Background: Tolerance enables bacteria to survive intermittent antibiotic exposure without an increase in antimicrobial susceptibility. In this study, we investigated the presence of tolerance to three antimicrobials, ceftriaxone, azithromycin and ciprofloxacin, in clinical isolates and the WHO (World Health Organization) reference panel of Neisseria gonorrhoeae. Methods: We used the modified tolerance disk (TD test) to assess for tolerance to ceftriaxone, azithromycin and ciprofloxacin in 14 WHO reference strains and 62 N. gonorrhoeae clinical isolates—evenly divided between anorectal and urogenital infections. The isolates underwent a three-step incubation process wherein the isolates were exposed to an antibiotic disk for 20 h of incubation (Step I), followed by the replacement of the antibiotic disk with a nutrient disk for overnight incubation (Step II) and additional overnight incubation with extra nutrients (Step III). Results: A total of 4 of the 62 clinical anorectal isolates and none of the urogenital isolates exhibited tolerance to azithromycin (p = 0.033). Tolerance to ceftriaxone and ciprofloxacin was observed in eight and four isolates, respectively, with no difference between infection sites. Tolerance was also detected in 8 (K, M, N, O, P, U, V, W) out of the 14 WHO reference strains, with varying patterns of tolerance to ceftriaxone (n = 8), ciprofloxacin (n = 2) and azithromycin (n = 1). Conclusions: This study identified ceftriaxone, azithromycin and ciprofloxacin tolerance in clinical and WHO reference N. gonorrhoeae isolates. Azithromycin tolerance was more common in anorectal than urogenital infections.

Background: The use of antimicrobials to treat food animals leaves antimicrobial residues in foodstuffs. The World Health Organization (WHO) defines the acceptable daily intakes (ADIs) of these residues as the dose of these antimicrobials that is safe for an average human to consume on a daily basis. We hypothesized that the lowest dose of ciprofloxacin classified as safe by the WHO could select for ciprofloxacin-resistant strains of Klebsiella pneumoniae in a Galleria mellonella model. Objectives: We aimed to evaluate if the consumption of peri-ADI doses of ciprofloxacin could select for ciprofloxacin-resistant (Ser464Phe, GyrB, ciprofloxacin MIC of 4 µg/mL) compared to -susceptible (isogenic, ciprofloxacin MIC of 0.047 µg/mL) strains of K. pneumoniae in a Galleria mellonella model. Results: A significant increase was seen in the proportion of resistance for the 1× ADI and 1/10th ADI concentrations on day 2 compared to the positive control. Methods: A model of K. pneumoniae infection in G. mellonella larvae was used for the experiment. The larvae were inoculated with K. pneumoniae followed by 10× ADI, 1× ADI, 1/10th ADI, 1/100th ADI, and 1/1000th ADI doses of ciprofloxacin. The isolation of K. pneumoniae colonies was then performed on selective agar plates with and without ciprofloxacin (1 µg/mL). The proportion of colonies with ciprofloxacin resistance was then calculated for each group at 24 and 48 h. Conclusions: We found that, at 48 h, there was an enrichment of K. pneumoniae colonies with ciprofloxacin resistance in the larvae receiving 1× ADI and 1/10th ADI concentrations of ciprofloxacin. These results suggest that the ciprofloxacin MSC_select for K. pneumoniae in this model is 1/10th of the acceptable daily concentration (ADI) dose of ciprofloxacin, which is equivalent to 0.239 ng/µL.

The growing global threat of antimicrobial resistance is reaching a crisis point as common bacterial infections, including those caused by pathogenic Neisseria species, are becoming increasingly untreatable. This is compelling the scientific community to search for new antimicrobial agents, taking advantage of computational mining and using whole genome sequences to discover natural products from the human microbiome with antibiotic effects. In this study, we investigated the crude extract from a Rothia dentocariosa strain with demonstrated antimicrobial activity against pathogenic Neisseria spp. by spot-on-lawn assay. The genomic DNA of the R. dentocariosa strain was sequenced, and bioinformatic evaluation was performed using antiSMASH and PRISM to search for biosynthetic gene clusters (BGCs). The crude extract with potential antimicrobial activity was run on Tricine-SDS-PAGE, and the putative peptides were characterised using liquid chromatography–tandem mass spectrometry (LC-MS). The crude extract inhibited the growth of the pathogenic Neisseria spp. Six BGCs were identified corresponding to non-ribosomal peptide synthases (NRPSs), polyketide synthases (PKSs), and ribosomally synthesised and post-translationally modified peptides. Three peptides were also identified corresponding to Actinorhodin polyketide putative beta-ketoacyl synthase 1. These findings serve as a useful reference to facilitate the research and development of NRPS and PKS as antimicrobial products against multidrug-resistant N. gonorrhoeae.

Neisseria gonorrhoeae can acquire antimicrobial resistance (AMR) through horizontal gene transfer (HGT) from other Neisseria spp. such as commensals like Neisseria subflava. Low doses of antimicrobials in food could select for AMR in N. subflava, which could then be transferred to N. gonorrhoeae. In this study, we aimed to determine the lowest concentration of ciprofloxacin that can induce ciprofloxacin resistance (minimum selection concentration—MSC) in a N. subflava isolate (ID-Co000790/2, a clinical isolate collected from a previous community study conducted at ITM). In this study, Neisseria subflava was serially passaged on gonococcal (GC) medium agar plates containing ciprofloxacin concentrations ranging from 1:100 to 1:10,000 below its ciprofloxacin MIC (0.006 µg/mL) for 6 days. After 6 days of serial passaging at ciprofloxacin concentrations of 1/100th of the MIC, 24 colonies emerged on the plate containing 0.06 µg/mL ciprofloxacin, which corresponds to the EUCAST breakpoint for N. gonorrhoeae. Their ciprofloxacin MICs were between 0.19 to 0.25 µg/mL, and whole genome sequencing revealed a missense mutation T91I in the gyrA gene, which has previously been found to cause reduced susceptibility to fluoroquinolones. The N. subflava MSC_{de novo} was determined to be 0.06 ng/mL (0.00006 µg/mL), which is 100×-fold lower than the ciprofloxacin MIC. The implications of this finding are that the low concentrations of fluoroquinolones found in certain environmental samples, such as soil, river water, and even the food we eat, may be able to select for ciprofloxacin resistance in N. subflava.

Antibiotic tolerance is associated with antibiotic treatment failure, and molecular mechanisms underlying tolerance are poorly understood. We recently succeeded in inducing tolerance to ceftriaxone (CRO) in an N. gonorrhoeae reference isolate. In a prior in vitro study, six biological replicates of WHO P strains were exposed to CRO (10× the MIC) followed by overnight growth, and tolerance was assessed using a modified Tolerance Disc (T.D.) test. In the current study, we characterized the mutation profile of these CRO-tolerant phenotypes. The whole genome was sequenced from isolates from different replicates and time points. We identified mutations in four genes that may contribute to ceftriaxone tolerance in N. gonorrhoeae, including a mutation in the enolase (eno) gene that arose independently in three lineages.

Background Neisseria gonorrhoeae can acquire antimicrobial resistance (AMR) through horizontal gene transfer (HGT) from other Neisseria spp. such as commensals like Neisseria subflava. The prevalence of Neisseria subflava in the oropharynx is close to 100%. Low doses of antimicrobials in food could select for AMR in N. subflava, which could then be transferred to N. gonorrhoeae. In this study, we aimed to determine the lowest concentration of ciprofloxacin that can induce ciprofloxacin resistance (minimum selection concentration – MSC) in N. subflava. Methods Neisseria subflava Co000790/2 was serially passaged on GC agar plates containing ciprofloxacin concentrations ranging from 1:100- to 1:10,000-below its ciprofloxacin MIC (0.006 µg/ml) for six days. Results After 6 days of serial passaging at ciprofloxacin concentrations 1/100th of the MIC, 24 colonies emerged on the 0.06 µg/ml ciprofloxacin plate. Their ciprofloxacin MICs were between 0.19 to 0.25 µg/ml, and whole genome sequencing revealed a missense mutation T91I in the gyrA gene, which has previously been found to cause reduced susceptibility to fluoroquinolones. Conclusion The N. subflava MSCde novo was determined to be 0.06 ng/mL or 1:100 below the MIC. The implications of this finding are that the low concentrations of antibiotics found in certain environmental samples and even the food we eat may be able to select for ciprofloxacin resistance in N. subflava.