The Prevalence of Antibiotic Tolerance in Neisseria gonorrhoeae Varies by Anatomical Site.

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.

Effect of erythromycin residuals in food on the development of resistance in Streptococcus pneumoniae: an in vivo study in Galleria mellonella.

Background: The use of antimicrobials to treat food animals may result in antimicrobial residues in foodstuffs of animal origin. The European Medicines Association (EMA) and World Health Organization (WHO) define safe antimicrobial concentrations in food based on acceptable daily intakes (ADIs). It is unknown if ADI doses of antimicrobials in food could influence the antimicrobial susceptibility of human-associated bacteria. Objectives: This aim of this study was to evaluate if the consumption of ADI doses of erythromycin could select for erythromycin resistance in a Galleria mellonella model of Streptococcus pneumoniae infection. Methods: A chronic model of S. pneumoniae infection in G. mellonella larvae was used for the experiment. Inoculation of larvae with S. pneumoniae was followed by injections of erythromycin ADI doses (0.0875 and 0.012 µg/ml according to EMA and WHO, respectively). Isolation of S. pneumoniae colonies was then performed on selective agar plates. Minimum inhibitory concentrations (MICs) of resistant colonies were measured, and whole genome sequencing (WGS) was performed followed by variant calling to determine the genetic modifications. Results: Exposure to single doses of both EMA and WHO ADI doses of erythromycin resulted in the emergence of erythromycin resistance in S. pneumoniae. Emergent resistance to erythromycin was associated with a mutation in rplA, which codes for the L1 ribosomal protein and has been linked to macrolide resistance in previous studies. Conclusion: In our in vivo model, even single doses of erythromycin that are classified as acceptable by the WHO and EMA induced significant increases in erythromycin MICs in S. pneumoniae. These results suggest the need to include the induction of antimicrobial resistance (AMR) as a significant criterion for determining ADIs.

Ciprofloxacin Concentrations 100-Fold Lower than the MIC Can Select for Ciprofloxacin Resistance in Neisseria subflava: An In Vitro Study.

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 MSCde 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.

Could traces of fluoroquinolones in food induce ciprofloxacin resistance in Escherichia coli and Klebsiella pneumoniae? An in vivo study in Galleria mellonella with important implications for maximum residue limits in food.

We hypothesized that the residual concentrations of fluoroquinolones allowed in food (acceptable daily intake-ADIs) could select for ciprofloxacin resistance in our resident microbiota. We developed models of chronic Escherichia coli and Klebsiella pneumoniae infection in Galleria mellonella larvae and exposed them to ADI doses of ciprofloxacin via single dosing and daily dosing regimens. The emergence of ciprofloxacin resistance was assessed via isolation of the target bacteria in selective agar plates. Exposure to as low as one-tenth of the ADI dose of the single and daily dosing regimens of ciprofloxacin resulted in the selection of ciprofloxacin resistance in K. pneumoniae but not E. coli. This resistance was associated with cross-resistance to doxycycline and ceftriaxone. Whole genome sequencing revealed inactivating mutations in the transcription repressors, ramR and rrf2, as well as mutations in gyrA and gyrB. We found that ciprofloxacin doses 10-fold lower than those classified as acceptable for daily intake could induce resistance to ciprofloxacin in K. pneumoniae. These results suggest that it would be prudent to include the induction of antimicrobial resistance as a significant criterion for determining ADIs and the associated maximum residue limits in food.IMPORTANCEThis study found that the concentrations of ciprofloxacin/enrofloxacin allowed in food can induce de novo ciprofloxacin resistance in Klebsiella pneumoniae. This suggests that it would be prudent to reconsider the criteria used to determine "safe" upper concentration limits in food.

Protective effect of microbisporicin (NAI-107) against vancomycin resistant Enterococcus faecium infection in a Galleria mellonella model.

Increasing antimicrobial resistance in Enterococcus faecium necessitates the search for novel treatment agents, such as bacteriocins. In this study, we conducted an in vivo assessment of five bacteriocins, namely Lacticin Z, Lacticin Q, Garvicin KS (ABC), Aureocin A53 and Microbisporicin (NAI-107), against vanB-resistant Enterococcus faecium using a Galleria mellonella model. Our in vitro experiments demonstrated the efficacy of all five bacteriocins against vanB-resistant E. faecium with only NAI-107 demonstrating in vivo efficacy. Notably, NAI-107 exhibited efficacy across a range of tested doses, with the highest efficacy observed at a concentration of 16 µg/mL. Mortality rates in the group treated with 16 µg/mL NAI-107 were lower than those observed in the linezolid-treated group. These findings strongly suggest that NAI-107 holds promise as a potential alternative therapeutic agent for treating infections caused by resistant E. faecium and warrants further investigation.

Chronic High-Level Parasitemia in HIV-Infected Individuals With or Without Visceral Leishmaniasis in an Endemic Area in Northwest Ethiopia: Potential Superspreaders?

BACKGROUND: People with human immunodeficiency virus (PWH) with recurrent visceral leishmaniasis (VL) could potentially drive Leishmania transmission in areas with anthroponotic transmission such as East Africa, but studies are lacking. Leishmania parasitemia has been used as proxy for infectiousness. METHODS: This study is nested within the Predicting Visceral Leishmaniasis in HIV-InfectedPatients (PreLeisH) prospective cohort study, following 490 PWH free of VL at enrollment for up to 24-37 months in northwest Ethiopia. Blood Leishmania polymerase chain reaction (PCR) was done systematically. This case series reports on 10 PWH with chronic VL (≥3 VL episodes during follow-up) for up to 37 months, and 3 individuals with asymptomatic Leishmania infection for up to 24 months. RESULTS: All 10 chronic VL cases were male, on antiretroviral treatment, with 0-11 relapses before enrollment. Median baseline CD4 count was 82 cells/µL. They displayed 3-6 VL treatment episodes over a period up to 37 months. Leishmania blood PCR levels were strongly positive for almost the entire follow-up (median cycle threshold value, 26 [interquartile range, 23-30]), including during periods between VL treatment. Additionally, we describe 3 PWH with asymptomatic Leishmania infection and without VL history, with equally strong Leishmania parasitemia over a period of up to 24 months without developing VL. All were on antiretroviral treatment at enrollment, with baseline CD4 counts ranging from 78 to 350 cells/µL. CONCLUSIONS: These are the first data on chronic parasitemia in PWH from Leishmania donovani-endemic areas. PWH with asymptomatic and symptomatic Leishmania infection could potentially be highly infectious and constitute Leishmania superspreaders. Xenodiagnosis studies are required to confirm infectiousness.

Gonococcal resistance to zoliflodacin could emerge via transformation from commensal Neisseria species. An in-vitro transformation study.

One of the most promising new treatments for gonorrhoea currently in phase 3 clinical trials is zoliflodacin. Studies have found very little resistance to zoliflodacin in currently circulating N. gonorrhoeae strains, and in-vitro experiments demonstrated that it is difficult to induce resistance. However, zoliflodacin resistance may emerge in commensal Neisseria spp., which could then be transferred to N. gonorrhoeae via transformation. In this study, we investigated this commensal-resistance-pathway hypothesis for zoliflodacin. To induce zoliflodacin resistance, ten wild-type susceptible isolates belonging to 5 Neisseria species were serially passaged for up to 48 h on gonococcal agar plates containing increasing zoliflodacin concentrations. Within 7 to 10 days, all strains except N. lactamica, exhibited MICs of ≥ 4 µg/mL, resulting in MIC increase ranging from 8- to 64-fold. The last passaged strains and their baseline were sequenced. We detected mutations previously reported to cause zoliflodacin resistance in GyrB (D429N and S467N), novel mutations in the quinolone resistance determining region (QRDR) (M464R and T472P) and mutations outside the QRDR at amino acid positions 28 and 29 associated with low level resistance (MIC 2 µg/mL). Genomic DNA from the laboratory evolved zoliflodacin-resistant strains was transformed into the respective baseline wild-type strain, resulting in MICs of ≥ 8 µg/mL in most cases. WGS of transformants with decreased zoliflodacin susceptibility revealed presence of the same zoliflodacin resistance determinants as observed in the donor strains. Two inter-species transformation experiments were conducted to investigate whether zoliflodacin resistance determinants of commensal Neisseria spp. could be acquired by N. gonorrhoeae. N. gonorrhoeae strain WHO P was exposed to (i) pooled genomic DNA from the two resistant N. mucosa strains and (ii) a gyrB amplicon of the resistant N. subflava strain 45/1_8. Transformants of both experiments exhibited an MIC of 2 µg/mL and whole genome analysis revealed uptake of the mutations detected in the donor strains. This is the first in-vitro study to report that zoliflodacin resistance can be induced in commensal Neisseria spp. and subsequently transformed into N. gonorrhoeae.

Ramoplanin as a novel therapy for Neisseria gonorrhoeae infection: an in vitro and in vivo study in Galleria mellonella.

Neisseria gonorrhoeae is a bacterial pathogen that causes gonorrhoea, a sexually transmitted infection. Increasing antimicrobial resistance in N. gonorrhoeae is providing motivation to develop new treatment options. In this study, we investigated the effectiveness of the antibiotic ramoplanin as a treatment for N. gonorrhoeae infection. We tested the effectiveness of ramoplanin in vitro against 14 World Health Organization (WHO) reference strains of N. gonorrhoeae and found that it was active against all 14 strains tested. Furthermore, in a Galleria mellonella infection model of N. gonorrhoeae WHO P, we demonstrated that ramoplanin was active in vivo without any evidence of toxicity. This suggests that ramoplanin might be a new promising antibiotic treatment for gonorrhoea.