Resolution of clonal subgroups among Neisseria gonorrhoeae IB-2 and IB-6 serovars by pulsed-field gel electrophoresis.

OBJECTIVE--Analysis of macrorestriction patterns by PFGE to resolve the relatedness of clonal subgroups amongst N gonorrhoeae IB-2 and IB-6 serovar strains. MATERIALS AND METHODS--Nineteen IB-2 and eight IB-6 serovar strains that differed in either auxotype or penicillin sensitivity were isolated over a two and a half-year period from patients attending several STD clinics in Sydney. During this period, a major clone, Wt/IB-2 (FS), established on epidemiological grounds, was circulating amongst homosexual males. The genetic relation of this major clone to the other strains present in the community was determined by pulsed-field gel electrophoretic (PFGE) analysis of DNA restriction fragments. Genomic DNA from the 27 isolates were prepared, digested with SpeI and BglII and the restriction patterns were analysed by contour-clamped homogeneous electric field electrophoresis (CHEF) in a CHEF DRIII equipment. RESULTS--Phenotypic characterisation of the 27 isolates by the combined use of auxotype, serological characterisation and penicillin sensitivity indicated the presence of subgroups within each of the two serovars. In the present study, PFGE analysis of SPeI and BglII-generated genomic DNA restriction patterns from six of the ten Wt/IB-2 (FS) correlated well with phenotypic characterisation of this major clone. Four of the ten Wt/IB-2 (FS) were found to be clonally-derived variants of this major clone as minor genome variations (less than 3 DNA fragments) were observed. Distinct clones were represented by three Wt/IB-2 (LS) isolates as the DNA fingerprints generated from these were unrelated to the major clone. Analysis of PFGE patterns of 6 Pro/IB-2 isolates showed that one was genotypically identical to the major clone, two were clonal variants and three had significantly different patterns to indicate that they were genotypically unrelated. Wt/IB-6 isolates had heterogenous PFGE patterns that were clearly unrelated to the Wt/IB-2 serovar strains. Within the IB-6 serovar, there were three isolates with the Wt/IB-6 (FS) phenotype that could be considered as clonal variants whilst the rest were genotypically distinct. CONCLUSIONS--PFGE analysis of macrorestriction patterns generated from SpeI- and BglII-cleavage of genomic DNA has enabled the establishment of clonal origins of strains present in the Sydney community during the period of study. The delineation of strains belonging to major A/S groups by PFGE analysis presents a clearer epidemiological picture than phenotypic characterisation alone.     

聚丙烯酰胺凝胶电泳和毛细管电泳用于淋球菌多位点串联重复序列分型的评估

淋球菌是引起淋病的致病菌,淋球菌感染不仅可引起新生儿失明等严重的并发症,还能够加速其他病原体及HIV感染［1］。近年来淋球菌分型方法报道较多,目前最常用的是淋球菌多抗原序列分型（NG-MAST）和多位点序列分型（MLST）分型,但这两种分型方法费用较高,不适合资源匮乏地区应用。多位点串联重复序列分型（multiple-locus variable number tandem repeat analysis,MLVA）是近年来用于淋球菌分型的一种方法,该分型方法是一种简单、价格便宜、易操作、结果客观的分型方法。本文的目的是比较聚丙烯酰胺凝胶电泳和毛细管电泳两种方法进行淋球菌MLVA分型的异同,以便选择最佳的实验方法……     

Rapid carbohydrate utilization test for the identification of Neisseria gonorrhoeae.

A rapid carbohydrate utilization test for the identification of N. gonorrhoeae was investigated, with reference to its use in a routine diagnostic laboratory. The rapid test was shown to give accurate results in agreement with those of a conventional serum-free sugar medium. Because of the shorter time taken for the confirmation of an isolate, and several other advantages, it is proposed that the rapid test is an extremely useful alternative to conventional sugar tests. Immunofluorescence was also used to identify isolates of N. gonorrhoeae and the rapid carbohydrate utilization test was found to assist in differentiating between N. gonorrhoeae and N. meningitidis when equivocal or negative immunofluorescence results were obtained.     

Stable serum resistance of Neisseria gonorrhoeae as an epidemiological marker.

Using a simple and rapid microassay, we tested 100 strains of Neisseria gonorrhoeae isolated from 81 patients (41 men and 40 women) for their sensitivity to killing by normal human serum (NHS). The reproducibility of the test was good when the bactericidal end points were taken as the dilution of fresh NHS that killed more than 95% of the test organisms. The bactericidal end points of strains isolated either from different anatomical sites or from sexual partners correlated well with the levels of sensitivity to serum of corresponding isolates, as well as with auxotypes. When the strains were not highly resistant to killing by NHS, this marker gave a precise definition of each strain and permitted the differentiation of isolates belonging to common auxotypes.     

Stable serum resistance of Neisseria gonorrhoeae as an epidemiological marker

Using a simple and rapid microassay, we tested 100 strains of Neisseria gonorrhoeae isolated from 81 patients (41 men and 40 women) for their sensitivity to killing by normal human serum (NHS). The reproducibility of the test was good when the bactericidal end points were taken as the dilution of fresh NHS that killed more than 95% of the test organisms. The bactericidal end points of strains isolated either from different anatomical sites or from sexual partners correlated well with the levels of sensitivity to serum of corresponding isolates, as well as with auxotypes. When the strains were not highly resistant to killing by NHS, this marker gave a precise definition of each strain and permitted the differentiation of isolates belonging to common auxotypes.     

淋病奈瑟菌对阿奇霉素的耐药性及其研究进展

为提高治疗效果和延缓淋球菌对广谱头孢菌素（extended?spectrum cephalosporins,ESC）耐药性的发展速度,美国、澳大利亚、加拿大和欧洲等国家或地区均推荐采用头孢曲松0.25或0.5 g单次肌内注射联合阿奇霉素（azithromycin, AZM）1或2 g单次口服作为无并发症淋病的一线治疗方案［1?3］。而且ESC联合AZM治疗可能对咽部淋球菌感染具有更好的疗效［4］。然而,AZM耐药淋球菌的流行,特别是AZM高度耐药菌株在多个国家或地区出现,对联合治疗方案的长期有效性造成了威胁。在缺乏新的治疗淋病有效药物前提下,有必要增加对AZM耐药淋球菌的耐药现状、耐药机制及产生原因的了解,以更有效地筛查或监控AZM耐药菌株,控制或延缓其耐药性的蔓延……     

Analysis of a recent epidemic due to penicillinase-producing Neisseria gonorrhoeae: epidemiologic and medical considerations

In Norfolk, Virginia, two epidemics of disease due to proved penicillinase-producing Neisseria gonorrhoeae were investigated intensively, both epidemiologically and medically. The first epidemic appeared in October 1976 and was controlled in one month; it was followed by a hiatus of four years before the emergence of the second epidemic in October 1980. The latter apparently was brought under control by December 1980, since no more patients with penicillinase-producing N. gonorrhoeae have been discovered to date (March 1982). Certain interesting medical aspects emerged from the investigation. Resistant organisms were cultured from asymptomatic as well as symptomatic patients and from all potentially infected sites. At times, resistant organisms were cultured from only one of several sites in a given patient or both resistant and sensitive organisms were cultured from a single site. It was concluded that this type of gonorrhea can be contained by intensive epidemiologic investigation combined with adequate diagnosis and treatment.     

Evaluation of the coagglutination test for the identification of Neisseria gonorrhoeae in primary cultures.

The coagglutination (COA) test for the identification of Neisseria gonorrhoeae was compared with immunofluorescence and sugar degradation tests on 1710 gonococcal isolates, 72 of which produce beta-lactamase. The COA test gave a positive result for 98.6% of the strains. Treatment of suspensions with Streptomyces enzyme reduced the incidence of inconclusive results due to autoagglutination to 1.2%. Cross-reactivity of the gonococcal antiserum was minimised by absorption with meningococci and Moraxella species. The COA provide a simple, quick, and reliable method for identifying N gonorrhoeae in culture.     

Neisseria gonorrhoeae culture: development of an easy method]

An easy and cheap method for culturing Neisseria is developed. The medium for gonococci is prepared as proposed by the producer (BBL, Oxoid, Hoechst). About 8 ml of the medium are poured in sterile air-tight stool tubes of 25 ml volume. Materials to be examined for gonococci are taken from the cervix, ureter or anus and are inoculated on the medium. A small piece (20-30 mg) of the GasPak tablet (BBL) is then deposited in the tube and closed immediately. The GasPak tablet consists of sodium bicarbonate and citric acid which, if they come in contact with humidity, produce CO2 gas. The inoculated tube is then put in an incubator at 37 degrees C for 14-24 h. This method gives a good microbiological result. With the aid of the oxidase reaction the colonies take a brown-black color. For further differentiation of the species the sugar fermentation method is necessary.     

The goat mammary gland as a model infection site for Neisseria gonorrhoeae.

Live and formalin-killed gonococci were instilled into the mammary glands of lactating and nonlactating goats. In lactating goats viable gonococci elicited a limited inflammatory process whereas in non-lactating goats, severe inflammation and swelling appeared and peaked on the 3rd day after instillation and persisted for about 10 days. No viable gonococci were recovered after the first day, but fluorescent antibody staining showed gonococci in the exudate from non-lactating goats up to 7 days after instillation.     

Reverse passive haemagglutination test for the rapid identification of Neisseria gonorrhoeae and detection of penicillinase production.

In a comparison of the reverse passive haemagglutination test (RPHA) with the direct immunofluorescent and rapid carbohydrate utilisation tests for the identification of Neisseria gonorrhoeae isolated from clinical specimens, 315 isolates of oxidase-positive Gram-negative diplococci were tested as pure 24-hour-old subcultures and samples from 108 similar organisms were taken directly from primary isolation plates. A similar test system was used to detect penicillinase production. Results showed agreement in 97.8% of organisms tested with the RPHA and conventional methods for identification of N. gonorrhoeae; similarly there was good agreement with conventional methods for detection of penicillinase production. The test was reliable and could be read within four hours; a result was therefore available on the same day the clinical specimen was received. The time and work involved in identifying N. gonorrhoeae using the RPHA was less than with conventional methods, but differentiation between N. gonorrhoeae and other Neisseria species from throat swabs proved difficult.     

Reverse passive haemagglutination test for the rapid identification of Neisseria gonorrhoeae and detection of penicillinase production.

In a comparison of the reverse passive haemagglutination test (RPHA) with the direct immunofluorescent and rapid carbohydrate utilisation tests for the identification of Neisseria gonorrhoeae isolated from clinical specimens, 315 isolates of oxidase-positive Gram-negative diplococci were tested as pure 24-hour-old subcultures and samples from 108 similar organisms were taken directly from primary isolation plates. A similar test system was used to detect penicillinase production. Results showed agreement in 97.8% of organisms tested with the RPHA and conventional methods for identification of N. gonorrhoeae; similarly there was good agreement with conventional methods for detection of penicillinase production. The test was reliable and could be read within four hours; a result was therefore available on the same day the clinical specimen was received. The time and work involved in identifying N. gonorrhoeae using the RPHA was less than with conventional methods, but differentiation between N. gonorrhoeae and other Neisseria species from throat swabs proved difficult.     

Neisseria gonorrhoeae: subdivision of auxogroups by genetic transformation.

Genetic transformation was used in an attempt to subdivide the most prevalent auxotypes of Neisseria gonorrhoeae in local isolates. The large proline requiring (Pro-) group could be divided into two genetic types, as could the less common arginine requiring (Arg-) group. The large arginine, hypoxanthine, and uracil requiring (Arg- Hyp- Ura-) group could not be subdivided by this method. The genetic relation between these and other auxotypes was investigated.     

Comparison of methods for the detection of penicillinase-producing Neisseria gonorrhoeae.

In an evaluation of four methods for detecting penicillinase-producing Neisseria gonorrhoeae the chromogenic cephalosporin, rapid iodometric, and penicillin disc diffusion methods gave complete agreement for all the 202 strains of gonococci tested. No false-positive or false-negative results occurred. The filter paper iodometric method detected 99% of the penicillinase-producing strains without any false-positive result.