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The purpose of this study was to compare the microbial profiles on the tongue dorsa of healthy subjects and subjects with halitosis using polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE). Tongue dorsum scrapings from five healthy subjects and six subjects with halitosis were analyzed by PCR-DGGE using primers specific for the V6 to V8 region of the eubacterial 16S rRNA gene. A dendrogram was derived from the PCR-DGGE fingerprints and the prominent DGGE bands of interest were identified through DNA sequencing. The similarity of the PCR-DGGE fingerprints was determined using the unweighted pair group method with an arithmetic mean dendrogram derived using Dice's Coefficient of Similarity. The sequence of PCR amplicons indicated that the microbial species most associated with halitosis wereHaemophilus parainfluenzae and a phylotype of Lachnospiraceae (Lachnospiraceaegenomosp. C1), whereas Streptococcus salivarius subsp. salivarius, Neisseria mucosaand Neisseria cinerea were species that did not appear to be associated with halitosis and are likely part of the healthy tongue flora. These results suggest that the presence ofH. parainfluenzae and Lachnospiraceae genomosp. C1 may be associated with a shift in the balance of oral microbes in subjects with halitosis. Key words: Polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE), 16S rRNA, halitosis, microbial profiles.

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.

The authors conducted this study to determine the etiologic agents of conjunctivitis in early infancy. From 1985 to 1990, 630 infants enrolled in a randomized, controlled, double-masked study of eye prophylaxis were observed for 60 days after delivery for signs of conjunctivitis. The following isolates were categorized as pathogens: Haemophilus influenzae, Streptococcus pneumoniae, Neisseria cinerea, Klebsiella pneumoniae, and Chlamydia trachomatis. Using conditional logistic regression for analysis of 97 infant pairs, the authors identified isolates categorized as pathogens almost exclusively among cases (odds ratio (OR) = 18.0, 95% confidence interval (CI) 2.3-128). Among the microorganisms which have not usually been regarded as pathogens in the etiology of infant conjunctivitis, Streptococcus mitis was the only microorganism associated with an increased risk of conjunctivitis (OR = 5.3, 95% CI 1.8-15.0). The findings concerning the species of bacteria most often associated with conjunctivitis, as well as the finding that method of delivery is unimportant, suggest that bacteria were transmitted to the infants' eyes after birth and not from the birth canal.

Neisseria gonorrhoeae strains with reduced susceptibility to cefixime and ceftriaxone, with minimum inhibitory concentrations (MICs) of cefixime of 0.125-0.25 microg/ml and ceftriaxone of 0.031-0.125 microg/ml, were isolated from male urethritis patients in Tokyo, Japan, in 2006. The amino acid sequences of PenA, penicillin-binding protein 2, in these strains were of two types: PenA mosaic and nonmosaic strains. In the PenA mosaic strain, some regions in the transpeptidase-encoding domain in PenA were similar to those of Neisseria perflava/sicca, Neisseria cinerea, Neisseria flavescens, Neisseria polysaccharea, and Neisseria meningitidis. In the PenA nonmosaic strain, there was a mutation of Ala-501 to Val in PenA. In addition, we performed homology modeling of PenA wild-type and mosaic strains and compared them. The results of the modeling studies suggested that reduced susceptibility to cephems such as cefixime and ceftriaxone is due to a conformational alteration of the beta-lactam-binding pocket. These results also indicated that the mosaic structures and the above point mutation in PenA make a major contribution to the reduced susceptibility to cephem antibiotics.

To better understand Neisseria meningitidis genomes and virulence, microarray comparative genome hybridization (mCGH) data were collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491 and FAM18, and N. gonorrhoeae FA1090. By comparing hybridization data to genome sequences, the core N. meningitidis genome and insertions/deletions (e.g. capsule locus, type I secretion system) related to pathogenicity were identified, including further characterization of the capsule locus, bioinformatics analysis of a type I secretion system, and identification of some metabolic pathways associated with intracellular survival in pathogens. Hybridization data clustered meningococcal isolates from similar clonal complexes that were distinguished by the differential presence of six distinct islands of horizontal transfer. Several of these islands contained prophage or other mobile elements, including a novel prophage and a transposon carrying portions of a type I secretion system. Acquisition of some genetic islands appears to have occurred in multiple lineages, including transfer between N. lactamica and N. meningitidis. However, island acquisition occurs infrequently, such that the genomic-level relationship is not obscured within clonal complexes. The N. meningitidis genome is characterized by the horizontal acquisition of multiple genetic islands; the study of these islands reveals important sets of genes varying between isolates and likely to be related to pathogenicity.

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.

Background Horizontal gene transfer is an important source of genetic variation among Neisseria species and has contributed to the spread of resistance to penicillin and sulfonamide drugs in the pathogen Neisseria meningitidis. Sulfonamide resistance in Neisseria meningitidis is mediated by altered chromosomal folP genes. At least some folP alleles conferring resistance have been horizontally acquired from other species, presumably from commensal Neisseriae. In this work, the DNA sequence surrounding folP in commensal Neisseria species was determined and compared to corresponding regions in pathogenic Neisseriae, in order to elucidate the potential for inter-species DNA transfer within this region. Results The upstream region of folP displayed differences in gene order between species, including an insertion of a complete Correia element in Neisseria lactamica and an inversion of a larger genomic segment in Neisseria sicca, Neisseria subflava and Neisseria mucosa. The latter species also had DNA uptake signal sequences (DUS) in this region that were one base different from the DUS in pathogenic Neisseriae. Another interesting finding was evidence of a horizontal transfer event from Neisseria lactamica or Neisseria cinerea that introduced a novel folP allele to the meningococcal population. Conclusion Genetic recombination events immediately upstream of folP and horizontal transfer have resulted in sequence differences in the folP region between the Neisseria species. This variability could be a consequence of the selective pressure on this region exerted by the use of sulfonamide drugs.

Mast cells often are found in a perivascular location but especially in mucosae, where they may response to various stimuli. They typically associate with immediate hypersensitive responses and are likely to play a critical role in host defense. In this chapter, a common airway pathogen, Moraxella catarrhalis, and a commensal bacterium, Neiserria cinerea, are used to illustrate activation of human mast cells. A human mast cell line (HMC-1) derived from a patient with mast cell leukemia was activated with varying concentrations of heat-killed bacteria. Active aggregation of bacteria over mast cell surfaces was detected by scanning electron microscopy. The activation of mast cells was analyzed by nuclear factor-kappaB (NF-kappaB) activation and cytokine production in culture supernatants. Both M. catarrhalis and N. cinerea induce mast cell activation and the secretion of two key inflammatory cytokines, interleukin-6 and MCP-1. This is accompanied by NF-kappaB activation. Direct bacterial contact with mast cells appears to be essential for this activation because neither cell-free bacterial supernatants nor bacterial lipopolysaccharide induce cytokine secretion.

The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes – nmb0278, nmb0294 and nmb0407 – encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.

Neisseria gonorrhoeae, first described by Neisser in 1879, is a Gram-negative, nonmotile, nonspore-forming diplococcus, belonging to the family Neisseriaceae. It is the etiologic agent of gonorrhea. The other pathogenic species is Neisseria meningitidis, to which N. gonorrhoeae is genetically closely related. Although N. meningitidis is not usually considered to be a sexually transmitted disease, it may infect the mucous membranes of the anogenital area of homosexual men (1). The other members of the genus, which include Neisseria lactamic a, Neisseriapolysaccharea, Neisseria cinerea, and Neisseria flavescens, which are related to Neisseria gonorrhoeae, and saccharolytic strains, such as Neisseria subflava, Neisseria sicca, and Neisseria mucosa, which are less genetically related to the aforementioned, are considered to be nonpathogenic, being normal flora of the nasopharyngeal mucous membranes (2).

The availability of new polysaccharide-protein conjugate vaccines against Neisseria meningitidis serogroup C prompted European National Health authorities to carefully monitor isolate characteristics. In Italy, during 1999–2001, the average incidence was 0.4 cases per 100,000 inhabitants. Serogroup B was predominant and accounted for 75% of the isolates, followed by serogroup C with 24%. Serogroup C was isolated almost twice as frequently in cases of septicemia than in cases of meningitis, and the most common phenotypes were C: 2a:P1.5 and C:2b:P1.5. Among serogroup B meningococci, the trend of predominant phenotypes has changed from year to year, with a recent increase in the frequency of B:15:P1.4. Only a few meningococci had decreased susceptibility to penicillin, and, in the penA gene, all of these strains had exogenous DNA blocks deriving from the DNA of commensal Neisseria flavescens, Neisseria cinerea, and Neisseria perflava/sicca. Fluorescent amplified fragment–length polymorphism analysis revealed the nonclonal nature of the strains with decreased susceptibility to penicillin. Infections caused by Neisseria meningitidis are still an important public health problem because of the severity and highly contagious nature of the disease. The level of interest related to the issue can be inferred from the number of reports published over the years and from

We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ Ng encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZNg is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ Ng on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZNg was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ Ng gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZNg-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZNg-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize ¶at the middle of the cell. Expression of the FtsZNg-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZNg-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector.

Twenty-two strains of Neisseria cinerea were recovered from paediatric patients over a 7-year period and forwarded to the Microbial Diseases Laboratory for biochemical identification and/or confirmation. Eighteen of these 22 strains (82%) were recovered from the eyes of very young children (≤1 year), >50% occurring during the neonatal period. The majority of eye isolates were involved in a variety of ocular infections including orbital cellulitis, conjunctivitis, and eye discharge (most common); in four of the 13 instances (31%) where laboratory data was available, Neisseria cinerea was recovered in pure culture. Neisseria cinerea isolates were often submitted to the Microbial Diseases Laboratory as possible ‘ N. gonorrhoeae ' or ‘ Neisseria species ' due to problems resulting from the use of commercial assays or unfamiliarity with the organism. These observations indicate that N. cinerea can produce eye infections in very young children, who presumably acquire this organism vertically from the mother during birth. Accurate identification of N. cinerea in such infants can preclude the social trauma and possible legal ramifications which can initially result from its misidentification as N. gonorrhoeae .

The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52-expressing gonococci on human neutrophils. Here we show that Opa52-expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src-like protein tyrosine kinases, Rac1 and PAK to Jun-N-terminal kinase. The induced signal is distinct from Fcgamma-receptor-mediated signalling and is specific for Opa52, since piliated Opa- gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src-like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin-independent, Opa52/CD66-mediated phagocytosis of pathogenic Neisseria.

Neisseria cinerea는 1906년 처음 기술되었으나 그 당시에는 Branhamella catarrhalis로 잘못 분류되었고 이후 수회의 명칭 변 경을 거쳐 현재 Neisseria species로 분류되었다. N. cinerea는 주 로 인두구부에 상재하는 비병원성 그람 음성 쌍알균으로 N. gonorrhoeae와 배양적 측면 및 생화학적 측면에서 유사한 특징을 가 진다[1]. 비병원성 균이지만 N. cinerea는 원내 폐렴, 임파선염, 직 장염과 관련이 있다고 보고되어 있고[2-5], N. cinerea에 의한 뇌 수막염은 매우 드물며 국내에서 보고된 사례는 없었다. 급성 세 균성 뇌수막염의 원인균은 주로 Streptococcus pneumoniae, N. meningitidis인 것으로 알려져 있다[6-9]. 저자들은 기저질환이 없는 환자가 발열, 두통, 구토 및 의식저하를 주소로 내원하여 N. cinerea에 의한 급성 세균성 뇌수막염 진단하에 항생제 치료 후 완치된 증례를 경험하였기에 국내 첫 증례로 보고하고자 한다.

Neisseria cinerea is bacteria known as non-pathogenic strain. However, in rare cases, it can cause opportunistic infections. Those diseases caused by N. cinerea include neonatal ophthalmia, proctitis, pneumonia, peritonitis in patients with continuous ambulatory peritoneal dialysis, endocarditis and meningitis. In this report, we describe a patient with septic arthritis and skin abscess of finger joints that was caused by N. cinerea . A 27-year-old man visited the hospital due to swelling, redness and pain of proximal interphalangeal joint of the left second finger. After blood culture test, ceftriaxone was administered on admission and debridement was performed the affected joints. N. cinerea was identified in the blood culture. The patient was improved with ceftriaxone.

Micro-organisms commonly present in human saliva and three DSM strains (Helicobacter pylori, Campylobacter jejuni and Neisseria cinerea), which can be isolated from the human gastro-intestinal tract, were assayed in vitro for their capacity to catalyse N-nitrosation of a series of medicinal drugs and other compounds. Following incubation at pH 7.2 in the presence of nitrate (or nitrite) for up to 24 (48) h, the yield of N-nitroso compounds (NOC) was quantified by HPLC equipped with a post-column derivatization device, allowing the sensitive detection of acid-labile and acid-stable NOC. Eleven out of the 23 test compounds underwent bacteria-catalysed nitrosation by salivary bacteria, the yield of the respective nitrosation products varying 800-fold. 4-(Methylamino)antipyrine exhibited the highest rate of nitrosation, followed by dichlofenac > metamizole > piperazine > five other drugs, whilst L-proline and L-thioproline had the lowest nitrosation rate. Ten drugs including aminophenazone, cimetidine and nicotine, did not inhibit bacterial growth, allowing transitory nitrite to be formed, but no N-nitroso derivatives were detected. Three drugs inhibited the proliferation of bacteria and neither nitrite nor any NOC were formed. Using metamizole as an easily nitrosatable precursor, two strains, Campylobacter jejuni and Helicobacter pylori, were shown to catalyse nitrosation in the presence of nitrite at pH 7.2. As compared to Neisseria cinerea used as a nitrosation-proficient control strain, H. pylori was 30-100 times less effective, whilst C. jejuni had intermediary activity. The results of our sensitive nitrosation assay further confirm that bacteria isolated from human sources, possessing nitrate reductase and/or nitrosating enzymes such as cytochrome cd1-nitrite reductase (Calmels et al., Carcinogenesis, 17, 533-536, 1996), can contribute to intragastric nitrosamine formation in the anacidic stomach when nitrosatable precursors from exogenous and endogenous sources are present.

We present an unusual case of recurrent (chronic ambulatory peritoneal dialysis) CAPD-associated peritonitis caused by Neisseria cinerea. Using DNA restriction fragment length polymorphism (RFLP) analysis, we determined that the recurrent infection was caused by reinfection with a different N. cinerea strain rather than relapse with the index strain and that the probable origin of the reinfecting organism was the patient's upper respiratory tract.

Reported here is a case of infective endocarditis caused by the saprophytic species Neisseria cinerea. To the best of our knowledge, this etiology has not been documented in the medical literature previously. The patient was an intravenous drug addict who developed tricuspid endocarditis with lung embolism. The disease was cured after treatment with ampicillin/clavulanate that was changed to ceftriaxone after an embolic event.

Studies of natural populations of Neisseria meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0-0.6% for fbp, 0-1.3% for argF, and 0-3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli 'housekeeping' genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcal argF gene: two regions of the gene differed by greater than 13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by less than 1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.

Branhamella catarrhalis was formerly regarded as a common, essentially harmless inhabitant of the pharynx. This misapprehension was caused, in part, by confusion with another pharyngeal resident, Neisseria cinerea. The two organisms can now be differentiated by the positive reactions of B. catarrhalis in tests for nitrate reduction and hydrolysis of tributyrin and DNase. B. catarrhalis is currently recognized as the third most frequent cause of acute otitis media and acute sinusitis in young children. It often causes acute exacerbations of chronic bronchopulmonary disease in older or immunocompromised adults and is incriminated occasionally in meningitis, endocarditis, bacteremia, conjunctivitis, keratitis, and urogenital infections. Virulence-associated factors, such as pili, capsules, outer membrane vesicles, iron acquisition proteins, histamine-synthesizing ability, resistance to the bactericidal action of normal human serum, and binding to the C1q complement component, have been identified in some strains. beta-Lactamase producing strains, first detected in 1976, have risen to approximately 75% worldwide. Thus far, however, practically all American strains of B. catarrhalis remain susceptible to alternative antibiotics. A possible selective advantage of recent isolates is their reportedly heightened tendency for adherence to oropharyngeal cells from patients with chronic bronchopulmonary disease.

Neisseria cinerea is an organism that has only recently been implicated as a human pathogen. In this case, N. cinerea was identified as the cause of ophthalmia neonatorum (conjunctivitis) in a 2-day-old girl.

Deoxyribonucleic acid relatedness studies (S1 nuclease method with DE-81 filters method) indicated that Neisseria gonorrhoeae, N. meningitidis, N. lactamica and "N. polysaccharea" form a single genospecies, in which four subspecies can be delineated. However, from a clinical and practical viewpoint, it seems desirable to maintain N. gonorrhoeae, N. meningitidis, N. lactamica and "N. polysaccharea" as separate species. N. cinerea is a valid species, closely related to N. gonorrhoeae, N. meningitidis, N. lactamica and "N. polysaccharea". These five species were O to 46% related to the other known species of the genus Neisseria.

Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids.

Little is known concerning the mechanism involved in cell contact between the donor and recipient during conjugation in Neisseria gonorrhoeae. The formation of stable mating pairs during conjugation in Escherichia coli appears to require a specific protein as well as LPS in the outer membrane of the recipient cell. To attempt to identify the cell surface components necessary for conjugation in the neisseriae, we began a comparison of the outer membrane of Neisseria cinerea strains that can (Con+) and cannot (Con-) serve as recipients in conjugation with N. gonorrhoeae. There were no differences in outer-membrane protein profiles on SDS-polyacrylamide gel electrophoresis between Con+ and Con- strains that could be correlated with the ability to conjugate. However, whole outer membrane isolated from Con+ strains specifically inhibited conjugation while those from Con- strains did not. Proteolytic cleavage of outer-membrane proteins by trypsin, pronase or alpha-chymotrypsin abolished the inhibitory effect of Con+ outer membranes, suggesting that these outer membranes contained a protease-sensitive protein(s) involved in conjugation. Although periodate oxidation of Con+ outer-membrane carbohydrates did not abolish the inhibitory action of these membranes, purified LPS from both Con+ and Con- strains inhibited conjugation when added at low concentrations. These results suggest that conjugation requires the presence of a specific conjugal receptor that consists of both LPS and one or more outer-membrane proteins. Both Con+ and Con- strains contain the necessary LPS, but only Con+ strains contain the required protein(s).

We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.

We report two cases of bacteremia due to Neisseria cinerea. One was a 2.5-yr-old boy with otitis media and pneumonia, who responded to treatment with amoxicillin. The other was a 47-yr-old man with underlying ethanol abuse who developed severe polymicrobial sepsis due to apparent intraabdominal disease. This man died despite extensive antimicrobial therapy.

Es wurden 30 Stamme asaccharolytischer Neisserien aus dem menschlichen Nasopharynx untersucht. Die Stamme liesen sich in zwei Gruppen einteilen, von denen die eine mitN. catarrhalis, die andere mitN. cinerea identisch ist. Neue Beschreibungen der beiden Arten werden gegeben. Seit ihrer Entdeckung vor der Jahrhundertwende istN. catarrhalis offenbar von einem allgemein verbreiteten zu einem seltenen Keim geworden.

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.

Neisseria cinerea and Neisseria gonorrhoeae may occur at the same body sites and may have similar colony morphologies. Ideally, systems used for rapid identification of N. gonorrhoeae should be able to differentiate N. cinerea from gonococci. We tested seven N. cinerea strains using the Gonochek II (Du Pont Diagnostics), Minitek (BBL Microbiology Systems), RapID-NH (Innovative Diagnostics, Inc.), RIM-N (American Microscan), and Phadebact (Pharmacia Diagnostics) systems. We found that the reactions produced by N. cinerea in Gonochek II, Minitek, and RapID-NH kits could be confused with the results produced by some strains of N. gonorrhoeae. The susceptibility of N. cinerea to colistin, its ability to grow on tryptic soy or Mueller-Hinton agar, and its inability to grow on modified Thayer-Martin medium help differentiate it from gonococci.

Sarkosyl-insoluble outer membrane preparations from Neisseria cinerea strains that can and cannot serve as recipients in conjugation with Neisseria gonorrhoeae were examined by SDS-PAGE. No striking differences existed between outer membrane protein profiles from those strains that can serve as recipients and those that cannot. When whole outer membranes from N. cinerea strains which can act as recipients were added to conjugation mixtures, inhibition of conjugation was observed. Whole outer membranes from N. cinerea strains which cannot act as recipients did not specifically inhibit conjugation. Proteolytic cleavage of outer membranes by trypsin, pronase or α-chymotrypsin completely abolished the inhibitory action of the whole outer membranes from N. cinerea strains which can act as recipients, whereas periodate oxidation did not. These results suggest that N. cinerea strains that can conjugate with N. gonorrhoeae contain a specific component, possibly a protein, in the outer membrane that is not present in the strains unable to serve as recipients in conjugation.

An asaccharolytic, gram-negative, oxidase-positive diplococcus was isolated on Martin-Lewis medium from the cervix of a patient attending an arthritis clinic at Seattle Public Health Hospital, Seattle, Wash. This strain, NRL 32165, did not produce detectable acid from glucose, maltose, sucrose, fructose, mannitol, or lactose in either cystine Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) or modified oxidation-fermentation medium and was identified presumptively as a glucose-negative Neisseria gonorrhoeae strain, but was identified later as Neisseria cinerea on the basis of its biochemical reactions. Nitrate was not reduced, nitrite (0.001%, wt/vol) was reduced, and polysaccharide was not produced from sucrose. Proline, arginine, and cystine-cysteine were required for growth on defined medium. Strain NRL 32165 did not react with antigonococcal protein I monoclonal antibodies and did not produce immunoglobulin A protease. In DNA:DNA homology studies with N. gonorrhoeae NRL 8038 (F62) and N. cinerea type strain NRL 30003, strain NRL 32165 showed 95% homology relative to N. cinerea and 44% homology relative to N. gonorrhoeae. Thus, the identity of strain NRL 32165 was confirmed as N. cinerea (von Lingelsheim 1906) Murray 1939. Of all Neisseria spp., N. cinerea is most likely to be misidentified as a glucose-negative N. gonorrhoeae strain.

Antibodies against the Gc2 serotype determinant of gonococcal lipopolysaccharides (LPS) and antisera against strains of meningococci were tested by ELISA against the Gc2 LPS, and the antibodies examined for inhibition by bacteria of prototype strains of gonococci and meningococci. From one of the anti-meningococcal sera and anti-lactose (anti-lac) type of antibody was isolated. The results showed that antigenic sites belonging to the serotype, variable, and common sets of determinants as defined for gonococcal LPSs, may cross-react with meningococci. The anti-lac antibody combined with all of 34 strains of gonococci, with 41 out of 44 strains of meningococci tested, and with a Neisseria cinerea strain. The anti-lac showed no reactivity with any of a number of other Gram-negative cocci or bacilli examined. The results indicate that LPS from most strains of the pathogenic Neisseria species share a lactosyl moiety, presumably an inner core structure, of similar or identical configuration.

Neisseria lactamica, a common human pharyngeal commensal, contributes to acquired immunity to Neisseria meningitidis. To define the surface antigens shared between these two species, we used monoclonal antibodies (MAbs) to study 35 N. lactamica strains isolated in various parts of the world for cross-reactivity with meningococcal capsules, outer membrane proteins, and lipooligosaccharides (LOS). No N. lactamica strain reacted significantly with MAbs specific for capsular group A, B, C, Y, or W, and we were unable to extract capsular polysaccharide from them. Only 2 of 33 strains reacted weakly with MAbs against class 2 serotype proteins P2b and P2c. None reacted with MAbs specific for meningococcal class 1 protein P1.2 or P1.16 or class 2/3 serotype protein P2a or P15. Most N. lactamica strains (30 of 35) bound one or more of seven LOS-specific MAbs. Two LOS epitopes, defined by MAbs O6B4 and 3F11, that are commonly found on pathogenic Neisseria species were found on 25 of 35 N. lactamica. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the LOS of N. lactamica are composed of multiple components that are physically and antigenically similar to the LOS of pathogenic Neisseria species. Among four other commensal neisserial species, only Neisseria cinerea shared LOS epitopes defined by MAbs O6B4 and 3F11. Previous studies have shown that pharyngeal colonization with N. lactamica induces bactericidal antibodies against the meningococcus. We postulate that shared N. lactamica and meningococcal LOS epitopes may play an important role in the development of natural immunity to the meningococcus.

Growth of Neisseria perflava, Neisseria cinerea, and Neisseria sicca strain Kirkland in media supplemented with sucrose (0.5 to 5.0% w/v) resulted in the formation of giant cells. Response to sucrose was specific in that a variety of other carbohydrates did not mediate giant cell formation. Giant cells appeared only under growth conditions and did not lyse upon transfer to medium lacking sucrose or upon resuspension in hypotonic media. Reversion of giant to normal cells occurred when giant cells were used as inocula and allowed to multiply in media lacking sucrose.

The discovery that penicillinase production in Neisseria gonorrhoeae was plasmid mediated and the spread of the beta-lactamase encoding plasmids in gonococcal isolates since 1976, raise the possibility that a nonpathogenic indigenous bacterium could serve as a reservoir for these plasmids. We initiated studies to define the ability of commensal Neisseria species and Branhamella catarrhalis strains, as well as strains of the pathogen Neisseria meningitidis, to serve as recipients in conjugation with Neisseria gonorrhoeae. We found that with N. gonorrhoeae as the donor, 3 of 5 Neisseria cinerea, 2 of 5 Neisseria flava, 0 of 1 Neisseria flavescens, 1 of 3 Neisseria subflava, 0 of 6 B. catarrhalis, 0 of 7 Neisseria lactamica, 1 of 5 Neisseria mucosa, 1 of 7 Neisseria perflava/sicca, and 0 of 13 N. meningitidis strains gave detectable conjugation frequencies (greater than 10(-8). N. cinerea was the only species found to maintain the gonococcal conjugal plasmid (pLE2451). A N. cinerea transconjugant containing pLE2451 was observed to transfer both the beta-lactamase plasmid and pLE2451 to N. gonorrhoeae at high frequency.

A three-month survey revealed 29 patients at our hospital with symptoms of acute pulmonary infection, from whom Branhamella cartarrhalis was isolated from lower respiratory tract specimens, in 18 cases in pure culture. Approximately 2% of all respiratory tract specimens examined during the period yielded growth of B. catarrhalis. All except one patient suffered from chronic pulmonary disease, notably chronic bronchitis. A phenotypic comparison was made between 55 strains of B. catarrhalis, of which 50 were recent isolates from lower respiratory tract specimens, and 23 Neisseria strains representing Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria cinerea, Neisseria flavescens, Neisseria mucosa, Neisseria pharyngis, and Neisseria lactamica. The morphology of B. catarrhalis colonies is very characteristic, and when the diagnosis is suspected, testing for the ability to hydrolyze tributyrin may confirm it within hours. Ability to produce deoxyribonuclease is another property which differentiates B. catarrhalis from the Neisseria species. Otherwise, the combination of nitrate reduction and failure to produce acid from glucose, maltose, and sucrose establishes the diagnosis.

The Haemophilus-Neisseria identification (HNID) panel (American MicroScan, Sacramento, Calif.) is a 4-h microdilution format system for identification of Haemophilus and Neisseria spp., Branhamella (Moraxella) catarrhalis, and Gardnerella vaginalis. The HNID panel was evaluated by using 423 clinical isolates and stock strains of these organisms, and HNID identifications were compared with those obtained by conventional methods. In addition, 32 isolates representing six genera not included in the HNID data base were tested to determine whether these organisms would produce unique biotype numbers for possible inclusion in the data base. The HNID panel correctly identified 95.3% of 86 Neisseria gonorrhoeae strains, 96% of 25 G. vaginalis strains, and 100% of 28 Neisseria lactamica strains and 48 B. catarrhalis strains. Only 64.7% of 68 Neisseria meningitidis isolates were identified correctly owing to false-negative or equivocal carbohydrate and/or aminopeptidase reactions. Among the Haemophilus spp., 98.8% of 83 H. influenzae strains, 97.1% of 34 H. parainfluenzae strains, and 80% of 15 H. aphrophilus and H. paraphrophilus strains were correctly identified. Eight strains of Neisseria cinerea, a species not included in the data base, produced profiles identical with those for B. catarrhalis and N. gonorrhoeae. Isolates of other species not included in the data base, including Eikenella corrodens, Kingella spp., and Cardiobacterium hominis, produced unique biochemical reaction patterns on the panel. Modification of interpretative criteria for certain tests, expansion of the data base to include other species, and suggestions for additional confirmatory tests will increase the accuracy and utility of the HNID panel.

In pathogenic Neisseria species the type IV pili (Tfp) are of primary importance in host–pathogen interactions. Tfp mediate initial bacterial attachment to cell surfaces and formation of microcolonies via pilus–pilus interactions. Based on genome analysis, many non-pathogenic Neisseria species are predicted to express Tfp, but aside from studies on Neisseria elongata, relatively little is known about the formation and function of pili in these organisms. Here, we have analysed pilin expression and the role of Tfp in Neisseria cinerea. This non-pathogenic species shares a close taxonomic relationship to the pathogen Neisseria meningitidis and also colonizes the human oropharyngeal cavity. Through analysis of non-pathogenic Neisseria genomes we identified two genes with homology to pilE, which encodes the major pilin of N. meningitidis. We show which of the two genes is required for Tfp expression in N. cinerea and that Tfp in this species are required for DNA competence, similar to other Neisseria. However, in contrast to the meningococcus, deletion of the pilin gene did not impact the association of N. cinerea to human epithelial cells, demonstrating that N. cinerea isolates can adhere to human epithelial cells by Tfp-independent mechanisms.