A Persistent Tuberculosis Outbreak in the UK Is Characterized by Hydrophobic fadB4-Deficient Mycobacterium tuberculosis That Replicates Rapidly in Macrophages.

The genetic diversity of Mycobacterium tuberculosis can influence disease severity and transmissibility. To better understand how this diversity influences individuals and communities, we phenotyped M. tuberculosis that was causing a persistent outbreak in the East Midlands, United Kingdom. Compared to nonoutbreak isolates, bacilli had higher lipid contents and more hydrophobic cell surfaces. In macrophage infection models, the bacteria increased more rapidly, provoked the enhanced accumulation of macrophage lipid droplets and enhanced the secretion of IL-1ß. Natural deletions in fadB4, nrdB, and plcC distinguished the outbreak isolates from other lineage 3 isolates in the region. fadB4 is annotated with a putative role in cell envelope biosynthesis, so the loss of this gene has the potential to alter the interactions of bacteria with immune cells. Reintroduction of fadB4 to the outbreak strain led to a phenotype that more closely resembled those of nonoutbreak strains. The improved understanding of the microbiological characteristics and the corresponding genetic polymorphisms that associate with outbreaks have the potential to inform tuberculosis control. IMPORTANCE Tuberculosis (TB) killed 1.5 million people in 2020 and affects every country. The extent to which the natural genetic diversity of Mycobacterium tuberculosis influences disease manifestation at both the individual and epidemiological levels remains poorly understood. Insights into how pathogen polymorphisms affect patterns of TB have the potential to translate into clinical and public health practice. Two distinct lineage 3 strains isolated from local TB outbreaks, one of which (CH) was rapidly terminated and the other of which (Lro) persistently transmitted for over a decade, provided us with an opportunity to study these issues. We compared genome sequences, microbiological characteristics, and early immune responses that were evoked upon infection. Our results indicate that the natural lack of fadB4 in the Lro strain contributes to its unique features.

Pathogenic Differences of Type 1 Restriction-Modification Allele Variants in Experimental Listeria monocytogenes Meningitis.

Background:L. monocytogenes meningoencephalitis has a mortality rate of up to 50% and neurofunctional sequelae are common. Type I restriction-modification systems (RMS) are capable of adding methyl groups to the host genome. Some contain multiple sequence recognition (hsdS) genes that recombine, resulting in distinct DNA methylation patterns and patterns of gene expression. These phenotypic switches have been linked to virulence and have recently been discovered in multiple clonal complexes of L. monocytogenes. In the present study, we investigated the significant of RMS on L. monocytogenes virulence during the acute phase of experimental meningitis. Methods:L. monocytogenes strains containing RMS systems were identified, and purified clones enriched for single hsdS alleles were isolated. In vivo, 11-day old Wistar rats were infected with an inoculum containing (a) one of 4 single RMS allele variants (A, B, C, D) treated with amoxicillin (AMX 50 mg/kg/dosis, q8h), (b) a mixture of all 4 variants with or without AMX treatment, or (c) different mixtures of 2 RMS allele variants. At selected time points after infection, clinical and inflammatory parameters, bacterial titers and brain damage were determined. Changes in the relative frequency of the occurring RMS alleles in the inoculum and in CSF or cerebellum of infected animals were analyzed by capillary electrophoresis. Results: We have identified a phase variable RMS locus within L. monocytogenes CC4 and generated stocks that stably expressed each of the possible hsdS genes within that loci. Generation of these allele variants (A, B, C, D) allowed us to determine the methylation pattern associated with each hsdS through SMRT sequencing. In vivo infections with these single allele variants revealed differences in disease severity in that C induced the worst clinical outcome and more pronounced hippocampal apoptosis; D showed the most pronounced weight loss and the highest bacterial titer in the cerebellum. A caused the least severe disease. Conclusion: We identified that L. monocytogenes expressing hsdS (A) causes less damage than when other hsdS genes are expressed. While expression of hsdSC and D worsened the outcome in L. monocytogenes meningitis. We also demonstrate a competitive advantage of variants C and B over variant A in this model. Phenotypical switching may therefore represent a mechanism of virulence regulation during the acute phase of CNS infections with L. monocytogenes.

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages.

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.

Deletion of the Zinc Transporter Lipoprotein AdcAII Causes Hyperencapsulation of Streptococcus pneumoniae Associated with Distinct Alleles of the Type I Restriction-Modification System.

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated ΔadcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the ΔadcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated ΔadcAII strains. However, transformation of ΔadcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated ΔadcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of ΔadcAII Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the ΔadcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and ΔadcAII, further investigation of which could further characterize mechanisms of capsule regulation.

Methylation Warfare: Interaction of Pneumococcal Bacteriophages with Their Host.

Virus-host interactions are regulated by complex coevolutionary dynamics. In Streptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of the nrdR nucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogen S. pneumoniaeIMPORTANCE With antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogen Streptococcus pneumoniae and explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.

Determination of Repeat Number and Expression States of Phase-Variable Loci Through Next Generation Sequencing and Bioinformatic Analysis.

Phase variation (PV) enables high frequency, reversible switches in expression of genetic loci across numerous species of bacteria. A major mechanism of PV in bacteria is the use of slipped strand mispairing across simple sequence repeats (SSRs). The generation and online availability of genomic datasets enables a comprehensive analysis of the distribution and composition of SSRs across multiple bacterial genomes of a species. PhasomeIt is a program that was developed to rapidly identify SSRs, to determine whether these SSRs mediate PV and to find homologous PV loci across multiple genomes. We describe use of this program for analysis of neisserial genomes. We further describe a method to reassemble specific PV loci to allow analysis of large repeat tracts which are often poorly assembled due to inherent drawbacks of the Illumina next generation sequencing (NGS) platform. These methodologies allow for rapid analysis of a major mechanism of PV across numerous species of Neisseria and other bacterial species.

An ex vivo porcine spleen perfusion as a model of bacterial sepsis.

An ex vivo, porcine spleen perfusion model was established to study the early events occurring in the spleen prior to the onset of bacterial sepsis, using organs retrieved from animals slaughtered for food production. Porcine spleens were harvested from adult pigs and connected to a normothermic extracorporeal perfusion circuit. A constant perfusion of heparinized blood was performed for 6 hours. After injection of Streptococcus pneumoniae to the circuit serial samples of both blood and spleen biopsies were collected and analysed. Functionality of the perfused organs was assessed by monitoring the blood-gas parameters, flow rate and filtering capability of the organ. Interestingly, we observed full clearance of bacteria from the blood and an increase in bacterial counts in the spleen. Classical histology and immunohistochemistry on biopsies also confirmed no major damages in the organ architecture and changes in the immune cell distribution, other than the presence of clusters of pneumococci. A time-course study confirmed that each focus of infection derived from the replication of single pneumococcal cells within splenic macrophages. The model proposed - in line with the 3Rs principles - has utility in the replacement of experimental animals in infection research. Murine models are prevalently used to study pneumococcal infections, but are often not predictive for humans due to substantial differences in the immune systems of the two species. This model is designed to overcome these limitations, since porcine immunology and splenic architecture in particular, closely resemble those of humans.

Sputum Moraxella catarrhalis strains exhibit diversity within and between COPD subjects.

PURPOSE: Moraxella catarrhalis is implicated in the pathogenesis of some COPD exacerbations. We sought to investigate whether the M. catarrhalis strain is variable between COPD subjects; that an exacerbation is associated with acquisition of a new strain and that certain strains are more commonly associated with exacerbations. PATIENTS AND METHODS: Sputum samples were collected at stable and exacerbation visits from COPD subjects from a single center as part of the COPDMAP consortium. Samples identified as M. catarrhalis positive by qPCR were recultured in liquid cultures grown to extract genomic DNA; underwent Illumina MiSeq and bacterial genome sequences were de novo assembled and Multi Locus Sequence Type (MLST) was determined. RESULTS: Thirty-five samples were obtained from 18 subjects. These included 13 stable and 22 exacerbation samples. The diversity between samples was very large with 25 different M. catarrhalis MLSTs being identified out of the 35 samples of which 12 MSLTs have not been described previously. Change and persistence of M. catarrhalis strain were observed between stable visits, from stable to exacerbation and vice-a-versa, and between exacerbation visits. CONCLUSION: Sputum M. catarrhalis strains exhibit marked diversity within and between COPD subjects. Acquisition of a new strain is common between stable and exacerbation events such that no strain is specifically associated with an exacerbation.

Draft Whole-Genome Sequences of Periodontal Pathobionts Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia Contain Phase-Variable Restriction-Modification Systems.

Periodontal disease comprises mild to severe inflammatory host responses to oral bacteria that can cause destruction of the tooth-supporting tissue. We report genome sequences for 18 clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia, Gram-negative obligate anaerobes that play a role in the periodontal disease process.

Identification and characterization of a new ferric enterobactin receptor, CfrB, in Campylobacter.

The ferric enterobactin (FeEnt) receptor CfrA is present in the majority of Campylobacter jejuni isolates and is responsible for high-affinity iron acquisition. Our recent work and that of others strongly suggested the existence of another FeEnt uptake system in Campylobacter. Here we have identified and characterized a new FeEnt receptor (designated CfrB) using both in vitro and in vivo systems. CfrB, a homolog of C. jejuni NCTC 11168 Cj0444, shares approximately 34% of amino acid identity with CfrA. Alignment of complete CfrB sequences showed that the CfrB is highly conserved in Campylobacter. Immunoblotting analysis using CfrB-specific antiserum demonstrated that CfrB was dramatically induced under iron-restricted conditions and was produced in the majority of Campylobacter coli (41 out of 45) and in some C. jejuni (8 out of 32) primary strains from various sources and from geographically diverse areas. All of the CfrB-producing C. coli strains also produced CfrA, which was rarely observed in the tested C. jejuni strains. Isogenic cfrB, cfrA, and cfrA cfrB double mutants were constructed in 43 diverse Campylobacter strains. Growth promotion assays using these mutants demonstrated that CfrB has a major role in FeEnt iron acquisition in C. coli. Chicken colonization experiments indicated that inactivation of the cfrB gene alone greatly reduced and even abolished Campylobacter colonization of the intestines. A growth assay using CfrB-specific antiserum strongly suggested that specific CfrB antibodies could block the function of CfrB and diminish FeEnt-mediated growth promotion under iron-restricted conditions. Together, this work reveals the complexity of FeEnt systems in the two closely related Campylobacter species and demonstrates the important role of the new FeEnt receptor CfrB in Campylobacter iron acquisition and in vivo colonization.

Elucidation of the mechanism by which catecholamine stress hormones liberate iron from the innate immune defense proteins transferrin and lactoferrin.

The ability of catecholamine stress hormones and inotropes to stimulate the growth of infectious bacteria is now well established. A major element of the growth induction process has been shown to involve the catecholamines binding to the high-affinity ferric-iron-binding proteins transferrin (Tf) and lactoferrin, which then enables bacterial acquisition of normally inaccessible sequestered host iron. The nature of the mechanism(s) by which the stress hormones perturb iron binding of these key innate immune defense proteins has not been fully elucidated. The present study employed electron paramagnetic resonance spectroscopy and chemical iron-binding analyses to demonstrate that catecholamine stress hormones form direct complexes with the ferric iron within transferrin and lactoferrin. Moreover, these complexes were shown to result in the reduction of Fe(III) to Fe(II) and the loss of protein-complexed iron. The use of bacterial ferric iron uptake mutants further showed that both the Fe(II) and Fe(III) released from the Tf could be directly used as bacterial nutrient sources. We also analyzed the transferrin-catecholamine interactions in human serum and found that therapeutically relevant concentrations of stress hormones and inotropes could directly affect the iron binding of serum-transferrin so that the normally highly bacteriostatic tissue fluid became significantly more supportive of the growth of bacteria. The relevance of these catecholamine-transferrin/lactoferrin interactions to the infectious disease process is considered.

Catecholamine inotrope resuscitation of antibiotic-damaged staphylococci and its blockade by specific receptor antagonists.

The increasing use of antibiotic-coated catheters, such as those containing rifampin or minocycline, has led to a decrease in catheter colonization by staphylococci but not to a decrease in the incidence of catheter-related bloodstream infection (BSI). Because catheters are used for the administration of catecholamine inotropes to maintain cardiac function, we examined whether 2 commonly employed inotropes, dopamine and norepinephrine, could affect bacterial viability after exposure to rifampin and minocycline. Rifampin inhibition and minocycline inhibition of staphylococcal growth could be reversed by exposure to dopamine or norepinephrine as a result, in part, of catecholamine-mediated increased provision of host-sequestered iron. The simultaneous addition of inotropes with an antibiotic did not affect antibiotic susceptibility. Inotrope-induced growth in bacteria previously exposed to antibiotics was blocked by the inclusion in culture media of specific catecholamine-receptor antagonists. Considered collectively, these results provide a mechanistic basis for understanding how host-related factors, such as inotrope-based therapeutics, may influence the recovery of antibiotic-stressed bacteria in clinical settings.

Microbial endocrinology: how stress influences susceptibility to infection.

A holistic approach to understanding the mechanisms by which stress influences the pathogenesis of infectious disease has resulted in the development of the field of microbial endocrinology. This transdisciplinary field represents the intersection of microbiology with mammalian endocrinology and neurophysiology, and is based on the tenet that microorganisms have evolved systems for using neurohormones, which are widely distributed throughout nature, as environmental cues to initiate growth and pathogenic processes. This review reveals that responsiveness to human stress hormones is widespread in the microbial world and documents recent advances in microbial endocrinology.

Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China.

BACKGROUND: We are profoundly ignorant about the diversity of viruses that infect the domain Archaea. Less than 100 have been identified and described and very few of these have had their genomic sequences determined. Here we report the genomic sequence of a previously undescribed archaeal virus. RESULTS: Haloarchaeal strains with 16S rRNA gene sequences 98% identical to Halorubrum saccharovorum were isolated from a hypersaline lake in Inner Mongolia. Two lytic viruses infecting these were isolated from the lake water. The BJ1 virus is described in this paper. It has an icosahedral head and tail morphology and most likely a linear double stranded DNA genome exhibiting terminal redundancy. Its genome sequence has 42,271 base pairs with a GC content ofapproximately 65 mol%. The genome of BJ1 is predicted to encode 70 ORFs, including one for a tRNA. Fifty of the seventy ORFs had no identity to data base entries; twenty showed sequence identity matches to archaeal viruses and to haloarchaea. ORFs possibly coding for an origin of replication complex, integrase, helicase and structural capsid proteins were identified. Evidence for viral integration was obtained. CONCLUSION: The virus described here has a very low sequence identity to any previously described virus. Fifty of the seventy ORFs could not be annotated in any way based on amino acid identities with sequences already present in the databases. Determining functions for ORFs such as these is probably easier using a simple virus as a model system.

Influence of dietary catechols on the growth of enteropathogenic bacteria.

The dietary constituents that may act, in the broadest sense, as co-factors to enable bacterial enteropathogens to replicate in gastrointestinal environments are still largely unknown. Recent work has demonstrated that certain non-nutritional components of food, such as the catecholamines, can contribute to the ability of Gram-negative pathogens to replicate in iron-restrictive media that may be reflective of gastrointestinal environments. The present report examines whether other, non-catecholamine, dietary catechols, which occur widely in plant foods, can also influence enteropathogen growth in an iron-restrictive environment such as might be found in the gastrointestinal tract. In the present study, we have examined the ability of a range of catechol-rich foodstuffs, ranging from beverages (tea and coffee) to fruit and vegetable extracts, as well as purified preparations of commonly consumed dietary catechols (catechins, chlorogenic acid, caffeic acid and tannic acid), to modulate the growth of the Gram-negative enteric pathogens Escherichia coli O157:H7 and Salmonella enterica SV Enteriditis. Time-dependent growth in response to dietary catechols (0.05-5.0% v/v of beverage or fruit/vegetable extracts; 10-200 microM of purified catechols) was examined in an iron-replete, rich medium as well as in an iron-limited, basal medium designed to reflect the iron-restricted environment that is more characteristic of human and animal tissues. Results obtained in iron-replete, rich medium demonstrated dose-dependent bacteriostatic effects for certain catechols, consistent with previous studies. However, in iron-restricted medium, all of the dietary catechols produced marked growth stimulation of up to 4 logs greater than non-supplemented controls. Mechanistic studies measuring the uptake of radiolabelled (55)Fe from (55)Fe-labelled lactoferrin and transferrin in bacteria grown in the presence or absence of dietary catechols demonstrated that the ability of catechols to stimulate bacterial growth was dependent on the provision of iron from iron-sequestering glycoproteins. Urea gel analysis of transferrin incubated in the presence of the dietary catechols confirmed that these compounds were directly chelating and removing transferrin-complexed iron. Analysis using E. coli O157:H7 entA and tonB mutants further showed that a functional siderophore synthesis and uptake system was required for the growth-stimulatory response. In contrast to previous studies, which have reported the anti-microbial activity of dietary catechols, the present study demonstrates that these non-nutritional components of foods can, under iron-restrictive conditions, provide iron and enable the growth of enteric bacterial pathogens.

Blockade of catecholamine-induced growth by adrenergic and dopaminergic receptor antagonists in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica.

BACKGROUND: The ability of catecholamines to stimulate bacterial growth was first demonstrated just over a decade ago. Little is still known however, concerning the nature of the putative bacterial adrenergic and/or dopaminergic receptor(s) to which catecholamines (norepinephrine, epinephrine and dopamine) may bind and exert their effects, or even whether the binding properties of such a receptor are similar between different species. RESULTS: Use of specific catecholamine receptor antagonists revealed that only alpha, and not beta, adrenergic antagonists were capable of blocking norepinephrine and epinephrine-induced growth, while antagonism of dopamine-mediated growth was achieved with the use of a dopaminergic antagonist. Both adrenergic and dopaminergic antagonists were highly specific in their mechanism of action, which did not involve blockade of catecholamine-facilitated iron-acquisition. Use of radiolabeled norepinephrine suggested that the adrenergic antagonists could be acting by inhibiting catecholamine uptake. CONCLUSION: The present data demonstrates that the ability of a specific pathogen to respond to a particular hormone is dependent upon the host anatomical region in which the pathogen causes disease as well as the neuroanatomical specificity to which production of the particular hormone is restricted; and that both are anatomically coincidental to each other. As such, the present report suggests that pathogens with a high degree of exclusivity to the gastrointestinal tract have evolved response systems to neuroendocrine hormones such as norepinephrine and dopamine, but not epinephrine, which are found with the enteric nervous system.

Specificity of catecholamine-induced growth in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica.

The present study demonstrates that catecholamine responsiveness in Yersinia enterocolitica, a bacterial pathogen whose infectious spectrum is principally limited to the gut, is limited to norepinephrine and dopamine, and not epinephrine; this behavior contrasts with observations for two pathogens with a wider extra-gastrointestinal spectrum, Escherichia coli O157:H7 and Salmonella enterica, which respond to all three catecholamines. Epinephrine showed lower potency than norepinephrine and dopamine in inducing growth of E. coli and S. enterica, and was a potent antagonist of norepinephrine and dopamine growth responsiveness in Y. enterocolitica. Given that only norepinephrine and dopamine and not epinephrine-containing neurons are found with the enteric nervous system, the results suggest that certain of the more exclusive enteric pathogens may have developed response systems preferentially for those neuroendocrine hormones that are produced by the enteric nervous system as host-derived signals by which to sense the environment and initiate pathogenic processes.

Involvement of enterobactin in norepinephrine-mediated iron supply from transferrin to enterohaemorrhagic Escherichia coli.

Exposure of bacteria to members of the stress-associated family of catecholamine hormones, principally norepinephrine, has been demonstrated to increase both growth and production of virulence-related factors. Mutation of genes for enterobactin synthesis and uptake revealed an absolute requirement for enterobactin in norepinephrine-stimulated growth of Escherichia coli O157:H7. The autoinducer produced by norepinephrine-stimulated E. coli could not substitute for enterobactin. We also demonstrate that norepinephrine promotes iron shuttling between transferrin molecules, thereby enabling the bacterial siderophore enterobactin to more readily acquire iron for growth. These results suggest one of the possible mechanisms by which the hormonal output of stress may affect enterohaemorrhagic E. coli pathogenicity.

Stimulation of Staphylococcus epidermidis growth and biofilm formation by catecholamine inotropes.

BACKGROUND: Bacterial colonisation of indwelling medical devices by coagulase-negative staphylococci is a prevalent risk in intensive-care units. Factors determining biofilm formation and progression to catheter- related infection are incompletely understood. We postulated that administration of inotropic agents via indwelling intravenous catheters may stimulate growth and formation of biofilms by Staphylococcus epidermidis. METHODS: Inocula representing physiologically relevant infecting doses of S epidermidis were incubated in a minimum medium supplemented with fresh human plasma in the presence or absence of pharmacological concentrations of norepinephrine or dobutamine. Biofilm formation on polystyrene and medical-grade silicone was examined. After incubation, cultures were assessed for growth and formation of biofilms by colony counting and scanning electronmicroscopy. The production of exopolysaccharide, a major constituent of S epidermidis biofilms, was also assessed by use of immunofluorescence microscopy. FINDINGS: Incubation of S epidermidis with catecholamine inotropes in the presence of human plasma resulted in a significant increase in growth compared with control on both polystyrene and silicone surfaces, with pronounced increases in biofilm formation as visualised by scanning electronmicroscopy. Experiments with transferrin labelled with radioactive iron showed the ability of catecholamine inotropes to facilitate acquisition of iron by S epidermidis. Immunofluorescence microscopy revealed extensive exopolysaccharide production associated with S epidermidis biofilms. INTERPRETATION: The ability of catecholamine inotropic drugs to stimulate bacterial proliferation and biofilm formation may be an aetiological factor in the development of intravascular catheter colonisation and catheter-related infection. The removal of iron from transferrin for subsequent use by S epidermidis is a possible mechanism by which catecholamine inotropes stimulate bacterial growth as biofilms.

Growth stimulation of intestinal commensal Escherichia coli by catecholamines: a possible contributory factor in trauma-induced sepsis.

Trauma is well recognized to result in the immediate and sustained release of stress-related neurochemicals such as the catecholamine norepinephrine. Past work has shown that in addition to their ability to function as neurotransmitters, catecholamines can also directly stimulate the growth of a number of pathogenic bacteria. The development of trauma-associated sepsis has often been linked to the ability of otherwise normal commensal bacteria to invade and penetrate the gut mucosal barrier. Therefore, the aim of our study was to examine whether catecholamines could also stimulate the growth of commensal Escherichia coli strains of the type present in the intestinal tract at the time of a traumatic event. Herein we report that the growth of a range of non-pathogenic isolates of E. coli of human and environmental origin was significantly increased in the presence of catecholamines. A primary mechanism by which catecholamines increase bacterial growth was shown to be iron removal from lactoferrin and transferrin and subsequent acquisition by bacteria. The 3,4-dihydroxybenzoyl (catechol) structure of the catecholamines was further demonstrated to be critical to iron acquisition. The synthetic catecholamine inotropes dobutamine and isoprenaline, as well as norepinephrine metabolites that retained the catechol structure were also active, whereas norepinephrine metabolites in which the catechol moiety had been modified were not. A role for catecholamine-mediated bacterial iron supply in the pathophysiology of gut-derived sepsis due to trauma is proposed.