RESUMO
Clinical resistance to the currently recommended extended-spectrum cephalosporins (ESCs), the last remaining treatment options for gonorrhea, is being reported. Gonorrhea may become untreatable, and new treatment options are crucial. We investigated the in vitro activity of ertapenem, relative to ceftriaxone, against N. gonorrhoeae isolates and the effects of ESC resistance determinants on ertapenem. MICs were determined using agar dilution technique or Etest for international reference strains (n = 17) and clinical N. gonorrhoeae isolates (n = 257), which included the two extensively drug-resistant (XDR) strains H041 and F89 and additional isolates with high ESC MICs, clinical ESC resistance, and other types of clinical high-level and multidrug resistance (MDR). Genetic resistance determinants for ESCs (penA, mtrR, and penB) were sequenced. In general, the MICs of ertapenem (MIC(50) = 0.032 µg/ml; MIC(90) = 0.064 µg/ml) paralleled those of ceftriaxone (MIC(50) = 0.032 µg/ml; MIC(90) = 0.125 µg/ml). The ESC resistance determinants mainly increased the ertapenem MIC and ceftriaxone MIC at similar levels. However, the MIC ranges for ertapenem (0.002 to 0.125 µg/ml) and ceftriaxone (<0.002 to 4 µg/ml) differed, and the four (1.5%) ceftriaxone-resistant isolates (MIC = 0.5 to 4 µg/ml) had ertapenem MICs of 0.016 to 0.064 µg/ml. Accordingly, ertapenem had in vitro advantages over ceftriaxone for isolates with ceftriaxone resistance. These in vitro results suggest that ertapenem might be an effective treatment option for gonorrhea, particularly for the currently identified ESC-resistant cases and possibly in a dual antimicrobial therapy regimen. However, further knowledge regarding the genetic determinants (and their evolution) conferring resistance to both antimicrobials, and clear correlates between genetic and phenotypic laboratory parameters and clinical treatment outcomes, is essential.
Assuntos
Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Gonorreia/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , beta-Lactamas/farmacologia , Farmacorresistência Bacteriana , Ertapenem , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/patogenicidadeRESUMO
Molecular detection of Neisseria gonorrhoeae in extragenital samples may result in false-positive results due to cross-reaction with commensal Neisseria species or Neisseria meningitidis. This study examined 450 characterized clinical culture isolates, comprising 216 N. gonorrhoeae isolates and 234 isolates of nongonococcal Neisseria species (n = 218) and 16 isolates of other closely related bacteria, with six commercial nucleic acid amplification tests (NAATs). The six NAATs tested were Gen-Probe APTIMA COMBO 2 and APTIMA GC, Roche COBAS Amplicor CT/NG and COBAS 4800 CT/NG tests, BD ProbeTec GC Qx amplified DNA assay, and Abbott RealTime CT/NG test. All assays except COBAS Amplicor CT/NG test where four (1.9%) isolates were not detected showed a positive result with all N. gonorrhoeae isolates (n = 216). Among the 234 nongonococcal isolates examined, initial results from all assays displayed some false-positive results due to cross-reactions. Specifically, the COBAS Amplicor and ProbeTec tests showed the highest number of false-positive results, detecting 33 (14.1%) and 26 (11%) nongonococcal Neisseria isolates, respectively. On the first testing, APTIMA COMBO 2, APTIMA GC, Abbott RealTime, and Roche COBAS 4800 showed lower level of cross-reactions with five (2.1%), four (1.7%), two (1%), and two (1%) of the isolates showing low-level positivity, respectively. Upon retesting of these nine nongonococcal isolates using freshly cultured colonies, none were positive by the APTIMA COMBO 2, Abbott RealTime, or COBAS 4800 test. In conclusion, the COBAS Amplicor and ProbeTec tests displayed high number of false-positive results, while the remaining NAATs showed only sporadic low-level false-positive results. Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites.
Assuntos
Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Neisseria/classificação , Neisseria/isolamento & purificação , Infecções por Neisseriaceae/diagnóstico , Infecções por Neisseriaceae/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Reações Falso-Positivas , Humanos , Neisseria/genética , Sensibilidade e EspecificidadeRESUMO
Antimicrobial resistance (AMR) in Neisseria gonorrhoeae remains a global public health problem. Susceptibility to first-line treatment extended-spectrum cephalosporins (ESCs) is decreasing worldwide resulting in therapeutic failures with oral ESCs. This study describes a cefpodoxime 10 µg disc test for screening for gonococci containing a penA mosaic allele encoding a mosaic penicillin-binding protein 2 (PBP2) and decreased ESC susceptibility. Selected clinical gonococcal isolates (n = 315), containing a high proportion of gonococci with decreased ESC susceptibility and high geographical, temporal and genetic diversity, were examined using agar dilution (n = 149; cefpodoxime and ceftriaxone) and Etest (n = 315; cefixime), and disc diffusion using a commercially available cefpodoxime 10 µg disc (n = 315). penA sequencing was performed on all isolates. The 2008 WHO gonococcal reference strains (n = 8) were included as quality controls. Using a ≤11 mm annular radius of growth inhibition as the breakpoint for the cefpodoxime 10 µg disc, all 78, with exception of one isolate (13 mm), mosaic PBP2-containing isolates, which also displayed decreased susceptibility to oral ESCs, were identified. In addition, 85 non-mosaic PBP2-containing isolates (44% of which contained a PBP2 A501 alteration) had annular radii ≤11 mm and raised minimal inhibitory concentrations to the ESCs. Screening for detection of mosaic PBP2-containing gonococci and decreased ESC susceptibility, most pronounced to oral ESCs, using a commercially available cefpodoxime 10 µg disc was rapid, inexpensive and sensitive. This test can be used in AMR surveillance programmes for public health purposes especially in less-resourced settings. Further studies to refine this disc testing-based approach are in progress.
Assuntos
Antibacterianos/farmacologia , Ceftizoxima/análogos & derivados , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Alelos , Cefixima/farmacologia , Ceftizoxima/farmacologia , Ceftriaxona/farmacologia , Relação Dose-Resposta a Droga , Gonorreia/microbiologia , Testes de Sensibilidade Microbiana/métodos , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Análise de Sequência de DNA , CefpodoximaAssuntos
Antibacterianos/farmacologia , Ceftriaxona/farmacologia , Gonorreia/microbiologia , Gonorreia/transmissão , Neisseria gonorrhoeae/efeitos dos fármacos , Resistência beta-Lactâmica , Técnicas de Tipagem Bacteriana , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Neisseria gonorrhoeae/classificação , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificaçãoRESUMO
With increasing concerns regarding diminishing treatment options for gonorrhea, maintaining the efficacy of currently used treatments and ensuring optimal Neisseria gonorrhoeae antimicrobial resistance surveillance are of the utmost importance. Penicillin is still used to treat gonorrhea in some parts of the world. In this study, we developed and validated a real-time PCR assay for the detection of penicillinase-producing N. gonorrhoeae (PPNG) in noncultured clinical samples with the aim of enhancing penicillin resistance surveillance. The assay (PPNG-PCR2) was designed to be an indirect marker of penicillinase activity, by targeting a region of sequence predicted to be conserved across all N. gonorrhoeae plasmid types harboring the beta-lactamase gene while not specifically targeting the actual beta-lactamase-encoding sequence. The assay was evaluated by using a total of 118 N. gonorrhoeae clinical isolates and 1,194 clinical specimens, including 239 N. gonorrhoeae-positive clinical samples from which N. gonorrhoeae cells were isolated and for which phenotypic penicillinase results are available. Overall, the PPNG-PCR2 assay provided 100% sensitivity and 98.7% specificity compared to bacterial culture results for the detection of PPNG in clinical specimens. PPNG-PCR2 false-positive results, presumably due to cross-reactions with unrelated bacterial species, were observed for up to 1.3% of clinical samples but could be distinguished on the basis of high cycle threshold values. In tandem with phenotypic surveillance, the PPNG-PCR2 assay has the potential to provide enhanced epidemiological surveillance of N. gonorrhoeae penicillin resistance and is of particular relevance to regions where penicillin is still used to treat gonorrhea.
Assuntos
Farmacorresistência Bacteriana , Gonorreia/microbiologia , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Penicilinase/biossíntese , Reação em Cadeia da Polimerase/métodos , Sequência Conservada , Primers do DNA/genética , DNA Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/isolamento & purificação , Penicilinase/genética , Plasmídeos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: Gonorrhoea remains a global public health problem and the treatment options are diminishing through the emergence of gonococci resistant to most antimicrobials. Previous in vitro studies have indicated a role for Neisseria gonorrhoeae pilQ alterations in conferring resistance to antimicrobials, including penicillin. In this study, we investigated whether pilQ polymorphisms were associated with decreased susceptibility to extended-spectrum cephalosporins (ESCs) in clinical gonococcal strains. METHODS: Full-length pilQ nucleotide and PilQ amino acid sequences from geographically and temporally diverse gonococcal clinical isolates (nâ=â63), including the 2008 WHO reference strains, representing a range of ceftriaxone and cefixime MICs (≤0.008-0.25 and <0.016-0.5 mg/L, respectively) and 38 N. gonorrhoeae multiantigen sequence types, were examined. Previously described alterations associated with decreased ESC susceptibility (mosaic penA, mtrR and penB alterations) were also examined. RESULTS: Fifteen different pilQ nucleotide sequence types and nine different PilQ amino acid sequence types were observed, with two PilQ types accounting for 53 (84%) of the isolates. Independent of other genetic resistance determinants (penA mosaic, mtrR promoter deletion and penB), only one pilQ alteration, a D526N substitution, provided a statistically significant association with ceftriaxone (Pâ<â0.01) and cefixime (Pâ<â0.05) MICs. However, the two isolates exhibiting D526N lacked all three previously described alterations associated with decreased ESC susceptibility, thereby providing an alternative basis for the low MICs (≤0.008 mg/L) observed for these strains. The previously described E666K (pilQ2) and F595L (pilQ1) mutations were absent in all 63 isolates. CONCLUSIONS: pilQ polymorphisms are unlikely contributors to decreased susceptibility to ESCs in clinical gonococcal strains.
Assuntos
Antibacterianos/farmacologia , Cefixima/farmacologia , Ceftriaxona/farmacologia , Resistência às Cefalosporinas/genética , Proteínas de Fímbrias/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Sequência de Aminoácidos , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Neisseria gonorrhoeae/genéticaRESUMO
OBJECTIVES: Reduced susceptibility to extended-spectrum cephalosporins in Neisseria gonorrhoeae has, to date, been associated with three alterations: a mosaic penA allele encoding the penicillin-binding protein 2 (PBP2); A-del-mtrR, an adenine deletion in the mtrR promoter; and penB, comprising mutated alleles of PorBIb. In this study, we examined an association between reduced susceptibility to ceftriaxone and additional mutations in gonococcal PBP2. METHODS: N. gonorrhoeae isolates (n = 76) exhibiting reduced susceptibility to ceftriaxone but lacking the mosaic penA sequence were investigated for A-del-mtrR and penB as well as substitutions at PBP2 501, 542 and 551 using a previously described real-time PCR approach. To further investigate PBP2 542 and 551 substitutions, we reanalysed penA sequence data from a previous study of 98 gonococci exhibiting a range of ceftriaxone MICs. RESULTS: Of 76 N. gonorrhoeae isolates exhibiting reduced susceptibility to ceftriaxone and lacking the mosaic penA sequence, a 501 (A501V or A501T) substitution was present in 9/76, a 542 substitution in 39/76 and a 551 substitution in 26/76 isolates. Reanalysis of 98 gonococcal isolates from a previous study showed that substitutions at PBP2 542 (G542S) and 551 (P551S or P551L) were significantly associated with raised MICs to ceftriaxone (P = 0.0186 and 0.001, respectively) and penicillin (P = 0.0231 and 0.0007, respectively). CONCLUSIONS: Our findings provide strong evidence for the involvement of PBP2 G542S and P551S/P551L in reduced susceptibility to ceftriaxone and to penicillin. Further studies are needed to investigate the precise and relative roles played by these mutations.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Ceftriaxona/farmacologia , Mutação de Sentido Incorreto , Neisseria gonorrhoeae/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/genética , Resistência beta-Lactâmica , Substituição de Aminoácidos/genética , Testes de Sensibilidade Microbiana , Análise de Sequência de DNARESUMO
Eighteen hundred Neisseria gonorrhoeae isolates collected in Sydney, Australia, in 2007 and 2008 were examined for mosaic penA alleles that mediated cephalosporin resistance, and the genotypes of the isolates were evaluated. In 2008, there were substantial increases in numbers (from 15 to 85) and proportions (from 1.5 to 10.3%) of mosaic-containing gonococci and major shifts in genotypic patterns, with 10 new genotypes representing 74 of the 85 mosaic-containing isolates and genotypes detected between 2001 and 2005 having disappeared. Enhanced surveillance of gonococcal resistance to cephalosporins is necessary.
Assuntos
Genes Bacterianos/genética , Gonorreia/microbiologia , Neisseria gonorrhoeae/genética , Proteínas de Ligação às Penicilinas/genética , Alelos , Antibacterianos/farmacologia , Austrália/epidemiologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana/genética , Genótipo , Gonorreia/epidemiologia , Humanos , Estudos LongitudinaisRESUMO
OBJECTIVES: The Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) system, based on PCR amplification and sequence analysis of the gonococcal porB and tbpB genes, is widely used for molecular typing of gonococcal isolates but is not validated for non-cultured clinical samples. This study sought to examine the performance of the NG-MAST system on a range of non-cultured samples. METHODS: Nucleic acid extracts of 73 N gonorrhoeae-positive samples, comprising eight cervical swabs, nine urethral swabs, 35 urine samples, one vaginal swab, 13 rectal swabs and seven throat swabs, were analysed by NG-MAST. For 27 specimens, corresponding gonococcal isolates were also analysed and the results compared. A panel of 44 non-gonococcal Neisseria strains and 100 N gonorrhoeae-negative clinical samples were used to investigate further the specificity of the NG-MAST PCR reactions. RESULTS: PCR amplification and DNA sequencing of gonococcal porB and tbpB genes was successful for all N gonorrhoeae-positive urogenital specimens, 11 of 13 rectal swabs and four of seven throat swabs. For the 27 N gonorrhoeae-positive specimens with corresponding gonococcal isolates, the porB and tbpB sequences obtained from the non-cultured specimen were identical to those obtained from the isolate. Cross-reaction with non-gonococcal Neisseria species was observed for both the porB and tbpB PCR reactions, and proved to be problematical for NG-MAST typing of throat swab specimens. CONCLUSIONS: The NG-MAST system can successfully be applied directly to non-cultured urogenital samples, but is less suitable for extragenital specimens, particularly throat swabs, due to cross-reaction with commensal Neisseria species.
Assuntos
Antígenos de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Gonorreia/microbiologia , Neisseria gonorrhoeae/classificação , Colo do Útero/microbiologia , DNA Bacteriano/genética , Feminino , Humanos , Masculino , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/isolamento & purificação , Faringe/microbiologia , Reação em Cadeia da Polimerase/métodos , Reto/microbiologia , Sensibilidade e Especificidade , Uretra/microbiologia , Urina/microbiologiaRESUMO
Globally, antimicrobial resistance (AMR) in Neisseria gonorrhoeae is increasing in prevalence, both within and across antibiotic classes, including extended-spectrum cephalosporins, raising concerns that gonorrhea may become untreatable in certain circumstances. The AMR surveillance that is essential to optimize standard treatments is often lacking or of poor quality in countries with high disease rates. Recent initiatives by the WHO to enhance global AMR surveillance that focus on multidrug- and extensively drug-resistant N. gonorrhoeae through revision of surveillance standards and use of a new panel of N. gonorrhoeae control strains are described. Keys to meeting these new challenges posed by gonococcal AMR remain the reduction in global burden of gonorrhea combined with implementation of wider strategies for general AMR control, and better understanding of mechanisms of emergence and spread of AMR.
Assuntos
Antibacterianos , Cefalosporinas , Farmacorresistência Bacteriana Múltipla , Gonorreia , Neisseria gonorrhoeae/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cefalosporinas/farmacologia , Cefalosporinas/uso terapêutico , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Humanos , Vigilância da População/métodos , Prevalência , Saúde Pública , Organização Mundial da SaúdeRESUMO
In 2007 there were 281 laboratory-confirmed cases of invasive meningococcal disease analysed by the National Neisseria Network, a nationwide network of reference laboratories. The phenotypes (serogroup, serotype and serosubtype) and antibiotic susceptibility of 154 isolates of Neisseria meningitidis from invasive cases of meningococcal disease were determined and an additional 127 cases were confirmed by non-culture based methods. Nationally, 223 (85%) confirmed cases where a serogroup was determined were infected with serogroup B and 17 (6.5%) with serogroup C meningococci. The total number of confirmed cases was 10 more than the 271 cases identified in 2006. Queensland and New South Wales recorded slight increases in case numbers and Victoria recorded a decline. Typical primary and secondary disease peaks were observed in those aged 4 years or less and in adolescents and young adults respectively. Serogroup B cases predominated in all age groups and jurisdictions. The common phenotypes circulating in Australia were B:15:P1.7, B:4:P1.4 and C:2a:P1.5. No evidence of meningococcal capsular 'switching' was detected. About three-quarters of all isolates showed decreased susceptibility to the penicillin group of antibiotics (MIC 0.06-0.5 mg/L). All isolates remained susceptible to rifampicin. A single serogroup B isolate had decreased susceptibility to ciprofloxacin (MIC 0.06 mg/L). This was the first local isolate of this type since the original report of this phenomenon in Australia in 2000.
Assuntos
Infecções Meningocócicas/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Idoso , Austrália/epidemiologia , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Neisseria meningitidis/classificação , Neisseria meningitidis/efeitos dos fármacos , Estações do Ano , Sorotipagem , Fatores de TempoRESUMO
PURPOSE OF REVIEW: Antibiotic resistance in Neisseria gonorrhoeae poses on-going problems for individual case management and disease control for gonorrhoea. Considerable reliance is now placed on third-generation cephalosporins for the treatment of gonorrhoea following the loss of efficacy of penicillins and quinolones. Current clinical and laboratory perspectives on N. gonorrhoeae with decreased susceptibility to third-generation cephalosporins are provided. RECENT FINDINGS: Treatment failures following therapy with the oral third-generation cephalosporins cefixime and ceftibuten have been reported, but not with the injectable ceftriaxone. The gonococci involved have raised minimal inhibitory concentrations to these agents, including to ceftriaxone. The presence of multiple chromosomal changes form the basis for this 'resistance', prominent among which is a mosaic penicillin-binding protein 2 found in association with additional known and unknown mutations in other genes. The imprecise nature of laboratory criteria for detecting these gonococci means that the distribution and prevalence of these strains is also uncertain. SUMMARY: Concerns regarding the appearance of gonococci associated with treatment failure with oral cephalosporins are increasing. The origins, causes and patterns of spread of these clinically resistant gonococci are reminiscent of the earlier experiences with quinolone-resistant gonococci. Preventive measures require simultaneous implementation of disease-control principles, coupled with those for antimicrobial resistance.
Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Gonorreia/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Resistência beta-Lactâmica , Antibacterianos/uso terapêutico , Cefalosporinas/uso terapêutico , Gonorreia/tratamento farmacológico , Humanos , Falha de TratamentoRESUMO
Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials. There is now growing concern that without the availability of appropriate public health strategies to combat this problem, gonorrhea could become untreatable. For this reason, surveillance for gonococcal antimicrobial resistance must be optimal both in terms of obtaining a representative sample of gonococcal isolates and in terms of having the appropriate tools to identify resistance. To aid with this surveillance, molecular tools are increasingly being used. In the present study, we investigated the use of a simple heat denaturation protocol for isolate DNA preparation combined with SYBR green-based real-time PCR for the identification of mutations associated with N. gonorrhoeae antimicrobial resistance. A total of 109 clinical gonococcal isolates were tested by high-resolution melting (HRM) curve analysis for chromosomal mutations associated with gonococcal resistance to beta-lactam antibiotics: a penA 345A insertion, ponA L421P, mtrR G45D, substitutions at positions 120 and 121 in porB1b, and an adenine deletion in the mtrR promoter. An allele-specific PCR assay was also investigated for its ability to detect the adenine deletion in the mtrR promoter. The results were compared to those obtained by DNA sequencing. Our HRM assays provided the accurate discrimination of heat-treated isolates in which the sequence types differed in GC content, including isolates with the penA 345A insertion and the ponA L421P and mtrR G45D mutations. The allele-specific PCR assay accurately identified isolates with the adenine deletion in the mtrR promoter. Heat-denatured DNA combined with SYBR green-based real-time PCR offers a simple, rapid, and inexpensive means of detecting gonococcal resistance mechanisms. These methods may have broader application in the detection of polymorphisms associated with phenotypes of interest.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Neisseria gonorrhoeae/genética , Reação em Cadeia da Polimerase/métodos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Sequência de Bases , Dados de Sequência Molecular , Mutação , Neisseria gonorrhoeae/efeitos dos fármacos , Reprodutibilidade dos Testes , Homologia de Sequência do Ácido NucleicoRESUMO
Diagnostic, genotypic and antibiotic-resistance determinants of Neisseria gonorrhoeae were analysed by molecular methods to verify the failure of ceftriaxone treatment in two cases of pharyngeal gonorrhoea. Monoplex assays were needed to define competitive inhibition of a positive Chlamydia PCR in a duplex assay. Different penA changes were detected in the N. gonorrhoeae isolated from the two cases. These were associated with raised ceftriaxone MICs of 0.03 and 0.016 mg l(-1), which may have contributed to the treatment failures in these cases.
Assuntos
Ceftriaxona/uso terapêutico , Chlamydia trachomatis/isolamento & purificação , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/isolamento & purificação , Doenças Faríngeas/tratamento farmacológico , Doenças Faríngeas/microbiologia , Adulto , Antibacterianos/uso terapêutico , Chlamydia trachomatis/genética , Feminino , Homossexualidade , Humanos , Masculino , Reação em Cadeia da Polimerase , Falha de TratamentoRESUMO
OBJECTIVES: Emergence and spread of antimicrobial resistance (AMR) in Neisseria gonorrhoeae remain a major global problem and expanded, but valid, AMR surveillance is crucial for public health purposes. The World Health Organization (WHO) Collaborating Centre in Sydney, Australia, continually evaluates N. gonorrhoeae strains used in quality control and assurance aspects of the national, WHO regional and international programmes for AMR surveillance it conducts. Here we phenotypically and genetically characterized the 2008 WHO N. gonorrhoeae reference panel, widely used under existing WHO AMR surveillance protocols. MATERIALS AND METHODS: The eight N. gonorrhoeae WHO reference strains were phenotypically characterized by antibiogram, auxotype, serovar and prolyliminopeptidase screening; and genetically with regard to resistance plasmid types, polymorphisms in divergent genetic resistance-mediating loci (n = 9), porB sequencing and N. gonorrhoeae multi-antigen sequence typing. RESULTS: The 2008 WHO reference strains represented all the important susceptible and resistant phenotypes, including corresponding resistance genotypes, and the range of resistances currently seen for relevant antimicrobials. Several pertinent additional phenotypic and genotypic markers, for example, epidemiological markers, were also determined. CONCLUSIONS: The 2008 WHO N. gonorrhoeae reference strain panel was extensively characterized, which is crucial for the expansion of gonococcal AMR surveillance nationally and internationally. The panel is available through WHO sources for quality assurance and quality control aspects of current phenotypic testing protocols, to allow valid comparison of AMR data derived by divergent methods, and also for the control of present and future molecular assays for AMR detection. Additional WHO reference strains can be included as required by the emergence of additional resistant phenotypes and/or genotypes.
Assuntos
DNA Bacteriano/genética , Testes de Sensibilidade Microbiana/normas , Neisseria gonorrhoeae/fisiologia , Padrões de Referência , Antígenos de Bactérias/genética , Austrália , Técnicas de Tipagem Bacteriana , Humanos , Neisseria gonorrhoeae/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Plasmídeos , Garantia da Qualidade dos Cuidados de Saúde/métodos , Controle de Qualidade , Análise de Sequência de DNA , SorotipagemRESUMO
We report a duplex real-time PCR assay for the simultaneous screening of mutations involved in fluoroquinolone resistance within gyrA and parC quninolone resistance-determining regions (QRDRs) in Neisseria gonorrhoeae. Our assay clearly detects all mutated QRDRs and allows the identification of common genotypes, whether the QRDRs contain single or double mutations, providing valuable epidemiological tools. When this method is used in conjunction with similar assays and in vitro analyses, essential antibiotic resistance surveillance can be performed for public health purposes.
Assuntos
DNA Girase/genética , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Neisseria gonorrhoeae/genética , Quinolonas/farmacologia , DNA Bacteriano/química , DNA Bacteriano/genética , Suscetibilidade a Doenças , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Quinolonas/uso terapêuticoRESUMO
The Australian Gonococcal Surveillance Programme monitors the antibiotic susceptibility of Neisseria gonorrhoeae isolated in all states and territories. In 2008 the in vitro susceptibility of 3,110 isolates of gonococci from public and private sector sources was determined by standardised methods. Different antibiotic susceptibility patterns were again seen in the various jurisdictions and regions. Resistance to the penicillins nationally was at 44% and ranged between 25% in Queensland and 73% in South Australia with the exception of the Northern Territory, where the proportion of drug resistant strains was 4%. Quinolone resistance in gonococci isolates also continued to increase so that nationally 54% of all isolates were ciprofloxacin-resistant, and most of this resistance was at high minimal inhibitory concentrations (MIC) levels. The proportions of quinolone resistant gonococci detected ranged between 80% in South Australia and 31% in Western Australia. All isolates remained sensitive to spectinomycin. Approximately 1.1% of isolates showed some decreased susceptibility to ceftriaxone (MIC 0.06 mg/L or more) and azithromycin resistance was also present in low numbers of gonococci with MICs up to 16 mg/L. A high proportion of gonococci examined in larger urban centres were from male patients and rectal and pharyngeal isolates were common in men. In other centres and in rural Australia the male to female ratio of cases was lower, and most isolates were from the genital tract.
Assuntos
Gonorreia/epidemiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Antibacterianos/farmacologia , Austrália/epidemiologia , Farmacorresistência Bacteriana , Feminino , Gonorreia/microbiologia , Humanos , Masculino , Vigilância da PopulaçãoRESUMO
In 2008, there were 260 laboratory-confirmed cases of invasive meningococcal disease (IMD) analysed by the National Neisseria Network, a nationwide network of reference laboratories. One hundred and forty-nine isolates of Neisseria meningitidis from invasive cases of meningococcal disease were available for which the phenotypes (serogroup, serotype and serosubtype) and antibiotic susceptibility were determined. An additional 111 cases were confirmed by non-culture based methods. Nationally, 223 (85%) laboratory-confirmed cases where a serogroup was determined were infected with serogroup B and 17 (6.5%) infected with serogroup C meningococci. Nationally, the total number of confirmed cases has remained relatively stable since 2006, but the number of cases in each jurisdiction may vary from year to year. Queensland had the highest number of recorded cases in 2008. Typical primary and secondary disease peaks were observed in those aged 4 years or less and in adolescents and young adults respectively. Serogroup B cases predominated in all age groups and jurisdictions. The common phenotypes circulating in Australia were again B:15:P1.7 and B:4:P1.4. Although serogroup C cases were numerically low, phenotype C:2a:P1.5 predominated in this group. No evidence of meningococcal capsular 'switching' was detected. About three-quarters of all isolates showed decreased susceptibility to the penicillin group of antibiotics (MIC 0.06-0.5 mg/L). All isolates remained susceptible to ceftriaxone. One isolate had reduced susceptibility to rifampicin and two to ciprofloxacin.
Assuntos
Farmacorresistência Bacteriana , Infecções Meningocócicas/epidemiologia , Adolescente , Adulto , Idoso , Antibacterianos/uso terapêutico , Austrália/epidemiologia , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Infecções Meningocócicas/tratamento farmacológico , Infecções Meningocócicas/microbiologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Neisseria meningitidis/classificação , Sorotipagem , Adulto JovemRESUMO
OBJECTIVES: To investigate the presence of resistance genes in nosocomial multidrug-resistant (MDR) Acinetobacter baumannii isolated from outbreak and sporadic settings. METHODS: Thirty-two A. baumannii isolates were collected, 13 of which were involved in two outbreaks from different hospitals, which resulted in four deaths. The remaining 19 isolates were collected sporadically over 5 years from two other hospitals. The MICs of 25 antibiotics were determined for each isolate. PCR screening was carried out to identify possible genes that contributed to each resistance phenotype. Repetitive extragenic palindromic-PCR (REP-PCR) was performed to assess isolate clonality in conjunction with genotype data. RESULTS: Between eight and 12 resistance determinants were detected in the 32 MDR A. baumannii isolates examined. These resistance determinants included the genes blaOXA-23 and ampC, with the upstream element ISAba1 promoting increased gene expression and subsequent resistance to carbapenems and cephalosporins, respectively. In all isolates, resistance to quinolones and fluoroquinolones was conferred by an S83L mutation in GyrA. Twenty-eight of the 32 isolates were also positive for tet(B), a tetracycline resistance determinant, blaTEM-1, which contributed to beta-lactam resistance, and strB, which contributed to aminoglycoside resistance. Class 1 integrons that harboured aacC1, aadA1, qacEDelta1 and sul1 were identified in 10 of the 32 isolates (31%) together with the kanamycin resistance gene, aphA1. A putative trimethoprim resistance gene, folA, was also identified in all isolates. REP-PCR together with genotyping identified three main clonal types. CONCLUSIONS: Isolates of A. baumannii from both outbreak and sporadic cases possess at least eight resistance gene determinants that give rise to the MDR phenotype.