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Detection of Haemophilus ducreyi in genital ulcer specimens by culture lacks sensitivity. To enhance detection, a heminested polymerase chain reaction (PCR) assay was developed targeting the nucleotide sequence of a gene, designated p27, which encodes for a 27 kDa H. ducreyi-specific protein. The p27 PCR assay detected all (37/37) H. ducreyi strains tested and gave no amplified product from DNA extracts of any of 31 other microorganisms, from 30 non-genital ulcer specimens, or from 29 urethral and vaginal swab specimens collected from non-chancroid STD patients. In genital ulcer disease specimens, compared to combined positive results obtained by culture and a previously described PCR assay, the p27 PCR assay showed a sensitivity of 91% (48/53). The p27 PCR assay provides a specific and a sensitive detection of H. ducreyi in clinical specimens. [ABSTRACT FROM AUTHOR]

Abstract Background The vaginal microbiome plays an important role in urogenital health. Quantitative real time Polymerase Chain Reaction (qPCR) assays for the most prevalent vaginal Lactobacillus species and bacterial vaginosis species G. vaginalis and A. vaginae exist, but qPCR information regarding variation over time is still very limited. We set up qPCR assays for a selection of seven species and defined the temporal variation over three menstrual cycles in a healthy Caucasian population with a normal Nugent score. We also explored differences in qPCR data between these healthy women and an ‘at risk’ clinic population of Caucasian, African and Asian women with and without bacterial vaginosis (BV), as defined by the Nugent score. Results Temporal stability of the Lactobacillus species counts was high with L. crispatus counts of 108 copies/mL and L. vaginalis counts of 106 copies/mL. We identified 2 types of ‘normal flora’ and one ‘BV type flora’ with latent class analysis on the combined data of all women. The first group was particularly common in women with a normal Nugent score and was characterized by a high frequency of L. crispatus, L. iners, L. jensenii, and L. vaginalis and a correspondingly low frequency of L. gasseri and A. vaginae. The second group was characterized by the predominance of L. gasseri and L. vaginalis and was found most commonly in healthy Caucasian women. The third group was commonest in women with a high Nugent score but was also seen in a subset of African and Asian women with a low Nugent score and was characterized by the absence of Lactobacillus species (except for L. iners) but the presence of G. vaginalis and A. vaginae. Conclusions We have shown that the quantification of specific bacteria by qPCR contributes to a better description of the non-BV vaginal microbiome, but we also demonstrated that differences in populations such as risk and ethnicity also have to be taken into account. We believe that our selection of indicator organisms represents a feasible strategy for the assessment of the vaginal microbiome and could be useful for monitoring the microbiome in safety trials of vaginal products.

Background: The ResistAZM randomized controlled trial found that the receipt of ceftriaxone/azithromycin, compared to ceftriaxone was not associated with an increase in the proportion of oral commensal Neisseria spp. and streptococci with azithromycin resistance fourteen days after treatment., Methods: We repeated the analyses by measuring the minimum inhibitory concentrations (MICs) of azithromycin and ceftriaxone for individual colonies of commensal Neisseria spp. and streptococci at day 0 and day 14 in both arms., Results: The receipt of ceftriaxone/azithromycin but not ceftriaxone was associated with an increase in azithromycin MIC for both Neisseria spp. (P < 0.0001) and streptococci (P = 0.0076). Likewise, ceftriaxone/azithromycin but not ceftriaxone monotherapy was associated with an increase in ceftriaxone MICs in Neisseria spp. (P = 0.0035)., Conclusions: Whereas the proportion-method failed to detect an association between the receipt of azithromycin and increased macrolide resistance, the MIC-distribution method detected this effect. The MIC-distribution method is thus a more sensitive method to assess the effect of antimicrobials on antimicrobial susceptibility., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Background: Targeted preventive strategies in persons living with HIV (PLWH) require markers to predict visceral leishmaniasis (VL). We conducted a longitudinal study in a HIV-cohort in VL-endemic North-West Ethiopia to 1) describe the pattern of Leishmania markers preceding VL; 2) identify Leishmania markers predictive of VL; 3) develop a clinical management algorithm according to predicted VL risk levels., Methods: The PreLeisH study followed 490 adult PLWH free of VL at enrolment for up to two years (2017-2021). Blood RT-PCR targeting Leishmania kDNA, Leishmania serology and Leishmania urine antigen test (KAtex) were performed every 3-6 months. We calculated the sensitivity/specificity of the Leishmania markers for predicting VL and developed an algorithm for distinct clinical management strategies, with VL risk categories defined based on VL history, CD4 count and Leishmania markers (rK39 RDT & RT-PCR)., Findings: At enrolment, 485 (99%) study participants were on antiretroviral treatment; 360/490 (73.5%) were male; the median baseline CD4 count was 392 (IQR 259-586) cells/μL; 135 (27.5%) had previous VL. Incident VL was diagnosed in 34 (6.9%), with 32 (94%) displaying positive Leishmania markers before VL. In those without VL history, baseline rK39 RDT had 60% sensitivity and 84% specificity to predict VL; in patients with previous VL, RT-PCR had 71% sensitivity and 95% specificity. The algorithm defined 442 (92.3%) individuals at low VL risk (routine follow-up), 31 (6.5%) as moderate risk (secondary prophylaxis) and six (1.2%) as high risk (early treatment)., Interpretation: Leishmania infection markers can predict VL risk in PLWH. Interventional studies targeting those at high risk are needed., Funding: The PreLeisH study was supported by grants from the Department of Economy, Science and Innovation of the Flemish Government, Belgium (757013) and the Directorate-General for Development Cooperation and Humanitarian Aid (DGD), Belgium (BE-BCE&#95;KBO-0410057701-prg2022-5-ET)., Competing Interests: Declaration of interests None to declare., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

Background. Commensal streptococci are common inhabitants of the oral microbiome and regulate its structure and function in beneficial ways for human health. They can, however, also be opportunistic pathogens and act as a reservoir of resistance genes that can be passed on to other bacteria, including pathogens. Little is known about the prevalence of these commensals in parents and their children and their antimicrobial susceptibilities in the Belgian general population. Gap Statement. The macrolide susceptibility of commensal oral Streptococci in Belgium is unknown. Methods. We assessed the prevalence and azithromycin susceptibility of commensal streptococcal species in the parents ( n =38) and children ( n =50) of 35 families in Belgium. Results. The most frequently detected taxonomic grouping was Streptococcus mitis/oralis , which was detected in 78/181 (43.1%) of the children's isolates and 66/128 (51.6%) of the parents' isolates. Of the 311 isolates collected in this study, 282 isolates (90.7%) had an azithromycin MIC value greater than the breakpoint of 0.25 mg l -1 and 146 isolates (46.9%) had azithromycin MICs greater than 2 mg l -1 . There was no difference in the azithromycin MIC distribution of all streptococcal isolates between children and parents. All individuals were colonized by streptococci with azithromycin MICs greater than 0.25 mg l -1 , and 87.5% of individuals had streptococci with MICs greater than 2 mg l -1 . Interpretation. The most prevalent species identified in both age groups was S. mitis/oralis . All individuals harboured streptococci with macrolide resistance. This highlights the need for additional antimicrobial stewardship initiatives to reduce the consumption of macrolides in the general population.

Background: The emergence of ceftriaxone-resistant Neisseria gonorrhoeae poses a significant threat to existing treatment regimens. Our study aimed to assess the efficacy of antimicrobials that could be combined with ceftriaxone to reduce the probability of ceftriaxone resistance emerging and spreading in N. gonorrhoeae ., Methods and Results: Broth microdilution was used to determine the minimal inhibitory concentrations (MICs) for a panel of ceftriaxone-resistant (WHO X, Y, Z) and ceftriaxone-susceptible (WHO L, N, P) N. gonorrhoeae WHO reference strains for the following antimicrobials: ceftriaxone, doxycycline, azithromycin, zoliflodacin, fosfomycin, pristinamycin, ramoplanin, gentamicin and NAI-107. The MICs for zoliflodacin and pristinamycin for all strains were lower than or equal to the available breakpoints. A checkerboard assay was used to determine the drug-drug combination effect, which showed either an indifferent or an additive effect for all the combinations tested with ceftriaxone., Conclusions: The low MICs of zoliflodacin and pristinamycin for the three ceftriaxone-resistant strains suggest that these antimicrobials could be used as partner drugs with ceftriaxone to reduce the probability of ceftriaxone resistance spreading in areas with a high prevalence of ceftriaxone resistance., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Introduction. Commensal Neisseria spp. are highly prevalent in the oropharynx as part of the healthy microbiome. N. meningitidis can colonise the oropharynx too from where it can cause invasive meningococcal disease. To identify N. meningitidis , clinical microbiology laboratories often rely on Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS). Hypothesis/Gap statement. N. meningitidis may be misidentified by MALDI-TOF MS. Aim. To conduct genomic surveillance of oropharyngeal Neisseria spp. in order to: (i) verify MALDI-TOF MS species identification, and (ii) characterize commensal Neisseria spp. genomes. Methodology. We analysed whole genome sequence (WGS) data from 119 Neisseria spp. isolates from a surveillance programme for oropharyngeal Neisseria spp. in Belgium. Different species identification methods were compared: (i) MALDI-TOF MS, (ii) Ribosomal Multilocus Sequence Typing (rMLST) and (iii) rplF gene species identification. WGS data were used to further characterize Neisseria species found with supplementary analyses of Neisseria cinerea genomes. Results. Based on genomic species identification, isolates from the oropharyngeal Neisseria surveilence study were composed of the following species: N. meningitidis ( n =23) , N. subflava ( n =61), N. mucosa ( n =15), N. oralis ( n =8), N. cinerea ( n =5), N. elongata ( n =3), N. lactamica ( n =2), N. bacilliformis ( n =1) and N. polysaccharea ( n =1). Of these 119 isolates, four isolates identified as N. meningitidis ( n =3) and N. subflava ( n =1) by MALDI-TOF MS , were determined to be N. polysaccharea ( n =1) , N. cinerea ( n =2) and N. mucosa ( n =1) by rMLST. Phylogenetic analyses revealed that N. cinerea isolates from the general population ( n =3, cluster one) were distinct from those obtained from men who have sex with men (MSM, n =2, cluster two). The latter contained genomes misidentified as N. meningitidis using MALDI-TOF MS. These two N. cinerea clusters persisted after the inclusion of published N. cinerea WGS ( n =42). Both N. cinerea clusters were further defined through pangenome and Average Nucleotide Identity (ANI) analyses. Conclusion. This study provides insights into the importance of genomic genus-wide Neisseria surveillance studies to improve the characterization and identification of the Neisseria genus.

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., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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.

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.

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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: We have no competing interests., (© 2024 Baranchyk et al.)

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., (© 2024. The Author(s).)

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&#95;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., (© 2024. The Author(s).)

Background: commensal Neisseria species are part of the oropharyngeal microbiome and play an important role in nitrate reduction and protecting against colonization by pathogenic bacteria. They do, however, also serve as a reservoir of antimicrobial resistance. Little is known about the prevalence of these species in the general population, how this varies by age and how antimicrobial susceptibility varies between species., Methods: we assessed the prevalence and antimicrobial susceptibility of commensal Neisseria species in the parents (n = 38) and children (n = 50) of 35 families in Belgium., Results: various commensal Neisseria (n = 5) could be isolated from the participants. Most abundant were N. subflava and N. mucosa. Neisseria subflava was detected in 77 of 88 (87.5%) individuals and N. mucosa in 64 of 88 (72.7%). Neisseria mucosa was more prevalent in children [41/50 (82%)] than parents [23/38 (60.5%); P < .05], while N. bacilliformis was more prevalent in parents [7/36 (19.4%)] than children [2/50 (4%); P < .05]. Neisseria bacilliformis had high ceftriaxone minimum inhibitory concentrations (MICs; median MIC 0.5 mg/l; IQR 0.38-0.75). The ceftriaxone MICs of all Neisseria isolates were higher in the parents than in the children. This could be explained by a higher prevalence of N. bacilliformis in the parents., Interpretation: the N. bacilliformis isolates had uniformly high ceftriaxone MICs which warrant further investigation., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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.

Importance: We screened 66 bacteriocins to see if they exhibited anti-gonococcal activity. We found 12 bacteriocins with anti-gonococcal effects, and 4 bacteriocins showed higher anti-gonococcal activity. Three bacteriocins, lacticin Z, lacticin Q, and Garvicin KS (ABC), showed in vitro anti-gonococcal activity but no in vivo inhibitory effects against the Neisseria gonorrhoeae (WHO-P) isolate. On the other hand, NAI-107 showed in vivo anti-gonococcal activity. The findings suggest that NAI-107 is a promising alternative to treat gonorrhea infections., Competing Interests: The authors declare no conflict of interest.

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.

Background: Four randomized controlled trials have now established that doxycycline post exposure (sex) prophylaxis (PEP) can reduce the incidence of chlamydia and syphilis in men who have sex with men. These studies have concluded that the risk of selecting for antimicrobial resistance is low. We evaluated this risk in vitro and in vivo using a Galleria mellonella infection model., Methods: We evaluated how long it took for doxycycline resistance to emerge during passage on doxycycline containing agar plates in 4 species - Escherichia coli , Klebsiella pneumoniae , Neisseria gonorrhoeae and Neisseria subflava . We then assessed if K. pneumoniae could acquire resistance to doxycycline (and cross resistance to other antimicrobials) during intermittent exposure to doxycycline in a Galleria mellonella model of doxycycline PEP., Results: In our passage experiments, we found that resistance first emerged in K. pneumoniae . By day 7 the K. pneumoniae MIC had increased from 2 mg/L to a median of 96 mg/L (IQR 64-96). Under various simulations of doxycycline PEP in the G. mellonella model, the doxycycline MIC of K. pneumoniae increased from 2 mg/L to 48 mg/L (IQR 48-84). Ceftriaxone and ciprofloxacin MICs increased over ten-fold. Whole genome sequencing revealed acquired mutations in ramR which regulates the expression of the AcrAB-TolC efflux pump., Conclusion: Doxycycline PEP can select for doxycycline, ceftriaxone and ciprofloxacin resistance in K. pneumoniae in a G. mellonella model. The emergent ramR mutations were similar to those seen in circulating strains of K. pneumoniae . These findings suggest that we need to assess the effect of doxycycline PEP on resistance induction on a broader range of bacterial species than has hitherto been the case., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kenyon, Gestels, Vanbaelen, Abdellati, Van Den Bossche, De Baetselier, Xavier and Manoharan-Basil.)

Background: Phenotypic studies have found high levels of antimicrobial resistance to cephalosporins, macrolides and fluoroquinolones in commensal Neisseria species in the oropharynx of men who have sex with men (MSM) using HIV pre-exposure prophylaxis (PrEP). These species include Neisseria subflava and Neisseria mucosa. This may represent a risk to pathogens like Neisseria gonorrhoeae which tend to take up antibiotic resistance genes (ARGs) from other bacteria. We aimed to explore to what extent the oropharyngeal resistome of MSM using PrEP differed from the general population., Methods: We collected oropharyngeal swabs from 32 individuals of the general population and from 64 MSM using PrEP. Thirty-two MSM had consumed antibiotics in the previous six months, whereas none of the other participants had. Samples underwent shotgun metagenomic sequencing. Sequencing reads were mapped against MEGARes 2.0 to estimate ARG abundance. ARG abundance was compared between groups by zero-inflated negative binomial regression., Findings: ARG abundance was significantly lower in the general population than in MSM (ratio 0.41, 95% CI 0.26-0.65). More specifically, this was the case for fluoroquinolones (0.33, 95% CI 0.15-0.69), macrolides (0.37, 95% CI 0.25-0.56), tetracyclines (0.41, 95% CI 0.25-0.69), and multidrug efflux pumps (0.11, 95% CI 0.03-0.33), but not for beta-lactams (1.38, 95% CI 0.73-2.61). There were no significant differences in ARG abundance between MSM who had used antibiotics and those that had not., Interpretation: The resistome of MSM using PrEP is enriched with ARGs, independent of recent antibiotic use. Stewardship campaigns should aim to reduce antibiotic consumption in populations at high risk for STIs., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2023 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Visceral leishmaniasis (VL) is a disease caused by Leishmania parasites. While predominantly transmitted by sandflies, cases of VL transmitted through blood transfusion have been reported, particularly in immunocompromised recipients. Although Leishmania parasites have been found in blood donors in some VL endemic areas, this has never been studied in East-Africa, where HIV prevalence is relatively high. We established the prevalence of asymptomatic Leishmania infection and associated socio-demographic factors among blood donors presenting at two blood bank sites (Metema and Gondar) in northwest Ethiopia between June and December 2020. Metema is located in a VL-endemic area; Gondar has historically been considered VL non-endemic but as an outbreak of VL has occurred around Gondar, it was defined as previously VL non-endemic. Blood samples were tested by the rK39 rapid diagnostic test (RDT), rK39 ELISA, direct agglutination test (DAT) and qPCR targeting kinetoplast DNA (kDNA). Asymptomatic infection was defined as positive by any of these tests in a healthy person. A total of 426 voluntary blood donors were included. The median age was 22 years (IQR, 19-28 years); 59% were male and 81% resided in urban areas. Only one participant had a history of VL and three had a family history of VL. Asymptomatic infection was detected in 15.0% (n = 32/213) in Metema and 4.2% (n = 9/213) in Gondar. The rK39 ELISA was positive in 5.4% (n = 23/426), the rK39 RDT in 2.6% (11/426), PCR in 2.6% (11/420) and DAT in 0.5% (2/426). There were six individuals with two positive tests: one positive on rK39 RDT and PCR and five positive on rK39 RDT and ELISA. The prevalence of asymptomatic infection was higher in Metema (VL-endemic) and males but was not associated with age, a history of VL amongst family members or living in a rural area. Antibodies against Leishmania and parasite DNA was detected in a substantial number of blood donors. Future research should be directed at better defining the risk to recipients, including parasite viability studies and longitudinal studies amongst recipients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mohammed et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

With increasing incidence of pathogenic Neisseria infections coupled with emerging resistance to antimicrobials, alternative approaches to limit the spread are sought. We investigated the inhibitory effect of oropharyngeal microbiota on the growth of N. gonorrhoeae and N. meningitidis and the impact of the essential oil-based mouthwash Listerine Cool Mint ® (Listerine). Oropharyngeal swabs from 64 men who have sex with men ( n = 118) from a previous study (PReGo study) were analysed (ClinicalTrials.gov, NCT03881007). These included 64 baseline and 54 samples following three months of daily use of Listerine. Inhibition was confirmed by agar overlay assay, and inhibitory bacteria isolated using replica plating and identified using MALDI-TOF. The number of inhibitory isolates were compared before and after Listerine use. Thirty-one pharyngeal samples (26%) showed inhibitory activity against N. gonorrhoeae and/or N. meningitidis , and 62 inhibitory isolates were characterised. Fourteen species belonging to the genera Streptococci and Rothia were identified. More inhibitory isolates were observed following Listerine use compared to baseline, although this effect was not statistically significant ( p = 0.073). This study isolated and identified inhibitory bacteria against pathogenic Neisseria spp. and established that daily Listerine use did not decrease their prevalence. These findings could provide a new approach for the prevention and treatment of pharyngeal Neisseria infections.

Background: The effect of sequential exposure to different antibiotics is an underexplored topic. Azithromycin can be detected in humans for up to 28 days post-ingestion and may prime bacterial responses to subsequently ingested antibiotics. Methods: In this in vitro study, we assessed if preexposure to azithromycin could accelerate the acquisition of resistance to ciprofloxacin in Neisseria gonorrhoeae reference strain, WHO-F. In a morbidostat, we set two conditions in 3 vials each: mono-exposure (preexposure to Gonococcal Broth followed by exposure to ciprofloxacin) and dual sequential exposure (preexposure to azithromycin followed by exposure to ciprofloxacin).The growth of the cultures was measured by a software (MATLAB). The program decided if gonococcal broth or antibiotics were added to the vials in order to keep the evolution of the cultures. Samples were taken twice a week until the end of the experiment i.e. until resistance was achieved or cellular death. Additionally, six replicates of WHO-F WT and WHO-F with rplV mutation, caused by azithromycin, were exposed to increasing concentrations of ciprofloxacin in plates to assess if there were differences in the rate of resistance emergence. Results: We found that after 12 hours of pre-exposure to azithromycin, N. gonorrhoeae's resilience to ciprofloxacin exposure increased. Pre-exposure to azithromycin did not, however, accelerate the speed to acquisition of ciprofloxacin resistance. Conclusions:   We found that azithromycin does not accelerate the emergence of ciprofloxacin resistance, but there were differences in the molecular pathways to the acquisition of ciprofloxacin resistance: the strains preexpossed to azithromycin followed a different route (GyrA: S91F pathway) than the ones without antibiotic preexposure (GyrA:D95N pathway). However, the number of isolates is too small to draw such strong conclusions., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 González N et al.)

In addition to antimicrobial resistance, bacteria contain other mechanisms to survive antibiotic exposure such as tolerance, defined as the ability to slow metabolism by the extension of the lag phase without altering antimicrobial susceptibility. In a number of bacterial species, tolerance has been associated with treatment failure and infection chronicity and is found to precede and facilitate antimicrobial resistance. It is unknown if tolerance can be induced in Neisseria gonorrhoeae . In this study, we determined if tolerance to ceftriaxone (CRO) can be induced in N. gonorrhoeae and detected in clinical isolates. To induce tolerance, WHO P N. gonorrhoeae reference strain samples were grown under daily 3 h intermittent CRO exposure (10× the MIC), partitioned by overnight growth in GC broth. This cyclic exposure was performed for 7 consecutive days in sextuplicate, with two control cultures to which GC medium without antibiotics was added. To detect tolerance and assess CRO susceptibility, modified Tolerance Disc (TD) and Epsilometer tests were performed on isolates after each CRO exposure cycle. Additionally, this experiment was carried out on 18 clinical N. gonorrhoeae isolates. Tolerance was first detected after two CRO exposure cycles in five out of six samples. The phenotype differed per cycle with no clear pattern. No tolerance was found in control samples but was detected in 10 out of 18 clinical isolates. The present study is the first to demonstrate the induction of tolerance to CRO in N. gonorrhoeae through antibiotic exposure. In addition, tolerance to CRO was found in clinical samples.

Background: Concentrations of fluoroquinolones up to 200-fold lower than the minimal inhibitory concentration (MIC) have been shown to be able to select for antimicrobial resistance in E. coli and Salmonella spp. (the minimum selection concentration-MSC). We hypothesized that the low concentrations of quinolones found in meat may play a role in the genesis of quinolone resistance in Neisseria gonorrhoeae . We aimed to (i) establish the ciprofloxacin MSC for N. gonorrhoeae and (ii) assess if, at the ecological level, the prevalence of gonococcal ciprofloxacin resistance is associated with the concentration of quinolones used in food animal production, which is an important determinant of long-term low-dose exposure to ciprofloxacin in humans., Methods: (i) To assess if subinhibitory ciprofloxacin concentrations could select for de novo generated resistant mutants, a susceptible WHO-P N. gonorrhoeae isolate was serially passaged at 1, 1:10, 1:100 and 1:1000 of the ciprofloxacin MIC of WHO-P (0.004 mg/L) on GC agar plates. (ii) Spearman's correlation was used to assess the association between the prevalence of ciprofloxacin resistance in N. gonorrhoeae and quinolone use for animals and quinolone consumption by humans., Results: Ciprofloxacin concentrations as low as 0.004 µg/L (1/1000 of the MIC of WHO-P) were able to select for ciprofloxacin resistance. The prevalence of ciprofloxacin resistance in N. gonorrhoeae was positively associated with quinolone use for food animals (ρ = 0.47; p = 0.004; N = 34)., Conclusion: Further individual level research is required to assess if low doses of ciprofloxacin from ingested foodstuffs are able to select for ciprofloxacin resistance in bacteria colonizing humans and other species.