Ciprofloxacin Concentrations 100-Fold Lower than the MIC Can Select for Ciprofloxacin Resistance in Neisseria subflava: An In Vitro Study.

Neisseria gonorrhoeae can acquire antimicrobial resistance (AMR) through horizontal gene transfer (HGT) from other Neisseria spp. such as commensals like Neisseria subflava. Low doses of antimicrobials in food could select for AMR in N. subflava, which could then be transferred to N. gonorrhoeae. In this study, we aimed to determine the lowest concentration of ciprofloxacin that can induce ciprofloxacin resistance (minimum selection concentration-MSC) in a N. subflava isolate (ID-Co000790/2, a clinical isolate collected from a previous community study conducted at ITM). In this study, Neisseria subflava was serially passaged on gonococcal (GC) medium agar plates containing ciprofloxacin concentrations ranging from 1:100 to 1:10,000 below its ciprofloxacin MIC (0.006 µg/mL) for 6 days. After 6 days of serial passaging at ciprofloxacin concentrations of 1/100th of the MIC, 24 colonies emerged on the plate containing 0.06 µg/mL ciprofloxacin, which corresponds to the EUCAST breakpoint for N. gonorrhoeae. Their ciprofloxacin MICs were between 0.19 to 0.25 µg/mL, and whole genome sequencing revealed a missense mutation T91I in the gyrA gene, which has previously been found to cause reduced susceptibility to fluoroquinolones. The N. subflava MSCde novo was determined to be 0.06 ng/mL (0.00006 µg/mL), which is 100×-fold lower than the ciprofloxacin MIC. The implications of this finding are that the low concentrations of fluoroquinolones found in certain environmental samples, such as soil, river water, and even the food we eat, may be able to select for ciprofloxacin resistance in N. subflava.

Lack of Association between Antimicrobial Consumption and Antimicrobial Resistance in a HIV Preexposure Prophylaxis Population: A Cross-Sectional Study.

BACKGROUND: In antibiotic naïve populations, there is a strong association between the use of an antimicrobial and resistance to this antimicrobial. Less evidence is available as to whether this relationship is weakened in populations highly exposed to antimicrobials. Individuals taking HIV preexposure prophylaxis (PrEP) have a high intake of antimicrobials. We previously found that there was no difference in the prevalence of pheno- and genotypic antimicrobial resistance between two groups of PrEP clients who had, and had not, taken antimicrobials in the prior 6 months. Both groups did, however, have a higher prevalence of resistance than a sample of the general population. METHODS: In the current study, we used zero-inflated negative binomial regression models to evaluate if there was an individual level association between the consumption of antimicrobials and 1. the minimum inhibitory susceptibilities of oral Neisseria subflava and 2. the abundance of antimicrobial resistance genes in the oropharynges of these individuals. RESULTS: We found no evidence of an association between the consumption of antimicrobials and the minimum inhibitory susceptibilities of oral Neisseria subflava or the abundance of antimicrobial resistance genes in these individuals. CONCLUSIONS: We conclude that in high-antimicrobial-consumption populations, the association between antimicrobial consumption and resistance may be attenuated. This conclusion would not apply to lower-consumption populations.

Successful Intra- but Not Inter-species Recombination of msr(D) in Neisseria subflava.

Resistance acquisition via natural transformation is a common process in the Neisseria genus. Transformation has played an important role in the emergence of resistance to many antimicrobials in Neisseria gonorrhoeae and Neisseria meningitidis. In a previous study, we found that currently circulating isolates of Neisseria subflava had acquired an msr(D) gene that has been found to result in macrolide resistance in other bacteria but never found in Neisseria species before. To determine if this resistance mechanism is transferable among Neisseria species, we assessed if we could transform the msr(D) gene into other commensal and pathogenic Neisseria under low dose azithromycin pressure. Intraspecies recombination in commensal N. subflava was confirmed with PCR and resulted in high-level macrolide resistance. Whole-genome sequencing of these transformed strains identified the complete uptake of the msr(D) integration fragment. Sequence analysis showed that a large fragment of DNA (5 and 12 kb) was transferred through a single horizontal gene transfer event. Furthermore, uptake of the msr(D) gene had no apparent fitness cost. Interspecies transformation of msr(D) from N. subflava to N. gonorrhoeae was, however, not successful.

Confirmation of the Need for Reclassification of Neisseria mucosa and Neisseria sicca Using Average Nucleotide Identity Blast and Phylogenetic Analysis of Whole-Genome Sequencing: Hinted by Clinical Misclassification of a Neisseria mucosa Strain.

The taxonomy of the genus Neisseria remains confusing, particularly regarding Neisseria mucosa and Neisseria sicca. In 2012, ribosomal multi-locus sequence typing reclassified both as N. mucosa, but data concerning 17 N. sicca strains remain available in GenBank. The continuous progress of high-throughput sequencing has facilitated ready accessibility of whole-genome data, promoting vigorous development of average nucleotide identity (ANI) and high-resolution phylogenetic analysis. Here, we report that a Neisseria isolate, which caused native-valve endocarditis and multiple embolic brain infarcts in a patient with congenital heart disease, was misidentified as N. sicca by VITEK MS. This isolate was reclassified as N. mucosa by ANI blast (ANIb) and by phylogenetic analysis using whole-genome data yielded by the PacBio Sequel and Illumina NovaSeq PE150 platforms. The confusion evident in the GenBank and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) databases suggests that N. mucosa (n = 13) and N. sicca (n = 16) in GenBank should be reclassified using ANIb and high-resolution phylogenetic analysis. The whole-genome data of 30 strains (including the clinical isolate) were compared with the data of 27 type Neisseria strains (including one N. sicca and two N. mucosa type strains) as a genomic index. In total, 25 (8 originally identified as N. mucosa and 17 originally identified as N. sicca) and 7 (1 originally identified as N. sicca and 6 originally identified as N. mucosa) strains were reclassified into the N. mucosa and Neisseria subflava groups, respectively; 1 residual N. mucosa strain was reclassified as Neisseria meningitidis. In conclusion, a combination of ANIb and robust phylogenetic analysis reclassified strains originally identified as N. mucosa and N. sicca into (principally) the N. mucosa group and the N. subflava group. The misclassified N. sicca and N. mucosa strains in the GenBank and MALDI-TOF MS databases were supposed to be corrected. Updated genomic classification strategy for originally identified N. mucosa and N. sicca strains was recommended to be adopted in GenBank.

Virulence genes and previously unexplored gene clusters in four commensal Neisseria spp. isolated from the human throat expand the neisserial gene repertoire.

Commensal non-pathogenic Neisseria spp. live within the human host alongside the pathogenic Neisseria meningitidis and Neisseria gonorrhoeae and due to natural competence, horizontal gene transfer within the genus is possible and has been observed. Four distinct Neisseria spp. 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 as Neisseria subflava biovar perflava and one as Neisseria 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 other Neisseria spp., 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 pathogenic Neisseria spp. In addition, the N. subflava biovar perflava possess 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 a Neisseria spp. gene pool and highlight the presence of systems within the commensals with functions still to be explored.

Production and characterization of low-calorie turanose and digestion-resistant starch by an amylosucrase from Neisseria subflava.

This study was intended to produce turanose and resistant starch (RS) using recombinant amylosucrase from Neisseria subflava (NsAS). Turanose production yield maximally reached to 76% of sucrose substrate at 40 °C by NsAS treatment. To evaluate turanose as a low-calorie functional sweetener, its hydrolysis pattern was investigated in continuous artificial digestion system. When turanose was consecutively exposed through small intestinal phase, only 8% of disaccharide was hydrolyzed. Structural modification of gelatinized corn or rice starch was carried out by NsAS with sucrose as a glucosyl donor. Non-digestibility of enzyme-modified starches increased to 47.3% maximally through branch-chain elongation, enough for chain-chain association and recrystallization. Obviously, NsAS-modified starches had higher gelatinization peak temperatures than native counterparts, and their paste viscosity was inversely related to their digestibility due to elongated-chain induced retrogradation. These results suggested that NsAS could be a vital biocatalyst candidate in food industry to produce next generation low-calorie carbohydrate food materials.

Unusual Neisseria species as a cause of infection in patients taking eculizumab.

BACKGROUND: Non-meningococcal, non-gonococcal Neisseria spp. are typically commensal and rarely cause invasive disease. Eculizumab is a terminal complement inhibitor that increases susceptibility to meningococcal disease, but data on disease caused by typically-commensal Neisseria spp. are lacking. This series describes postmarketing reports of typically-commensal Neisseria spp. disease in patients receiving eculizumab. METHODS: We searched the FDA Adverse Event Reporting System (FAERS) and medical literature for reports of commensal Neisseria spp. disease in patients receiving eculizumab, from eculizumab U.S. approval (2007) through January 31, 2018. RESULTS: We identified seven FAERS reports (including one case also reported in the literature) of non-meningococcal, non-gonococcal Neisseria disease, including N. sicca (mucosa)/subflava (n = 2), N. cinerea (n = 2), N. sicca (mucosa) (n = 1), N. mucosa (n = 1, with concurrent alpha-hemolytic Streptococcus bacteremia), and N. flavescens (subflava) (n = 1). Four cases had sources of patient immunosuppression in addition to eculizumab. Three patients had sepsis (n = 2) or septic shock (n = 1). Five patients were bacteremic. All patients were hospitalized; the infections resolved with antibiotics. CONCLUSIONS: Our search identified seven cases of disease from typically commensal Neisseria spp. in eculizumab recipients. These findings suggest that any Neisseria spp. identified from a normally sterile site in an eculizumab recipient could represent true infection warranting prompt treatment.

Meningitis due to a Combination of Streptococcus mitis and Neisseria subflava: A Case Report.

We report a rare case of meningitis due to a combination of Streptococcus mitis and Neisseria subflava. An 80-year-old female had a 4-year history of type II diabetes mellitus (DM) and an 11-year history of rheumatoid arthritis, which was treated with prednisolone, tacrolimus, and methotrexate. One month after the removal of a dental implant, she complained of a disturbance of consciousness and suffered a convulsion. A cerebrospinal fluid culture was found to be positive for both S. mitis and N. subflava. After 14 days of antibiotic treatment with 4 g/day ceftriaxone, her stiff neck, somnolence, and laboratory data greatly improved, and she was successfully discharged at 27 days after admission. Although both S. mitis and N. subflava are generally considered to be benign bacteria, they can cause meningitis in patients with the following risk factors: older age, on immunosuppressive treatment, DM, or dental treatment.

Expression, purification, and characterization of a novel amylosucrase from Neisseria subflava.

Amylosucrase (ASase) is a glucosyltransferase, which catalyzes the de novo synthesis of amylose-like polymers from sucrose. In the present study, ASase from Neisseria subflava (NsAS) was cloned, sequenced, and expressed in Escherichia coli. The production of NsAS was achieved by inducting gene expression with 0.2 mM isopropyl-ß-d-thiogalactopyranoside. The molecular mass of the Ni-NTA column purified NsAS analyzed by SDS-PAGE was determined to be 72 kDa. NsAS exhibited maximal activity at 45 °C and pH 8.0, and showed strong thermal stability at 40 °C with a half-life of 385 h. The reaction pattern of NsAS at [sucrose] range of 0.1-1.0 M showed that at 0.7 M of [sucrose], the production yield of insoluble linear α-(1,4)-glucans reached 24% maximum, and any further increase in [sucrose] resulted in a slight decrease in yield. Meanwhile, the production yield of turanose significantly increased from 16 to 29% by increasing [sucrose] from 0.1 to 1.0 M. The synthesized glucan had degrees of polymerization (DP); for 0.1, 0.4, 0.7, and 1.0 M sucrose, the DP values were 77, 49, 39, and 31 respectively. These results suggested that NsAS would be a promising candidate for food industrial production of linear α-(1,4)-glucans and turanose as a next generation sweetener.

Bacteriemia por Neisseria subflava en un recién nacido / Neisseria subflava bacteremia in newborns: case report and review of the literature

Resumen Neisseria subflava es una especie considerada comensal dentro de la familia Neisseriaceae; sin embargo, en algunos hospederos inmunocomprometidos y en niños se ha documentado como agente causal de infecciones invasoras. Se presenta el caso clínico de un recién nacido con bacteriemia por N. subflava con buena respuesta a terapia con cefotaxima. Las bacterias del género Neisseria más frecuentes de causar infecciones en recién nacidos son N. meningitidis, de alta mortalidad y N. gonorrhoeae, que se manifiesta principalmente con oftalmia neonatorum. Se destaca la importancia de realizar un diagnóstico microbiológico adecuado dado que las pruebas biquímicas pueden ser no concluyentes. La técnica de espectrometría de masas MALDITOF resulta una herramienta de utilidad.

Neisseria subflava belongs to Neisseriaceae family, is considered a comensal specie, however in certain host, mainly inmunosuppresed patientes and children, the literature has documented invasive infections. We present a case of a bacteriemia due to N. subflava in a newborn, treated with cefotaxime with good outcome. In newborns, the most common Neisseria bacteria to cause invasive infections are N. meningitidis, with highly fatal clinical course and N. gonorrhoeae which compromise the eye, oftalmia neonatorum, with uncommon invasive infections. It's very important the adequate microbiological diagnosis because the biochemical tests may be inconclusive. MALDITOF mass spectrometry technique is a useful tool.