Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance, a Novel Antimicrobial Resistance Multilocus Typing Scheme for Tracking Global Dissemination of N. gonorrhoeae Strains.

A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible (https://ngstar.canada.ca). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, and 23S rRNA) associated with resistance to ß-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA, porB, gyrA, and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs (n = 109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 (n = 100; 13.0%), ST-42 and ST-91 (n = 45; 5.9%), ST-64 (n = 44; 5.72%), and ST-139 (n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 (n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 (n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 (n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains.

Using strain typing to characterise a fluoroquinolone-resistant Neisseria gonorrhoeae transmission network in southern California.

OBJECTIVE: We investigated the initial outbreak of fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) in southern California with analysis of transmission using strain typing. METHODS: Surveillance for QRNG was conducted between 2000 and 2002 in southern California, including epidemiology and strain typing by a combination of antibiogram, auxotype, serovar, Lip type and amino acid alteration patterns in the quinolone-resistance determining region of GyrA and ParC. Combining epidemiological data with strain typing, we describe the emergence of QRNG outbreak strains using risk factor analysis and transmission networks. RESULTS: Two outbreak strains accounted for 82% of isolates. Both strains required proline, were Lip type 17c, had amino acid alterations 91> Phe in GyrA and 87> Arg in ParC, but they differed by their serovar, IB-3C8 versus IB-2H7, 2G2. Outbreak strains were positively associated with men who have sex with men (MSM), adjusted odds ratio (AOR) 23.9 (95% confidence interval (CI) 2.2 to 261) and negatively associated with travel history: AOR 0.05, (95% CI 0.0 to 0.6). Network analysis demonstrated that 17 cases were connected by sexual contacts and/or public venues including bars, bathhouses/sex clubs, and internet sites. CONCLUSIONS: QRNG may have become established among Californian MSM through an identified transmission network of southern Californian bars, bathhouses and internet sites.

Molecular epidemiology of Neisseria gonorrhoeae exhibiting decreased susceptibility and resistance to ciprofloxacin in Hawaii, 1991-1999.

BACKGROUND: Clinically significant resistance to Centers for Disease Control and Prevention (CDC)-recommended doses of fluoroquinolones (ciprofloxacin and ofloxacin) has been reported for Neisseria gonorrhoeae. In Hawaii, fluoroquinolone-resistant gonococcal isolates were first identified in 1991. GOAL: To assess the diversity, based on phenotypic and genotypic characterization, of gonococcal isolates exhibiting decreased susceptibility (CipI; MICs = 0.125-0.5 microg/ml) or clinically significant resistance (CipR; MICs > or = 1 microg/ml) to ciprofloxacin in Hawaii from 1991 through 1999. STUDY DESIGN: Antimicrobial susceptibilities, auxotype/serovar (A/S) class, GyrA/ParC alteration patterns, and plasmid profiles were determined for gonococci isolated in Honolulu from 1991 through 1999 that exhibited intermediate or clinically significant resistance to ciprofloxacin. Strain phenotypes were defined by A/S class, GyrA/ParC alteration pattern, and penicillin-tetracycline resistance phenotype supplemented with plasmid profiles for beta-lactamase-producing isolates. RESULTS: Altogether, 68 isolates exhibiting intermediate or clinically significant resistance to ciprofloxacin belonged to 23 and 19 strain phenotypes, respectively. Among the CipI and CipR isolates, 4 and 13 GyrA/ParC alterations patterns were identified, respectively. The 91,95/Asp-86 alteration pattern occurred most frequently among CipR isolates. Forty-four strain phenotypes were represented by only one isolate. In addition, seven pairs and two clusters of isolates were identified. CONCLUSIONS: From 1991 through 1997, few gonococcal strains exhibiting intermediate or clinically significant resistance to CDC-recommended doses of fluoroquinolones were identified from Hawaii. Isolates belonged to a large number of phenotypic and genotypic types, suggesting that most cases were imported, with only a few instances in which isolate pairs indicated that secondary transmission of infections had occurred in Hawaii. Beginning in 1998, the number of CipR isolates increased markedly, and more isolates belonged to fewer phenotypic and genotypic types, suggesting either more frequent importation of fewer strain types or the possibility that the endemic spread of a few strains is beginning to occur.

Use of the neisserial lipoprotein (Lip) for subtyping Neisseria gonorrhoeae.

The pathogenic Neisseria species N. meningitidis and N. gonorrhoeae possess an outer membrane lipoprotein, designated Lip, which is present in all strains tested. The predicted protein sequence of Lip consists of a consensus AAEAP amino acid repeat. The objective of this study was to determine the feasibility of using the Lip repeat number and sequence for subtyping of Neisseria gonorrhoeae. The lip genes of each isolate were amplified by PCR and sequenced to determine the repeat number and sequence. Among the 46 strains we examined, eight different Lip repeat numbers were identified, with lengths of 11 (1 strain), 12 (14 strains), 13 (2 strains), 14 (10 strains), 15 (5 strains), 16 (10 strains), 17 (3 strains), and 20 (1 strain) repeats. Analysis indicated differences in the sequences within the repeats that resulted in amino acid alterations in repeat classes that contained multiple strains. Among the 46 isolates examined, we were able to identify 17 unique Lip subtyping patterns.

Syphilis in Atlanta during an era of declining incidence.

BACKGROUND: Syphilis transmission in Atlanta is ongoing despite declining incidence. OBJECTIVES: To identify risk factors and missed opportunities for prevention. STUDY DESIGN: A case-control study design was used. Twenty-five sexually transmitted disease (STD) clinic patients with primary or secondary syphilis by polymerase chain reaction and serology and 49 matched controls were interviewed. RESULTS: Persons with syphilis more frequently had HIV infection (24% versus 2%; P = 0.005), crack-using sex partners (52% versus 18%; odds ratio [OR] = 5.1; 95% CI = 1.7-15.5), and a history of incarceration (80% versus 57%; OR = 3.0; CI = 1.0-9.3). Many cases (48%) and controls (31%) had received drug-abuse treatment. Only 40% of previously incarcerated patients and 74% of those with a history of drug treatment reported receiving STD/HIV education in those settings. Among all patients reporting recent HIV education, 41% were told about STD recognition and treatment. Unprotected sex and delay in seeking care were common. CONCLUSION: To prevent syphilis and associated HIV, more extensive STD education is needed in jails and drug-treatment centers.

Alterations within the quinolone resistance-determining regions of GyrA and ParC of Neisseria gonorrhoeae isolated in the Far East and the United States.

The genetic mutations within the quinolone resistance-determining regions (QRDRs) of gyrA and parC of 234 Neisseria gonorrhoeae strains isolated in the Far East and the United States, which exhibited either clinically significant ciprofloxacin resistance (CipR) or intermediate ciprofloxacin resistance (CipI) were characterized. A number of GyrA/ParC amino acid alteration patterns were identified, the most prevalent alteration pattern among CipR isolates being GyrA-91,95/ParC-Asp-86- > Asn (91,95/Asp-86- > Asn). Isolates containing 91,95/Asp-86- > Asn belonged to a number of A/S classes, penicillin/tetracycline resistance phenotypes, and plasmid profiles. These results strongly suggest that the continuing emergence of ciprofloxacin-resistant gonococci is not due to the spread of a single or a few strains but to numerous factors such as 'spread of existing strains, importation of new strains and, possibly, de novo development of ciprofloxacin resistance in previously susceptible strains.

Identification of novel mutation patterns in the parC gene of ciprofloxacin-resistant isolates of Neisseria gonorrhoeae.

Of 65 ciprofloxacin-resistant, clinical isolates of Neisseria gonorrhoeae, 5 isolates exhibited ParC mutations previously undescribed in the gonococcus. For isolates containing two ParC mutations (the Ser-87-->Ile and Glu-91-->Gly mutations and the Gly-85-->Cys and Arg116-->Leu mutations) the MICs of ciprofloxacin (8.0 to 64.0 microg/ml) were higher than those for the isolate containing the single ParC mutation (Arg-116-->Leu; MIC, 1.0 microg/ml).

Comparison of clinically directed, disease specific, and syndromic protocols for the management of genital ulcer disease in Lesotho.

OBJECTIVE: To evaluate two protocols for the syndromic management of genital ulcer disease (GUD) in Lesotho, southern Africa and to compare the performance of these protocols with that of a conventional disease specific approach. METHODS: A cross sectional study was conducted among consecutive patients with GUD attending an STD clinic in Maseru, Lesotho. The clinical diagnoses were made by using predefined criteria at the initial visit before the performance of laboratory tests. Attempts were made to detect the specific aetiology of the genital ulcers using PCR assays and syphilis serology. The results of PCR assays and syphilis serology were used as the gold standard against which the performance of the management approaches were applied. RESULTS: Of 100 patients initially recruited into the study, Haemophilus ducreyi infection was detected in 56%, herpes simplex virus in 26%, Treponema pallidum in 23%, and lymphogranuloma venereum in 7%. No pathogens were detected in 6% of patients. 17% of patients had mixed infections. Sensitivity, specificity, positive and negative predictive values of the three management protocols for GUD were compared after applying each to the study population. Theoretically, the lowest correct treatment rate would have been obtained by using the disease specific protocol (62%) compared with more than 90% in both syndromic management protocols. Considerable overtreatment for primary syphilis would occur following application of both syndromic protocols. This would be the result of the overdiagnosis of chancroid, in particular the misdiagnosis of genital herpes as chancroid, which would receive treatment for syphilis unnecessarily. The HIV seroprevalence among these patients was 36%. A significantly higher rate of HIV seropositivity was detected among the patients with herpes simplex virus infection when compared with those patients having other causes of genital ulcer disease (58% v 27%; odds ratio 3.73; 95% CI 1.26-11.26; p = 0.01). CONCLUSIONS: Poor sensitivity, specificity, and predictive values were recorded when the disease specific protocol was applied to the study population. In contrast, the syndromic management protocols provided adequate treatment for more than 90% of patients with GUD. Protocol C, which identified a minority of cases of genital herpes, was found to have an advantage when compared with protocol B (all patients with genital ulcer disease treated for both syphilis and chancroid) in that 29% of genital herpes cases would receive appropriate counselling.

Chancroid.

Chancroid is a genital ulcerative disease GUD). These diseases are common throughout the world and include syphilis, genital herpes, chancroid, lymphogranuloma venereum, and donovanosis. Chancroid is particularly common an Africa, Asia, and Latin America where its incidence may exceed that of syphilis as a cause of genital ulceration (1,2). However, chancroid is considered an uncommon sexually transmitted infection in the United States.

Comparison of clinical diagnosis and standard laboratory and molecular methods for the diagnosis of genital ulcer disease in Lesotho: association with human immunodeficiency virus infection.

A multiplex polymerase chain reaction (M-PCR) assay for Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) was compared with clinical and standard laboratory methods for the diagnosis of genital ulcer disease (GUD) in 105 patients; 36% were human immunodeficiency virus (HIV)-seropositive. Chancroid (80%), syphilis (8%), and genital herpes (8%) were the most frequent diagnoses. H. ducreyi and HSV were isolated from ulcers of 43% and 18% of patients, respectively; in 35%, all cultures were negative and the laboratory diagnosis indeterminate. M-PCR detected H. ducreyi, T. pallidum, and HSV in 56%, 23%, and 26% of patients, respectively; (no definitive diagnosis, 6%). The proportion of patients with more than one agent was 4% by culture and 17% by M-PCR (P = .002). Resolved sensitivities of M-PCR for H. ducreyi and HSV cultures were 95% and 93%, respectively. The sensitivities of H. ducreyi and HSV cultures were 75% and 60%, respectively. HSV, detected in 47% of specimens from HIV-infected versus 16% from HIV-uninfected patients (P < .001), may be emerging as a more frequent cause of GUD.

Fluoroquinolone resistance in Neisseria gonorrhoeae.

Fluoroquinolones and broad-spectrum cephalosporins are the most effective antimicrobial agents for the treatment of gonorrhea. However, clinically significant resistance to fluoroquinolones has emerged in Neisseria gonorrhoeae. Fluoroquinolone-resistant strains account for approximately 10% of all gonococcal strains in Hong Kong and the Republic of the Philippines. As many as 50% of strains from some Far Eastern countries exhibit decreased susceptibility (intermediate resistance) to fluoroquinolones. Strains with intermediate resistance and clinically significant resistance are being isolated sporadically in North America, where resistant strains have been associated with an outbreak and with failure of infections to respond to treatment with doses of ciprofloxacin and ofloxacin recommended by the Centers for Disease Control and Prevention; strains exhibiting decreased susceptibility to these agents are endemic in at least one metropolitan area. Monitoring for fluoroquinolone resistance is now critical for ensuring adequate treatment of infections with resistant strains and for maximizing the time during which fluoroquinolones may be used to treat gonorrhea.

Chancroid and Haemophilus ducreyi: an update.

Haemophilus ducreyi is a fastidious gram-negative bacillus that causes the sexually transmitted infection chancroid. Chancroid is a major genital ulcerative disease in Africa, Southeast Asia, the Caribbean, and Latin America and is of increasing concern in the United States. Genital ulcerative disease and chancroid in particular have been associated with facilitating the transmission of human immunodeficiency virus. The diagnosis of chancroid based on the clinical appearance of the genital lesion or on the isolation of H. ducreyi on selective medium is relatively insensitive. However, recent advances in nonculture diagnostic tests have enhanced our ability to diagnose chancroid. There has been renewed interest in understanding the pathogenesis of H. ducreyi. In vitro and in vivo models have been developed to help identify important virulence determinants. Through the use of biochemical and molecular techniques, macromolecular components that may be important in virulence have been identified.

Genetic diversity of the iron-binding protein (Fbp) gene of the pathogenic and commensal Neisseria.

The pathogenic Neisseria and most commensal Neisseria species produce an iron-binding protein (Fbp) when grown under iron-limited conditions. In the current study, we confirmed the presence of Fbp, as well as DNA sequences homologous to the gonococcal fbp, in strains of N. gonorrhoeae, N. meningitidis, N. cinerea, N. lactamica, N. subflava, N. kochii and N. polysaccharea. The fbp genes from these strains were amplified by the polymerase chain reaction, digested with StuI or RsaI, and the restriction patterns examined. The patterns for the gonococcal and meningococcal fbp were virtually identical; however, variations were observed in the fbp sequences of the commensal Neisseria species. N. flavescens, N. mucosa, N. sicca, N. ovis and Branhamella catarrhalis, did not produce Fbp as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity with an Fbp specific monoclonal antibody, nor did they hybridize to an fbp-specific DNA probe.

Identification and cloning of a fur homolog from Neisseria gonorrhoeae.

The promoter region of the major iron-regulated protein of Neisseria gonorrhoeae, Fbp, has two regions that exhibit homology with the Escherichia coli consensus Fur-binding sequences. Gel retardation assays suggested that purified E. coli Fur bound to two sites within the Fbp promoter. The presence of a gonococcal Fur homolog was suggested by Southern hybridization under conditions of low stringency, which revealed a DNA locus that exhibited homology to the E. coli fur gene. Oligonucleotides derived from the conserved regions of fur genes of extremely diverse bacteria were used to amplify a 140-bp fragment of a putative gonococcal fur gene. This fragment was used to identify clones containing the entire gonococcal fur gene. After sequencing the gonococcal fur gene and its promoter region, we found that gonococcal Fur exhibited 50% identity with E. coli Fur at the amino acid level; however, it complemented two E. coli Fur- mutants. The presence of a Fur homolog in N. gonorrhoeae suggests that Fur-regulated genes are widely distributed among extremely diverse bacteria.

Mouse subcutaneous chamber model for in vivo growth of Haemophilus ducreyi.

The ability of Haemophilus ducreyi, the causative agent of chancroid, to grow in subcutaneous chambers implanted in mice was studied. All seven H. ducreyi strains tested were able to maintain a long-term infection in ICR mice; one mouse remained infected with strain Hd175 for more than 4 months. Growth curves obtained following the removal of chamber fluid at various time points from infected mice demonstrated that H. ducreyi was growing during the course of the infection. In addition, all three mouse strains tested (ICR, CBA and BALB/c) were able to maintain long-term H. ducreyi infections. This model will be valuable in studying the effects of in vivo growth on the antigenic composition of H. ducreyi as well as for the identification of virulence factors.

Localization of and immune response to the lipid-modified azurin of the pathogenic Neisseria.

The development of vaccines to prevent Neisseria infections has been impeded by antigenic diversity of most Neisseria surface components. The lipid-modified azurin (Laz), one of two distinct surface proteins recognized by the H.8 monoclonal antibody, is present in all pathogenic Neisseria. The mature protein has two domains; one contains an H.8 epitope and the other has extensive homology to azurins, a class of bacterial copper-binding proteins. The cellular location of Laz and the serum immune response to Lax were examined in patients with disseminated Neisseria infections. The data demonstrated that Laz is probably contained in the Neisseria outer membrane, although unlike most outer membrane proteins it is Sarkosyl soluble. By probing recombinant bacteriophages encoding the H.8 and azurin domains of Laz, results showed that whereas the H.8 epitope is immunogenic in patients with disseminated Neisseria infections, the azurin domain of Laz plays little role in eliciting an antibody response in these patients.

Identification of a Staphylococcus aureus transposon (Tn4291) that carries the methicillin resistance gene(s).

We isolated a transposon (Tn4291) that carries the resistance gene(s) for methicillin in a secondary insertion site on the penicillinase plasmid pI524. Transposition of Tn4291 into pI524 occurred during the transduction of the tetracycline resistance plasmid pSN1 from a methicillin-resistant donor into a recipient that carried the mec allele in the primary site on the chromosome. Insertion of Tn4291 caused extensive rearrangement of pI524 and resulted in the formation of a 27.9-kilobase-pair plasmid (pIT103) which coded for resistance to methicillin and cadmium, but not penicillin. Although resistance to methicillin and cadmium were always linked, Tn4291 was stably maintained only in the presence of a chromosomal mec allele, while in its absence the plasmid was unstable and transposition to the primary site occurred. Subsequently, a 20.1-kilobase-pair plasmid, pIT203, was formed which retained cadmium resistance and regained the ability to express beta-lactamase activity.