Short-term exposure to antibiotics begets long-term disturbance in gut microbial metabolism and molecular ecological networks.

BACKGROUND: Antibiotic exposure can occur in medical settings and from environmental sources. Long-term effects of brief antibiotic exposure in early life are largely unknown. RESULTS: Post a short-term treatment by ceftriaxone to C57BL/6 mice in early life, a 14-month observation was performed using 16S rRNA gene-sequencing technique, metabolomics analysis, and metagenomics analysis on the effects of ceftriaxone exposure. Firstly, the results showed that antibiotic pre-treatment significantly disturbed gut microbial α and ß diversities (P < 0.05). Both Chao1 indices and Shannon indices manifested recovery trends over time, but they didn't entirely recover to the baseline of control throughout the experiment. Secondly, antibiotic pre-treatment reduced the complexity of gut molecular ecological networks (MENs). Various network parameters were affected and manifested recovery trends over time with different degrees, such as nodes (P < 0.001, R2 = 0.6563), links (P < 0.01, R2 = 0.4543), number of modules (P = 0.0672, R2 = 0.2523), relative modularity (P = 0.6714, R2 = 0.0155), number of keystones (P = 0.1003, R2 = 0.2090), robustness_random (P = 0.79, R2 = 0.0063), and vulnerability (P = 0.0528, R2 = 0.28). The network parameters didn't entirely recover. Antibiotic exposure obviously reduced the number of key species in gut MENs. Interestingly, new keystones appeared during the recovery process of network complexity. Changes in network stability might be caused by variations in network complexity, which supports the ecological theory that complexity begets stability. Besides, the metabolism profiles of the antibiotic group and control were significantly different. Correlation analysis showed that antibiotic-induced differences in gut microbial metabolism were related to MEN changes. Antibiotic exposure also caused long-term effects on gut microbial functional networks in mice. CONCLUSIONS: These results suggest that short-term antibiotic exposure in early life will cause long-term negative impacts on gut microbial diversity, MENs, and microbial metabolism. Therefore, great concern should be raised about children's brief exposure to antibiotics if the results observed in mice are applicable to humans. Video Abstract.

Impact of molecular ciprofloxacin resistance testing in management of gonorrhoea in a large urban clinic.

OBJECTIVES: Antibiotic resistance in gonorrhoea is of significant public health concern with the emergence of resistance to last-line therapies such as ceftriaxone. Despite around half of Neisseria gonorrhoeae isolates tested in the UK being susceptible to ciprofloxacin, very little ciprofloxacin is used in clinical practice. Testing for the S91F mutation associated with ciprofloxacin resistance is now available in CE-marked assays and may reduce the requirement for ceftriaxone, but many patients are treated empirically, or as sexual contacts, which may limit any benefit. We describe the real-world impact of such testing on antimicrobial use and clinical outcomes in people found to have gonorrhoea in a large urban UK sexual health clinic. METHODS: Molecular ciprofloxacin resistance testing (ResistancePlus GC assay (SpeeDx)) was undertaken as an additional test after initial diagnosis (m2000 Realtime CT/NG assay (Abbott Molecular)) in those not already known to have had antimicrobial treatment. Data from a 6-month period (from March to September 2022) were analysed to determine treatment choice and treatment outcome. RESULTS: A total of 998 clinical samples tested positive for N. gonorrhoeae in 682 episodes of infection. Of the 560 (56%) samples eligible for resistance testing, 269 (48.0%) were reported as wild-type, 180 (32.1%) were predicted to be resistant, 63 (11.3%) had an indeterminate resistance profile, and in 48 (8.6%) samples, N. gonorrhoeae was not detected. Ciprofloxacin was prescribed in 172 (75%) of 228 episodes in which the wild-type strain was detected. Four (2%) of those treated with ciprofloxacin had a positive test-of-cure sample by NAAT, with no reinfection risk. All four had ciprofloxacin-susceptible infection by phenotypic antimicrobial susceptibility testing. CONCLUSIONS: In routine practice in a large UK clinic, molecular ciprofloxacin resistance testing led to a significant shift in antibiotic use, reducing use of ceftriaxone. Testing can be targeted to reduce unnecessary additional testing. Longer term impact on antimicrobial resistance requires ongoing surveillance.

Clinical outcomes in patients with piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae bloodstream infection.

BACKGROUND: A small proportion of Escherichia coli and Klebsiella pneumoniae demonstrate in vitro non-susceptibility to piperacillin/tazobactam but retain susceptibility to ceftriaxone. Uncertainty remains regarding how best to treat these isolates. OBJECTIVES: We sought to compare clinical outcomes between patients with piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae bloodstream infection receiving definitive therapy with ceftriaxone versus an alternative effective antibiotic. METHODS: We retrospectively identified patients with a positive blood culture for piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae between 1 January 2013 and 31 December 2022. Patients were divided into one of two definitive treatment groups: ceftriaxone or alternative effective antibiotic. Our primary outcome was a composite of 90 day all-cause mortality, hospital readmission, or recurrence of infection. We used Cox proportional hazards models to compare time with the composite outcome between groups. RESULTS: Sixty-two patients were included in our analysis. Overall, median age was 63 years (IQR 49.5-71.0), the most common source of infection was intra-abdominal (25/62; 40.3%) and the median total duration of therapy was 12.0 days (IQR 9.0-16.8). A total of 9/22 (40.9%) patients in the ceftriaxone treatment group and 18/40 (45.0%) patients in the alternative effective antibiotic group met the composite endpoint. In an adjusted time-to-event analysis, there was no difference in the composite endpoint between groups (HR 0.67, 95% CI 0.30-1.50). The adjusted Bayesian posterior probability that the HR was less than or equal to 1 (i.e. ceftriaxone is as good or better than alternative therapy) was 85%. CONCLUSIONS: These findings suggest that ceftriaxone can be used to effectively treat bloodstream infections with E. coli or K. pneumoniae that are non-susceptible to piperacillin/tazobactam but susceptible to ceftriaxone.

Minimum inhibitory concentrations of Neisseria gonorrhoeae strains in clients of the Amsterdam sexual health clinic with a Dutch versus an international sexual network.

OBJECTIVES: International travel combined with sex may contribute to dissemination of antimicrobial-resistant (AMR) Neisseria gonorrhoeae (Ng). To assess the role of travel in Ng strain susceptibility, we compared minimum inhibitory concentrations (MICs) for five antibiotics (ie, azithromycin, ceftriaxone, cefotaxime, cefixime and ciprofloxacin) in strains from clients with an exclusively Dutch sexual network and clients with an additional international sexual network. METHODS: From 2013 to 2019, we recorded recent residence of sexual partners of clients (and of their partners) with Ng at the Center for Sexual Health of Amsterdam. We categorised clients as having: (1) exclusively sexual partners residing in the Netherlands ('Dutch only') or (2) at least one partner residing outside the Netherlands. We categorised the country of residence of sexual partners by World Bank/EuroVoc regions. We analysed the difference of log-transformed MIC of Ng strains between categories using linear or hurdle regression for each antibiotic. RESULTS: We included 3367 gay and bisexual men who had sex with men (GBMSM), 516 women and 525 men who exclusively had sex with women (MSW) with Ng. Compared with GBMSM with a 'Dutch only' network, GBMSM with: (1) a Western European network had higher MICs for ceftriaxone (ß=0.19, 95% CI=0.08 to 0.29), cefotaxime (ß=0.19, 95% CI=0.08 to 0.31) and cefixime (ß=0.06, 95% CI=0.001 to 0.11); (2) a Southern European network had a higher MIC for cefixime (ß=0.10, 95% CI=0.02 to 0.17); and (3) a sub-Saharan African network had a lower MIC for ciprofloxacin (ß=-1.79, 95% CI=-2.84 to -0.74). In women and MSW, higher MICs were found for ceftriaxone in clients with a Latin American and Caribbean network (ß=0.26, 95% CI=0.02 to 0.51). CONCLUSIONS: For three cephalosporin antibiotics, we found Ng strains with slightly higher MICs in clients with partner(s) from Europe or Latin America and the Caribbean. International travel might contribute to the spread of Ng with lower susceptibility. More understanding of the emergence of AMR Ng is needed.

Comparative study of oral microbiota in the experimental long-term opioid exposure, after its withdrawal and the use of complex drug correction.

OBJECTIVE: Aim: To study changes of dental biofilm microbiota composition during experimental opioid exposure, after its withdrawal and when using of complex drug correction.. PATIENTS AND METHODS: Materials and Methods: Microbiological studies (48 rats) included microscopic and bacteriological methods, as well as determination of antibiotic susceptibility of microbial isolates. Ceftriaxone and pentoxifylline were used to correction the changes. RESULTS: Results: The action of opioid for 10 weeks caused considerable changes in the microbiocenosis, which was illustrated by a significant increasing of the opportunistic pathogens quantitative indicators and the emergence of pathogenic microbiota. Changes in the microbiocenosis at 6 weeks of opioid exposure and after its withdrawal for 4 weeks were expressed in the appearance of pathogenic microbiota and the absence of significant differences in quantitative indicators of saprophytic and opportunistic microflora compared to similar indicators in animals with 10 weeks opioid exposure. This indicated a slow progression of dysbiotic changes and the inflammatory process in the oral cavity of rats. CONCLUSION: Conclusions: After 10 weeks of experiment with opioid administration for 6 weeks and the use of ceftriaxone and pentoxifylline on the background of 4-week opioid withdrawal, a significant reduction of quantitative indicators of opportunistic bacteria and elimination of pathogenic species of microorganisms was determined. The use of complex drug correction on the background of 10 weeks of opioid exposure led to a significant reduction in the quantitative indicators of opportunistic pathogens and contributed to the elimination of most pathogenic species of microbiota under the action of ceftriaxone.

Characterization of Ceftriaxone-Resistant Haemophilus influenzae Among Korean Children.

BACKGROUND: Haemophilus influenzae is a frequently encountered pathogen responsible for respiratory tract infections in children. Following the detection of ceftriaxone-resistant H. influenzae at our institution, we aimed to investigate the resistance mechanisms of ceftriaxone in H. influenzae, with a particular focus on alterations in penicillin-binding protein 3 (PBP3) and ß-lactamase production. METHODS: Among H. influenzae isolates collected at Asan Medical Center Children's Hospital from March 2014 to April 2019, ceftriaxone-resistant strains by the disk-diffusion test were included. Ceftriaxone minimum inhibitory concentrations (MICs) were determined using the E-test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The presence of ß-lactamase was assessed through cefinase test and TEM-1/ROB-1 polymerase chain reaction (PCR). PBP3 alterations were explored via ftsI gene sequencing. RESULTS: Out of the 68 collected strains, 21 exhibited resistance to ceftriaxone in disk diffusion tests. Two strains were excluded due to failed subculture. Among 19 ceftriaxone-resistant H. influenzae isolates, eighteen were non-typeable H. influenzae, and twelve were positive for TEM-1 PCR. Isolates were classified into groups II (harboring only N526K, n = 3), III (N526K+S385T, n = 2), III+ (S385T+L389F+N526K, n = 11), and III-like+ (S385T+L389F+R517H, n = 3) according to the PBP3 alteration pattern. With a median ceftriaxone MIC of 0.190 mg/L (range, 0.008-0.750), the median ceftriaxone MIC was the highest in group III-like+ (0.250 mg/L), followed by groups III+ (0.190 mg/L), III (0.158 mg/L), and II (0.012 mg/L). All three strains belonging to group II, which did not harbor the S385T substitution, had ceftriaxone MICs of ≤ 0.125 mg/L. CONCLUSION: The emergence of ceftriaxone-resistant H. influenzae with ceftriaxone MIC values of up to 0.75 mg/L was observed even in children in South Korea, with most associated with S385T and L389F substitutions. The N526K mutation alone does not significantly impact ceftriaxone resistance. Further large-scale studies are essential to investigate changes in antibiotic resistance patterns and factors influencing antibiotic resistance in H. influenzae isolated from pediatric patients in Korea.

Dissemination of IncC plasmids in Salmonella enterica serovar Thompson recovered from seafood and human diarrheic patients in China.

Salmonella Thompson is a prevalent foodborne pathogen and a major threat to food safety and public health. This study aims to reveal the dissemination mechanism of S. Thompson with co-resistance to ceftriaxone and ciprofloxacin. In this study, 181 S. Thompson isolates were obtained from a retrospective screening on 2118 serotyped Salmonella isolates from foods and patients, which were disseminated in 12 of 16 districts in Shanghai, China. A total of 10 (5.5 %) S. Thompson isolates exhibited resistance to ceftriaxone (MIC ranging from 8 to 32 µg/mL) and ciprofloxacin (MIC ranging from 2 to 8 µg/mL). The AmpC ß-lactamase gene blaCMY-2 and plasmid-mediated quinolone resistance (PMQR) genes of qnrS and qepA were identified in the 9 isolates. Conjugation results showed that the co-transfer of blaCMY-2, qnrS, and qepA occurred on the IncC plasmids with sizes of ∼150 (n = 8) or ∼138 (n = 1) kbp. Three typical modules of ISEcp1-blaCMY-2-blc-sugE, IS26-IS15DIV-qnrS-ISKpn19, and ISCR3-qepA-intl1 were identified in an ST3 IncC plasmid pSH11G0791. Phylogenetic analysis indicated that IncC plasmids evolved into Lineages 1, 2, and 3. IncC plasmids from China including pSH11G0791 in this study fell into Lineage 1 with those from the USA, suggesting their close genotype relationship. In conclusion, to our knowledge, it is the first report of the co-existence of blaCMY-2, qnrS, and qepA in IncC plasmids, and the conjugational transfer contributed to their dissemination in S. Thompson. These findings underline further challenges for the prevention and treatment of Enterobacteriaceae infections posed by IncC plasmids bearing blaCMY-2, qnrS, and qepA.

Ureidopenicillins Are Potent Inhibitors of Penicillin-Binding Protein 2 from Multidrug-Resistant Neisseria gonorrhoeae H041.

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido ß-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R1 group. Crystal structures show that the ureido ß-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of ß-lactams with improved activity against ESC-resistant N. gonorrhoeae.

Multidrug-resistant conjugative plasmid carrying mphA confers increased antimicrobial resistance in Shigella.

Shigellosis remains a common gastrointestinal disease mostly in children < 5 years of age in developing countries. Azithromycin (AZM), a macrolide, is currently the first-line treatment for shigellosis in Bangladesh; ciprofloxacin (CIP) and ceftriaxone (CRO) are also used frequently. We aimed to evaluate the current epidemiology of antimicrobial resistance (AMR) and mechanism(s) of increasing macrolide resistance in Shigella in Bangladesh. A total of 2407 clinical isolates of Shigella from 2009 to 2016 were studied. Over the study period, Shigella sonnei was gradually increasing and become predominant (55%) over Shigella flexneri (36%) by 2016. We used CLSI-guided epidemiological cut-off value (ECV) for AZM in Shigella to set resistance breakpoints (zone-diameter ≤ 15 mm for S. flexneri and ≤ 11 mm for S. sonnei). Between 2009 and 2016, AZM resistance increased from 22% to approximately 60%, CIP resistance increased by 40%, and CRO resistance increased from zero to 15%. The mphA gene was the key macrolide resistance factor in Shigella; a 63MDa conjugative middle-range plasmid was harboring AZM and CRO resistance factors. Our findings show that, especially after 2014, there has been a rapid increase in resistance to the three most effective antibiotics. The rapid spread of macrolide (AZM) resistance genes among Shigella are driven by horizontal gene transfer rather than direct lineage.

Pharmacodynamic evaluation of ceftriaxone single-dose therapy (0.125-1 g) to eradicate ceftriaxone-susceptible and ceftriaxone-resistant Neisseria gonorrhoeae strains in a hollow fibre infection model for gonorrhoea.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is threatening the gonorrhoea treatment, and optimizations of the current ceftriaxone-treatment regimens are crucial. We evaluated the pharmacodynamics of ceftriaxone single-dose therapy (0.125-1 g) against ceftriaxone-susceptible and ceftriaxone-resistant gonococcal strains, based on EUCAST ceftriaxone-resistance breakpoint (MIC > 0.125 mg/L), in our hollow fibre infection model (HFIM) for gonorrhoea. METHODS: Gonococcal strains examined were WHO F (ceftriaxone-susceptible, MIC < 0.002 mg/L), R (ceftriaxone-resistant, MIC = 0.5 mg/L), Z (ceftriaxone-resistant, MIC = 0.5 mg/L) and X (ceftriaxone-resistant, MIC = 2 mg/L). Dose-range HFIM 7 day experiments simulating ceftriaxone 0.125-1 g single-dose intramuscular regimens were conducted. RESULTS: Ceftriaxone 0.125-1 g single-dose treatments rapidly eradicated WHO F (wild-type ceftriaxone MIC). Ceftriaxone 0.5 and 1 g treatments, based on ceftriaxone human plasma pharmacokinetic parameters, eradicated most ceftriaxone-resistant gonococcal strains (WHO R and Z), but ceftriaxone 0.5 g failed to eradicate WHO X (high-level ceftriaxone resistance). When simulating oropharyngeal gonorrhoea, ceftriaxone 0.5 g failed to eradicate all the ceftriaxone-resistant strains, while ceftriaxone 1 g eradicated WHO R and Z (low-level ceftriaxone resistance) but failed to eradicate WHO X (high-level ceftriaxone resistance). No ceftriaxone-resistant mutants were selected using any ceftriaxone treatments. CONCLUSIONS: Ceftriaxone 1 g single-dose intramuscularly cure most of the anogenital and oropharyngeal gonorrhoea cases caused by the currently internationally spreading ceftriaxone-resistant gonococcal strains, which should be further confirmed clinically. A ceftriaxone 1 g dose (±azithromycin 2 g) should be recommended for first-line empiric gonorrhoea treatment. This will buy countries some time until novel antimicrobials are licensed. Using ceftriaxone 1 g gonorrhoea treatment, the EUCAST ceftriaxone-resistance breakpoint is too low.

Recent dynamics in Neisseria gonorrhoeae genomic epidemiology in Brazil: antimicrobial resistance and genomic lineages in 2017-20 compared to 2015-16.

OBJECTIVES: Regular quality-assured WGS with antimicrobial resistance (AMR) and epidemiological data of patients is imperative to elucidate the shifting gonorrhoea epidemiology, nationally and internationally. We describe the dynamics of the gonococcal population in 11 cities in Brazil between 2017 and 2020 and elucidate emerging and disappearing gonococcal lineages associated with AMR, compare to Brazilian WGS and AMR data from 2015 to 2016, and explain recent changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: WGS was performed using Illumina NextSeq 550 and genomes of 623 gonococcal isolates were used for downstream analysis. Molecular typing and AMR determinants were obtained and links between genomic lineages and AMR (determined by agar dilution/Etest) examined. RESULTS: Azithromycin resistance (15.6%, 97/623) had substantially increased and was mainly explained by clonal expansions of strains with 23S rRNA C2611T (mostly NG-STAR CC124) and mtr mosaics (mostly NG-STAR CC63, MLST ST9363). Resistance to ceftriaxone and cefixime remained at the same levels as in 2015-16, i.e. at 0% and 0.2% (1/623), respectively. Regarding novel gonorrhoea treatments, no known zoliflodacin-resistance gyrB mutations or gepotidacin-resistance gyrA mutations were found. Genomic lineages and sublineages showed a phylogenomic shift from sublineage A5 to sublineages A1-A4, while isolates within lineage B remained diverse in Brazil. CONCLUSIONS: Azithromycin resistance, mainly caused by 23S rRNA C2611T and mtrD mosaics/semi-mosaics, had substantially increased in Brazil. This mostly low-level azithromycin resistance may threaten the recommended ceftriaxone-azithromycin therapy, but the lack of ceftriaxone resistance is encouraging. Enhanced gonococcal AMR surveillance, including WGS, is imperative in Brazil and other Latin American and Caribbean countries.

Ceftriaxone-Resistant Gonorrhea - China, 2022.

Gonorrhea is a widespread sexually transmitted infection; in 2022, China reported 96,313 cases of gonorrhea, making it the fourth most common notifiable infectious disease in the country after viral hepatitis, pulmonary tuberculosis, and syphilis. The rise in prevalence in antimicrobial-resistant strains, particularly the international spread of ceftriaxone-resistant clones, poses a formidable challenge to gonorrhea control. The China Gonococcal Resistance Surveillance Program (China-GRSP), established in 1987 and covering 19 of 34 provincial-level administrative units, continuously monitors gonococcal antimicrobial resistance. In 2022, 13 China-GRSP sentinel sites collected 2,804 gonococcal isolates, representing 2.9% of all cases reported in China, and 4.1% of cases reported in the 13 participating provinces. The prevalence of Neisseria gonorrhoeae resistance to ceftriaxone was 8.1%, approximately three times the 2017 rate of 2.9%; five provinces reported >10% ceftriaxone resistance. Resistance prevalences to cefixime, azithromycin, tetracycline, penicillin, and ciprofloxacin were 16.0%, 16.9%, 77.1%, 77.8%, and 97.6%, respectively. Only one case of spectinomycin resistance was reported. These data highlight a substantial increase in ceftriaxone resistance from 2017 to 2022. Effective diagnosis and treatment and appropriate management of sex partners are essential to protect the health of infected persons and prevent ongoing transmission of gonorrhea, including transmission of resistant strains. Identifying reasons for the spread of ceftriaxone-resistant N. gonorrhoeae in China could guide strategies, such as antibiotic stewardship, to mitigate the rising resistance rate and curb the spread of resistant strains.

Combining antibiotic with anti-TLR2/TLR13 therapy prevents brain pathology in pneumococcal meningitis.

Despite effective antibiotic therapy, brain-destructive inflammation often cannot be avoided in pneumococcal meningitis. The causative signals are mediated predominantly through TLR-recruited myeloid differentiation primary response adaptor 88 (MyD88), as indicated by a dramatic pneumococcal meningitis phenotype of Myd88-/- mice. Because lipoproteins and single-stranded RNA are crucial for recognition of Gram-positive bacteria such as Streptococcus pneumoniae by the host immune system, we comparatively analyzed the disease courses of Myd88-/- and Tlr2-/- Tlr13-/- mice. Their phenotypic resemblance indicated TLR2 and -13 as master sensors of S. pneumoniae in the cerebrospinal fluid. A neutralizing anti-TLR2 antibody (T2.5) and chloroquine (CQ) - the latter applied here as an inhibitor of murine TLR13 and its human ortholog TLR8 - abrogated activation of murine and human primary immune cells exposed to antibiotic-treated S. pneumoniae. The inhibitory effect of the T2.5/CQ cocktail was stronger than that of dexamethasone, the current standard adjunctive drug for pneumococcal meningitis. Accordingly, TLR2/TLR13 blockade concomitant with ceftriaxone application significantly improved the clinical course of pneumococcal meningitis compared with treatment with ceftriaxone alone or in combination with dexamethasone. Our study indicates the importance of murine TLR13 and human TLR8, besides TLR2, in pneumococcal meningitis pathology, and suggests their blockade as a promising antibiotic therapy adjunct.

Phenotypic and genotypic characterization of Neisseria gonorrhoeae isolates among individuals at high risk for sexually transmitted diseases in Zurich, Switzerland.

BACKGROUND: While ceftriaxone resistance remains scarce in Switzerland, global Neisseria gonorrhoeae (NG) antimicrobial resistance poses an urgent threat. This study describes clinical characteristics in MSM (men who have sex with men) diagnosed with NG infection and analyses NG resistance by phenotypic and genotypic means. METHODS: Data of MSM enrolled in three clinical cohorts with a positive polymerase chain reaction test (PCR) for NG were analysed between January 2019 and December 2021 and linked with antibiotic susceptibility testing. Bacterial isolates were subjected to whole genome sequencing (WGS). RESULTS: Of 142 participants, 141 (99%) were MSM and 118 (84%) living with HIV. Participants were treated with ceftriaxone (N = 79), azithromycin (N = 2), or a combination of both (N = 61). No clinical or microbiological failures were observed. From 182 positive PCR samples taken, 23 were available for detailed analysis. Based on minimal inhibitory concentrations (MICs), all isolates were susceptible to ceftriaxone, gentamicin, cefixime, cefpodoxime, ertapenem, zoliflodacin, and spectinomycin. Resistance to azithromycin, tetracyclines and ciprofloxacin was observed in 10 (43%), 23 (100%) and 11 (48%) of the cases, respectively. Analysis of WGS data revealed combinations of resistance determinants that matched with the corresponding phenotypic resistance pattern of each isolate. CONCLUSION: Among the MSM diagnosed with NG mainly acquired in Switzerland, ceftriaxone MICs were low for a subset of bacterial isolates studied and no treatment failures were observed. For azithromycin, high occurrences of in vitro resistance were found. Gentamicin, cefixime, cefpodoxime, ertapenem, spectinomycin, and zoliflodacin displayed excellent in vitro activity against the 23 isolates underscoring their potential as alternative agents to ceftriaxone.

Doxycycline post-exposure prophylaxis could theoretically select for resistance to various antimicrobials in 19 pathobionts: an in silico analysis.

OBJECTIVES: Doxycycline post exposure prophylaxis (PEP) has been shown to reduce the incidence of bacterial STIs. However, if there is genetic linkage between resistance to tetracycline and other antimicrobials, then it could also select for resistance to these other antimicrobials. We therefore undertook to evaluate if there is an association between the minimum inhibitory concentrations (MICs) of tetracycline and other antimicrobials in 19 clinically important bacterial species. METHODS: Mixed-effects linear regression was used to assess if minocycline MICs were associated with the MICs of eight other antimicrobials (ceftriaxone, ampicillin, oxacillin, vancomycin, erythromycin, levofloxacin, amikacin, and trimethoprim-sulfamethoxazole) in 19 bacterial species in the Antimicrobial Testing Leadership and Surveillance (ATLAS) database. RESULTS: With the notable exception of vancomycin, where no association was found, strong positive associations were typically found between the MICs of minocycline and each of the eight antimicrobials in each of the species assessed. For example, the minocycline MICs of all the Gram-positive species were positively associated with ampicillin, ceftriaxone, oxacillin and erythromycin MICs (all P-values < 0.001). The only exceptions were ampicillin for Streptococcus pyogenes and ceftriaxone for S. dysgalactiae, where no significant associations were found. Similarly in the Gram-negative species, the minocycline MICs of all the species except Haemophilus influenzae and Stenotrophomonas maltophilia were positively associated with the MICs of ceftriaxone, ampicillin, levofloxacin and amikacin (all P-values < 0.001). CONCLUSIONS: There is a theoretical risk that doxycycline PEP could select for resistance not only to tetracyclines but to a range of other antimicrobials in each of the 19 pathobionts assessed.

Detection of antimicrobial resistance in <5 h in Neisseria gonorrhoeae isolates using flow cytometry-proof of concept for seven clinically relevant antimicrobials.

INTRODUCTION: Antimicrobial resistance in Neisseria gonorrhoeae compromises gonorrhoea treatment and rapid antimicrobial susceptibility testing (AST) would be valuable. We have developed a rapid and accurate flow cytometry method (FCM) for AST of gonococci. METHODS: The 2016 WHO gonococcal reference strains, and WHO Q, R and S (n = 17) were tested against seven clinically relevant antibiotics (ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and gentamicin). After 4.5 h incubation of inoculated broth, the fluorescent dye Syto™ 9 was added, followed by FCM analysis. After gating, the relative remaining population of gonococci, compared with unexposed growth control samples, was plotted against antimicrobial concentration, followed by non-linear curve regression analysis. Furthermore, the response at one single concentration/tested antibiotic was evaluated with the intention to use as a screening test for detection of resistant gonococci. RESULTS: A dose-dependent response was seen in susceptible isolates for all tested antimicrobials. There was a clear separation between susceptible/WT and resistant/non-WT isolates for ceftriaxone, cefixime, spectinomycin, ciprofloxacin and tetracycline. In contrast, for azithromycin, only high-level-resistant isolates were distinguished, while resistant isolates with MICs of 4 mg/L were indistinguishable from WT (MIC ≤ 1 mg/L) isolates. For gentamicin, all tested 17 isolates were WT and FCM analysis resulted in uniform dose-response curves. Using a single antibiotic concentration and a 50% remaining cell population cut-off, the overall sensitivity and specificity for resistance detection were 93% and 99%, respectively. CONCLUSIONS: By providing results in <5 h for gonococcal isolates, FCM-based AST can become a rapid screening method for antimicrobial resistance or antimicrobial susceptibility in gonococci.

Ceftriaxone averts neuroinflammation and relieves depressive-like behaviors via GLT-1/TrkB signaling.

The beneficial effect of a beta-lactam antibiotic, Ceftriaxone (CEF), to improve depressive-like symptoms has been documented previously, attributed to its modulation of glutamate neurotransmission. Here, we aimed to determine whether CEF could improve LPS-altered glutamatergic signaling associated with neuroinflammation-allied depression. To assess our goals, we established a neuroinflammation-allied depression mice model by injecting lipopolysaccharides (LPS), followed by behavioral and biochemical analysis. LPS-treated mice displayed depressive symptoms, neuroinflammation, dysregulated glutamate and its transporter (GLT-1) expression, altered expression of astrocyte reactive markers (GFAP, cxcl10, steap4, GBP2, and SRGN), and dysregulated BDNF/TrkB signaling. However, these changes were rescued by CEF treatment, as we found decreased neuroinflammation, relief of depression symptoms, and improved GLT-1 and BDNF/TrkB signaling upon CEF treatment. Moreover, GLT-1 and BDNF/TrkB regulation role of CEF was validated by K252a and DHK treatment. In summary, the anti-depressive effects of glutamate modulators, like CEF, are closely related to their anti-inflammatory role.

[Establishment and Evaluation of a Resazurin-Based Microdilution Assay for Microbial Sensitivity Test of Neisseria gonorrhoeae]. / æ·‹ç— å¥ˆç‘ŸèŒè¯æ•è¯•éªŒåˆƒå¤©é’å¾®é‡ç¨€é‡Šæ³•çš„å»ºç«‹ä¸Žè¯„ä»·.

Objective: To establish and evaluate a microbial sensitivity test method for Neisseria gonorrhoeae based on resazurin coloration. Methods: Based on the broth microdilution method, resazurin was added as a live bacteria indicator. WHO G, a WHO gonococcal reference strain, was used to optimize the incubation time for resazurin-stained bacteria and the color change was visually observed to obtain the results. Agar dilution method (the gold standard) and resazurin-based microdilution assay were used to determine the minimum inhibitory concentration (MIC) of azithromycin, ceftriaxone, and spectinomycin for 3 reference strains and 32 isolates of Neisseria gonorrhoeae. The results were analyzed based on essential agreement (EA), which reflected the consistency of the MIC values, category agreement (CA), which reflected the consistency in the determination of drug resistance, intermediary, and sensitivity, very major error (VME), which reflected false sensitivity, and major error (ME), which reflected pseudo drug resistance, to evaluate the accuracy of resazurin-based microdilution assay as a microbial sensitivity test of of Neisseria gonorrhoeae. CA and EA rates≥90% and VME and ME rates≤3% were found to be the acceptable performance rates. Results: The results obtained 6 hours after resazurin was added were consistent with those of the agar dilution method and the resazurin-based microdilution assay was established accordingly based on this parameter. The EA of resazurin-based microdilution assay for measuring the MIC results of azithromycin, ceftriaxone, and spectinomycin was 97.1%, 91.5%, and 94.3%, respectively, and the CA was 88.6%, 94.3%, and 94.3%, respectively. The VME was 0% for all three antibiotics, while the ME was 11.4%, 5.7%, and 5.7%, respectively. Conclusion: The resazurin-based microdilution assay established in this study showed good agreement with agar dilution method for measuring the MIC of antibiotics against Neisseria gonorrhoeae. Moreover, the sensitivity results of this method were highly reliable and could be easily obtained through naked eye observation. Nonetheless, the results of drug resistance should be treated with caution and the optimization of parameters should be continued.

Differential in vitro susceptibility to ampicillin/ceftriaxone combination therapy among Enterococcus faecalis infective endocarditis clinical isolates.

OBJECTIVES: To investigate the genomic diversity and ß-lactam susceptibilities of Enterococcus faecalis collected from patients with infective endocarditis (IE). METHODS: We collected 60 contemporary E. faecalis isolates from definite or probable IE cases identified between 2018 and 2021 at the University of Pittsburgh Medical Center. We used whole-genome sequencing to study bacterial genomic diversity and employed antibiotic checkerboard assays and a one-compartment pharmacokinetic-pharmacodynamic (PK/PD) model to investigate bacterial susceptibility to ampicillin and ceftriaxone both alone and in combination. RESULTS: Genetically diverse E. faecalis were collected, however, isolates belonging to two STs, ST6 and ST179, were collected from 21/60 (35%) IE patients. All ST6 isolates encoded a previously described mutation upstream of penicillin-binding protein 4 (pbp4) that is associated with pbp4 overexpression. ST6 isolates had higher ceftriaxone MICs and higher fractional inhibitory concentration index values for ampicillin and ceftriaxone (AC) compared to other isolates, suggesting diminished in vitro AC synergy against this lineage. Introduction of the pbp4 upstream mutation found among ST6 isolates caused increased ceftriaxone resistance in a laboratory E. faecalis isolate. PK/PD testing showed that a representative ST6 isolate exhibited attenuated efficacy of AC combination therapy at humanized antibiotic exposures. CONCLUSIONS: We find evidence for diminished in vitro AC activity among a subset of E. faecalis IE isolates with increased pbp4 expression. These findings suggest that alternate antibiotic combinations against diverse contemporary E. faecalis IE isolates should be evaluated.