Bacterial hemagglutination by Neisseria gonorrhoeae

Direct bacterial hemagglutination was investigated with 20 clinical isolates of Neisseria gonorrhoeae. The hemagglutination tests were performed by both a macrotechnique with glass slides and a microtechnique with autotrays. Only organisms from form type 1 or 2 colonies caused hemagglutination. There was no statistical difference at a 10% or higher level in hemagglutination powers of type 1 and type 2 organisms, of male urethral and female cervical isolates, and of the eight major human blood types (ABO-Rh). Of seven erythrocyte species tested, only human cells were agglutinated. D-Mannose did not prevent the agglutination. Rabbit antigonococcal serum and high-titer antigonococcal human sera inhibited the hemagglutination. The results suggest the pili are the mediators of hemagglutination and that their specific agglutination of human erythrocytes may be a correlate of their adherence to human mucosal cells in natural infection. Also, although the procedure is presently insensitive, it is possible to detect human antigonococcal antibody by inhibition of direct bacterial hemagglutination.     

The anticomplement activity of Neisseria gonorrhoeae

Bacteria of the species N. gonorrhoeae have anticomplementary activity whose absolute values exceed the level of this activity both in other representatives of the genus Neisseria and in microorganisms of other taxonomic groups found in the microbiocenosis of the reproductive tract. The specificity of the anticomplementary action of N. gonorrhoeae extracellular products with respect to individual components of the complement system, as well as their role in the formation of the state of seroresistance and in the determination of the effectiveness of the interaction of gonococci with neutrophilic phagocytes in the system of opsonic cooperation, have been characterized.     

Persistence of multiple serovars of Neisseria gonorrhoeae

The nm23 gene that encodes nucleoside diphosphate (NDP) kinase has been proposed as a candidate tumor metastasis suppressor in rodent experimental carcinoma models and several types of human carcinomas. The present studies were designed to investigate, by immunohistochemical analysis, whether thyroid tissues express the nm23-H1/NDP kinase and, if so, whether the nm23-H1/NDP kinase expression is correlated to tumor metastasis suppressor potential in thyroid tumors. We found that normal thyroid epithelial cells were stained weakly but homogeneously by mouse monoclonal anti-nm23-H1/NDP kinase antibody. The staining intensity of the nm23-H1/NDP kinase in benign thyroid tumors tended to be weaker than that in normal thyroid tissues. In contrast, a series of malignant thyroid tumors expressed differently the nm23-H1/NDP kinase: the intensity of the nm23-H1/NDP kinase staining was stronger in 22 of 49 malignant tumors (45%), similar in 24 (49%), and weaker in 3 (6%) compared to that in normal tissues. The comparison of the staining intensity of the nm23-H1/NDP kinase in primary lesions of tumors with lymph node metastases and those without metastases revealed no statistically significant difference. In addition, there was also no significant difference in the nm23-H1/NDP kinase staining intensity of primary and metastatic lymph node lesions of tumors with lymph node metastases. These data indicate that the nm23-H1/NDP kinase may not be a good predictive marker for tumor regional metastatic potential in malignant thyroid tumors. We conclude that in thyroid tumors the nm23-H1/NDP kinase expression may be dissociated from metastasis suppressor activity.     

Regression-line analysis of trimethoprim-sulfamethoxazole activity against Neisseria gonorrhoeae

A standardized disk diffusion test was developed and used to test the susceptibility of 102 strains of Neisseria gonorrhoeae to combinations of trimethoprim and sulfamethoxazole (TMP/SMX) by relating zone diameters of inhibition to minimal inhibitory concentrations (MIC's). MIC's for TMP/SMX in ratios of 1:20 ranged from 0.08/1.52 to 2.5/47.5 mug/ml and zones of inhibition ranged from 34 to 10 mm. The coefficient of correlation (r) was -0.75. For comparison, a regression line was similarly calculated for ampicillin. MIC's ranged from 0.02 to 0.32 mug/ml and zones of inhibition ranged from 50 to 31 mm; r was -0.71. With establishment of MIC breakpoints to define the categories, susceptible, intermediate, and resistant, the disk duffusion test would be as reliable for estimating susceptibility of gonococci to TMP/SMX as for estimating susceptiblity to ampicillin.     

Prevalence of multiple antibiotic resistance in Neisseria gonorrhoeae

The susceptibility of 100 strains of Neisseria gonorrhoeae to five antimicrobial agents (penicillin, streptomycin, oxytetracycline, sulphamethoxazole, and gentamicin) was examined. Three strains were resistant to each antimicrobial, fourteen exhibited resistance to three or four of the five compounds tested, and 49 were resistant to a single agent, or to pairs of the selected agents. 34 strains were found to be fully sensitive to all of the compounds tested. It is suggested that resistance to gentamicin and sulphamethoxazole may be determined by a multiple resistance gene. The overall frequency of penicillin resistance was found to be 26-5 per cent (MIC greater than 0-06 mug/ml.), suggesting a significant decrease in resistance since 1968.     

Drift in susceptibility of Neisseria gonorrhoeae to ciprofloxacin and emergence of therapeutic failure

Ciprofloxacin, 500 mg, was introduced as the first-line therapy for gonorrhea at St. Mary's Hospital, London, in 1989, when a surveillance program was initiated to detect the emergence of resistance. Isolates of Neisseria gonorrhoeae from consecutive patients attending the Jefferiss Wing, Genitourinary Medicine Clinic at St. Mary's Hospital, between 1989 and 1997 have been tested for susceptibility to ciprofloxacin by using an agar dilution breakpoint technique. Isolates considered potentially resistant (MIC, >0.12 microg/ml) were further characterized by determination of the MICs of ciprofloxacin, nalidixic acid, and penicillin, auxotyped and serotyped, and screened for mutations in the DNA gyrase gene, gyrA, and the topoisomerase IV gene, parC. A total of 4,875 isolates were tested. While the majority of isolates were highly susceptible (MIC, 0.12 microg/ml); all of these belonged to serogroup B, and NR/IB-1 was the most common auxotype/serovar class. The infections in 14 of the 18 patients were known to be acquired abroad, and 5 were known to result in therapeutic failure. The surveillance program has established that ciprofloxacin is still a highly effective antibiotic against N. gonorrhoeae in this population. However, it has identified a drift in susceptibility which may have resulted from increased usage of ciprofloxacin. High-level resistance has now emerged, although treatment failure is still uncommon.     

Antibiotic susceptibility of Neisseria gonorrhoeae in Trinidad and Tobago

The authors studied the state of humoral immunity in thirty six female individuals with normal pregnancy, 52 ones presenting with mild anemia, 33 with moderately severe, and 14 with severe anemia, as well as in twelve healthy non-pregnant women. The studies were made by trimesters in the time course of pregnancy. Those pregnant with no anemia demonstrated lowering of B-lymphocytes, rise in circulating immune complexes (CIC) as compared to the non-pregnant individuals; no difference in the immunoglobulins content was noted. In anemia of the pregnant women the immunity B-system gets suppressed with progression of anemia, CIG tend to be on the increase, IgM and IgG get augmented during the second and third trimesters of pregnancy. The above changes suggest some inadequacy of humoral immunity in anemia of pregnancy.     

Emergence of in vitro resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Japan

To investigate emerging fluoroquinolone resistance in Neisseria gonorrhoeae isolated in Japan, we compared the in vitro antimicrobial susceptibilities of 79 gonococcal isolates from 1992 through 1993 to 14 fluoroquinolones and 14 other antibiotics with those of 27 isolates from between 1981 and 1984. The MICs at which 90% of the isolates were inhibited by nine fluroquinolones, including norfloxacin, enoxacin, ofloxacin, ciprofloxacin, tosufloxacin, lomefloxacin, fleroxacin, levofloxacin, and sparfloxacin, for isolates from 1992 to 1993 were 8- or 16-fold higher than those for isolates from 1981 to 1984. Furthermore, the MICs at which 90% of the isolates were inhibited by five fluroquinolones, including OPC-17116, T-3761, DU-6859a, AM-1155, and Q-35, that have recently been synthesized but have not yet been introduced for clinical use in Japan for isolates from 1992 to 1993 were also 2- to 16-fold higher than those for isolates from 1981 to 1984. The gonococcal isolates from 1992 to 1993 showed no significant decreases in susceptibility to beta-lactams, tetracyclines, macrolides, and spectinomycin, compared with those for isolates from 1981 to 1984. Our data indicate that the incidence of gonococcal strains with decreased susceptibilities to fluoroquinolones is increasing in Japan.     

Characterization of multiresistant strains of Neisseria gonorrhoeae isolated in Nicaragua

The extensive use of antibiotics in Nicaragua raises concerns about the resulting levels of susceptibility of pathogenic bacteria. This is the first study that characterizes 18 strains of N. gonorrhoeae isolated in Nicaragua (1989), for their antibiotic susceptibility. Strains were predominantly of the auxotype/serotype Proto/PIB. There was no difference in lipopolysaccharides profiles obtained after SDS-PAGE for all strains. Variable expression of the PII outer membrane protein was not associated to antimicrobial resistance. All strains were susceptible to ceftriaxone, spectinomycin, rifampin and cefoxitin. The strains were classified in five groups based on plasmid profiles. A total of 78% of the isolates were penicillinase-producing (PPNG) and 22% were tetracycline-resistant N. gonorrhoeae (TRNG). One PPNG strain showed a concomitant decreased of penicillin binding to penicillin-binding protein 2. These randomly chosen isolates of N. gonorrhoeae from Nicaragua possess high levels of resistance to multiple families of drugs.     

Characteristics of atypical Neisseria gonorrhoeae from disseminated and localized infections

Approximately 6% of 1,200 clinical isolates of Neisseria gonorrhoeae were atypical because they produced smaller than normal colonies on conventioal chocolate agar and fermented glucose weakly. Auxotyping studies indicated that these atypical strains required for growth arginine, uracil, and, in most instances, hypoxanthine. In addition, all of them were susceptible to 0.02 U of penicillin/ml. None of the normal colony isolates, including those susceptible to the same low concentration of penicillin, had the same nutritional characteristics. Atypical strains comprised almost half of the isolates from disseminated infections, but only 5% of those from localized infections. Auxotyping was used to identify the contact of a patient who became reinfected nine times with an atypical gonoccal strain. In addition to its usefulness in such epidemiological studies, this technique has enabled us to distinguish a subgroup of gonococci with apparent increased pathogenicity.     

Ligase chain reaction for detection of Neisseria gonorrhoeae in urogenital swabs

The ligase chain reaction (LCR) is an in vitro nucleic acid amplification technique that exponentially amplifies targeted DNA sequences. In a multicenter study, we evaluated the use of a 4-h LCR-based assay for the diagnosis of Neisseria gonorrhoeae infection of the cervix and male urethra. The LCR results were compared with those of culture for N. gonorrhoeae by using selective media. This assay amplifies target sequences within the N. gonorrhoeae opacity gene. Discordant LCR-positive and culture-negative specimens were further evaluated by testing by another LCR assay which used N. gonorrhoeae-specific pilin probe sets. A total of 1,539 female endocervical specimens and 808 male urethral swab specimens were evaluated in the study. An expanded "gold standard" was defined to include all culture-positive as well as culture-negative, confirmed LCR-positive specimens. After resolution of discrepant samples, the sensitivities of the N. gonorrhoeae LCR assays for the female and male specimens were 97.3 and 98.5%, respectively, with specificities of 99.6 and 99.8%, respectively. Resolved culture sensitivities were 83.9 and 96.5% for the female and male specimens, respectively. The LCR assay for gonorrhea is a rapid, highly sensitive nonculture method for detecting gonococcal infection of the cervix and male urethra.     

Identification and functional characterization of the Neisseria gonorrhoeae lbpB gene product

We cloned lbpB, encoding a predicted 80-kDa lipoprotein, upstream of lbpA. A nonpolar mutant (LbpB- LbpA+) had normal lactoferrin (LF) binding and grew normally with LF as an iron source, whereas LbpB- LbpA- and LbpB+ LbpA- strains had reduced binding of LF and did not grow with LF as an iron source. LbpB bound LF directly in an affinity purification, suggesting that LbpB might play a still-uncharacterized role in the LF iron utilization.     

Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.     

Complement processing and immunoglobulin binding to Neisseria gonorrhoeae determined in vitro simulates in vivo effects

Local inflammation elicited by Neisseria gonorrhoeae correlates closely with sensitivity to killing by normal human serum. Serum-sensitive (SS) isolates are rendered resistant in vitro by lipooligosaccharide sialylation. Differences in C3b processing on N. gonorrhoeae in vitro were found to match findings at the cervical level in vivo. Nonsialylated SS gonococci bound 5-fold more C3b than did stably serum-resistant (SR) gonococci; most was processed to iC3b, yet significant C3b persisted. Sialylated SS gonococci bound 4-fold less total C3 antigen than did SR gonococci, which was promptly converted to iC3b. C3b bound later on stably SR gonococci but again was processed swiftly to iC3b. In vivo, the iC3b/C3 ratio of SS isolates more closely resembled nonsialylated SS isolates in vitro, implying heterogeneous sialylation or desialylation in vivo. In vitro, total IgM bound was unchanged by sialylation of SS isolates, but total C4 bound decreased by 75%, suggesting that sialylation may indirectly regulate the classical complement pathway.     

Neisseria gonorrhoeae that infect men have lipooligosaccharides with terminal N-acetyllactosamine repeats

Infectious Neisseria gonorrhoeae make relatively large lipooligosaccharides (LOS) that structurally resemble human glycosphingolipids. MS11mkC is an LOS variant of N. gonorrhoeae strain MS11 which was isolated from men at the onset of dysuria (Schneider, H., Griffiss, J. M., Boslego, J. W., Hitchcock, P. J., Zahos, K. M., and Apicella, M. A. (1991) J. Exp. Med. 174, 1601-1605). Delayed extraction matrix-assisted laser desorption and ionization and electrospray ionization mass spectrometry of O-deacylated MS11mkC LOS produced ions consistent with known LOS which have lacto-N-neotetraose (Galbeta1-->4GlcNAcbeta1-->3Galbeta1-->4Glc; paraglobosyl; monoclonal antibodies (mAbs) 1B2(+) and 06B4(+)) and GalNAc-->lacto-N-neotetraose (gangliosyl; mAb 1-1-M+) oligosaccharides. Ion peaks for a larger LOS which also bound mAb 1B2 indicated the addition of a hexose (+162 Da) to gangliosyl LOS or the addition of a hexose and a N-acetylhexosamine (+365 Da) to paraglobosyl LOS. Analysis of HF-treated and O-deacylated LOS revealed three major components present in a phosphoethanolamine (PEA)0 and a PEA1 series. Digestion of MS11mkC LOS by beta-N-acetylhexosaminidase and beta-galactosidase, alone and sequentially, combined with mAb binding patterns, confirmed the presence of a nonreducing terminal repeating LacNAc ((Galbeta1-->4GlcNAc)2) on the largest LOS, rather than a parallel oligosaccharide structure.     

Roles of PilC and PilE proteins in pilus-mediated adherence of Neisseria gonorrhoeae and Neisseria meningitidis to human erythrocytes and endothelial and epithelial cells

Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from Escherichia coli interacted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated Neisseria gonorrhoeae and Neisseria meningitidis to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type N. gonorrhoeae also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type N. gonorrhoeae, the piliated PilC-deficient mutant, and N. gonorrhoeae pili assembled in Pseudomonas aeruginosa agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that N. gonorrhoeae PilE is responsible for pilus-mediated hemagglutination.