A requirement for flow to enable the development of Ureaplasma parvum biofilms in vitro.

AIMS: To use a flow-based method to establish, quantify and visualize biofilms of Ureaplasma parvum. METHODS AND RESULTS: Absorbance readings of a U. parvum HPA5 culture were taken at 550 nm every 3 h for 30 h in order to establish a growth curve, with viability determined by the number of colour changing units (CCUs). Biofilms were established using the DTU flow-cell with a flow rate of 0·01 ml min-1 and compared to the static control. Titres of bacteria were determined by CCU and biofilm biomass was quantified by Syto9 staining and COMSTAT analysis. High-resolution images were obtained by scanning electron microscopy (SEM). Flow resulted in significantly more biofilm and higher cell titre (0·599 µm3 /µm2  ± 0·152 and 4 × 108  CCU per ml, respectively) compared with static conditions (0·008 µm3 /µm2  ± 0·010 and no recoverable cells, respectively). SEM revealed pleomorphic cells, with signs of budding and possible membrane vesicle formation. CONCLUSIONS: Flow is an essential requirement for the establishment of U. parvum biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first quantification of biofilm biomass formed by U. parvum. It is now possible to establish viable biofilms of U. parvum which will allow for future testing of antimicrobial agents and understanding of virulence-associated with adhesion.

Antibiotic resistance among Ureaplasma spp. isolates: cause for concern?

There is growing global concern regarding the rise of antibiotic-resistant organisms. Many of these reports have focused on various Gram-positive and Gram-negative pathogens, with little attention to the genus Ureaplasma. Ureaplasma spp. are associated with numerous infectious diseases affecting pregnant women, neonates and the immunocompromised. Treatment options are extremely limited due to high levels of intrinsic resistance resulting from the unique physiology of these organisms and further restricted in cases of the developing fetus or neonate, often limiting therapeutic options to predominantly macrolides or rarely fluoroquinolones. The increasing presence of macrolide- and fluoroquinolone-resistant strains among neonatal infections may result in pan-drug resistance and potentially untreatable conditions. Here, we review the requirements for accurate measurement of antimicrobial susceptibility, provide a comprehensive review of the antimicrobial resistance (AMR) for Ureaplasma species in the literature and contextualize these results relative to some investigators' reliance on commercial kits that are not CLSI compliant when determining AMR. The dramatic variation in the resistance patterns and impact of high levels of AMR amongst neonatal populations suggests the need for continued surveillance. Commercial kits represent an excellent tool for initial antibiotic susceptibility determination and screening. However, AMR reporting must utilize internationally standardized methods, as high-titre samples, or Mycoplasma hominis-contaminated samples routinely give false AMR results. Furthermore, there is a requirement for future reports to determine the underlying AMR mechanisms and determine whether expanding AMR is due to spontaneous mutation, transmission of resistance genes on mobile elements or selection and expansion of resistant clones.

Antimicrobial activity of Manuka honey against antibiotic-resistant strains of the cell wall-free bacteria Ureaplasma parvum and Ureaplasma urealyticum.

The susceptibility of the cell wall-free bacterial pathogens Ureaplasma spp. to Manuka honey was examined. The minimum inhibitory concentration (MIC) of Manuka honey for four Ureaplasma urealyticum and four Ureaplasma parvum isolates was determined. Sensitivity to honey was also compared to clinical isolates with resistance to tetracycline, macrolide and fluoroquinolone antibiotics. Finally step-wise resistance training was utilized in an attempt to induce increased tolerance to honey. The MIC was dependent on the initial bacterial load with 7·5 and 18·0% w/v honey required to inhibit U. urealyticum at 1 and 106 colour changing units (CCU), respectively, and 4·8 and 15·3% w/v required to inhibit U. parvum at 1 and 106  CCU respectively. MIC values were consistently lower for U. parvum compared with U. urealyticum. Antimicrobial activity was seen against tetracycline-resistant, erythromycin-resistant and ciprofloxacin-resistant isolates at 105  CCU. No resistance to honey was observed with 50 consecutive challenges at increasing concentrations of honey. This is the first report of the antimicrobial activity of Manuka honey against a cell wall-free bacterial pathogen. The antimicrobial activity was retained against antibiotic-resistant strains and it was not possible to generate resistant mutants. SIGNIFICANCE AND IMPACT OF THE STUDY: Manuka honey is known to have a broad spectrum of antimicrobial activity, with the bacterial cell wall being suggested as a predominant site of action. This study has demonstrated that Manuka honey has activity against Ureaplasma spp., a genus of cell wall-free bacteria which are intrinsically resistant to many available antibiotics making treatment inherently difficult. This is the first report of the antimicrobial activity of Manuka honey against a bacterial pathogen, in the absence of a cell well and opens scope for the use of components of Manuka honey as a therapeutic among Ureaplasma infections.

Characterisation of colistin resistance in Gram-negative microbiota of pregnant women and neonates in Nigeria.

A mobile colistin resistance gene mcr was first reported in 2016 in China and has since been found with increasing prevalence across South-East Asia. Here we survey the presence of mcr genes in 4907 rectal swabs from mothers and neonates from three hospital sites across Nigeria; a country with limited availability or history of colistin use clinically. Forty mother and seven neonatal swabs carried mcr genes in a range of bacterial species: 46 Enterobacter spp. and single isolates of; Shigella, E. coli and Klebsiella quasipneumoniae. Ninety percent of the genes were mcr-10 (n = 45) we also found mcr-1 (n = 3) and mcr-9 (n = 1). While the prevalence during this collection (2015-2016) was low, the widespread diversity of mcr-gene type and range of bacterial species in this sentinel population sampling is concerning. It suggests that agricultural colistin use was likely encouraging sustainment of mcr-positive isolates in the community and implementation of medical colistin use will rapidly select and expand resistant isolates.

Role of pulmonary infection in the development of chronic lung disease of prematurity.

We studied the role of ante- and post-natal infection in the development of chronic lung disease (CLD) of prematurity. 192 newborn infants (61 term and 131 pre-term of <34 weeks gestation: 88 with respiratory distress syndrome, 35 developed CLD and eight died) were recruited. 16S ribosomal RNA (rRNA) genes were identified by PCR of DNA isolated from 840 gastric and lung fluid samples. Ureaplasma spp. were also cultured. Presence of 16S rRNA genes (OR 1.6, 95% CI 1.2-2.2) and Ureaplasma spp. (OR 3.6, 95% CI 1.7-7.7) was significantly associated with the development of CLD. This association remained if the 16S rRNA genes and Ureaplasma spp. were first identified within the first 3 days of life (OR 2.4 (95% CI 1.4-4.1) and 3.8 (95% CI 1.4-10.0), respectively) or if first identified after 3 days of age (OR 1.7 (95% CI 1.1-2.8) and OR 5.1 (95% CI 1.3-19.8), respectively). Peak lung fluid interleukin (IL)-6 and IL-8 were significantly associated with presence of microbes (p<0.0001 and p=0.0001, respectively) and development of CLD (p=0.003 and 0.001, respectively). Both early and late microbial presence in neonatal lung fluid samples was significantly associated with the development of CLD suggesting that both ante- and post-natal infection play a role in the development of CLD.

Efficient generation of monoclonal antibodies against surface-expressed proteins by hyperexpression in rodent cells.

The generation of antibodies is one of the first requirements in the characterisation of a newly cloned protein. However, this requires expression and purification of the protein in sufficient yield and purity for immunisation of animals and screening of fusion wells. Even with the development of highly efficient protocols based upon incorporation of specific peptide tags, this can be a tedious and time-consuming process. In an effort to improve the speed and efficiency of obtaining antibodies reactive with newly cloned proteins we have developed an approach based upon the expression of the protein at high level in cell lines originating from the species to be used for immunisation. To illustrate this approach we describe the generation of antibodies against two recently cloned proteins that are normally expressed at the membrane, the rat and mouse analogues of human decay accelerating factor (DAF; CD55). However, the strategy is applicable not only to membrane proteins but also to other proteins which can be expressed on the cell membrane by incorporating at the carboxy terminus the signal sequence for glycosyl phosphatidylinositol (GPI) anchor addition derived from DAF or another GPI-anchored protein. The strategy also permits rapid and efficient screening using flow cytometry on expressing cells.

Complement expression on astrocytes and astrocytoma cell lines: failure of complement regulation at the C3 level correlates with very low CD55 expression.

Primary fetal human astrocytes and an astrocytoma cell line, U373-MG, expressed membrane cofactor protein (CD46), CD59, and low levels of decay-accelerating factor (CD55). Astrocyte CD55 was capable of regulating C3 deposition on the cell surface; albeit at a lower level than primary human fibroblasts. Negligible complement-mediated lysis of primary astrocytes and the U373-MG cell line was observed, even when large amount of astrocyte-specific, complement-activating antibodies were bound to the cells. Blocking the function of CD59 on astrocytes resulted in a > 90% cell lysis, while equivalent lysis of fibroblasts could only be achieved with additional blocking of CD55.

The viral aetiology of croup and recurrent croup.

BACKGROUND: Historically croup was subdivided into classic "viral" croup with associated viral upper respiratory tract infections, and recurrent or spasmodic croup where asthma and allergies were thought more important. METHODS: All children admitted to the University Hospital of Wales with croup in 2003 were eligible. Baseline demographics including croup score were recorded and per-nasal swabs taken for virus detection by RT-PCR. Recurrent croup was defined as at least one other admission for croup in the preceding or following 3 years. RESULTS: Sixty (29.4%) children entered the study, and a viral pathogen was detected in 41 (68%). There was no significant difference in the rate of virus detection between those with single episode croup and recurrent croup. CONCLUSIONS: The aetiologies of viral and recurrent croup appear similar.

Improved gene delivery to intestinal mucosa by adenoviral vectors bearing subgroup B and d fibers.

A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses.

Dissecting the regions of virion-associated Kaposi's sarcoma-associated herpesvirus complement control protein required for complement regulation and cell binding.

Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.

Antibody-independent activation of the classical complement pathway by cytomegalovirus-infected fibroblasts.

Human fibroblasts weakly activated the alternative complement pathway, as assessed by C3b deposition, while 4- to 5-fold more C3b was observed 4 days after infection on cytomegalovirus (CMV)-infected fibroblasts when incubated with human serum. CMV-infected fibroblasts activated via the classical complement pathway independent of specific anti-CMV antibody and incubation of CMV-infected fibroblasts with serum deficient in complement components revealed that C1q, but not mannan-binding lectin, was required for complement activation. The enhanced complement activation by CMV-infected cells was observed as early as 4 h after infection and required the active transcription of CMV genes. No difference in the complement activation by CMV-infected cells was observed with the use of CMV-seropositive or -seronegative serum as a complement source, suggesting that CMV infection induces or up-regulates a protein that binds directly to C1q in a complement-activating conformation.

Human and rodent decay-accelerating factors (CD55) are not species restricted in their complement-inhibiting activities.

Homologous complement activation is restricted on cells by the complement regulators, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. These proteins act in concert with other membrane structures to protect cells from homologous complement attack. In contrast, cells are usually sensitive to heterologous complement attack. It has been suggested that species-specific restriction of complement activation can be attributed to the inability of regulators to inhibit across species. We have investigated the capacities of human, rat and mouse analogues of DAF to regulate homologous and heterologous complement. Cells transfected with cDNA encoding these analogues were protected from heterologous complement attack. C3b-deposition experiments indicated that whilst cells were best protected by DAF from the same species, all three analogues inhibited human, rat and mouse complement. Comparable results were obtained in haemolysis assays using soluble, recombinant forms of the proteins. Inhibition of the classical pathway (CP) was best achieved with homologous DAF, although human DAF also inhibited rat complement, rat DAF also inhibited human complement and mouse DAF inhibited complement from all species. Human DAF was the best inhibitor of alternative pathway (AP)-mediated attack, inhibiting complement from all species. Mouse DAF inhibited mouse and rat AP, whilst rat DAF inhibited only rat AP. These data indicate that human and rodent analogues of DAF are not species restricted and highlights interesting differences in the capacity to regulate AP and CP. This has implications in broader fields of research, such as xenotransplantation, where cross-species regulation of complement is of paramount importance.

Development of a model for cytomegalovirus infection of oligodendrocytes.

The well-characterized human oligodendroglioma (HOG) cell line, cells of which resemble immature oligodendrocytes, was used to investigate the level of permissiveness to human cytomegalovirus (CMV) infection. Expression of CMV genes was incomplete following exposure of HOG cells to CMV, in contrast with results observed with the astroglioma cell line U373-MG (used as a positive control). However, treatment with phorbol 12-myristate 13-acetate (PMA) or with dibutryl cAMP (dbcAMP) plus the phosphatase inhibitor 1-isobutyl-3,3-methyl xanthine (IBMX) rendered the HOG cells fully permissive to CMV; down-regulation of HLA class I and production of virions were only observed under these conditions. In contrast to the findings seen with the HOG cell line, treatment of U373-MG cells with dbcAMP/IBMX or PMA did not interfere with CMV-induced down-regulation of HLA class I. However, these chemical stimulators reduced virus production in U373-MG cells by 30 and 70%, respectively.

Tissue distribution of the rat analogue of decay-accelerating factor.

In humans, decay-accelerating factor (DAF) is a widely distributed, cell-bound inhibitor of the complement activation enzymes and plays a key role in regulating complement activation, preventing the generation of anaphylotoxins and opsonins, and protecting against complement-mediated lysis. Rodent analogues of DAF have recently been identified, providing a new avenue for the analysis of function. Rat DAF was cloned in our laboratory. Here we describe the generation of monoclonal antibodies (mAbs) against rat DAF, using transfected cells as immunogen, and their use in the analysis of the distribution of DAF in the rat by flow cytometry, Western blot analysis and immunohistochemistry. One of the mAbs was found to block the complement inhibitory function of rat DAF, offering the prospect of neutralization of DAF function in vivo. The antibodies have also been used for purification of DAF from rat erythrocytes by affinity chromatography. Rat DAF purified in this manner was similar in molecular mass to human DAF. The purified protein incorporated into lipid membranes, confirming the presence of a glycolipid anchor, and incorporated protein strongly inhibited the rat C3 convertase. Rat DAF was strongly expressed on endothelia throughout the animal and was also present in most tissues and organs. DAF expression was weak or absent in the brain and on circulating and spleen-resident T cells. Strong DAF expression observed in the kidney was restricted to the glomerulus and Bowman's capsule. DAF expression in the testis was found only in association with the later stages of spermatogenesis.

Neutralization of cytomegalovirus virions: the role of complement.

Complement provides a key immunologic defense against invading pathogens; thus, a clear understanding of the interactions between cytomegalovirus (CMV) and complement may permit the development of strategies to enhance CMV neutralization. In the presence of specific anti-CMV antibodies, complement enhanced the neutralizing ability of serum by 2- to 3-fold. However, in the absence of specific anti-CMV antibodies, complement was ineffective in neutralizing CMV virions by plaque assay. Although complement alone did not mediate any neutralizing effect, CMV consumed complement activity from seronegative serum, resulting in the deposition of C3 on the virion. However, only in the presence of specific anti-CMV antibody did complement activation continue to the deposition of C9 on the virions. These results strongly suggest complement regulation by CMV virions that is modulated by anti-CMV antibody; this regulation may be attributed to three host complement regulators on the virions: CD55, CD46, and CD59.

Cytokine-mediated up-regulation of CD55 and CD59 protects human hepatoma cells from complement attack.

Hepatic parenchymal cells respond in many different ways to acute-phase cytokines. Some responses may protect against damage by liver-derived inflammatory mediators. Previous investigations have shown that cytokines cause increased secretion by hepatoma cells of soluble complement regulatory proteins, perhaps providing protection from complement attack. More important to cell protection are the membrane complement regulators. Here we examine, using flow cytometry and Northern blotting, the effects of different cytokines, singly or in combination, on expression of membrane-bound complement regulators by a hepatoma cell line. The combination of tumour necrosis factor-alpha, IL-1beta, and IL-6 caused increased expression of CD55 (three-fold) and CD59 (two-fold) and decreased expression of CD46 at day 3 post-exposure. Interferon-gamma reduced expression of CD59 and strongly antagonized the up-regulatory effects on CD59 mediated by the other cytokines. Complement attack on antibody-sensitized hepatoma cells following a 3-day incubation with the optimum combination of acute-phase cytokines revealed increased resistance to complement-mediated lysis and decreased C3b deposition. During the acute-phase response there is an increased hepatic synthesis of the majority of complement effector proteins. Simultaneous up-regulation of expression of CD55 and CD59 may serve to protect hepatocytes from high local concentrations of complement generated during the acute-phase response.

Altered expression of host-encoded complement regulators on human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV)-infected cells persist in the presence of anti-HCMV antibody, suggesting that HCMV has evolved mechanisms to evade host immune defenses. Insofar as no virus-encoded complement inhibitors have been identified for HCMV, we hypothesized that HCMV infection may alter the expression of host-encoded cell surface complement inhibitors. Herein, we report that cell surface expression of two complement regulator proteins, CD55 and CD46, which are members of the regulators of complement activation (RCA) gene cluster, increased up to eightfold following infection of fibroblasts or glioblastoma cells with HCMV, but not after infection with HSV-1 or adenovirus. However, the cell surface expression of a third complement regulator, CD59, which is not a member of the RCA gene cluster, was not altered during HCMV infection. Functional studies using purified complement components demonstrated that up-regulation of CD55 suppressed the activity of cell-associated C3 convertases on HCMV-infected cells. Furthermore, increased CD55 expression protected infected cells from complement-mediated lysis, an effect which directly correlated with the length of HCMV infection. Increased expression of host-encoded complement regulator proteins may provide protection of HCMV-infected cells from the host immune response in vivo, through increasing the resistance of infected cells to complement-mediated lysis and decreasing the deposition of C3-derived products on the cell surface.

Development of adenovirus vectors encoding rat complement regulators for use in therapy in rodent models of inflammatory diseases.

C activation has been implicated in the pathogenesis of numerous inflammatory human diseases and disease models. A therapy based on C inhibition might therefore be of benefit to reduce inflammation and ameliorate disease. C inhibition in vivo can be accomplished by the delivery of soluble recombinant C regulators either systemically or directly to a target site, but effects are transitory. We have developed a strategy for the efficient delivery of the membrane-bound rat C inhibitors, CD59, Crry, and decay-accelerating factor (DAF), using replication-deficient adenovirus vectors with the intention of treating rat models of disease in which C is implicated. The adenovirus recombinants(RAd), RAdCD59, RAdCrry, and RAdDAF, respectively, have been tested for expression and function of the transgene in vitro. Infection of human fetal foreskin fibroblasts resulted in high levels of expression of each of the rat inhibitors. The constructs were also tested for inhibition of rat C-mediated cell lysis and C3b deposition. In a cell lysis assay, each inhibited to varying degrees of efficiency in the order RAdCD59 = RAdDAF > RAdCrry. In a C3b deposition assay, RAdDAF caused a greater reduction in C3b deposition than RAdCrry and RAdCD59 was ineffective. These agents, individually or in combination, provide the tools for testing the effects of prolonged inhibition of C at a target site on the progress of experimental models of disease.