M ycobacterium avium complex genomics and transmission in a London hospital.

BACKGROUND: Non-tuberculous mycobacteria (NTM) are environmental microorganisms and opportunistic pathogens in individuals with pre-existing lung conditions such as cystic fibrosis (CF) and non-CF bronchiectasis. While recent studies of Mycobacterium abscessus have identified transmission within single CF centres as well as nationally and globally, transmission of other NTM species is less well studied. METHODS: To investigate the potential for transmission of the Mycobacterium avium complex (MAC) we sequenced 996 isolates from 354 CF and non-CF patients at the Royal Brompton Hospital (London, UK; collected 2013-2016) and analysed them in a global context. Epidemiological links were identified from patient records. Previously published genomes were used to characterise global population structures. RESULTS: We identified putative transmission clusters in three MAC species, although few epidemiological links could be identified. For M. avium, lineages were largely limited to single countries, while for Mycobacterium chimaera, global transmission clusters previously associated with heater-cooler units (HCUs) were found. However, the immediate ancestor of the lineage causing the major HCU-associated outbreak was a lineage already circulating in patients. CONCLUSIONS: CF and non-CF patients shared transmission chains, although the lack of epidemiological links suggested that most transmission is indirect and may involve environmental intermediates or asymptomatic carriage in the wider population.

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.

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.

VNTR analysis reveals unexpected genetic diversity within Mycoplasma agalactiae, the main causative agent of contagious agalactia.

BACKGROUND: Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. Previous studies of M. agalactiae have shown it to be unusually homogeneous and there are currently no available epidemiological techniques which enable a high degree of strain differentiation. RESULTS: We have developed variable number tandem repeat (VNTR) analysis using the sequenced genome of the M. agalactiae type strain PG2. The PG2 genome was found to be replete with tandem repeat sequences and 4 were chosen for further analysis. VNTR 5 was located within the hypothetical protein MAG6170 a predicted lipoprotein. VNTR 14 was intergenic between the hypothetical protein MAG3350 and the hypothetical protein MAG3340. VNTR 17 was intergenic between the hypothetical protein MAG4060 and the hypothetical protein MAG4070 and VNTR 19 spanned the 5' end of the pseudogene for a lipoprotein MAG4310 and the 3' end of the hypothetical lipoprotein MAG4320. We have investigated the genetic diversity of 88 M. agalactiae isolates of wide geographic origin using VNTR analysis and compared it with pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. Simpson's index of diversity was calculated to be 0.324 for PFGE and 0.574 for VNTR analysis. VNTR analysis revealed unexpected diversity within M. agalactiae with 9 different VNTR types discovered. Some correlation was found between geographical origin and the VNTR type of the isolates. CONCLUSION: VNTR analysis represents a useful, rapid first-line test for use in molecular epidemiological analysis of M. agalactiae for outbreak tracing and control.

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.

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.

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.

A high level of strain variation within the Mycoplasma ovipneumoniae population of the UK has implications for disease diagnosis and management.

Mycoplasma ovipneumoniae is one of only two mycoplasma species associated with small ruminant disease in Britain and has been associated with an increasing number of disease outbreaks since 2002. This investigation used well-defined techniques to assess the variability of UK M. ovipneumoniae isolates, in an attempt to identify strain clusters within the population. Strains received for routine diagnosis between 2002 and 2004 were analysed using random amplified polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE). Of the 43 samples screened 40 RAPD Hum-1, 41 RAPD Hum-4 and 40 PFGE profiles were observed. Composite data analysis divided strains into 10 similarity clusters with SDS-PAGE and Western blotting indicating that this DNA variability is translated into a pattern of variable protein expression. In order to assess the strains isolated within flocks two sets of samples, from diverse locations, were included in this test panel. The presence of variable isolates existing on the same farm may reflect animal movement and the introduction of asymptomatic, carrier, animals where M. ovipneumoniae is already established within a flock. These findings have significant implications regarding disease diagnosis and management.

Identification and differentiation of European and African/Australian strains of Mycoplasma mycoides subspecies mycoides small-colony type using polymerase chain reaction analysis.

Mycoplasma mycoides subspecies mycoides small-colony type (M. m. m. SC) is the cause of the economically important contagious bovine pleuropneumonia. Isolates from Africa and Australia have previously been documented to have a fragment of approximately 8.84 kb, which is absent in European strains. A set of polymerase chain reaction (PCR) primers over this region was designed to identify M. m. m. SC isolates and separate European strains from those of Africa/Australia. Specificity of the PCR assay was achieved through the positioning of an oligonucleotide within the insertion sequence IS1296, upstream of this deletion, which then was paired with a reverse primer, upstream of the deletion, within the 8.84 kb-deleted region or downstream of the deletion, generating fragments of 1.1 kb (all M. m. m. SC strains), 1.4 kb (African/Australian strains only) and 1.3 kb (European strains only), respectively. Identification and differentiation was specific for DNA from M. m. m. SC with no amplification of DNA from other cluster members or closely related species. The PCR products did not require differentiation by use of a restriction endonuclease, and have potential for use in detection of this organism in clinical samples.

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.

Immunoproteomic characterisation of Mycoplasma mycoides subspecies capri by mass spectrometry analysis of two-dimensional electrophoresis spots and western blot.

OBJECTIVES: Mycoplasma mycoides subspecies capri is one of the causative agents of contagious agalactia in goats. The disease is characterised by mastitis, pneumonia, arthritis, keratitis and in acute cases septicaemia. No vaccine is currently available that has been demonstrated to prevent disease. METHODS: This study used two-dimensional electrophoresis to separate proteins from whole-cell preparations and tandem mass spectrometry to identify them. KEY FINDINGS: In total, 145 spots were successfully identified corresponding to 74 protein identities. Twenty of these proteins were found to be immunogenic by western blot analysis using a pooled serum sample from experimentally infected goats. CONCLUSIONS: Six proteins were found to have a less than 95% amino acid similarity to a closely related Mycoplasma species showing that they warrant further evaluation in development of diagnostic tests. These proteins were a dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex, phosphoglycerate kinase, pyrimidine-nucleoside phosphorylase, 30S ribosomal protein S6, ribulose-phosphate 3-epimerase and D-lactate dehydrogenase.

A simplified PCR method for genotyping Mycoplasma mycoides subspecies mycoides small colony: the aetiologic agent of contagious bovine pleuropneumonia.

Mycoplasma mycoides subspecies mycoides small colony is the aetiological agent of contagious bovine pleuropneumonia, a cattle disease endemic to areas of sub-Saharan Africa. Twenty isolates from various geographical locations and the type strain were analysed by multi-locus sequence analysis (MLSA). The data generated was then used to develop three PCR primer sets to differentiate these isolates. The PCRs differentiated the isolates into four groups; the type strain (T); isolates of European origin (Eu); isolates from Tanzania (Af1) with a final group consisting of isolates from Namibia and Botswana (Af2). These PCRs offers a rapid and efficient post-identification typing method without the need to sequence and analyse multiple genes.

Characterization of Mycoplasma isolated from an ibex (Capra ibex) suffering from keratoconjunctivitis in northern Italy.

In 2005 a Mycoplasma species was isolated from ocular-conjunctival swabs from an adult male Alpine ibex (Capra ibex) from the Valle d'Aosta Region, Northern Italy. The animal suffered from bilateral ocular discharge with diffuse inflammation, severe corneal involvement of the left eye and mild corneal opacity of the right eye. Histologic examination revealed a keratoconjunctivitis characterized by lymphocytic and plasmacellular infiltration. Laboratory investigations of the isolate included culture, transmission electron microscopy, PCR, and denaturing gradient gel electrophoresis, as well as DNA sequencing of the 16S rDNA gene. These tests identified the isolate as Mycoplasma mycoides subsp. capri large-colony serovar, an organism that has occasionally been associated with keratoconjunctivitis in goats. For a correct diagnosis, it is necessary to carry out laboratory investigations, as clinical cases of keratoconjunctivitis in wild ruminants are not always ascribable to Mycoplasma conjunctivae.