Unveiling azole resistance mechanisms in Candida glabrata clinical isolates encoding wild-type or gain-of-function CgPdr1 alleles

The relevance of C. glabrata as a human pathogen is linked with its poor susceptibility to azoles as well as its extreme genomic plasticity that allows the rapid acquisition of resistance. Extensive characterization of azole-resistant C. glabrata strains unveiled the central role of the transcriptional regulator CgPdr1 in the resistance phenotype, with many strains encoding hyperactive (or gain-of-function; GOF) CgPdr1 alleles. Large scale profiling of a collection of clinical C. glabrata isolates recovered in hospitals of the Lisbon area, in Portugal, led to the identification of 11 strains exhibiting resistance to fluconazole and voriconazole, while 2 were only resistant to fluconazole. Among these strains, 10 were found to encode alleles of the CgPDR1 gene harbouring multiple non-synonymous SNPs that were not found in the alleles encoded by susceptible strains, including K274Q, I392M and I803T not previously described as GOF mutations. The isolates encoding these alleles were found to over-express several CgPdr1 target genes including the azole efflux pump CgCDR1 sustaining the idea that these represent new gain-of-function CgPdr1 alleles. Only one of the identified azole-resistant strains was found to encode a CgPDR1 allele fully identical to the one encoded by susceptible strains. To better understand the resistance phenotype of this strain, its transcriptome was compared with the one of a susceptible strain and of strains encoding CgPdr1 GOF alleles. The results of this comparative transcriptomic analysis will be discussed shedding light into the different azole-resistance mechanisms evolved by C. glabrata, including those independent of CgPdr1 GOF strains.

Phenotypic detection, antimicrobial susceptibility and virulence profile of staphylococci in the pig production setting, Makurdi, Nigeria

Livestock, particularly pigs, have increasingly been recognized as important reservoirs for zoonotic transmission of pathogenic bacteria, including staphylococci. Livestock production systems in developing countries of sub-Saharan Africa, including Nigeria, are characterized by high misuse/abuse of antimicrobials and a close association between humans and these animals, which promotes the emergence and transmission of resistant and potentially virulent bacteria. In the present study, we investigated the occurrence and characteristics (species distribution, virulence and resistance profile) of staphylococci from smallholder backyard pig farms, slaughter slabs and pig handlers in Makurdi, Nigeria. A total of 330 nasal swabs originating from 300 pigs and 30 in-contact humans were collected and processed. One hundred and thirteen samples [34.2 %; 95 % confidence interval (CI): 29.1–39.6] comprising 103 (34.3 %; 95 % CI: 29.0–40.0) and 10 (33.3 %; 95 % CI: 17.3–52.8 %) samples from pigs and humans, respectively, were positive for staphylococci, yielding 120 isolates (pigs n=110, humans n=10). The 120 isolates were distributed into 15 species with Staphylococcus aureus (n=25) followed by Staphylococcus cohnii (n=19) and Staphylococcus sciuri (n=14) occurring more frequently. All isolates were resistant to β-lactam (100 %) antibiotics. Resistance to some critical antimicrobials, including linezolid (22 %), vancomycin (19.2 %), gentamicin (7.5%) and the fluoroquinolones ciprofloxacin (75.8 %) and enrofloxacin (66.7 %), was also observed. Majority (99.2 %) of the isolates displayed a multidrug resistance phenotype with the AMP-C-CIP-E-ENR-FOX-OX-P-S-SXT-TE phenotype being predominant. Overall, 70 % of the isolates expressed the methicillin resistance phenotype, out of which 20 % (n=17) were MRSA. Resistance to serum bactericidal activity and biofilm production were respectively observed in 45 (100 %) and 5 (11.3 %) of the coagulase-positive staphylococci. Our findings demonstrated the occurrence of a high diversity of staphylococci expressing multidrug resistance and potentially virulent phenotypes among healthy swine and pig handlers in small-scale backyard farms in North-Central Nigeria. These findings underscore the potential role of pig production settings in the emergence and dissemination of potentially virulent staphylococci and the importance of the development of antimicrobial resistance monitoring systems/implementation of control measures in developing countries. Proper hygienic practices and control of indiscriminate use and misuse of antibiotics are recommended.

Interrogating Novel Bromodomain Inhibition Resistance Mechanism in Mllr Leukemia

MLL-rearranged (MLLr) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLLr patients achieved an initial remission but ultimately relapsed. Bromo- and Extra-Terminal domain inhibitors (BETi) prevent the progression of many cancer types in preclinical studies, including MLLr leukemia. However, the mechanisms controlling drug response and resistance of BET inhibitors are not well understood. We have addressed this timely, crucial scientific question by completing genetic screens to explore potential BETi resistance mechanisms. By conducting genome-wide and targeted loss-of-function CRISPR screens using MLLr AML cell lines upon BETi treatment including ABBV-744, JQ1, and dBET1, we discovered that Speckle Type POZ (SPOP) gene deficiency leads to significant BETi resistance in in vitro cell culture systems (SEM, OCI-AMl2 and MV4,11), and by in vivo transplantation of human MLLr leukemia SEM cells into immune-deficient mice. However, no BETi resistance phenotype was seen in non-MLLr SPOP-deficient cells. SPOP was previously reported as an adaptor protein to bridge the E3 ubiquitination complex component CUL3 to the substrate proteins BRD4 and MYC in prostate and many other solid cancers. However, in SPOP knockout MLLr leukemia cells, TRIM24, not BRD4 and MYC, was identified as a substrate likely responsible for SPOP's role in drug resistance. Genetically blocking TRIM24 via CRISPR knockout in SPOP-knockout cells reversed the BETi resistance phenotype. Transcriptomic analysis of TRIM24-deficient cells identified the GSK3A signature as the top influenced pathway. Additionally, proteomics expression analysis and a kinase vulnerability CRISPR screen also indicated that resistant cells are sensitive to GSK3B inhibition. Further validation by CRISPR knockout and pharmaceutical blockage of GSK3A/3B (by ChIR-98014) sensitized the SPOP-deficient resistant cells to BETi treatment in vitro. In SEM xenograft models in NSG mice, ABBV-744 or CHIR-98014 minimally impacted human CD45 + leukemia cell proliferation while synergistic treatment significantly reduced the tumor progression. In summary, our data suggest the novel SPOP/TRIM24/GSK3A/3B axis plays an essential role in BETi therapy-resistant leukemia cells. Targeting GSK3A/3B pathways by ChIR-98014 can overcome SPOP-associated BETi resistance in in vivo preclinical models of MLLr leukemia. Successful outcomes following combination therapy using ChIR-98014 and BETi in PDX models would translate to a clinical application that holds the promise to cure MLLr leukemia. Disclosures No relevant conflicts of interest to declare.

Challenges of Phage Therapy as a Strategic Tool for the Control of Salmonella Kentucky and Repertoire of Antibiotic Resistance Genes in Africa

Salmonella Kentucky ST198 (S. Kentucky ST198) is the most ubiquitous multidrug resistant (MDR) strain posing the greatest threat to public health, livestock and food industry in Africa. The reinvention of bacteriophage (Phage) as a non-antibiotic alternative only gives a glimmer of hope in the control of MDR strains of Salmonellae. S. Kentucky ST198 posses’ chromosomal and plasmid factors capable of been co-opted into phage mediated transduction and co-transduction of antibiotic resistance genes (ARGs) as well as cross-serovar transduction of ARGs. Phage DT104, DT120 and P-22 like prophages like PDT17 and ES18 together have been shown to be capable of transducing and co-transducing the classical ACSSuT resistance phenotype identified in most S. Kentucky ST198 strain on the continent. Also, the institution of fluoroquinolones and third generation cephalosporin for salmonellosis treatment in animals or human infected by S. Kentucky ST198 strain resistant to these drugs can induce Salmonella phage transduction of kanamycin between different Salmonella serovars if present. This review highlights possible risk associated with the use of known Salmonella phages in the control of S. Kentucky ST198 and the need for chromosomal and plasmid tracking of genes prior to the institution of phage therapy on the continent.

Genomic epidemiology of rifampicin ADP-ribosyltransferase (Arr) in the Bacteria domain

Arr is an ADP-ribosyltransferase enzyme primarily reported in association with rifamycin resistance, which has been used to treat tuberculosis in addition to Gram-positive infections and, recently, pan-resistant Gram-negative bacteria. The arr gene was initially identified on the Mycolicibacterium smegmatis chromosome and later on Proteobacteria plasmids. This scenario raised concerns on the distribution and spread of arr, considering the Bacteria domain. Based on 198,082 bacterial genomes/metagenomes, we performed in silico analysis, including phylogenetic reconstruction of Arr in different genomic contexts. Besides, new arr alleles were evaluated by in vitro analysis to assess their association with rifampin resistance phenotype. The arr gene was prevalent in thousands of chromosomes and in hundreds of plasmids from environmental and clinical bacteria, mainly from the phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Furthermore, this gene was identified in other and new genomic contexts. Interestingly, Arr sequences associated with rifampin resistance were distributed across all phylogeny, indicating that, despite the diversity, their association with rifampin resistance phenotype were maintained. In fact, we found that the key residues were highly conserved. In addition, other analyzes have raised evidence of another Arr function, which is related to guanidine metabolism. Finally, this scenario as a whole also suggested the Actinobacteria phylum as a potential ancestral source of arr within the Bacteria domain.

The natural occurring Y129F polymorphism in Rhizopus oryzae (R. arrhizus) Cyp51Ap accounts for its intrinsic voriconazole resistance

Rhizopus oryzae (heterotypic synonym: R. arrhizus) intrinsic voriconazole and fluconazole resistance has been linked to its CYP51A gene. However, the amino acid residues involved in this phenotype have not yet been established. A comparison between R. oryzae and Aspergillus fumigatus Cyp51Ap sequences showed differences in several amino acid residues. Some of them were already linked with voriconazole resistance in A. fumigatus. The objective of this work was to analyze the role of two natural polymorphisms in the intrinsic voriconazole resistance phenotype of R. oryzae (Y129F and T290A, equivalent to Y121F and T289A seen in triazole-resistant A. fumigatus). We have generated A. fumigatus chimeric strains harboring different R. oryzae CYP51A genes (wild-type and mutants). These mutant R. oryzae CYP51A genes were designed to carry nucleotide changes that produce mutations at Cyp51Ap residues 129 and 290 (emulating the Cyp51Ap protein of azole susceptible A. fumigatus). Antifungal susceptibilities were evaluated for all the obtained mutants. The polymorphism T290A (alone or in combination with Y129F) had no impact on triazole MIC. On the other hand, a > 8-fold decrease in voriconazole MICs was observed in A. fumigatus chimeric strains harboring the RoCYP51Ap-F129Y. This phenotype supports the assumption that the naturally occurring polymorphism Y129F at R. oryzae Cyp51Ap is responsible for its voriconazole resistance phenotype. In addition, these chimeric mutants were posaconazole hypersusceptible. Thus, our experimental data demonstrate that the RoCYP51Ap-F129 residue strongly impacts VRC susceptibility and that it would be related with posaconazole-RoCYP51Ap interaction. Lay summary Rhizopus oryzae is intrinsically resistant to voriconazole, a commonly used antifungal agent. In this work, we analyze the role of two natural polymorphisms present in the target of azole drugs. We established that F129 residue is responsible of the intrinsic voriconazole resistance in this species.

Genomic characterisation of an mcr-1 and mcr-3-producing Escherichia coli strain isolated from pigs in France

The current study is about genomic characterisation of an atypical multidrug-resistant Escherichia coli harbouring two mobilised colistin resistance (mcr) genes isolated from pigs in France. Stool samples taken from a pig farm in Avignon in the department of the Vaucluse were subjected to a molecular screening for the detection of mcr gene variants. These samples were cultured on selective LBJMR medium. Growing bacteria were identified using MALDI-TOF, followed by antibiotic susceptibility testing (AST). Whole genome sequencing (WGS) and bioinformatic genome analysis was performed. The selective culture of stools revealed the presence of an E. coli strain named Q4552 which was simultaneously positive for the mcr-1.1 and mcr-3.5 genes. This strain exhibited resistance phenotype to fourteen antibiotics, including colistin. Genome sequencing revealed a circular chromosome and eight plasmids. Genomic analysis revealed a chromosomic integration of a mobile genetic element (MGE) harbouring the mcr-1.1 gene, while the mcr-3.5 gene was plasmidic (i.e., an IncFII plasmid). Its resistome exhibited twenty-two resistance genes, explaining its multidrug resistance phenotype. The Q4552 strain is an ST-843 clone belonging to the clonal complex Cplx-568 and is the only ST type of this cplx-568 which has been isolated from animals, humans, and the environment. Here, we report the first co-occurrence of the mcr-1 and mcr-3 genes in France from a pathogenic E. coli strain isolated from a pig farm. Since this clone (ST-843) has been reported in zoonotic transmissions, programs to monitor such colistin resistant bacterium are urgently required to avoid its spread and zoonotic transmission to humans.

Emergence of a tet(M) Variant Conferring Resistance to Tigecycline in Streptococcus suis

The aim of this study was to gain insight into the resistance determinants conferring resistance to tigecycline in Streptococcus (S.) suis and to investigate the genetic elements involved in their horizontal transfer. A total of 31 tetracycline-resistant S. suis isolates were screened for tigecycline resistance by broth microdilution. S. suis isolate SC128 was subjected to whole genome sequencing with particular reference to resistance determinants involved in tigecycline resistance. Transferability of genomic island (GI) GISsuSC128 was investigated by transformation. The roles of tet(L) or tet(M) in contributing to tigecycline resistance in S. suis were confirmed by transformation using different tet(L)- or tet(M)-carrying constructs. Only S. suis SC128 showed a tigecycline resistance phenotype. A tet(L)-tet(M) and catA8 co-carrying GISsuSC128 was identified in this isolate. After transfer of the novel GI into a susceptible recipient, this recipient showed the same tigecycline resistance phenotype. Further transfer experiments with specific tet(L)- or tet(M)-carrying constructs confirmed that only tet(M), but not tet(L), contributes to resistance to tigecycline. Protein sequence analysis identified a Tet(M) variant, which is responsible for tigecycline resistance in S. suis SC128. It displayed 94.8% amino acid identity with the reference Tet(M) of Enterococcus faecium DO plasmid 1. To the best of our knowledge, this is the first time that a tet(M) variant conferring resistance to tigecycline was identified in S. suis. Its location on a GI will accelerate its transmission among the S. suis population.

Newly identified parasitic nematode beta-tubulin alleles confer resistance to benzimidazoles

Infections by parasitic nematodes cause large health and economic burdens worldwide. We use anthelmintic drugs to reduce these infections. However, resistance to anthelmintic drugs is extremely common and increasing worldwide. It is essential to understand the mechanisms of resistance to slow its spread. Recently, four new parasitic nematode beta-tubulin alleles have been identified in benzimidazole (BZ) resistant parasite populations: E198I, E198K, E198T, and E198stop. These alleles have not been tested for the ability to confer resistance or for any effects that they might have on organismal fitness. We introduced these four new alleles into the sensitive C. elegans laboratory-adapted N2 strain and exposed these genome-edited strains to both albendazole and fenbendazole. We found that all four alleles conferred resistance to both BZ drugs. Additionally, we tested for fitness consequences in both control and albendazole conditions over seven generations in competitive fitness assays. We found that none of the edited alleles had deleterious effects on fitness in control conditions and that all four alleles conferred strong and equivalent fitness benefits in BZ drug conditions. Because it is unknown if previously validated alleles confer a dominant or recessive BZ resistance phenotype, we tested the phenotypes caused by five of these alleles and found that none of them conferred a dominant BZ resistance phenotype. Accurate measurements of resistance, fitness effects, and dominance caused by the resistance alleles allow for the generation of better models of population dynamics and facilitate control practices that maximize the efficacy of this critical anthelmintic drug class.