), but this trade-off could be mediated either by mutations in ompA or in LPS-core genes that likely altered OmpA conformation. Author Summary Shigella flexneri is a facultative intracellular pathogen of humans, and a leading cause of bacillary dysentery. With few effective treatments and rising antibiotic resistance in these bacteria, there is increasing interest in alternatives to classical infection management of S. flexneri infections. Phage therapy poses an attractive alternative, particularly if a therapeutic phage can be found that results in an evolutionary trade-off between phage resistance and bacterial virulence. Here, we isolate a novel lytic phage from water collected in Cuatro Cienegas, Mexico that uses the OmpA porin of S. flexneri as a receptor. We use phenotypic assays and genome sequencing to show that phage A1-1 selects for phage-resistant mutants that can be grouped into two categories: OmpA-deficient mutants and LPS-deficient mutants. Despite these underlying mechanistic differences, we confirmed that naturally-occurring phage A1-1 selected for evolved phage resistance that coincided with impaired intercellular spread of S. flexneri in a eukaryotic infection model.

Inhibitor Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition

Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated twenty Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (eight mutants), enniatin B (eight) or beauvericin (four). The twenty mutations were in just nine residues at the centres of transmembrane segment 1 (TMS1) (six mutations), TMS4 (four), TMS5 (four), TMS8 (one) and TMS11 (two) and in A713P (three), a previously reported FK506-resistant ‘hotspot 1’ mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) isolates were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25 resistant mutants were altered in four amino acids: G521R, M639I, A713P and T1355N. These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N (TMS11) possibly cause inhibitor-resistance by altering TMS-contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.

A Survey of Spontaneous Antibiotic-Resistant Mutants of the Halophilic, Thermophilic Bacterium Rhodothermus marinus

Rhodothermus marinus is a halophilic extreme thermophile, with potential as a model organism for studies of the structural basis of antibiotic resistance. In order to facilitate genetic studies of this organism, we have surveyed the antibiotic sensitivity spectrum of R. marinus and identified spontaneous antibiotic-resistant mutants. R. marinus is naturally insensitive to aminoglycosides, aminocylitols and tuberactinomycins that target the 30S ribosomal subunit, but is sensitive to all 50S ribosomal subunit-targeting antibiotics examined, including macrolides, lincosamides, streptogramin B, chloramphenicol, and thiostrepton. It is also sensitive to kirromycin and fusidic acid, which target protein synthesis factors. It is sensitive to rifampicin (RNA polymerase inhibitor) and to the fluoroquinolones ofloxacin and ciprofloxacin (DNA gyrase inhibitors), but insensitive to nalidixic acid. Drug-resistant mutants were identified using rifampicin, thiostrepton, erythromycin, spiramycin, tylosin, lincomycin, and chloramphenicol. The majority of these were found to have mutations that are similar or identical to those previously found in other species, while several novel mutations were identified. This study provides potential selectable markers for genetic manipulations and demonstrates the feasibility of using R. marinus as a model system for studies of ribosome and RNA polymerase structure, function, and evolution.