Identification of Amino Acids Conferring High-Level Resistance to Expanded-Spectrum Cephalosporins in the penA Gene from Neisseria gonorrhoeae Strain H041

The recent identification of a high-level-ceftriaxone-resistant (MIC = 2 to 4 μg/ml) isolate of Neisseria gonorrhoeae from Japan (H041) portends the loss of ceftriaxone as an effective treatment for gonococcal infections. This is of grave concern because ceftriaxone is the last remaining option for first-line empirical antimicrobial monotherapy. The penA gene from H041 (penA41) is a mosaic penA allele similar to mosaic alleles conferring intermediate-level cephalosporin resistance (Cephⁱ) worldwide but has 13 additional mutations compared to the mosaic penA gene from the previously studied Cephⁱ strain 35/02 (penA35). When transformed into the wild-type strain FA19, the penA41 allele confers 300- and 570-fold increases in the MICs for ceftriaxone and cefixime, respectively. In order to understand the mechanisms involved in high-level ceftriaxone resistance and to improve surveillance and epidemiology during the potential emergence of ceftriaxone resistance, we sought to identify the minimum number of amino acid alterations above those in penA35 that confer high-level resistance to ceftriaxone. Using restriction fragment exchange and site-directed mutagenesis, we identified three mutations, A311V, T316P, and T483S, that, when incorporated into the mosaic penA35 allele, confer essentially all of the increased resistance of penA41. A311V and T316P are close to the active-site nucleophile Ser310 that forms the acyl-enzyme complex, while Thr483 is predicted to interact with the carboxylate of the β-lactam antibiotic. These three mutations have thus far been described only for penA41, but dissemination of these mutations in other mosaic alleles would spell the end of ceftriaxone as an effective treatment for gonococcal infections.     

Emergence of cephem- and aztreonam-high-resistant Neisseria gonorrhoeae that does not produce β-lactamase

Regarding Neisseria gonorrhoeae, the National Committee for Clinical Laboratory Standards (NCCLS) has not defined the breakpoint minimum inhibitory concentration (MIC) for expanded spectrum cephems such as cefpodoxime and ceftizoxime, because of the absence of resistant strains to these antibiotics. To date, in gonococcal urethritis, after treatment with third generation cephems and aztreonam, clinical failures caused by resistant N. gonorrhoeae strains have not been reported. However, we experienced two clinical failures in patients with gonococcal urethritis treated with cefdinir and aztreonam. N. gonorrhoeae isolates from these two patients showed high-level MICs to these agents. The MIC of cefdinir was 1 μg/ml for both strains and that of aztreonam was 8 μg/ml for both strains, while the MICs of other β-lactams were also higher than the NCCLS value, except for ceftriaxone, for which the MIC was 0.125 μg/ml for both strains. Moreover, the MICs of fluoroquinolones, tetracyclines, and erythromycin against these two isolates were higher than the NCCLS susceptibility value. These isolates were susceptible to spectinomycin. In N. gonorrhoeae, the emergence of these β-lactam-resistant isolates is of serious concern. However, a more serious problem is that these isolates were already resistant to non-β-lactam antimicrobials. In Japan, ceftriaxone has not been permitted for clinical use against gonococcal infections. Therefore, in Japan, patients with gonococcal urethritis caused by these resistant N. gonorrhoeae strains should be treated with cefodizime or spectinomycin. Received: October 30, 2000 / Accepted: November 30, 2000     

High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure

Recently, the first Neisseria gonorrhoeae strain (H041) highly resistant to the expanded-spectrum cephalosporins (ESCs) ceftriaxone and cefixime, which are the last remaining options for first-line gonorrhea treatment, was isolated in Japan. Here, we confirm and characterize a second strain (F89) with high-level cefixime and ceftriaxone resistance which was isolated in France and most likely caused a treatment failure with cefixime. F89 was examined using six species-confirmatory tests, antibiograms (33 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of known gonococcal resistance determinants (penA, mtrR, penB, ponA, and pilQ). F89 was assigned to MLST sequence type 1901 (ST1901) and NG-MAST ST1407, which is a successful gonococcal clone that has spread globally. F89 has high-level resistance to cefixime (MIC = 4 μg/ml) and ceftriaxone (MIC = 1 to 2 μg/ml) and resistance to most other antimicrobials examined. A novel penA mosaic allele (penA-CI), which was penA-XXXIV with an additional A501P alteration in penicillin-binding protein 2, was the primary determinant for high-level ESC resistance, as determined by transformation into a set of recipient strains. N. gonorrhoeae appears to be emerging as a superbug, and in certain circumstances and settings, gonorrhea may become untreatable. Investigations of the biological fitness and enhanced understanding and monitoring of the ESC-resistant clones and their international transmission are required. Enhanced disease control activities, antimicrobial resistance control and surveillance worldwide, and public health response plans for global (and national) perspectives are also crucial. Nevertheless, new treatment strategies and/or drugs and, ideally, a vaccine are essential to develop for efficacious gonorrhea management.     

Antimicrobial resistance in Neisseria gonorrhoeae in the UK: surveillance and management

Successful antimicrobial therapy is fundamental to the public health control of gonorrhea, in the absence of a protective immune response. Neisseria gonorrhoeae, the causative agent, has presented a constant challenge for the provision of such therapy as it has demonstrated the ability to become resistant to successive highly active agents chosen for first-line treatment. Acquisition of plasmids from other bacteria and long-term use of a single agent has selected both single step high-level and low-level resistance due to multiple mutations. While therapeutic failure of the current recommended agents cefixime and ceftriaxone begins to emerge, choice of alternative therapies is limited. Guidelines for therapy will be dependent on surveillance programs but individual patient management will require a viable organism to detect emerging resistance. Advances in molecular detection, while advantageous for the diagnosis of gonorrhea, fail to provide a viable organism, posing even greater challenges for the definition of treatment failure, and appropriate end points for test of cure. Innovative and collaborative approaches will be essential to maintain gonorrhea as a treatable infection.     

Antimicrobial resistance in Neisseria gonorrhoeae in the UK: surveillance and management

Successful antimicrobial therapy is fundamental to the public health control of gonorrhea, in the absence of a protective immune response. Neisseria gonorrhoeae, the causative agent, has presented a constant challenge for the provision of such therapy as it has demonstrated the ability to become resistant to successive highly active agents chosen for first-line treatment. Acquisition of plasmids from other bacteria and long-term use of a single agent has selected both single step high-level and low-level resistance due to multiple mutations. While therapeutic failure of the current recommended agents cefixime and ceftriaxone begins to emerge, choice of alternative therapies is limited. Guidelines for therapy will be dependent on surveillance programs but individual patient management will require a viable organism to detect emerging resistance. Advances in molecular detection, while advantageous for the diagnosis of gonorrhea, fail to provide a viable organism, posing even greater challenges for the definition of treatment failure, and appropriate end points for test of cure. Innovative and collaborative approaches will be essential to maintain gonorrhea as a treatable infection.     

Predicting bacteremia by procalcitonin levels in patients evaluated for sepsis in the emergency department

Neisseria gonorrhoeae has retained antimicrobial resistance to drugs previously recommended for first-line empiric treatment of gonorrhea, and resistance to ceftriaxone, the last option for monotherapy, is evolving. Crucial actions to combat this developing situation include implementing response plans; considering use of dual antimicrobial regimens; enhancing surveillance of gonorrhea, gonococcal antimicrobial resistance, treatment failures and antimicrobial use/misuse and improving prevention, early diagnosis, contact tracing and treatment. The ways forward also include an intensified research to identify novel antimicrobial resistance determinants and develop and evaluate appropriate use of molecular antimicrobial resistance testing, ideally point-of-care and with simultaneous detection of gonococci, to supplement culture-based methods and ideally guide tailored treatment. It is crucial with an enhanced understanding of the dynamics of the national and international emergence, transmission and evolution of antimicrobial-resistant gonococcal strains. Genome sequencing combined with epidemiological metadata will detail these issues and might also revolutionize the molecular antimicrobial resistance testing. Ultimately, novel antimicrobials are essential and some antimicrobials in development have shown potent in vitro activity against gonococci. Several of these antimicrobials deserve further attention for potential future treatment of gonorrhea.     

Effect of dexamethasone on therapy of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis.

Treatment of pneumococcal meningitis has become problematic because of the emergence of penicillin- and cephalosporin-resistant strains and because of the concern that dexamethasone therapy might reduce penetration of antibiotics into the cerebrospinal fluid (CSF). We addressed these issues with our rabbit meningitis model by studying two pneumococcal isolates that were resistant to penicillin and ceftriaxone and susceptible to vancomycin and rifampin. Ceftriaxone, vancomycin, and rifampin were given alone or in combination, with or without coadministration of dexamethasone. Treatment was started 12 to 14 h after intracisternal inoculation of approximately 10(4) CFU of one of the organisms. Rifampin concentrations in serum and CSF were similar, regardless of whether dexamethasone was given, whereas those of ceftriaxone were somewhat lower at each time point in animals given dexamethasone. The penetration of vancomycin into CSF was consistently and substantially reduced with dexamethasone treatment, which resulted in a delay in CSF sterilization not observed in non-dexamethasone-treated animals. When rifampin was used with ceftriaxone for treatment of meningitis caused by the more resistant strain, bacteriologic cure occurred promptly, with or without dexamethasone therapy. In areas with high rates of occurrence of resistant pneumococcal strains, we believe initial empiric therapy of bacterial meningitis should include two antibiotics: ceftriaxone and either rifampin or vancomycin. When dexamethasone is used, the combination of ceftriaxone and rifampin is preferred for therapy.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点。方法采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度（MIC）,判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准。用纸片酸度法检测产β-内酰胺酶淋球菌菌株。结果107株淋球菌中,检出94株对青霉素耐药（87．9％）,产β-内酰胺酶淋球菌43株（40．2％）;四环素耐药率为88．8％,其中质粒介导高度耐四环素淋球菌（TRNG）63株（58．9％）;环丙沙星耐药率81．3％;未发现对大观霉素和头孢曲松耐药菌株。青霉素、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍。结论淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差。     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

淋球菌流行株对5种抗生素敏感性分析

目的 检测2008年度临床分离的淋球菌对青霉素、四环素、大观霉素、头孢曲松和环丙沙星的敏感性,分析耐药菌株的流行特点.方法 采用琼脂稀释法测定菌株对5种抗生素的最小抑菌浓度(MIC),判断其敏感性并按WHO西太平洋地区淋球菌耐药性监测统一标准.用纸片酸度法检测产β-内酰胺酶淋球菌菌株.结果 107株淋球菌中,检出94株对青霉素耐药(87.9%),产β-内酰胺酶淋球菌43株(40.2%);四环素耐药率为88.8%,其中质粒介导高度耐四环素淋球菌(TRNG)63株(58.9%);环丙沙星耐药率81.3%;未发现对大观霉素和头孢曲松耐药菌株.青霉索、四环素和环丙沙星的MIC50及MIC90均已超过耐药标准,尤以青霉素为甚,其MIC50及MIC90均超过耐药标准1、32倍.结论 淋球菌对大观霉素和头孢曲松的敏感性较高,可作为治疗的首选药物;对青霉素、四环素和环丙沙星耐药率较高,提示对淋病的治疗作用差.     

European Surveillance of Sexually Transmitted Infections (ESSTI): the first combined antimicrobial susceptibility data for Neisseria gonorrhoeae in Western Europe

OBJECTIVES: To conduct a sentinel surveillance study for antimicrobial resistance in Neisseria gonorrhoeae in Western Europe in 2004 as part of the European Surveillance of Sexually Transmitted Infections (ESSTI) Programme. METHODS: Gonococcal isolates were collected from centres in 12 countries and transferred to two reference centres for testing. The same methodology of agar dilution was used to determine susceptibility to a range of antimicrobials used for the treatment of gonorrhoea including azithromycin, ceftriaxone, ciprofloxacin, penicillin and tetracycline. Quality control between the two laboratories was assessed during the testing. RESULTS: A total of 1055 gonococcal isolates were collected, of which 965 (91.5%) were retrievable for susceptibility testing. Resistance was found to be high to ciprofloxacin (30.9%), but also present to penicillin (21.3%) and tetracycline (59.8%). Azithromycin resistance was above 5%, the first time this has been documented in Europe. Three isolates had a low level of resistance to ceftriaxone. With regard to quality control between the two reference laboratories, 92% of MIC results were within two dilutions. CONCLUSIONS: These are the first sentinel surveillance data for Western Europe for N. gonorrhoeae and they have implications for choice of antimicrobial for treatment of gonorrhoea on a European and a local level. This is the start of the formation of a European gonococcal antimicrobial surveillance programme (EURO-GASP).