Search

Mosaic penA alleles have caused most of the cephalosporin resistance in Neisseria gonorrhoeae , but their evolution is mostly unknown. The penA gene from Neisseria cinerea strain AM1601 (ceftriaxone MIC, 1.0 μg/ml) caused ceftriaxone resistance (MIC, 1 μg/ml) in a ceftriaxone-susceptible gonococcal strain. The 3′-terminal half of AM1601 penA was almost identical to that of the ceftriaxone-resistant gonococcal GU140106 and FC428 strains. N. cinerea can serve as a reservoir of ceftriaxone resistance-mediating penA sequences that can be transferred to gonococci.

Neisseria cinerea is a human commensal. The first known case of meningitis and bacteremia due to Neisseria cinerea following percutaneous glycerol instillation of the trigeminal ganglion is reported. Conventional phenotypic methods and complete 16S RNA gene sequencing accurately identified the pathogen. Difficulties in differentiation from pathogenic neisseriae are discussed.

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Capsular polysaccharide vaccines are available against meningococcal serogroups A, C, W and Y. More recently two protein based vaccines, Bexsero® and Trumenba®, have been licenced against meningococcal serogroup B strains; both vaccines contain meningococcal factor H binding protein (fHbp). fHbp is a surface exposed lipoprotein which binds the negative complement regulator, complement factor H (CFH), thereby inhibiting the alternative pathway of complement activation. Recent analysis of available genomes has indicated that some commensal Neisseria species also contain genes that potentially encode fHbp, although the function of these genes and how immunisation with fHbp-containing vaccines could affect the commensal flora have yet to be established. Here we show that the commensal species Neisseria cinerea expresses functional fHbp on its surface and is responsible for recruitment of CFH by the bacterium. N. cinerea fHbp binds CFH at similar affinity as meningococcal fHbp, and promotes survival of N. cinerea in human serum. We examined the potential impact of fHbp-containing vaccines on N. cinerea We found that immunisation with Bexsero® elicits serum bactericidal activity against N. cinerea, which is primarily directed against fHbp. The shared function of fHbp in N. cinerea and N. meningitidis, and cross reactive responses elicited by Bexsero® suggest that the introduction of fHbp-containing vaccines has the potential to affect carriage of N. cinerea and other commensal species.

In Neisseria gonorrhoeae , the mosaic structure of the penA gene (encoding penicillin-binding protein 2 [PBP 2]), which is composed of fragments of the penA genes from Neisseria cinerea and Neisseria perflava , has been significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to develop a rapid assay for the detection of mosaic PBP 2 of N. gonorrhoeae by real-time PCR. This assay successfully detected the mosaic penA gene of N. gonorrhoeae , and its sensitivity was ≥10 1 copies/reaction. Six hundred twenty-one clinical strains were examined by this assay for the presence of mosaic PBP 2, which was detected in 85 (39.4%) of 216 strains from 2002, 69 (40.6%) of 170 strains from 2003, 71 (44.4%) of 160 strains from 2004, and 31 (41.3%) of 75 strains from 2005. The MICs of cephalosporins for strains with the mosaic PBP 2 detected by the assay were statistically higher than those for strains without the mosaic PBP 2. One hundred sixty-six (64.8%) of 256 strains with the mosaic PBP 2 exhibited cefixime MICs of ≥0.5 μg/ml. The emergence and spread of strains with mosaic PBP 2 could be a threat to the cefixime treatment of gonorrhea. This real-time PCR assay for the detection of mosaic PBP 2 of N. gonorrhoeae is thus useful in the prediction of decreased susceptibilities to oral cephalosporins.

Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 μg/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene ( penA ) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 μg/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava ( N. sicca ), Neisseria cinerea , Neisseria flavescens , and Neisseria meningitidis . These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae .

The COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae cross-reacts with certain strains of nonpathogenic Neisseria species. In some strains, the target sequence is identical to that of N. gonorrhoeae, whereas other strains have a small number of mismatches within the regions recognized by the primers or probe used in the COBAS AMPLICOR NG test. These cross-reactive strains are occasionally present in urogenital specimens, causing false-positive results in the COBAS AMPLICOR NG test. Analysis of the data generated in a large multicenter clinical trial showed that 2.9% of the specimens gave signals between A660s of 0.2 and 3.5 but that one-half of these equivocal specimens did not contain N. gonorrhoeae. Most of these equivocal specimens were correctly classified as true positive or true negative by retesting in duplicate and defining a PCR-positive result as two of three results with an A660 of >2.0. If specimens had been classified as positive or negative based on a single test result using a cutoff of an A660 of 0.2, specificity would have ranged from 96.2 to 98.9% depending on specimen type, sex, and presence of symptoms. By employing the equivocal zoneretesting algorithm, specificity increased to 98.6 to 99.9% with little effect (0.1 to 4.9% decrease) on sensitivity in most specimen types, enabling the test to achieve a positive predictive value of at least 90% in populations with a prevalence of 4% or higher. In lower-prevalence populations, the test could be used to screen for presumptive infections that would have to be confirmed by an independent test. The COBAS AMPLICOR CT/NG test provides a powerful diagnostic tool for screening for both chlamydial and gonococcal infections. We and others have observed that the NG portion of the test (COBAS AMPLICOR NG) cross-reacts with some isolates of certain nonpathogenic Neisseria species (4). When the original, recommended cutoff (A660 of 0.2) is used, the COBAS AMPLICOR NG test can produce false-positive results for Neisseria gonorrhoeae, presumably due to the presence of cross-reactive Neisseria species in some urogenital specimens. In one population, approximately 26% of COBAS AMPLICOR NG-positive results were false positives, which corresponded to approximately 3% of the total population (4). In contrast, the same laboratory observed fewer than 1% falsepositive results among urogenital specimens from a second population (4). In this paper, we confirm that the test does cross-react with some isolates of Neisseria subflava (4) and Neisseria cinerea and compare the target sequences in these cross-reactive species with that of N. gonorrhoeae. Using data from a multicenter trial (n 4,173 patients; prevalence 13.1%) conducted at six sites in the United States (8), we show how test sensitivity and specificity vary with the cutoff value used. We then assess whether both sensitivity and specificity can be optimized by establishing a large equivocal zone and using an algorithm that involves additional testing of specimens yielding equivocal results. Implementation of the equivocal zone-retesting algorithm identified by this analysis produced good specificity (98.8 to 99.9%) without sacrificing sensitivity in the multicenter trial (8).

A full-length heme oxygenase gene from the gram-negative pathogen Neisseria meningitidis was cloned and expressed in Escherichia coli . Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the E. coli cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen Corynebacterium diphtheriae and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXα and carbon monoxide as the final products. Homologs of the hemO gene were identified and characterized in six commensal Neisseria isolates, Neisseria lactamica , Neisseria subflava , Neisseria flava , Neisseria polysacchareae , Neisseria kochii , and Neisseria cinerea . All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.

Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non- N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence (∼9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

The penicillin-binding protein 2 genes (penA) of penicillin-resistant Neisseria meningitidis have a mosaic structure that has arisen by the introduction of regions from the penA genes of Neisseria flavescens or Neisseria cinerea. Chromosomal DNA from both N. cinerea and N. flavescens could transform a penicillin-susceptible isolate of N. meningitidis to increased resistance to penicillin. With N. flavescens DNA, transformation to resistance was accompanied by the introduction of the N. flavescens penA gene, providing a laboratory demonstration of the interspecies recombinational events that we believe underlie the development of penicillin resistance in many meningococci in nature. Surprisingly, with N. cinerea DNA, the penicillin-resistant transformants did not obtain the N. cinerea penA gene. However, the region of the penA gene derived from N. cinerea in N. meningitidis K196 contained an extra codon (Asp-345A) which was not found in any of the four N. cinerea isolates that we examined and which is known to result in a decrease in the affinity of PBP 2 in gonococci.

Bacterial peritonitis is a well-recognized complication of chronic ambulatory peritoneal dialysis (CAPD) in patients with end-stage renal failure. We present a case of peritonitis due to an unusual pathogen, Neisseria cinerea , unresponsive to the standard intraperitoneal (i.p.) vancomycin and gentamicin, which responded rapidly to oral ciprofloxacin.

An 8-year-old boy developed proctitis. Rectal swabs yielded a Neisseria sp. that was repeatedly identified by API (Analytab Products, Plainview, N.Y.), Minitek (BBL Microbiology Systems, Cockeysville, Md.), and Bactec (Johnston Laboratories, Towson, Md.) methods as Neisseria gonorrhoeae. Subsequent testing in a reference laboratory yielded an identification of Neisseria cinerea. It is suggested that identification of a Neisseria sp. isolated from genital or rectal sites in a child be confirmed by additional serological, growth, and antibiotic susceptibility tests and, if necessary, by a reference laboratory. The implications of such misidentifications are discussed.

We describe what appears to be the first reported case of nosocomial pneumonia caused by Neisseria cinerea. The isolate metabolized glucose when tested in BACTEC Neisseria Differentiation Kits (Johnston Laboratories), but did not produce detectable acid in cystine-Trypticase (BBL Microbiology Systems) agar medium or in modified oxidation-fermentation medium. Clinical laboratories that rely on the BACTEC method for differentiation of pathogenic neisseriae should be aware of the fact that N. cinerea may mimic N. gonorrhoeae when tested in BACTEC Neisseria Differentiation kits. The ability of N. cinerea to grow well on tryptic soy and Mueller-Hinton agars and its inability to grow on modified Thayer-Martin medium are characteristics which help to distinguish N. cinerea from N. gonorrhoeae.

Six strains of Neisseria cinerea were tested in BACTEC Neisseria Differentiation kits (Johnston Laboratories, Inc., Towson, Md.), and all yielded positive glucose growth indices and negative maltose and fructose growth indices. These results were similar to those achieved with Neisseria gonorrhoeae. However, most of the N. cinerea isolates tested yielded 3-h glucose growth indices that were lower than those obtained with gonococci. 14C-labeled gas was produced significantly faster (P less than 0.02) by N. gonorrhoeae than by N. cinerea. Additional studies suggested that the 14C-labeled gas produced by N. cinerea was carbon dioxide. N. cinerea strains were similar to Branhamella catarrhalis strains because both species failed to produce detectable acid from glucose, maltose, sucrose, fructose, and lactose in cysteine-tryptic agar media. However, in contrast to N. cinerea strains, B. catarrhalis strains did not metabolize glucose in BACTEC Neisseria Differentiation kits.