Evaluation of four methods for detecting the beta-lactamase activity in Neisseria gonorrhoeae isolated in Cuba

Four methods (chromogenic, acidimetric, inhibition, and iodometric) for demonstration of the beta-lactamase production by 70 isolates of Neisseria gonorrhoeae, were evaluated in Cuba. There was 100% correlation between all beta-lactamase methods and the standardized penicillin dilution susceptibility test for penicillinase-non-producing N. gonorrhoeae. For penicillinase-producing N. gonorrhoeae strains, there was a perfect correlation between the chromogenic method and penicillin susceptibility testing, but one and two strains failed to give a positive result for beta-lactamase with the inhibition/acidimetric and the iodometric methods, respectively. There was a high concordance between the chromogenic method, considered as gold standard and the rest of penicillinase tests evaluated: Kappa Index (KI) = 0.98 for inhibition/acidimetric methods and KI = 0.97 for the iodometric method. The four methods evaluated were accurate, reproducible, easily readable, economical, and ease to use for screening primary isolates of N. gonorrhoeae in Cuba. We recommended the use of the inhibition method, when testing the penicillinase activity in gonococcal isolates in provincial and municipal reference laboratories.     

Spectinomycin as initial treatment for gonorrhoea.

The prevalence of penicillinase producing Neisseria gonorrhoeae at this hospital increased exponentially from less than 0.5% in 1978 to 6.5% of all isolates in 1982. In January 1983 first line treatment for uncomplicated heterosexual anogenital gonorrhoea was therefore changed from ampicillin and probenecid to spectinomycin. This subsequently cured 95% of cases seen at the Praed Street Clinic. Although there was an initial fall in the monthly isolation rate of penicillinase producing N gonorrhoeae after the introduction of spectinomycin, this was not maintained. The exponential increase in the prevalence of the strain did slow in 1983, rising to only 8.7%. This, however, may have reflected a general decline in the rate of increase of penicillinase producing N gonorrhoeae throughout Britain. The failure to influence the prevalence of penicillinase producing N gonorrhoeae to any great degree may have been due in part to spectinomycin resistance in both penicillinase producing and non-penicillinase-producing N gonorrhoeae. All of the isolates appeared identical, apart from the presence of the 4.4 megadalton plasmid in penicillinase producing N gonorrhoeae, but they could not be linked epidemiologically. Changing treatment in only one of the many venereal diseases clinics in London, where patients have open access to all such clinics, is unlikely to affect the prevalence of penicillinase producing N gonorrhoeae. This has probably been more important than spectinomycin resistance in limiting the effectiveness of spectinomycin in reducing the prevalence of the strain.     

Study of five penicillinase producing Neisseria gonorrhoeae isolated in Italy

Five penicillinase producing Neisseria gonorrhoeae (PPNG) were isolated from urethral specimens of men admitted to the "Santa Chiara" Hospital (Trento, Italy). All strains proved to be resistant to penicillin and ampicillin, and sensitive to cefuroxime, erythromycin, tetracycline, spectinomycin, nalidixic acid and ciprofloxacin. PPNG plasmid profiles showed that four of the isolates carried the 3.2 MDa "Africa" plasmid and one the 4.5 MDa "Asia" plasmid, the two well-known phenotypes reported in the USA and Europe as well as in Asian and African countries. Membrane matings were performed using N. gonorrhoeae carrying the 24.5 MDa conjugative plasmid as donors and E. coli K12 J 53 as recipient. The transfer of beta-lactamic antibiotic resistance was supported by the presence of 4.5 or 3.2 MDa plasmid bands and by beta-lactamase production in the transconjugants. Restriction analysis of Asian and African plasmids is reported.     

淋球菌AFLP基因分型的初步研究

采用扩增片段长度多态性技术(AFLP)对奈瑟氏淋球菌菌株进行基因分型研究。以EcoRI和MesI酶切26株淋球菌临床分离株基因组,并进行AFLP分析。同一地区的淋球菌分离株之间存在相当大的DNA多态性。AFLP是鉴别淋球菌临床分离株有用而敏感的基因分型技术,有助于了解流行淋球菌菌株的来源、流行菌株之间的克隆相关性,以及抗生素耐药性菌株的传播情况。     

Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance

Sparling, Philip F. (Communicable Disease Center, Atlanta, Ga.). Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bacteriol. 92:1364-1371. 1966.-Eight strains of Neisseria gonorrhoeae were transformed to streptomycin resistance by deoxyribonucleic acid (DNA) extracted from a streptomycin-resistant strain of N. gonorrhoeae. In all strains, competence was greatest in the naturally occurring, virulent clonal types 1 and 2, which gave transformation frequencies up to 1%. Clonal types 3 and 4, which arise on laboratory transfer and are avirulent, gave maximal transformation frequencies of 0.00005%. Competence was maximal in lag and early log phases of growth, but was maintained throughout the growth cycle. A complex broth was required for the physiological expression of competence. The kinetics of DNA uptake, dose-response curve of DNA versus transformants, time required for phenotypic expression, and other features were similar to those in other bacterial transformation systems.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

Characterization of Neisseria gonorrhoeae strains isolated from patients with conjunctivitis

The conjunctivitis produced by Neisseria gonorrhoeae is the less frequently reported clinical form of gonococcal infection. We aim to phenotypically characterize N. gonorrhoeae isolated from conjunctivae sites. A total of six cases of this disease were notified in the Camagüey province, Cuba. All the strains isolated were penicillin-producing, showed the serogroup WI and exhibited the same antimicrobial susceptibility pattern and plasmid profile (2.6-3. 2-24.5). The results contribute to the characterization of N. gonorrhoeae strains circulating in our environment.     

淋病流行株外排系统与外膜通透性和多重耐药性的关系

探讨外排系统、外膜通透性与淋病流行株多重耐药性的关系。应用K—B法和琼脂稀释法从湛江地区分离出62株淋球菌多重耐药株。利用SDS—PAGE测定淋球菌外膜孔蛋白的表达;应用直接荧光法测定能量抑制剂加入前后淋球菌对抗生素的摄入和积累情况,比较耐药菌与敏感菌内膜泵蛋白表达的差异;利用煮沸法提取细菌DNA,PCR扩增mtrR基因,并对扩增产物测序,比较敏感株与多重耐药株的差异。结果5株多重耐药菌均有外膜孔蛋白表达的缺失或下降,同时伴有外排泵蛋白的表达;5株敏感淋球菌无mtrR的突变,10株多重耐药株均有mtrR基因的突变。表明外排系统、外膜通透性与淋病流行株的多重耐药性密切相关。     

广州地区74株淋球菌耐药性结果分析

目的了解广州地区淋球菌对抗生素的耐药性及PPNG和TRNG的流行状况。方法用琼脂稀释法测定最低抑菌浓度（MIC）;用纸片碘量法检测β-内酰胺酶。结果74株淋球菌检出PPNG31株（41．9％）、TRNG19株（25．7％）、环丙沙星耐药率达97．3％,高度耐药株（MIC≥16mg／L）22株（29．7％）,未发现对头孢三嗪、壮观霉素耐药的菌株,且抗菌活性最强。结论持续监测淋球菌的耐药性十分重要。     

Lipids of Branhamella catarrhalis and Neisseria gonorrhoeae.

Three strains of Branhamella catarrhalis and three strains of Neisseria gonorrhoeae were analyzed with regard to their phospholipid and neutral lipid composition. B. catarrhalis (ATCC 23246) contained 5.12 +/- 0.34% lipid, determined gravimetrically, compared to 8.56 +/- 0.15% and 9.73 +/- 0.06% for two strains of N. gonorrhoeae. Cardiolipin, phosphatidylglycerol, and phosphatidyl-ethanolamine were identified in extracts of both species. In addition, B. catarrhalis contained small amounts of phosphatidylcholine, and N. gonorrhoeae contained small amounts of lyso-phosphatidylethanolamine, which accumulated with autolysis accompanying late cell culture growth. The kinetics of change of relative amounts of phospholipids in both species were measured and found to differ substantially. Neutral lipid accounted for 30.4% of the total lipid of B. catarrhalis (ATCC 23246) and 7.6% of the total lipid of N. gonorrhoeae NYH 002. Hydrocarbons, triglycerides, free fatty acids, coenzyme Q, diglycerides, and free hydroxy fatty acids were identified in the neutral lipid fraction of both species. The three strains of N. gonorrhoeae, sensitive, intermediate, and resistant to penicillin, exhibited no significant difference in the composition or metabolism of phospholipid.     

A study by high-resolution two-dimensional polyacrylamide gel electrophoresis of relationships between Neisseria gonorrhoeae and other bacteria

High-resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the soluble proteins from seven strains of Neisseria gonorrhoeae, six strains of Neisseria meningitidis and one or two strains of twelve other species. Approximately 200 individual polypeptides could be visualized as Coomassie Blue stained spots on an electrophoretogram of N. gonorrhoeae and similar numbers were found for the other bacteria. Each species of bacterium had a distinctly different pattern of spots which could be recognized. Quantitative comparisons of 48 selected spots derived from one strain of N. gonorrhoeae with those of five other strains of gonococcus, three strains of N. meningitidis and one of Branhamella catarrhalis, showed relationships in agreement with their current taxonomic classification but with a higher level of discrimination than that of previously used methods. It was also possible to distinguish the individual gonococcal strains. It is suggested that the method could be useful for bacterial classification and identification.     

Confirmatory identification of Neisseria gonorrhoeae by slide coagglutination

Neisseria gonorrhoeae was identified by the Phadebact gonococcus test, a rapid slide coagglutination technique, and the results obtained were compared with those obtained by conventional methods (Gram stain morphology, oxidase reaction, and carbohydrate utilization tests) for the confirmatory identification of gonococci. Of 308 clinical isolates examined, the coagglutination procedure correctly identified 97.8% of the isolates tested as N. gonorrhoeae and 93.9% of other bacteria as not N. gonorrhoeae. The coagglutination procedure also identified 29 laboratory strains correctly as not N. gonorrhoeae. The slide coagglutination test is easy to perform and offers a valuable alternative to other techniques for the confirmatory identification of N. gonorrhoeae.     

中药止痢灵对淋病流行株的体外抑菌作用

目的：了解止痢灵对淋病流行株的体外抑菌作用。方法：从湛江地区分离出22株淋病流行株,应用打孔法测定止痢灵及其单方的体外抑菌作用,根据抑菌圈直径大小,判定中药的敏感性。结果：复方止痢灵对淋病流行株有较强的抑菌作用;而其单方黄柏、白头翁、苦参对淋球菌的抑菌作用较弱。结论：止痢灵对淋球菌的抑菌作用,为临床治疗淋病及其他泌尿系感染开辟了新的途径,也为耐药菌株的控制提供了理论依据。     

In vitro anti-Neisseria gonorrhoeae activity of Terminalia macroptera leaves

We used the agar dilution method to evaluate the antibacterial effect of Terminalia macroptera leaf (Tml) extract against nine reference and clinical Neisseria gonorrhoeae strains, including penicillin- and tetracycline-resistant and -susceptible strains. Tml possesses anti-N. gonorrhoeae activity against all of the strains and the minimum inhibitory concentrations (MIC) were between 100 and 200 microg ml(-1). We then used a liquid-liquid partition method to divide the Tml extract into five fractions and determined the anti-N. gonorrhoeae activity of each of the fractions. All of the fractions showed antibacterial activity. The most active one was identified as the diethyl ether fraction and had MIC values of between 25 and 50 microg ml(-1) against all of the strains.     

Multivariate analysis of Neisseria DNA restriction endonuclease patterns

Chromosomal DNA was extracted from eleven Neisseria meningitidis and seven Neisseria gonorrhoeae isolates and cleaved with the restriction enzyme HindIII. The DNA fragments were separated according to their size, using a 4% polyacrylamide gel. The band patterns obtained were digitized and statistically analysed by the SIMCA method. To develop the models for N. meningitidis (class 1) and N. gonorrhoeae (class 2), all eleven meningococci and seven gonococci, were used. All strains were classified correctly and showed an extremely good class separation.     

Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function.

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.     

Genome analysis and strain comparison of correia repeats and correia repeat-enclosed elements in pathogenic Neisseria

Whole genome sequences of Neisseria meningitidis strains Z2491 and MC58 and Neisseria gonorrhoeae FA1090 were analyzed for Correia repeats (CR) and CR-enclosed elements (CREE). A total of 533, 516, and 256 copies of CR and 270, 261, and 102 copies of CREE were found in these three genomes, respectively. The lengths of CREE range from 28 to 348 bp, and the lengths of multicopy CREE appear mainly in the ranges of 154 to 156 bp and 105 to 107 bp. The distribution of CREE lengths is similar between the two N. meningitidis genomes, with a greater number of 154- to 156-bp CREE (163 and 152 copies in N. meningitidis strain Z2491 and N. meningitidis strain MC58, respectively) than 105- to 107-bp CREE (72 and 77 copies). In the N. gonorrhoeae strain FA1090 genome there are relatively more 105- to 107-bp CREE (51 copies) than 154- to 156-bp CREE (36 copies). The genomic distribution of 107-bp CREE also shows similarity between the two N. meningitidis strains (15 copies share the same loci) and differences between N. meningitidis strains and N. gonorrhoeae FA1090 (only one copy is located in the same locus). Detailed sequence analysis showed that both the terminal inverted repeats and the core regions of CREE are composed of distinct basic sequence blocks. Direct TA dinucleotide repeats exist at the termini of all CREE. A survey of DNA sequence upstream of the sialyltransferase gene, lst, in several Neisseria isolates showed that 5 N. meningitidis strains contain a 107-bp CREE in this region but 25 N. gonorrhoeae strains show an exact absence of a 105-bp sequence block (i.e., the 107-bp CREE without a 5' TA dinucleotide) in the same region. Whole-genome sequence analysis confirmed that this 105-bp indel exists in many homologous 107-bp CREE loci. Thus, we postulate that all CREE are made of target TA with indels of various lengths. Analysis of 107-bp CREE revealed that they exist predominantly in intergenic regions and are often near virulence, metabolic, and transporter genes. The abundance of CREE in Neisseria genomes suggests that they may have played a role in genome organization, function, and evolution. Their differential distribution in different pathogenic Neisseria strains may contribute to the distinct behaviors of each Neisseria species.     

Transfer of the gonococcal penicillinase plasmid: mobilization in Escherichia coli by IncP plasmids and isolation as a DNA-protein relaxation complex

A 4.4-megadalton penicillinase plasmid, pWD2, from Neisseria gonorrhoeae was transformed into Escherichia coli. pWD2 was efficiently mobilized by IncP plasmids in E. coli but not by Flac, R1drd-19, or R64drd-11. pWD2 could be isolated as a DNA-protein relaxation complex with properties similar to the well characterized ColE1 complex. The host range of pWD2 was shown to include gonococci, Enterobacteriaceae, and Hemophilus influenzae, but not Acinetobacter calcoaceticus or Pseudomonas aeruginosa. These findings suggest that P-group plasmids could have played a role in the dissemination of the TEM beta-lactamase to pathogenic gram-negative bacteria.     

应用单抗和流式细胞仪分析淋球菌的表面抗原

报告了利用流式细胞仪测定分析10株抗淋球菌脂寡糖单克隆抗体所识别的抗原分子表达的特点,定量地显示了这些抗原分子表达的稳定性和数量的多少,评价了单克隆抗体与不同血清型淋球菌的反应性。