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Commensal non-pathogenicNeisseriaspp. live within the human host alongside the pathogenicNeisseria meningitidisandNeisseria gonorrhoeaeand due to natural competence, horizontal gene transfer within the genus is possible and has been observed. Four distinctNeisseriaspp. isolates taken from the throats of two human volunteers have been assessed here using a combination of microbiological and bioinformatics techniques. Three of the isolates have been identified asNeisseria subflavabiovarperflavaand one asNeisseria cinerea. Specific gene clusters have been identified within these commensal isolate genome sequences that are believed to encode a Type VI Secretion System, a newly identified CRISPR system, a Type IV Secretion System unlike that in otherNeisseriaspp., a hemin transporter, and a haem acquisition and utilization system. This investigation is the first to investigate these systems in either the non-pathogenic or pathogenicNeisseriaspp. In addition, theN. subflavabiovarperflavapossess previously unreported capsule loci and sequences have been identified in all four isolates that are similar to genes seen within the pathogens that are associated with virulence. These data from the four commensal isolates provide further evidence for aNeisseriaspp. gene pool and highlight the presence of systems within the commensals with functions still to be explored.

The present study highlights challenges in the veterinary microbiology diagnostic laboratory in the identification of bacteria responsible for infections in veterinary settings, particularly when evidence-based data is lacking. A 1.8-year-old neutered male domestic cat (FIV/FeLV negative) was presented to a veterinary practice in April 2016 with a history of left unilateral mild conjunctivitis that was empirically treated with fusidic acid and chloramphenicol. In January 2017, the same animal was presented with chronic left unilateral conjunctivitis and an eye swab was submitted for microbiological culture and susceptibility testing. Significant growth was not detected in two samples tested. Finally, in February 2017 another eye swab produced a slow growing pure culture identified by VITEK 2 as Neisseria cinerea (94 % confidence). Given the morphology and multidrug resistance profile of the isolate a 16S rRNA PCR was performed for definitive identification. The nucleotide sequence of the PCR amplicon was 99 % homologous to Acinetobacter equi sp. nov. strain 114. Veterinary microbiology diagnostic laboratories play an important role worldwide, not only in preserving animal health and welfare but also in controlling the spread of zoonotic pathogens. The lack of evidence-based information on the ocular microbiome of healthy cats and the complexity of bacterial ecosystems renders the interpretation of results difficult. A further problem for both the laboratory and the clinician is the lack of interpretive criteria for antibiotic susceptibility test results for some types of infections in animals (including those caused by Acinetobacter ) and the complete unavailability of criteria for topical antibiotic preparations.

SinceNeisseria gonorrhoeaeandNeisseria meningitidisare obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensalNeisseriagenome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. OnlyNeisseria mucosacontained a nitrate reductase (Nar), and onlyNeisseria lactamica,Neisseria cinerea,Neisseria subflava,Neisseria flavescensandNeisseria siccacontained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested exceptN. mucosahad a two- to fourfold lower nitrite reductase activity thanN. gonorrhoeae, whileN. meningitidisand most of the commensalNeisseriaspecies had a two- to fourfold higher nitric oxide (NO) reductase activity. ForN. meningitidisand most of the commensalNeisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared withN. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that ofN. gonorrhoeae. The greatest difference between theNeisseriaspecies was the lack of a functional Nos system in the pathogenic speciesN. gonorrhoeaeandN. meningitidis.

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.