Diversity of the type VI secretion systems in the Neisseria spp.

Complete Type VI Secretion Systems were identified in the genome sequence data of Neisseria subflava isolates sourced from throat swabs of human volunteers. The previous report was the first to describe two complete Type VI Secretion Systems in these isolates, both of which were distinct in terms of their gene organization and sequence homology. Since publication of the first report, Type VI Secretion System subtypes have been identified in Neisseria spp. The characteristics of each type in N. subflava are further investigated here and in the context of the other Neisseria spp., including identification of the lineages containing the different types and subtypes. Type VI Secretion Systems use VgrG for delivery of toxin effector proteins; several copies of vgrG and associated effector / immunity pairs are present in Neisseria spp. Based on sequence similarity between strains and species, these core Type VI Secretion System genes, vgrG, and effector / immunity genes may diversify via horizontal gene transfer, an instrument for gene acquisition and repair in Neisseria spp.

Bacterial Adaptation to Venom in Snakes and Arachnida.

Animal venoms are considered sterile sources of antimicrobial compounds with strong membrane-disrupting activity against multidrug-resistant bacteria. However, venomous bite wound infections are common in developing nations. Investigating the envenomation organ and venom microbiota of five snake and two spider species, we observed venom community structures that depend on the host venomous animal species and evidenced recovery of viable microorganisms from black-necked spitting cobra (Naja nigricollis) and Indian ornamental tarantula (Poecilotheria regalis) venoms. Among the bacterial isolates recovered from N. nigricollis, we identified two venom-resistant, novel sequence types of Enterococcus faecalis whose genomes feature 16 virulence genes, indicating infectious potential, and 45 additional genes, nearly half of which improve bacterial membrane integrity. Our findings challenge the dogma of venom sterility and indicate an increased primary infection risk in the clinical management of venomous animal bite wounds. IMPORTANCE Notwithstanding their 3 to 5% mortality, the 2.7 million envenomation-related injuries occurring annually-predominantly across Africa, Asia, and Latin America-are also major causes of morbidity. Venom toxin-damaged tissue will develop infections in some 75% of envenomation victims, with E. faecalis being a common culprit of disease; however, such infections are generally considered to be independent of envenomation. Here, we provide evidence on venom microbiota across snakes and arachnida and report on the convergent evolution mechanisms that can facilitate adaptation to black-necked cobra venom in two independent E. faecalis strains, easily misidentified by biochemical diagnostics. Therefore, since inoculation with viable and virulence gene-harboring bacteria can occur during envenomation, acute infection risk management following envenomation is warranted, particularly for immunocompromised and malnourished victims in resource-limited settings. These results shed light on how bacteria evolve for survival in one of the most extreme environments on Earth and how venomous bites must be also treated for infections.

The safety profile of Bald's eyesalve for the treatment of bacterial infections.

The rise in antimicrobial resistance has prompted the development of alternatives to combat bacterial infections. Bald's eyesalve, a remedy used in the Early Medieval period, has previously been shown to have efficacy against Staphylococcus aureus in in vitro and in vivo models of chronic wounds. However, the safety profile of Bald's eyesalve has not yet been demonstrated, and this is vital before testing in humans. Here, we determined the safety potential of Bald's eyesalve using in vitro, ex vivo, and in vivo models representative of skin or eye infections. We also confirmed that Bald's eyesalve is active against an important eye pathogen, Neisseria gonorrhoeae. Low levels of cytotoxicity were observed in eyesalve-treated cell lines representative of skin and immune cells. Results from a bovine corneal opacity and permeability test demonstrated slight irritation to the cornea that resolved within 10 min. The slug mucosal irritation assay revealed that a low level of mucus was secreted by slugs indicating moderate mucosal irritation. We obtained promising results from mouse wound closure experiments; no visible signs of irritation or inflammation were observed. Our results suggest that Bald's eyesalve could be tested further on human volunteers to assess safety for topical application against bacterial infections.

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.

Monocaprin eye drop formulation to combat antibiotic resistant gonococcal blindness.

Neisseria gonorrhoeae bacteria are acknowledged as an urgent threat to human health because this species has developed resistances to all of the antibiotics used clinically to treat its infections. N. gonorrhoeae causes the sexually transmitted disease gonorrhoea, but also causes blindness when the bacteria infect the eyes. Infants are particularly susceptible, acquiring the infection from their mothers at birth. We have shown that the monoglyceride monocaprin rapidly kills N. gonorrhoeae and other bacterial species and is non-irritating in ocular assays. Here we show that the physical and chemical properties of monocaprin make it ideal for use in a thickened eye drop formulation to combat eye infections. Monocaprin-containing formulations were assessed using analytical techniques and for antimicrobial activity in vitro and in ex vivo infections. Monocaprin-containing formulations retained activity after three years and are non-irritating, unlike preparations of povidone iodine in our assays. A recommended formulation for further development and investigation is 0.25% monocaprin in 1% HPMC with 1% polysorbate 20.

In Vitro Models of Eye Infection with Neisseria gonorrhoeae.

Prophylaxis with silver nitrate and later antibiotics has significantly reduced the cases of infant blindness from gonococcal infection at birth to the point where it has all but been forgotten in the developed world as the devastating disease that it was in the pre-antibiotic era. As a result, while it is known that the bacteria are transmitted to the eyes during passage through the infected birth canal, little is known about Neisseria gonorrhoeae colonization of the eye and the establishment and progression of keratitis. Treatment failures due to rising antimicrobial resistance necessitate investigations into all aspects of gonococcal disease, including eye infections, so that new treatment strategies can be developed. Here we present models for N. gonorrhoeae eye infection using excised bovine corneas and coculture of gonococci with primary human corneal epithelial cells. These models can be used to explore the interactions of the bacteria with corneal tissues and cells and to investigate novel therapeutics against infection.

The Growing Threat of Gonococcal Blindness.

Antibiotic-resistant gonorrhea is now a reality, as well as the consequences of untreatable infections. Gonococcal eye infections result in blindness if not properly treated; they accounted for the vast majority of infections in children in homes for the blind in the pre-antibiotic era. Neisseria gonorrhoeae infects the eyes of infants born to mothers with gonorrhea and can also infect the eyes of adults. Changes in sexual practices may account for the rise in adult gonococcal eye infections, although some cases seem to have occurred with no associated genital infection. As gonorrhea becomes increasingly difficult to treat, the consequences for the treatment of gonococcal blindness must be considered as well. Monocaprin was shown to be effective in rapidly killing N. gonorrhoeae, and is non-irritating in ocular models. Repeated passage in sub-lethal monocaprin induces neither resistance in gonococci nor genomic mutations that are suggestive of resistance. Here, we show that 1 mM monocaprin kills 100% of N. gonorrhoeae in 2 min, and is equally effective against N. meningitidis, a rare cause of ophthalmia neonatorum that is potentially lethal. Monocaprin at 1 mM also completely kills Staphylococcus aureus after 60 min, and 25 mM kills 80% of Pseudomonas aeruginosa after 360 min. Previously, 1 mM monocaprin was shown to eliminate Chlamydia trachomatis in 5 min. Monocaprin is, therefore, a promising active ingredient in the treatment and prophylaxis of keratitis, especially considering the growing threat of gonococcal blindness due to antimicrobial resistance.

Alternative antimicrobials: the properties of fatty acids and monoglycerides.

With the rising antibiotic resistance of many bacterial species, alternative treatments are necessary to combat infectious diseases. The World Health Organization and the US Centres for Disease Control and Prevention have warned that some infections, such as those from Neisseria gonorrhoeae, may be untreatable within a few years. One avenue of exploration is the use of antimicrobial fatty acids and their derivatives for therapeutic prevention or treatment of bacterial infections. Several studies have explored the activity of fatty acids and their derivatives, including monoglycerides against a variety of bacterial species. These are reviewed here, assessing the antimicrobial properties that have been demonstrated and the feasibility of therapeutic applications.

Mutations in Neisseria gonorrhoeae grown in sub-lethal concentrations of monocaprin do not confer resistance.

Neisseria gonorrhoeae, due to its short lipooligosaccharide structure, is generally more sensitive to the antimicrobial effects of some fatty acids than most other Gram negative bacteria. This supports recent development of a fatty acid-based potential treatment for gonococcal infections, particularly ophthalmia neonatorum. The N. gonorrhoeae genome contains genes for fatty acid resistance. In this study, the potential for genomic mutations that could lead to resistance to this potential new treatment were investigated. N. gonorrhoeae strain NCCP11945 was repeatedly passaged on growth media containing a sub-lethal concentration of fatty acid myristic acid and monoglyceride monocaprin. Cultures were re-sequenced and assessed for changes in minimum inhibitory concentration. Of note, monocaprin grown cultures developed a mutation in transcription factor gene dksA, which suppresses molecular chaperone DnaK and may be involved in the stress response. The minimum inhibitory concentration after exposure to monocaprin showed a modest two-fold change. The results of this study suggest that N. gonorrhoeae cannot readily evolve resistance that will impact treatment of ophthalmia neonatorum with monocaprin.

Fatty Acid Based Microemulsions to Combat Ophthalmia Neonatorum Caused by Neisseria gonorrhoeae and Staphylococcus aureus.

The bacterial species Neisseria gonorrhoeae (N. gonorrhoeae) and Staphylococcus aureus (S. aureus) are amongst the main microorganisms that cause ophthalmia neonatorum. The current treatment involves the use of various antibiotics such as ciprofloxacin, cephalosporin, ceftriaxone and cefotaxime. However, this treatment strategy is becoming more ineffective due to the antibiotic resistance in N. gonorrhoeae. The current study explores the potential use of fatty acid based microemulsions (ME) to prevent N. gonorrhoeae and S. aureus infections in new-borns' eyes without harmful side effects such as corneal or conjunctiva irritation. Pseudo-ternary phase diagrams were constructed to evaluate microemulsion regions and six different α-linolenic acid based microemulsions were prepared. The prepared formulations were characterized for α-linolenic acid content, size, transparency, zeta potential, Polarized light Microscopy, antimicrobial activity and ex vivo ocular toxicity. The mean droplet size of the ME formulations was in the range of 190.4 to 350.5 nm and polydispersity index (PDI) values were in the range of 0.102 to 0.561. All formulations were found stable upon storage for at least 8 weeks. In addition, self-diffusion coefficients determined by nuclear magnetic resonance (NMR) reflected that the diffusability of water increased at higher than 30% w/w water, while that of fatty acids and surfactants was in reverse. The antimicrobial efficacy of microemulsions was determined against N. gonorrhoeae and S. aureus. It was concluded that all microemulsions have strong antimicrobial effects against N. gonorrhoeae and S. aureus. Finally, bovine corneal opacity permeability (BCOP) and hen's egg chorioallantoic (HET-CAM) tests results showed that all microemulsion formulations were not strong ocular irritants.

Investigating Potential Chromosomal Rearrangements during Laboratory Culture of Neisseria gonorrhoeae.

Comparisons of genome sequence data between different strains and isolates of Neisseria spp., such as Neisseria gonorrhoeae, reveal that over the evolutionary history of these organisms, large scale chromosomal rearrangements have occurred. Factors within the genomes, such as repetitive sequences and prophage, are believed to have contributed to these observations. However, the timescale in which rearrangements occur is not clear, nor whether it might be expected for them to happen in the laboratory. In this study, N. gonorrhoeae was repeatedly passaged in the laboratory and assessed for large scale chromosomal rearrangements. Using gonococcal strain NCCP11945, for which there is a complete genome sequence, cultures were passaged for eight weeks in the laboratory. The resulting genomic DNA was assessed using Pulsed Field Gel Electrophoresis, comparing the results to the predicted results from the genome sequence data. Three cultures generated Pulsed Field Gel Electrophoresis patterns that varied from the genomic data and were further investigated for potential chromosomal rearrangements.

Prevention of Ophthalmia Neonatorum Caused by Neisseria gonorrhoeae Using a Fatty Acid-Based Formulation.

Ophthalmia neonatorum, also called neonatal conjunctivitis, acquired during delivery can occur in the first 28 days of life. Commonly caused by the bacterial pathogen Neisseria gonorrhoeae, infection can lead to corneal scarring, perforation of the eye, and blindness. One approach that can be taken to prevent the disease is the use of an ophthalmic prophylaxis, which kills the bacteria on the surface of the eye shortly after birth. Current prophylaxes are based on antibiotic ointments. However, N. gonorrhoeae is resistant to many antibiotics and alternative treatments must be developed before the condition becomes untreatable. This study focused on developing a fatty acid-based prophylaxis. For this, 37 fatty acids or fatty acid derivatives were screened in vitro for fast antigonococcal activity. Seven candidates were identified as bactericidal at 1 mM. These seven were subjected to irritation testing using three separate methods: the bovine corneal opacity and permeability (BCOP) test; the hen's egg test-chorioallantoic membrane (HET-CAM); and the red blood cell (RBC) lysis assay. The candidates were also tested in artificial tear fluid to determine whether they were effective in this environment. Four of the candidates remained effective. Among these, two lead candidates, monocaprin and myristoleic acid, displayed the best potential as active compounds in the development of a fatty acid-based prophylaxis for prevention of ophthalmia neonatorum.

Inversion of Correia repeat enclosed elements in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Within the gonococcal genome are over 100 copies of the insertion sequence-like Correia repeat enclosed element (CREE), which has been predicted to be mobile within the neisserial genomes. Although there is evidence of ancestral movement of these elements, no previous study has provided evidence for current mobilization. CREE has the ability to alter gene expression and regulation in many ways: by insertional mutagenesis, by introducing promoter elements, by generating mRNA processing sites and by association with non-coding RNAs. Previous studies have compared the genomic locations of CREEs in the Neisseria spp., demonstrating that otherwise identical regions have either the element or the target TA insertion site. In this study, we report for the first time, to our knowledge, movement of CREEs, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from N. gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample, 7 were seen in the control sample and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements, potentially resulting in reversible phase-variable gene expression.

Fatty acid microemulsion for the treatment of neonatal conjunctivitis: quantification, characterisation and evaluation of antimicrobial activity.

Fatty acids (FAs) are used by many organisms as defence mechanism against virulent bacteria. The high safety profile and broad spectrum of activity make them potential alternatives to currently used topical antibiotics for the treatment of eye infections in neonates. The current study utilised a Design of Experiment approach to optimise the quantification of five fatty acids namely; lauric acid, tridecanoic acid, myristoleic acid, palmitoleic acid and α-linolenic acid. The significance of the influence of the experimental parameters such as volume of catalyst, volume of n-hexane, incubation temperature, incubation time and the number of extraction steps on derivatisation was established by statistical screening with a factorial approach. Derivatisation was confirmed using attenuated total reflectance infrared (ATR) and 1H NMR spectrum. A gas chromatographic method (GC-FID) was developed and validated according to ICH guidelines for the identification and quantification of fatty acids. The results were found to be linear over the concentration range studied with coefficient of variation greater than 0.99 and high recovery values and low intra-day and inter-day variation values for all FAs. Then, different α-linolenic acid-based microemulsions (MEs) were prepared using Tween 80 as surfactant, polyethylene glycol 400 (PEG 400) as co surfactant and water as aqueous phase. The developed GC method was used to quantify the FA content in ME formulations. The results indicated that the developed GC method is very effective to quantify the FA content in the ME formulations. The antimicrobial efficacy of FA-based MEs were tested against Staphylococcus aureus. It was concluded that the FA-based MEs have strong antimicrobial effect against S. aureus.

Correia Repeat Enclosed Elements and Non-Coding RNAs in the Neisseria Species.

Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs.

DNA uptake sequences in Neisseria gonorrhoeae as intrinsic transcriptional terminators and markers of horizontal gene transfer.

DNA uptake sequences are widespread throughout the Neisseria gonorrhoeae genome. These short, conserved sequences facilitate the exchange of endogenous DNA between members of the genus Neisseria. Often the DNA uptake sequences are present as inverted repeats that are able to form hairpin structures. It has been suggested previously that DNA uptake sequence inverted repeats present 3' of genes play a role in rho-independent termination and attenuation. However, there is conflicting experimental evidence to support this role. The aim of this study was to determine the role of DNA uptake sequences in transcriptional termination. Both bioinformatics predictions, conducted using TransTermHP, and experimental evidence, from RNA-seq data, were used to determine which inverted repeat DNA uptake sequences are transcriptional terminators and in which direction. Here we show that DNA uptake sequences in the inverted repeat configuration occur in N. gonorrhoeae both where the DNA uptake sequence precedes the inverted version of the sequence and also, albeit less frequently, in reverse order. Due to their symmetrical configuration, inverted repeat DNA uptake sequences can potentially act as bi-directional terminators, therefore affecting transcription on both DNA strands. This work also provides evidence that gaps in DNA uptake sequence density in the gonococcal genome coincide with areas of DNA that are foreign in origin, such as prophage. This study differentiates for the first time, to our knowledge, between DNA uptake sequences that form intrinsic transcriptional terminators and those that do not, providing characteristic features within the flanking inverted repeat that can be identified.

Phase variable DNA repeats in Neisseria gonorrhoeae influence transcription, translation, and protein sequence variation.

There are many types of repeated DNA sequences in the genomes of the species of the genus Neisseria, from homopolymeric tracts to tandem repeats of hundreds of bases. Some of these have roles in the phase-variable expression of genes. When a repeat mediates phase variation, reversible switching between tract lengths occurs, which in the species of the genus Neisseria most often causes the gene to switch between on and off states through frame shifting of the open reading frame. Changes in repeat tract lengths may also influence the strength of transcription from a promoter. For phenotypes that can be readily observed, such as expression of the surface-expressed Opa proteins or pili, verification that repeats are mediating phase variation is relatively straightforward. For other genes, particularly those where the function has not been identified, gathering evidence of repeat tract changes can be more difficult. Here we present analysis of the repetitive sequences that could mediate phase variation in the Neisseria gonorrhoeae strain NCCP11945 genome sequence and compare these results with other gonococcal genome sequences. Evidence is presented for an updated phase-variable gene repertoire in this species, including a class of phase variation that causes amino acid changes at the C-terminus of the protein, not previously described in N. gonorrhoeae.

Insight into proteomic investigations of Neisseria meningitidis serogroup C strain L91543 from analysis of its genome sequence.

Here, we describe the draft sequence of a virulent isolate of Neisseria meningitidis strain L91543, belonging to serogroup C. The findings from previous proteomic and metabolomic studies of this strain can now be further interpreted with genomic analysis. Comparative analysis of the genome sequence revealed close similarity and localized synteny with the genome sequence of N. meningitidis serogroup C strain, FAM18. Polymorphisms were identified in the signal peptide sequence of factor H binding protein, a target for new meningococcal vaccines, which may result in its inefficient translocation across the cytoplasmic membrane affecting its processing (lipidation and cleavage of the signal peptide) and transportation to the outer membrane in strain L91543. This would explain the unusual proteomic data for factor H binding protein for this strain. NadA, another target for new vaccines, and the MtrR regulator, which controls expression of NadA, both contain SNPs between strains L91543 and FAM18. The genome sequence data were generated using Ion Torrent PGM sequencing, assembled into 50 contigs with 64× coverage and annotated with 2262 genes, 14 rRNAs and 56 tRNAs. The availability of the genome of N. meningitidis strain L91543 will aid our understanding of the proteome of this organism, importantly its vaccine antigens.

Microarray analysis of the Ler regulon in enteropathogenic and enterohaemorrhagic Escherichia coli strains.

The type III protein secretion system is an important pathogenicity factor of enteropathogenic and enterohaemorrhagic Escherichia coli pathotypes. The genes encoding this apparatus are located on a pathogenicity island (the locus of enterocyte effacement) and are transcriptionally activated by the master regulator Ler. In each pathotype Ler is also known to regulate genes located elsewhere on the chromosome, but the full extent of the Ler regulon is unclear, especially for enteropathogenic E. coli. The Ler regulon was defined for two strains of E. coli: E2348/69 (enteropathogenic) and EDL933 (enterohaemorrhagic) in mid and late log phases of growth by DNA microarray analysis of the transcriptomes of wild-type and ler mutant versions of each strain. In both strains the Ler regulon is focused on the locus of enterocyte effacement - all major transcriptional units of which are activated by Ler, with the sole exception of the LEE1 operon during mid-log phase growth in E2348/69. However, the Ler regulon does extend more widely and also includes unlinked pathogenicity genes: in E2348/69 more than 50 genes outside of this locus were regulated, including a number of known or potential pathogenicity determinants; in EDL933 only 4 extra-LEE genes, again including known pathogenicity factors, were activated. In E2348/69, where the Ler regulon is clearly growth phase dependent, a number of genes including the plasmid-encoded regulator operon perABC, were found to be negatively regulated by Ler. Negative regulation by Ler of PerC, itself a positive regulator of the ler promoter, suggests a negative feedback loop involving these proteins.

Sequence features contributing to chromosomal rearrangements in Neisseria gonorrhoeae.

Through whole genome sequence alignments, breakpoints in chromosomal synteny can be identified and the sequence features associated with these determined. Alignments of the genome sequences of Neisseria gonorrhoeae strain FA1090, N.gonorrhoeae strain NCCP11945, and N. gonorrhoeae strain TCDC-NG08107 reveal chromosomal rearrangements that have occurred. Based on these alignments and dot plot pair-wise comparisons, the overall chromosomal arrangement of strain NCCP11945 and TCDC-NG08107 are very similar, with no large inversions or translocations. The insertion of the Gonococcal Genetic Island in strain NCCP11945 is the most prominent distinguishing feature differentiating these strains. When strain NCCP11945 is compared to strain FA1090, however, 14 breakpoints in chromosomal synteny are identified between these gonococcal strains. The majority of these, 11 of 14, are associated with a prophage, IS elements, or IS-like repeat enclosed elements which appear to have played a role in the rearrangements observed. Additional rearrangements of small regions of the genome are associated with pilin genes. Evidence presented here suggests that the rearrangements of blocks of sequence are mediated by activation of prophage and associated IS elements and reintegration elsewhere in the genome or by homologous recombination between IS-like elements that have generated inversions.