A Single Amino Acid Substitution in Elongation Factor G Can Confer Low-Level Gentamicin Resistance in Neisseria gonorrhoeae.

The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.

Gastrodin modified polyurethane conduit promotes nerve repair via optimizing Schwann cells function.

Peripheral nerve regeneration and functional recovery remain a major clinical challenge. Nerve guidance conduit (NGC) that can regulate biological behavior of Schwann cells (SCs) and facilitate axonal regeneration through microenvironmental remodeling is beneficial for nerve regeneration and functional recovery. Gastrodin, a main constituent of a Chinese traditional herbal medicine, has been known to display several biological and pharmacological properties, especially antioxidative, anti-inflammatory and nerve regeneration. Herein, polyurethane (PU) NGCs modified by different weight ratio of Gastrodin (0, 1 and 5 wt%) were designed for sequential and sustainable drug release, that created a favorable microenvironment for nerve regeneration. The scaffold showed suitable pore structure and biocompatibility in vitro, and evidently promoted morphological and functional recovery of regenerated sciatic nerves in vivo. Compared to the PU and 1%Gastrodin/PU scaffolds, the 5%Gastrodin/PU significantly enhanced the proliferation, migration and myelination of SCs and up-regulated expression of neurotrophic factors, as well as induction of the differentiation of PC12 cells. Interestingly, the obvious anti-inflammatory response was observed in 5%Gastrodin/PU by reduced expression of TNF-α and iNOS, which also evidenced by the few fibrous capsule formation in the subcutaneous implantation. Such a construct presented a similarity to autograft in vivo repairing a 10 mm sciatic nerve defects. It was able to not only boost the regenerated area of nerve and microvascular network, but also facilitate functional axons growth and remyelination, leading to highly improved functional restoration. These findings demonstrate that the 5%Gastrodin/PU NGC efficiently promotes nerve regeneration, indicating their potential for use in peripheral nerve regeneration applications.

Effects of Noninvasive Low-Intensity Focus Ultrasound Neuromodulation on Spinal Cord Neurocircuits In Vivo.

Although neurocircuits can be activated by focused ultrasound stimulation, it is unclear whether this is also true for spinal cord neurocircuits. In this study, we used low-intensity focused ultrasound (LIFU) to stimulate lumbar 4-lumbar 5 (L4-L5) segments of the spinal cord of normal Sprague Dawley rats with a clapper. The activation of the spinal cord neurocircuits enhanced soleus muscle contraction as measured by electromyography (EMG). Neuronal activation and injury were assessed by EMG, western blotting (WB), immunofluorescence, hematoxylin and eosin (H&E) staining, Nissl staining, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), somatosensory evoked potentials (SEPs), motor evoked potentials (MEPs), and the Basso-Beattie-Bresnahan locomotor rating scale. When the LIFU intensity was more than 0.5 MPa, LIFU stimulation induced soleus muscle contraction and increased the EMG amplitudes (P < 0.05) and the number of c-fos- and GAD65-positive cells (P < 0.05). When the LIFU intensity was 3.0 MPa, the LIFU stimulation led to spinal cord damage and decreased SEP amplitudes for electrophysiological assessment (P < 0.05); this resulted in coagulation necrosis, structural destruction, neuronal loss in the dorsal horn by H&E and Nissl staining, and increased expression of GFAP, IL-1ß, TNF-α, and caspase-3 by IHC, ELISA, and WB (P < 0.05). These results show that LIFU can activate spinal cord neurocircuits and that LIFU stimulation with an irradiation intensity ≤1.5 MPa is a safe neurostimulation method for the spinal cord.

Evaluation of Drugs with Therapeutic Potential for Susceptibility of Neisseria Gonorrhoeae Isolates from 8 Provinces in China from 2018.

PURPOSE: The study aimed to evaluate meropenem, fosfomycin, berberine hydrochloride, and doxycycline minimum inhibitory concentrations (MICs) of Neisseria gonorrhoeae collected from eight provinces in China in 2018. METHODS: The MICs of 540 Neisseria gonorrhoeae isolates (451 isolates selected randomly and 89 isolates selected with preference) were determined to meropenem, fosfomycin, berberine hydrochloride, and doxycycline using the agar dilution method, and the MICs of ceftriaxone and azithromycin were detected for comparison. RESULTS: Among 451 randomly selected isolates, the MIC90 was 0.06 mg/L for meropenem, 64 mg/L for fosfomycin, 64 mg/L for berberine hydrochloride, and 16 mg/L for doxycycline. All isolates showed the MIC ≤ 0.125 mg/L to meropenem, 13 isolates (2.9%) showed MIC > 64 mg/L to fosfomycin, 8 isolates (1.8%) demonstrated MIC > 64 mg/L to berberine hydrochloride, and 271 isolates (60.1%) demonstrated MIC > 1 mg/L to doxycycline. Comparing all 540 tested isolates, a correlation of r = 0.50 (P < 0.001) between meropenem and ceftriaxone MIC was observed. In 24 ceftriaxone-decreased susceptibility isolates, all isolates showed an MIC ≤ 0.125 mg/L for meropenem, 1 isolate (4.2%) showed an MIC > 64 mg/L for fosfomycin, 1 isolate (4.2%) showed an MIC > 64 mg/L for berberine hydrochloride, and 13 isolates (54.2%) showed an MIC > 1 mg/L for doxycycline. In 87 azithromycin resistant isolates, all isolates showed an MIC ≤ 0.125 mg/L for meropenem, 2 isolates (2.3%) showed an MIC > 64 mg/L for fosfomycin, 4 isolates (4.6%) showed an MIC > 64 mg/L for berberine hydrochloride, and 64 isolates (73.6%) showed an MIC > 1 mg/L for doxycycline. CONCLUSION: The in vitro results suggest that meropenem might be a promising treatment option for resistant gonococcal infections, while the effects of fosfomycin and berberine hydrochloride should be further evaluated as potential therapeutic agents. The effectiveness of these drugs in animal experiments and clinical use may need further study.

Widespread Use of High-dose Ceftriaxone Therapy for Uncomplicated Gonorrhea Without Reported Ceftriaxone Treatment Failure: Results From 5 Years of Multicenter Surveillance Data in China.

BACKGROUND: Antimicrobial resistance to Neisseria gonorrhoeae has emerged for each of the antibiotics recommended as first-line therapies following their introduction into clinical practice. To improve rational and effective clinical antibiotic treatment, we analyzed the prescription patterns of antibiotics and their therapeutic effect in the treatment of uncomplicated gonorrhea in China. METHODS: We obtained data from a follow-up multicenter surveillance program. Multinomial logistic regression analyses were conducted to explore the associations between demographic/clinical variables with the levels of sensitivity to ceftriaxone and prescription of high-dose ceftriaxone. RESULTS: In this study, 1686 patients infected with N. gonorrhoeae were recruited in a surveillance network during 1 January 2013 through 31 December 2017 in 7 hospitals distributed in 5 provinces. The prevalence of isolates with decreased susceptibility to ceftriaxone was 9.8% (131/1333), fluctuating between 5.6% and 12.1%. Injectable ceftriaxone was chosen as the first-line treatment among 83.1% of patients, and most of them (72.7% [1018/1401]) received >1000 mg dosage. Patients who were previously infected with gonorrhea or other sexually transmitted infections (adjusted odds ratio [AOR], 1.618 [95% confidence interval {CI}, 1.11-2.358]; AOR, 2.08 [95% CI, 1.41-3.069]) or who already used antibiotics for this infection (AOR, 1.599 [95% CI, 1.041-2.454]) were associated with a higher prescribed ceftriaxone dosage. All of the patients recruited in this study were cured regardless of the isolates' susceptibility to ceftriaxone or the dosage of ceftriaxone they received. CONCLUSIONS: No ceftriaxone treatment failure for uncomplicated gonorrhea was reported in China; however, high-dose ceftriaxone was widely used in China. Its impacts need further study.

Could Dampening Expression of the Neisseria gonorrhoeae mtrCDE-Encoded Efflux Pump Be a Strategy To Preserve Currently or Resurrect Formerly Used Antibiotics To Treat Gonorrhea?

Neisseria gonorrhoeae has developed resistance to every antibiotic introduced for treatment of gonorrhea since 1938, and concern now exists that gonorrheal infections may become refractory to all available antibiotics approved for therapy. The current recommended dual antibiotic treatment regimen of ceftriaxone (CRO) and azithromycin (AZM) is threatened with the emergence of gonococcal strains displaying resistance to one or both of these antibiotics. Non-beta-lactamase resistance to penicillin and third-generation cephalosporins, as well as low-level AZM resistance expressed by gonococci, requires overexpression of the mtrCDE-encoded efflux pump, which in wild-type (WT) strains is subject to transcriptional repression by MtrR. Since earlier studies showed that loss of MtrCDE renders gonococci hypersusceptible to beta-lactams and macrolides, we hypothesized that transcriptional dampening of mtrCDE would render an otherwise resistant strain susceptible to these antibiotics as assessed by antibiotic susceptibility testing and during experimental infection. In order to test this hypothesis, we ectopically expressed a WT copy of the mtrR gene, which encodes the repressor of the mtrCDE efflux pump operon, in N. gonorrhoeae strain H041, the first reported gonococcal strain to cause a third-generation-cephalosporin-resistant infection. We now report that MtrR production can repress the expression of mtrCDE, increase antimicrobial susceptibility in vitro, and enhance beta-lactam efficacy in eliminating gonococci as assessed in a female mouse model of lower genital tract infection. We propose that strategies that target the MtrCDE efflux pump should be considered to counteract the increasing problem of antibiotic-resistant gonococci.IMPORTANCE The emergence of gonococcal strains resistant to past or currently used antibiotics is a global public health concern, given the estimated 78 million infections that occur annually. The dearth of new antibiotics to treat gonorrhea demands that alternative curative strategies be considered to counteract antibiotic resistance expressed by gonococci. Herein, we show that decreased expression of a drug efflux pump that participates in gonococcal resistance to antibiotics can increase gonococcal susceptibility to beta-lactams and macrolides under laboratory conditions, as well as improve antibiotic-mediated clearance of gonococci from the genital tract of experimentally infected female mice.

Susceptibility of Neisseria gonorrhoeae to azithromycin and ceftriaxone in China: A retrospective study of national surveillance data from 2013 to 2016.

BACKGROUND: Gonorrhea remains one of the most common sexually transmitted diseases worldwide. Successful treatment has been hampered by emerging resistance to each of the antibiotics recommended as first-line therapies. We retrospectively analyzed the susceptibility of gonorrhea to azithromycin and ceftriaxone using data from the China Gonococcal Resistance Surveillance Programme (China-GRSP) in order to provide evidence for updating the treatment recommendations in China. METHODS AND FINDINGS: In this study, we included 3,849 isolates collected from patients with a confirmed positive Neisseria gonorrhoeae (N. gonorrhoeae) culture at clinic visits during the period of 1 January 2013 through 31 December 2016 in 7 provinces. Antimicrobial susceptibility testing of gonorrhea isolates using agar dilution was conducted to determine minimum inhibitory concentration (MIC). Resistance to azithromycin (RTA) was defined as MIC ≥ 1.0 mg/l, and decreased susceptibility to ceftriaxone (DSC) was defined as MIC ≥ 0.125 mg/l. The prevalence of isolates with RTA was 18.6% (710/3,827; 95% CI 17.4%-19.8%). The percentage of patients with DSC fluctuated between 9.7% and 12.2% over this period. The overall prevalence of isolates with both RTA and DSC was 2.3% (87/3,827; 95% CI 1.9%-2.8%) and it increased from 1.9% in 2013 to 3.3% in 2016 (chi-squared test for trend, P = 0.03). Study limitations include the retrospective study design and potential biases in the sample, which may overrepresent men with symptomatic infection, coastal residents, and people reporting as heterosexual. CONCLUSIONS: To our knowledge, this is the first national study on susceptibility of N. gonorrhoeae to azithromycin and ceftriaxone in China. Our findings indicate high rates of RTA and DSC from 2013 to 2016. Although dual therapy with azithromycin and ceftriaxone has been recommended by WHO and many countries to treat gonorrhea, reevaluation of this therapy is needed prior to its introduction in China.