In vitro evolution of ciprofloxacin resistance in Neisseria commensals and derived mutation population dynamics in natural Neisseria populations.

Commensal Neisseria are members of a healthy human oropharyngeal microbiome; however, they also serve as a reservoir of antimicrobial resistance for their pathogenic relatives. Despite their known importance as sources of novel genetic variation for pathogens, we still do not understand the full suite of resistance mutations commensal species can harbor. Here, we use in vitro selection to assess the mutations that emerge in response to ciprofloxacin selection in commensal Neisseria by passaging 4 replicates of 4 different species in the presence of a selective antibiotic gradient for 20 days; then categorized derived mutations with whole genome sequencing. 10/16 selected cells lines across the 4 species evolved ciprofloxacin resistance (≥ 1 ug/ml); with resistance-contributing mutations primarily emerging in DNA gyrase subunit A and B (gyrA and gyrB), topoisomerase IV subunits C and E (parC and parE), and the multiple transferable efflux pump repressor (mtrR). Of note, these derived mutations appeared in the same loci responsible for ciprofloxacin reduced susceptibility in the pathogenic Neisseria, suggesting conserved mechanisms of resistance across the genus. Additionally, we tested for zoliflodacin cross-resistance in evolved strain lines and found 6 lineages with elevated zoliflodacin minimum inhibitory concentrations. Finally, to interrogate the likelihood of experimentally derived mutations emerging and contributing to resistance in natural Neisseria, we used a population-based approach and identified GyrA 91I as a substitution circulating within commensal Neisseria populations and ParC 85C in a single gonococcal isolate. Small clusters of gonococcal isolates had commensal-like alleles at parC and parE, indicating recent cross-species recombination events.

Rolling the evolutionary dice: Neisseria commensals as proxies for elucidating the underpinnings of antibiotic resistance mechanisms and evolution in human pathogens.

Species within the genus Neisseria are adept at sharing adaptive allelic variation, with commensal species repeatedly transferring resistance to their pathogenic relative Neisseria gonorrhoeae. However, resistance in commensals is infrequently characterized, limiting our ability to predict novel and potentially transferable resistance mechanisms that ultimately may become important clinically. Unique evolutionary starting places of each Neisseria species will have distinct genomic backgrounds, which may ultimately control the fate of evolving populations in response to selection as epistatic and additive interactions coerce lineages along divergent evolutionary trajectories. Alternatively, similar genetic content present across species due to shared ancestry may constrain existing adaptive solutions. Thus, identifying the paths to resistance across commensals may aid in characterizing the Neisseria resistome-or the reservoir of alleles within the genus as well as its depth. Here, we use in vitro evolution of four commensal species to investigate the potential and repeatability of resistance evolution to two antimicrobials, the macrolide azithromycin and the ß-lactam penicillin. After 20 days of selection, commensals evolved resistance to penicillin and azithromycin in 11/16 and 12/16 cases, respectively. Almost all cases of resistance emergence converged on mutations within ribosomal components or the mtrRCDE efflux pump for azithromycin-based selection and mtrRCDE, penA, and rpoB for penicillin selection, thus supporting constrained adaptive solutions despite divergent evolutionary starting points across the genus for these particular drugs. Though drug-selected loci were limited, we do identify novel resistance-imparting mutations. Continuing to explore paths to resistance across different experimental conditions and genomic backgrounds, which could shunt evolution down alternative evolutionary trajectories, will ultimately flesh out the full Neisseria resistome.IMPORTANCENeisseria gonorrhoeae is a global threat to public health due to its rapid acquisition of antibiotic resistance to all first-line treatments. Recent work has documented that alleles acquired from close commensal relatives have played a large role in the emergence of resistance to macrolides and beta-lactams within gonococcal populations. However, commensals have been relatively underexplored for the resistance genotypes they may harbor. This leaves a gap in our understanding of resistance that could be rapidly acquired by the gonococcus through a known highway of horizontal gene exchange. Here, we characterize resistance mechanisms that can emerge in commensal Neisseria populations via in vitro selection to multiple antimicrobials and begin to define the number of paths to resistance. This study, and other similar works, may ultimately aid both surveillance efforts and clinical diagnostic development by nominating novel and conserved resistance mechanisms that may be at risk of rapid dissemination to pathogen populations.

Rolling the evolutionary dice: Neisseria commensals as proxies for elucidating the underpinnings of antibiotic resistance mechanisms and evolution in human pathogens.

Species within the genus Neisseria are especially adept at sharing adaptive allelic variation across species' boundaries, with commensal species repeatedly transferring resistance to their pathogenic relative N. gonorrhoeae. However, resistance in commensal Neisseria is infrequently characterized at both the phenotypic and genotypic levels, limiting our ability to predict novel and potentially transferable resistance mechanisms that ultimately may become important clinically. Unique evolutionary starting places of each Neisseria species will have distinct genomic backgrounds, which may ultimately control the fate of evolving populations in response to selection, as epistatic and additive interactions may coerce lineages along divergent evolutionary trajectories. However alternatively, similar genetic content present across species due to shared ancestry may constrain the adaptive solutions that exist. Thus, identifying the paths to resistance across commensals may aid in characterizing the Neisseria resistome - or the reservoir of alleles within the genus, as well as its depth. Here, we use in vitro evolution of four commensal species to investigate the potential for and repeatability of resistance evolution to two antimicrobials, the macrolide azithromycin and the ß-lactam penicillin. After 20 days of selection, commensals evolved elevated minimum inhibitory concentrations (MICs) to penicillin and azithromycin in 11/16 and 12/16 cases respectively. Almost all cases of resistance emergence converged on mutations within ribosomal components or the mtrRCDE efflux pump for azithromycin-based selection, and mtrRCDE or penA for penicillin selection; thus, supporting constrained adaptive solutions despite divergent evolutionary starting points across the genus for these particular drugs. However, continuing to explore the paths to resistance across different experimental conditions and genomic backgrounds, which could shunt evolution down alternative evolutionary trajectories, will ultimately flesh out the full Neisseria resistome.

Draft Genome Sequences of Three Antibiotic-Producing Soil Bacteria, Staphylococcus pasteuri WAM01, Peribacillus butanolivorans WAM04, and Micrococcus yunnanensis WAM06, with Growth-Inhibiting Effects against Commensal Neisseria Strains.

We report the isolation, identification, and assemblies of three antibiotic-producing soil bacteria (Staphylococcus pasteuri, Peribacillus butanolivorans, and Micrococcus yunnanensis) that inhibit the growth of Neisseria commensals in coculture. With pathogenic Neisseria strains becoming increasingly resistant to antibiotics, bioprospecting for novel antimicrobials using commensal relatives may facilitate discovery of clinically useful drugs.

Isolation, Whole-Genome Sequencing, and Annotation of Two Antibiotic-Producing and -Resistant Bacteria, Enterobacter roggenkampii RIT 834 and Acinetobacter pittii RIT 835, from Disposable Masks Collected from the Environment.

We report the whole-genome sequence and annotation of two antibiotic-resistant bacteria, Enterobacter roggenkampii RIT 834 and Acinetobacter pittii RIT 835, isolated from disposed masks. We found that these strains are resistant to five of seven commonly used antibiotics and that they produce bactericidal compounds against Escherichia coli.

Canary in the Coal Mine: How Resistance Surveillance in Commensals Could Help Curb the Spread of AMR in Pathogenic Neisseria.

Antimicrobial resistance (AMR) is widespread within Neisseria gonorrhoeae populations. Recent work has highlighted the importance of commensal Neisseria (cN) as a source of AMR for their pathogenic relatives through horizontal gene transfer (HGT) of AMR alleles, such as mosaic penicillin binding protein 2 (penA), multiple transferable efflux pump (mtr), and DNA gyrase subunit A (gyrA) which impact beta-lactam, azithromycin, and ciprofloxacin susceptibility, respectively. However, nonpathogenic commensal species are rarely characterized. Here, we propose that surveillance of the universally carried commensal Neisseria may play the role of the "canary in the coal mine," and reveal circulating known and novel antimicrobial resistance determinants transferable to pathogenic Neisseria. We summarize the current understanding of commensal Neisseria as an AMR reservoir, and call to increase research on commensal Neisseria species, through expanding established gonococcal surveillance programs to include the collection, isolation, antimicrobial resistance phenotyping, and whole-genome sequencing (WGS) of commensal isolates. This will help combat AMR in the pathogenic Neisseria by: (i) determining the contemporary AMR profile of commensal Neisseria, (ii) correlating AMR phenotypes with known and novel genetic determinants, (iii) qualifying and quantifying horizontal gene transfer (HGT) for AMR determinants, and (iv) expanding commensal Neisseria genomic databases, perhaps leading to the identification of new drug and vaccine targets. The proposed modification to established Neisseria collection protocols could transform our ability to address AMR N. gonorrhoeae, while requiring minor modifications to current surveillance practices. IMPORTANCE Contemporary increases in the prevalence of antimicrobial resistance (AMR) in Neisseria gonorrhoeae populations is a direct threat to global public health and the effective treatment of gonorrhea. Substantial effort and financial support are being spent on identifying resistance mechanisms circulating within the gonococcal population. However, these surveys often overlook a known source of resistance for gonococci-the commensal Neisseria. Commensal Neisseria and pathogenic Neisseria frequently share DNA through horizontal gene transfer, which has played a large role in rendering antibiotic therapies ineffective in pathogenic Neisseria populations. Here, we propose the expansion of established gonococcal surveillance programs to integrate a collection, AMR profiling, and genomic sequencing pipeline for commensal species. This proposed expansion will enhance the field's ability to identify resistance in and from nonpathogenic reservoirs and anticipate AMR trends in pathogenic Neisseria.

Evolutionary paths to macrolide resistance in a Neisseria commensal converge on ribosomal genes through short sequence duplications.

Neisseria commensals are an indisputable source of resistance for their pathogenic relatives. However, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (≥ 2 µg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n = 7/16), we find mutations that reduce susceptibility to azithromycin converge on the locus encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through short tandem duplication events. Interestingly, one of the laboratory evolved mutations in rpmH is identical (7LKRTYQ12), and two nearly identical, to those recently reported to contribute to high-level azithromycin resistance in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Ultimately, studies like this will be critical for predicting commensal alleles that could rapidly disseminate into pathogen populations via allelic exchange across recombinogenic microbial genera.

Isolation, Whole-Genome Sequencing, and Annotation of Three Unclassified Antibiotic-Producing Bacteria, Enterobacter sp. Strain RIT 637, Pseudomonas sp. Strain RIT 778, and Deinococcus sp. Strain RIT 780.

We report the isolation, whole-genome sequencing, and annotation of Enterobacter sp. strain RIT 637, Pseudomonas sp. strain RIT 778, and Deinococcus sp. strain RIT 780. Disk diffusion assays using spent medium demonstrated that all bacteria produced bactericidal compounds against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923.

Draft Genome Sequences of Three Penicillin-Resistant Neisseria gonorrhoeae Strains Isolated in Cincinnati, Ohio, in 1994.

Here, we report the draft genome sequences of three penicillin-resistant Neisseria gonorrhoeae isolates. We include associated data on MICs and genetic relationships to other N. gonorrhoeae strains collected from across the United States. Resistance mutations known to contribute to reduced penicillin susceptibility are annotated in each genome.

Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669-Potential Zoonotic Pathogens Isolated from Spotted Turtles.

Antimicrobial resistance (AMR) is one of the biggest challenges of the 21st century, and biofilm formation enables bacteria to resist antibiotic at much higher concentrations than planktonic cells. Earlier, we showed that the Gram-negative Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669 (closely related to C. freundii NBRC 12681) from infected spotted turtles (Clemmys guttata), formed biofilms and upregulated toxin expression on plastic surfaces, and were predicted to possess multiple antibiotic resistance genes. Here, we show that they each resist several antibiotics in the planktonic phase, but were susceptible to neomycin, and high concentrations of tetracycline and cotrimoxazole. The susceptibility of their biofilms to neomycin and cotrimoxazole was tested using the Calgary device. For A. hydrophila, the minimum inhibitory concentration (MIC) = 500-1000, and the minimum biofilm eradication concentration (MBEC) > 1000 µg/mL, using cotrimoxazole, and MIC = 32.3-62.5, and MBEC > 1000 µg/mL, using neomycin. For C. freundii MIC = 7.8-15.6, and, MBEC > 1000 µg/mL, using cotrimoxazole, and MIC = 7.8, and MBEC > 1000 µg/mL, using neomycin. Both A. hydrophila and C. portucalensis activated an acyl homoserine lactone (AHL) dependent biosensor, suggesting that quorum sensing could mediate biofilm formation. Their multidrug resistance in the planktonic form, and weak biofilm eradication even with neomycin and cotrimoxazole, indicate that A. hydrophila and C. portucalensis are potential zoonotic pathogens, with risks for patients living with implants.