Biology subjects

Gaballa, A.

Publications and source records attributed to Gaballa, A..

6 recordsLinked to original sources

bioRxiv2024

Lipid A phosphoethanolamine transferase-mediated site-selective modifications show association with colistin resistance phenotypes and fitness

Genes encoding lipid A modifying phosphoethanolamine transferases (PETs) are genetically diverse and can confer resistance to colistin and antimicrobial peptides. To better understand the functional diversity of PETs, we characterized three canonical mobile colistin resistance (mcr) alleles (mcr-1, -3, -9), one intrinsic pet (eptA), and two mcr-like genes (petB, petC). Using an isogenic expression system, we show that mcr-1 and mcr-3 are phenotypically similar by conferring colistin resistance with lower fitness costs. mcr-9, which is phylogenetically closely related to mcr-3, and eptA only provide fitness advantages in the presence of sub-inhibitory concentrations of colistin and significantly reduce fitness in media only. PET-B and PET-C were phenotypically distinct from bonafide PETs; neither conferred colistin resistance or caused considerable fitness cost in Escherichia coli. Strikingly, we found for the first time that different PETs selectively modify different phosphates of lipid A - MCR-1, MCR-3, and PET-C selectively modify the 4-phosphate, while MCR-9 and EptA modify the 1-phosphate. 4-phosphate modifications facilitated by MCR-1 and -3 are associated with high levels of colistin resistance and low toxicity. Our results suggest that PETs have a wide phenotypic diversity and that high level colistin resistance is associated with specific lipid A modification patterns that has been largely unexplored thus far. IMPORTANCERising levels of resistance to increasing numbers of antimicrobials has led to the revival of last resort antibiotic colistin. Unfortunately, resistance to colistin is also spreading in the form of mcr genes, making it essential to (i) improve identification of resistant bacteria to allow clinicians to prescribe effective drug regimens and (ii) develop new combination therapies effective at targeting resistant bacteria. Our results demonstrate that PETs, including MCR variants, are site-selective in E. coli, with site-selectivity correlating with the level of resistance and fitness costs conferred by certain PETs. Site-selectivity associated with a given PET may not only help predict colistin resistance phenotypes, but may also provide an avenue to (i) improved drug regimens and (ii) development of new combination therapies to better combat colistin resistant bacteria.

microbiology↗

bioRxiv2023

Escherichia coli B-strains are intrinsically resistant to colistin and not suitable for characterization and identification of mcr genes

Antimicrobial resistance is an increasing threat to human and animal health. Due to the rise of multi- and extreme drug resistance, last resort antibiotics, such as colistin, are extremely important in human medicine. While the distribution of colistin resistance genes can be tracked through sequencing methods, phenotypic characterization of putative antimicrobial resistance (AMR) genes is still important to confirm the phenotype conferred by different genes. While heterologous expression of AMR genes (e.g., in E. coli) is a common approach, so far, no standard method for heterologous expression and characterization of mcr genes exists. E. coli B-strains, designed for optimum protein expression, are frequently utilized. Here we report that four E. coli B-strains are intrinsically resistant to colistin (MIC 8 - 16 g/mL). Additionally, the three tested B-strains that encode T7 RNA show growth defects when transformed with empty or gene-expressing pET17b plasmids and grown in the presence of IPTG; K-12 or B-strains without T7 RNA polymerase do not show these growth defects. The E. coli Shuffle T7 express strain carrying empty pET17b also showed a heteroresistance phenotype when exposed to colistin in the presence of IPTG. These phenotypes could explain why B-strains were erroneously reported as colistin susceptible. Analysis of existing genome data identified one nonsynonymous change in each pmrA and pmrB in all four E. coli B-strains; the E121K change in PmrB has previously been linked to intrinsic colistin resistance. We conclude that E. coli B-strains are not appropriate heterologous expression hosts for identification and characterization of mcr genes.

microbiology↗

bioRxiv2022

More than mcr: Canonical Plasmid- and Transposon-Encoded Mobilized Colistin Resistance (mcr) Genes Represent a Subset of Phosphoethanolamine Transferases

Mobilized colistin resistance genes (mcr) may confer resistance to colistin, a last-resort, critically important antimicrobial for human health. mcr can often be transmitted horizontally (e.g., via mobile genetic elements); however, mcr encode phosphoethanolamine transferases (PET) closely related to chromosomally encoded, intrinsic lipid modification enzymes (e.g., EptA, EptB, CptA). To explore the genetic diversity of mcr within the context of intrinsic lipid modification PET, we identified 9,836 non-redundant protein accession numbers associated with mcr-like genes, representing a total of 69,814 mcr-like genes present across 256 bacterial genera. We subsequently identified 125 unique, putative novel mcr-like genes encoded on the same contig as a plasmid replicon and other antimicrobial resistance genes. Sequence similarity and a maximum likelihood phylogeny of mcr, putative novel mcr-like genes, and intrinsic lipid modification PET-encoding genes indicated that sequence similarity is insufficient to discriminate between genes involved in colistin resistance and genes encoding intrinsic lipid modification PET. A mixed-effect model of evolution (MEME) indicated that site- and branch-specific diversifying positive selection might have played a role in the evolution of subvariants within the mcr-2 and mcr-9 families. MEME suggested that positive selection played a role in the diversification of several residues in structurally important regions, including (i) a bridging region that connects the membrane-bound and catalytic periplasmic domains, and (ii) a periplasmic loop juxtaposing the substrate entry tunnel. These residues were found to be differentially conserved in different mcr families and thus may play a role in mcr subvariant phenotypic diversity. Moreover, we found that eptA and mcr are localized within different genomic contexts. Canonical eptA are typically chromosomally encoded in an operon with a two-component regulatory system or adjacent to a TetR-type regulator. In contrast, mcr are encoded as single-gene operons or adjacent to pap2 and dgkA, which encode a PAP2 family lipid A phosphatase and diacylglycerol kinase, respectively. Our data suggest that eptA can give rise to "colistin resistance genes" through various mechanisms, including selection and diversification of the genomic context, regulatory pathways, and mobilization. These mechanisms likely altered gene expression levels and enzyme activity, allowing bona fide eptA to evolve to function in colistin resistance.

microbiology↗

bioRxiv2021

Monitoring the Antimicrobial Resistance Dynamics of Salmonella enterica in Healthy Dairy Cattle Populations at the Individual Farm Level Using Whole-Genome Sequencing

Livestock represent a possible reservoir for facilitating the transmission of the zoonotic foodborne pathogen Salmonella enterica to humans; there is also concern that strains can acquire resistance to antimicrobials in the farm environment. Here, we use whole-genome sequencing (WGS) to characterize Salmonella strains (n = 128) isolated from healthy dairy cattle and their associated environments on 13 New York State farms to assess the diversity and microevolution of this important pathogen at the level of the individual herd. Additionally, the accuracy and concordance of multiple in silico tools are assessed, including: (i) two in silico serotyping tools, (ii) combinations of five antimicrobial resistance (AMR) determinant detection tools and one to five AMR determinant databases, and (iii) one antimicrobial minimum inhibitory concentration (MIC) prediction tool. For the isolates sequenced here, in silico serotyping methods outperformed traditional serotyping and resolved all un-typable and/or ambiguous serotype assignments. Serotypes assigned in silico showed greater congruency with the Salmonella whole-genome phylogeny than traditional serotype assignments, and in silico methods showed high concordance (99% agreement). In silico AMR determinant detection methods additionally showed a high degree of concordance, regardless of the pipeline or database used ([≥]98% agreement between susceptible/resistant assignments for all pipeline/database combinations). For AMR detection methods that relied exclusively on nucleotide BLAST, accuracy could be maximized by using a range of minimum nucleotide identity and coverage thresholds, with thresholds of 75% nucleotide identity and 50-60% coverage adequate for most pipeline/database combinations. In silico characterization of the microevolution and AMR dynamics of each of six serotype groups (S. Anatum, Cerro, Kentucky, Meleagridis, Newport, Typhimurium/Typhimurium variant Copenhagen) revealed that some lineages were strongly associated with individual farms, while others were distributed across multiple farms. Numerous AMR determinant acquisition and loss events were identified, including the recent acquisition of cephalosporin resistance-conferring blaCMY- and blaCTX-M-type beta-lactamases. The results presented here provide high-resolution insight into the temporal dynamics of AMR Salmonella at the scale of the individual farm and highlight both the strengths and limitations of WGS in tracking zoonotic pathogens and their associated AMR determinants at the livestock-human interface.

microbiology↗

bioRxiv2019

The Typhoid Toxin Produced by the Nontyphoidal Salmonella Serovar Javiana Can Utilize Multiple Binding Subunits, which Compete for Inclusion in the Holotoxin

Salmonella enterica encodes a wide array of virulence factors. One novel virulence factor, a DNA-damaging toxin known as the typhoid toxin (TT), was recently characterized in >40 nontyphoidal Salmonella (NTS) serovars. Interestingly, these NTS serovars, including S. enterica subsp. enterica serovar Javiana, also encode artB, a homolog of the binding subunit (PltB) of the TT. Here, we show that ArtB and PltB compete for inclusion in the pentameric binding subunit of the TT. Using a combination of in silico modeling, a bacterial two-hybrid system expressed in S. Javiana, and tandem affinity purification (TAP) of the holotoxin subunits, we show that ArtB and PltB interact in vivo. Furthermore, binding subunits composed of homo- and heteropentamers of ArtB and PltB are able to associate with CdtB and PltA to form biologically active toxins. As artB was, (i) conserved among S. Javiana isolates, and (ii) co-expressed with pltB and cdtB under Mg2+-limiting conditions, we hypothesized that ArtB and PltB compete for inclusion in the binding subunit. Using a novel competition assay, we show that PltB outcompetes ArtB for inclusion in the binding subunit, when cultured at neutral pH. Together, our results suggest that the TT produced by S. Javiana utilizes multiple configurations of the binding subunit, representing a novel toxin form and adaptation mechanism for the AB5 toxin family. Our work suggests that Salmonella serovars, including S. Javiana, evolved to encode and maintain multiple binding subunits that can be used to form an active toxin, which may enhance the variety of cells, tissues, or hosts susceptible to this novel form of the TT.

microbiology↗

bioRxiv2019

Identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible Salmonella enterica serotype Typhimurium isolate using a combination of high-throughput, in silico screening and functional analysis

Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcr homologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcr homologue, detected in a Salmonella enterica serotype Typhimurium (S. Typhimurium) genome using an in silico approach, followed by experimental functional analysis. The amino acid sequence of mcr-9, detected in a multidrug resistant (MDR) S. Typhimurium strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using translated nucleotide blast. The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2 mg/L European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. To determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host, mcr-9 was cloned in colistin-susceptible Escherichia coli NEB5 under an IPTG-induced promoter. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5 at 1, 2, and 2.5 mg/L colistin, albeit at a lower level when compared to mcr-3. Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, and Mcr-7 share a high degree of similarity at the structural level. The results of our approach indicate that mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance. Importance: Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a Highest Priority Critically Important Antimicrobial for human medicine (WHO, Critically Important Antimicrobials for Human Medicine, 5th Revision, 2017), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.

microbiology↗