CEACAM is not necessary for Neisseria gonorrhoeae to adhere to and invade female genital epithelial cells.

Neisseria gonorrhoeae has a repertoire of up to 11 opacity-associated (Opa) proteins that are adhesins. Most Opa proteins adhere to CEACAM antigens and when CEACAM molecules are present on the surface of transfected epithelial cells their binding by Opa is thought to induce invasion of these cells by gonococci. In this study, we investigated whether several malignant epithelial cell lines, normal cervical and fallopian tube epithelial cell cultures, as well as normal fallopian tube tissue express several of the CEACAM molecules, and whether gonococci use these molecules for adherence and invasion of these female genital epithelial cells. A primary cervical cell culture and metastatic cervical cell line ME180 both expressed CEACAM as shown by whole cell ELISA and flow cytometry, and increased the surface expression of total CEACAM during incubation with Opa+ gonococci. Opa+ gonococci both adhered to and invaded these cells; CEACAM-specific monoclonal antibody (MAb) partially abolished this interaction. Two primary fallopian epithelial tube cell cultures, a primary cervical cell culture and two malignant cell lines, HEC-1-B and HeLa, did not express CEACAM nor was CEACAM mRNA present. No evidence of either intracellular or secreted extracellular CEACAM was found with HEC-1-B and HeLa cells. Opa+ gonococci both adhered to and invaded CEACAM non-expressing cells; however, Opa+ gonococcal association with these non-expressing cell lines could not be inhibited with CEACAM-specific MAb. These data show that CEACAM is not always expressed on female genital epithelial cells and is not essential for gonococcal adherence and invasion. However, when CEACAM is expressed, Opa+ gonococci exploit it for the adherence to and invasion of these cells.

Neisseria gonorrhoeae coordinately uses Pili and Opa to activate HEC-1-B cell microvilli, which causes engulfment of the gonococci.

This study was undertaken to examine concomitant roles of pili and colony opacity-associated proteins (Opa) in promoting Neisseria gonorrhoeae adherence to and invasion of human endometrial HEC-1-B cells. Adherence of N. gonorrhoeae to cultured HEC-1-B cells was saturable, even though organisms adhered to <50% of the cells. During 4 to 6 h of incubation, adherent mono- and diplococci formed microcolonies on the surfaces of the cells. Microvilli of the HEC-1-B cells adhered by their distal ends to individual cocci within the microcolonies. When the microcolonies grew from isogenic pilus-negative (P-) Opa-, P- Opa+, or P+ Opa- gonococci, microvilli did not elongate, and the colonies were not engulfed. In contrast, the microvilli markedly elongated during exposure to P+ Opa+ gonococci. The microvilli adhered to the organisms along their full lengths and appeared to actively participate in the engulfment of the microcolonies. Internalized microcolonies, with P+ Opa+ gonococci, contained dividing cocci and appeared to be surrounded by cell membrane but were not clearly within vacuoles. In contrast, degenerate individual organisms were within vacuoles. Low doses of chloramphenicol, which inhibits protein synthesis by both prokaryotes and eukaryotes, prevented the microvillar response to and internalization of the P+ Opa+ gonococci; higher doses caused internalization without microvillus activation. Cycloheximide and anisomycin, which inhibit only eukaryotic protein synthesis, caused dose-dependent enhancement of uptake. Cytochalasins reduced engulfment; colchicine had no effect. These results show that gonococci must express both pili and Opa to be engulfed efficiently by HEC-1-B cells.

Characterization of antimicrobial resistance in Streptococcus pyogenes isolates from the San Francisco Bay area of northern California.

During 1994 and 1995, 157 isolates of Streptococcus pyogenes from patients with invasive disease were consecutively collected in the San Francisco Bay area to determine the frequency of antimicrobial resistance. Susceptibility testing was performed according to the guidelines of the National Committee for Clinical Laboratory Standards by the disk method and by broth microdilution. For comparison of susceptibility patterns, an additional 149 strains were randomly collected from patients with pharyngitis. For San Francisco County, 32% of the isolates from invasive-disease-related specimens but only 9% of the isolates from throat cultures from the same period were resistant to erythromycin (P = 0.0007). Alameda County and Contra Costa County had rates of resistance of

Risk factors for repeated gonococcal infections: San Francisco, 1990-1992.

Gonococcal (GC) infections are very common and are sustained by a core group of persons who often have repeated GC infections. Identifying individual risk factors for repeated GC infection is essential so that infection control programs can develop better strategies for decreasing the incidence of GC infection. A case-control study among high-risk persons found that being African American, having previous chlamydia infection, and having less than a high-school education were associated with repeated GC infections. Remarkably, measures of sexual behavior and access to health care were not associated with repeated GC infections. These findings suggest that among high-risk persons, the community prevalence of GC infection is more important in predicting risk for repeated GC infections than individual behavior. Interventions should include continued use of resources in high-prevalence communities and better understanding of the roles social and economic discrimination play in the risk for GC infections.

Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci.

Approximately 75% of coagulase-negative staphylococci are resistant to methicillin, but it is suspected that even more resistance exists that is not detected by standard susceptibility assays. To determine the most accurate assay for measuring resistance, we compared the detection of mecA by PCR with detection by National Committee for Clinical Laboratory Standards methods using oxacillin as the class drug. Strains from 11 species of coagulase-negative staphylococci were selected such that 84% were susceptible by the broth microdilution method. Of 45 mecA-positive strains, 1 strain was unable to express the mecA gene product after induction and was not included in further analyses. For microdilution with 2% NaCl, the disk test without salt, and agar screen containing 4% NaCl plus-6 micrograms of oxacillin per ml, the sensitivities in detecting the 44 mecA-positive strains were 50, 84, and 70%, respectively, at 24 h and 77, 82, and 100%, respectively, at 48 h. The specificities of microdilution, disk, and agar screen in detecting the 97 strains lacking mecA were 100, 89, and 100%, respectively, at 24 h. Only the disk test proved to be less specific at 48 h (81%). Furthermore, for 10 of the mecA-positive strains plus an additional 8 strains subsequently added to the analyses, the MICs were 2 micrograms/ml at 24 h by the broth microdilution method; all 18 strains were positive for mecA by PCR. Thus, an oxacillin MIC of > or = 2 micrograms/ml indicated resistance and is probably a more appropriate breakpoint than the current National Committee for Clinical Laboratory Standards breakpoint of 4 micrograms/ml for coagulase-negative staphylococci. Strains for which MICs are < 2 micrograms/ml may be methicillin resistant and should be verified as susceptible by oxacillin agar screening with incubation for 48 h.

Immunogenicity and evolutionary variability of epitopes within IgA1 protease from serogroup A Neisseria meningitidis.

Five murine epitopes were defined and mapped within IgA1 protease produced by Neisseria meningitidis. Epitopes 1 and 2 were present in IgA1 protease from all strains, and from Neisseria gonorrhoeae. Epitopes 3 through to 5 varied between subgroups of serogroup A meningococci, but have remained constant over decades within the subgroups, except for epitope 4, which changed between 1983 and 1987 during the spread of subgroup III meningococci from Asia to Africa. Binding of monoclonal antibodies to epitopes 1, 4 and 5 neutralized enzymatic function. Human sera containing antibodies to IgA1 protease as a result of natural infection inhibited binding of monoclonal antibodies to epitope 4 but not to the other epitopes.

Comparison of two BACTEC anaerobic culture media for recovery of anaerobic bacteria.

A selection of 22 anaerobic isolates, originally recovered from sterile body sites or wounds, was cultured in parallel in BACTEC anaerobic NR7A and lytic 37A bottles supplemented with volunteer human blood. The overall detection rate was 95% for the 37A medium and 75% for the NR7A medium (P = 0.003). The growth indices were consistently higher in the 37A bottles, and a positive result occurred sooner in the 37A medium in 11 of the 32 bottles positive in both media. In an analysis of patient specimens, the NR7A bottle had a 14% false-positive rate compared with 3% for the 37A bottle. The increase in recovery, coupled with the decrease in false-positive readings, make the lytic 37A bottle more efficacious than the NR7A bottle as an anaerobic culture medium.

Multistrain outbreak of chancroid in San Francisco, 1989-1991.

Restriction fragment length polymorphism (RFLP) and plasmid analyses were used to evaluate an outbreak of Haemophilus ducreyi in San Francisco. Fifty-four cases of culture-confirmed chancroid occurred between May 1989 and May 1991. Of these, 46 (96%) were in men and 35 (65%) were in blacks; the median age of patients was 34 years. Among the 32 isolates submitted for RFLP and plasmid analyses, six different HindIII RFLP patterns were identified. Two RFLP types were found in patients who had recently traveled to Los Angeles, Korea, or El Salvador. Four RFLP types appeared to be acquired locally and were more common among blacks (P = .002), in patients with a history of a sexually transmitted disease (P = .01), and in those who used drugs or exchanged drugs or money for sex (P = .08). The use of RFLP analysis confirmed that this outbreak was associated with multiple strains of H. ducreyi and allowed for the identification of risk factors for locally acquired chancroid.

In vitro model of Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin.

Electron microscopy was used to examine Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin. A clinical H. ducreyi isolate (90-244) adhered in snake-like whorls to the surfaces of cervical carcinoma cells (HeLa 229), endometrial adenocarcinoma cells (HEC-1-B), and human neonatal foreskin fibroblast (HFF) cells. A prototype strain of H. ducreyi (CIP542) adhered in randomly organized clumps on the surfaces of HFF. Strain 90-244 entered HFF and HEC-1-B cells but did not enter HeLa cells. The H. ducreyi in the HFF cells at 2 h were partly surrounded by a membrane consistent with that of a phagocytic vacuole. At 2 h, strain CIP542 was found in interstitial spaces between the HFF cells and also in the cytoplasm of the cells. After 7 and 24 h, both strains of H. ducreyi were found in the large interstitial spaces between the HFF cells, in the cytoplasm, and extracellularly. This model of in vitro H. ducreyi infection of eukaryotic cells will allow for more specific study of factors that determine the virulence of H. ducreyi.

Antibodies against IgA1 protease are stimulated both by clinical disease and asymptomatic carriage of serogroup A Neisseria meningitidis.

IgA1 protease was purified from a strain of serogroup A Neisseria meningitidis subgroup IV-1, representative of bacteria that caused an epidemic of meningococcal meningitis in The Gambia in 1982-1983. ELISAs and immunoblot assays were done using this protease as antigen with paired acute- and convalescent-phase sera from patients from that epidemic and from one in Finland caused by other serogroup A meningococci. Paired sera were also tested from healthy Gambians who were persistent nasopharyngeal carriers, persistent noncarriers, or persons who became carriers after the first serum sample was taken. The results correlated well between the two methods: Antibodies were stimulated by disease or acquisition of carriage, and they remained at a constant level upon continued carriage.

Evaluation of the autoSCAN-W/A rapid system for identification and susceptibility testing of gram-negative fermentative bacilli.

The autoSCAN-Walk-Away (W/A) system for identification and susceptibility testing was evaluated for 400 gram-negative fermentative bacteria by using the API 20E (366 isolates) and/or tube biochemical tests as the reference identification system and a frozen microdilution MIC tray system for susceptibility testing. The W/A system performed well for identification of this group of organisms representing 14 genera and 30 species, showing a sensitivity of 96% and results available in 2 h. Of the 16 misidentifications, 6 were with Serratia liquefaciens. A total of 63 isolates (17%) required further tests to complete the identification, compared with 106 (29%) of the isolates which required additional tests for the API 20E identification. Approximately half (32) of the additional tests with the W/A system were required in order to separate Citrobacter diversus from C. amalonaticus. For susceptibility determinations, the W/A system demonstrated an overall agreement of 93% (4,102 determinations) with 40 major errors (0.98%). However, of the 906 resistant organism-drug combinations in the study, there were 115 very major errors, for a false-susceptibility rate of 12.7% of the resistance determinations. Among these very major errors, 80% occurred with piperacillin and the cephalosporins. The W/A system completed the MIC determinations in 7 h; however, the difficulty in detecting resistance with some antimicrobial agents limited the advantages of the rapid susceptibility testing.

DNA amplification and reverse dot blot hybridization for detection and identification of mycobacteria to the species level in the clinical laboratory.

A method incorporating DNA amplification and reverse dot blot hybridization for the detection and identification of mycobacteria to the species level is described. The amplification procedure allowed for the incorporation of digoxigenin-labeled UTP, which was detected by chemiluminescence, removing the need for radioactivity. Using a set of primers and probes from the gene for the 65-kDa heat shock protein of mycobacteria, previously reported in the literature, the reverse dot blot method correctly identified 12 of the 12 M. tuberculosis isolates and 45 of the 50 M. avium complex isolates. Two of the nonhybridizing M. avium complex isolates were reidentified as M. xenopi. The other three nonhybridizing M. avium complex isolates, which were identified as M. intracellulare, hybridized with the probe for M. tuberculosis, as did two ATCC strains of M. intracellulare. The amplified DNA of M. intracellulare was sequenced, and the sequence was compared with the sequence from M. tuberculosis. The sequence for M. avium differed from M. tuberculosis by 5 of 20 bases. The sequence for M. intracellulare differed from M. tuberculosis by 2 of 20 bases, but this difference did not result in sufficient thermal instability to affect hybridization. The use of chemiluminescence allowed as few as 10(2) CFU to be detected. The format of the assay is readily applicable for implementation in the clinical laboratory.

Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae.

Opas (protein IIs) are a family of surface-exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10-11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserve 5' and 3' regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV-region-mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinical isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3' conserved-region probe reacted with 98% of the strains, and the 5' conserved-region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3-39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7-25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three-quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or one HV2 probe. The data indicate that some genes encoding HV regions of N. gonorrhoeae Opa proteins are widely distributed in nature.

Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane.

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.

Use of a siderophore detection medium, ethylene glycol degradation, and beta-galactosidase activity in the early presumptive differentiation of Nocardia, Rhodococcus, Streptomyces, and rapidly growing Mycobacterium species.

A simplified scheme for the presumptive early identification of Nocardia, Rhodococcus, rapidly growing Mycobacterium, and Streptomyces species is presented. The Nocardia and Streptomyces spp. and the Mycobacterium spp. were positive. The spp. were positive for siderophore activity, but only 25% of the Rhodococcus spp. were positive. The Rhodococcus and Mycobacterium spp. were negative for beta-galactosidase, while the Nocardia and Streptomyces spp. were positive. The Nocardia and Streptomyces spp. and the Mycobacterium spp. were negative for ethylene glycol degradation, while 75% of the Rhodococcus spp. were positive. In combination, these tests were useful for differentiating Mycobacterium, Rhodococcus, and Nocardia species but did not differentiate Nocardia from Streptomyces species.

Modification by sialic acid of Neisseria gonorrhoeae lipooligosaccharide epitope expression in human urethral exudates: an immunoelectron microscopic analysis.

Expression of lipooligosaccharide (LOS) antigenic determinants during human gonococcal infection was studied in secretions from seven men with gonococcal urethritis. Five monoclonal antibodies with distinct gonococcal LOS specificities and an H.8 lipoprotein monoclonal antibody were used in combination with immunogold electron microscopic analysis. The LOS epitope defined by antibody 6B7 was present on all seven strains in secretions and after in vitro growth. Gonococci from six of seven patients, when grown in vitro, expressed the 6B4 LOS epitope. The 6B4 epitope is a Gal beta 1-4-GlcNAc residue, which is immunochemically similar to the precursor of the human erythrocyte i antigen. This epitope was found unmodified on gonococcal LOS in urethral secretions from two patients. The unmodified epitope could not be demonstrated on organisms in five secretions. Neuraminidase digestion exposed the 6B4 epitope on organisms in these secretions and increased the 6B4 epitope density in the two secretions, which contained the unmodified epitope. These studies indicate that in vivo modification by sialylation of gonococcal LOS Gal beta 1-4-GlcNAc residue occurs during human infection.

Opa (protein II) influences gonococcal organization in colonies, surface appearance, size and attachment to human fallopian tube tissues.

Opa-expressing variants of Neisseria gonorrhoeae strain F62-SF and an Opa- variant, all non-piliated, were examined for differences in the interaction of the bacteria within colonies and in attachment to and damage of human fallopian tube mucosa. Expression of certain Opas was associated with the formation of transparent colonies where the bacteria were tightly packed and evenly spaced within the colonies. Expression of other Opas was associated with the formation of opaque colonies where the gonococci were less tightly packed and were unevenly spaced. Distinct differences in the size of the gonococci and in their surface characteristics were dependent upon the Opa being expressed. Certain Opas were associated with gonococci that had significantly larger cross-sectional areas and bigger perimeters. Scanning electron microscopy showed that OpaC- and OpaD-containing variants yielded greater mucosal damage than OpaB-containing and Opa- variants with the least damage caused by the OpaA-containing variant (clumped bacteria from dark opaque friable colonies). The mucosal damage after 60 min incubation included shortening and decreased numbers of microvilli on non-ciliated cells and invagination and sloughing of ciliated cells. Differences in the interactions of gonococci within colonies and in attachment to fallopian tube mucosa and damage to the mucosal cells occurred with different Opa-expressing variants of N. gonorrhoeae strain F62-SF.

Experience with selective reporting of susceptibility to antimicrobial agents.

Experience with a system of selective and hierarchical reporting of susceptibility to antimicrobial agents in the hospital setting is described. In March 1985 and January 1987 the medical records of all patients with susceptibility test results were reviewed; there were a total of 488 susceptibility reports. Antimicrobial therapy was evaluated for appropriateness on the basis of the reported susceptibility test results. Susceptibility reports would not have affected the choice of antimicrobial agents in the majority of cases because patients had already been discharged, infection had not been documented, or appropriate therapy had already been started. In approximately 40% of cases in which susceptibility reports could have influenced prescribing, physicians chose appropriate initial therapy after susceptibility results became available. If only the instances in which susceptibility reports could have influenced prescribing are considered, then therapy was appropriately changed 12.5% of the time in March 1985 and 24.2% of the time in January 1987. Selective reporting of susceptibility to antimicrobial agents should be viewed as an adjunct to, not a substitute for, other interventions to promote appropriate prescribing in cases of infection.

Expression of gonococcal protein II in Escherichia coli by translational fusion.

A protein II (P.II) gene from Neisseria gonorrhoeae was cloned in Escherichia coli and characterized by DNA sequence analysis. As with other reported P.II sequences, this gene contains an ATG initiation codon which is out of frame with respect to the remainder of the P.II amino acid sequence. A translational fusion was constructed in E. coli which linked the P.II sequence to the signal peptide of beta-lactamase. This P.II fusion differs from the gonococcal protein only in the first seven residues at the N terminus. In E. coli, the P.II fusion product exhibits properties analogous to those of P.II in N. gonorrhoeae. The P.II fusion product is a major component of the E. coli outer membrane and it is exposed on the cell surface. The P.II fusion protein also exhibits the heat-modifiable phenotype of gonococcal P.II.