Characterisation of the E. coli and Salmonella qseC and qseE mutants reveals a metabolic rather than adrenergic receptor role.

Catecholamine stress hormones (norepinephrine, epinephrine, and dopamine) are signals that have been shown to be used as environmental cues, which affect the growth and virulence of normal microbiota as well as pathogenic bacteria. It has been reported that Escherichia coli and Salmonella use the two-component system proteins QseC and QseE to recognise catecholamines and so act as bacterial adrenergic receptors. In this study, we mutated the E. coli O157:H7 and Salmonella enterica serovar Typhimurium genes encoding QseC and QseE and found that this did not block stress hormone responsiveness in either species. Motility, biofilm formation, and analysis of virulence of the mutants using two infection models were similar to the wild-type strains. The main differences in phenotypes of the qseC and qseE mutants were responses to changes in temperature and growth in different media particularly with respect to salt, carbon, and nitrogen salt sources. In this physiological respect, it was also found that the phenotypes of the qseC and qseE mutants differed between E. coli and Salmonella. These findings collectively suggest that QseC and QseE are not essential for E. coli and Salmonella to respond to stress hormones and that the proteins may be playing a role in regulating metabolism.

Host Stress Signals Stimulate Pneumococcal Transition from Colonization to Dissemination into the Lungs.

Streptococcus pneumoniae is an asymptomatic colonizer of the nasopharynx, but it is also one of the most important bacterial pathogens of humans, causing a wide range of mild to life-threatening diseases. The basis of the pneumococcal transition from a commensal to a parasitic lifestyle is not fully understood. We hypothesize that exposure to host catecholamine stress hormones is important for this transition. In this study, we demonstrated that pneumococci preexposed to a hormone released during stress, norepinephrine (NE), have an increased capacity to translocate from the nasopharynx into the lungs compared to untreated pneumococci. Examination of NE-treated pneumococci revealed major alterations in metabolic profiles, cell associations, capsule synthesis, and cell size. By systemically mutating all 12 two-component and 1 orphan regulatory systems, we also identified a unique genetic regulatory circuit involved in pneumococcal recognition and responsiveness to human stress hormones. IMPORTANCE Microbes acquire unique lifestyles under different environmental conditions. Although this is a widespread occurrence, our knowledge of the importance of various host signals and their impact on microbial behavior is not clear despite the therapeutic value of this knowledge. We discovered that catecholamine stress hormones are the host signals that trigger the passage of Streptococcus pneumoniae from a commensal to a parasitic state. We identify that stress hormone treatment of this microbe leads to reductions in cell size and capsule synthesis and renders it more able to migrate from the nasopharynx into the lungs in a mouse model of infection. The microbe requires the TCS09 protein for the recognition and processing of stress hormone signals. Our work has particular clinical significance as catecholamines are abundant in upper respiratory fluids as well as being administered therapeutically to reduce inflammation in ventilated patients, which may explain why intubation in the critically ill is a recognized risk factor for the development of pneumococcal pneumonia.

A Virulence Associated Siderophore Importer Reduces Antimicrobial Susceptibility of Klebsiella pneumoniae.

The accessory genomes of many pathogenic bacteria include ABC transporters that scavenge metal by siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug efflux. There are mechanistic and recently recognized structural similarities between siderophore importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ on antimicrobial resistance of Klebsiella pneumoniae. YbtPQ is encoded in the yersiniabactin cluster in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp increased the susceptibility of K. pneumoniae to all antimicrobials tested. The mechanism was dependent on the yersiniabactin importer YbtPQ and may involve antimicrobial efflux, since it was affected by the inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. pneumoniae is recognized as a reservoir of genes for the emergence of hospital outbreak strains and for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, to E. coli decreased its susceptibility to a broad range of antimicrobials. Thus a confirmed siderophore importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have a secondary function exporting antimicrobials.

A Retrospective Analysis of Peritoneal Dialysis-associated Peritonitis at a Single-care Children's Hospital in Saudi Arabia.

Peritoneal dialysis (PD) may lead to infection, which could cause dialysis failure. Estimates of infectious peritonitis rate, identification of causative microbes, and infection outcomes are scarcely reported in Saudi Arabia. We conducted this study to provide epidemiological data as a geographical reference for PD-associated peritonitis. Epidemiological, microbiological, and clinical data were retrospectively collected from pediatric patients' medical records who were on PD between 2009 and 2018. A total of 54 pediatric patients on PD were involved in the study. The median age of the patients was 3.6 years [interquartile range (IQR) 1.7-8.0]; 56% were female children. The median time spent on PD was 39.5 months (IQR 19.75-64.25), with an overall peritonitis rate of 0.52 episodes per patient-year. In terms of infection, 116 PD-associated peritonitis episodes were identified in 37 patients. There were 38 infection episodes (32.3%) due to skin microbiota, 22 (18.8%) of gut microbiota, 12 (10.2%) of environmental microbiota, three (2.6%) of polymicrobial, and 27 (23.2%) of culture negative peritonitis. There were 45 (39%) episodes of noncomplicated peritonitis and 71 (61%) complicated peritonitis episodes. The latter included 14 (19%) relapses, 45 (63%) repeated infections, and 12 (17%) catheter removals. Using multivariate analysis, the history of peritonitis [OD 7.59, 95% confidence interval (CI) 2.87-20.00] and the presence of cardiovascular disease (OD 3.24,95% CI 1.18-8.85) were independent predictors of a complicated peritonitis episode. History of infectious peritonitis and the presence of cardiovascular disease are potential predictors of complicated PD-associated peritonitis and may provide an identifier of high-risk patients.

The Iron Biology Status of Peritoneal Dialysis Patients May Be a Risk Factor for Development of Infectious Peritonitis.

Background:Infectious peritonitis is a clinically important condition contributing to the significant mortality and morbidity rates observed in peritoneal dialysis (PD) patients. Although some of the socioeconomic risk factors for PD-associated peritonitis have been identified, it is still unclear why certain patients are more susceptible than others to infection.Methods:We examined the molecular components of human peritoneal dialysate (HPD) in an attempt to identify factors that might increase patient susceptibility to infection. Characterization studies were performed on initial and follow-up dialysate samples collected from 9 renal failure patients on PD.Results:Our in vitro data showed that peritonitis-causing bacteria grew differently in the patient dialysates. Proteomic analysis identified an association between transferrin presence and infection risk, as peritoneal transferrin was discovered to be iron-saturated, which was in marked contrast to transferrin in blood. Further, use of radioactive iron-labeled transferrin showed peritoneal transferrin could act as a direct iron source for the growth of peritonitis-causing bacteria. We also found catecholamine stress hormones noradrenaline and adrenaline were present in the dialysates and were apparently involved in enhancing the growth of the bacteria via transferrin iron provision. This suggests the iron biology status of the PD patient may be a risk factor for development of infectious peritonitisConclusions:Collectively, our study suggests transferrin and catecholamines within peritoneal dialysate may be indicators of the potential for bacterial growth in HPD and, as infection risk factors, represent possible future targets for therapeutic manipulation.

Salad Leaf Juices Enhance Salmonella Growth, Colonization of Fresh Produce, and Virulence.

We show in this report that traces of juices released from salad leaves as they become damaged can significantly enhance colonization of salad leaves by Salmonella enterica Salad juices in water increased Salmonella growth by 110% over the level seen with the unsupplemented control and in host-like serum-based media by more than 2,400-fold over control levels. In serum-based media, salad juices induced growth of Salmonella via provision of Fe from transferrin, and siderophore production was found to be integral to the growth induction process. Other aspects relevant to salad leaf colonization and retention were enhanced, such as motility and biofilm formation, which were increased over control levels by >220% and 250%, respectively; direct attachment to salad leaves increased by >350% when a salad leaf juice was present. In terms of growth and biofilm formation, the endogenous salad leaf microbiota was largely unresponsive to leaf juice, suggesting that Salmonella gains a marked growth advantage from fluids released by salad leaf damage. Salad leaf juices also enhanced pathogen attachment to the salad bag plastic. Over 5 days of refrigeration (a typical storage time for bagged salad leaves), even traces of juice within the salad bag fluids increased Salmonella growth in water by up to 280-fold over control cultures, as well as enhancing salad bag colonization, which could be an unappreciated factor in retention of pathogens in fresh produce. Collectively, the study data show that exposure to salad leaf juice may contribute to the persistence of Salmonella on salad leaves and strongly emphasize the importance of ensuring the microbiological safety of fresh produce. IMPORTANCE: Salad leaves are an important part of a healthy diet but have been associated in recent years with a growing risk of food poisoning from bacterial pathogens such as Salmonella enterica Although this is considered a significant public health problem, very little is known about the behavior of Salmonella in the actual salad bag. We show that juices released from the cut ends of the salad leaves enabled the Salmonella cells to grow in water, even when it was refrigerated. Salad juice exposure also helped the Salmonella cells to attach to the salad leaves so strongly that washing could not remove them. Collectively, the results presented in this report show that exposure to even traces of salad leaf juice may contribute to the persistence of Salmonella on salad leaves as well as priming it for establishing an infection in the consumer.

Staphylococci, Catecholamine Inotropes and Hospital-Acquired Infections.

Patients in hospital intensive care units have long been recognized as being at high risk for developing infections from bacteria, fungi, and viruses from within the hospital locality. Risk factors for development of nosocomial infections have usually focussed on the patient's physical condition and the number and type of invasive medical procedures administered. Using the staphylococci as its focus, this chapter presents recent evidence that some of the medications routinely used in the treatment of acutely ill patients may also be a risk factor for the development of nosocomial infections.

Microbial endocrinology: host-bacteria communication within the gut microbiome.

The human body is home to trillions of micro-organisms, which are increasingly being shown to have significant effects on a variety of disease states. Evidence exists that a bidirectional communication is taking place between us and our microbiome co-habitants, and that this dialogue is capable of influencing our health in a variety of ways. This review considers how host hormonal signals shape the microbiome, and what in return the microbiome residents may be signalling to their hosts.

Host stress hormone norepinephrine stimulates pneumococcal growth, biofilm formation and virulence gene expression.

BACKGROUND: Host signals are being shown to have a major impact on the bacterial phenotype. One of them is the endogenously produced catecholamine stress hormones, which are also used therapeutically as inotropes. Recent work form our laboratories have found that stress hormones can markedly increase bacterial growth and virulence. This report reveals that Streptococcus pneumoniae, a commensal that can also be a major cause of community acquired and nosocomial pneumonia, is highly inotrope responsive. Therapeutic levels of the stress hormone norepinephrine increased pneumococcal growth via a mechanism involving provision of iron from serum-transferrin and inotrope uptake, as well as enhancing expression of key genes in central metabolism and virulence. Collectively, our data suggests that Streptococcus pneumoniae recognises host stress as an environmental cue to initiate growth and pathogenic processes. RESULTS: Effects of a clinically attainable concentration of norepinephrine on S. pneumoniae pathogenicity were explored using in vitro growth and virulence assays, and RT-PCR gene expression profiling of genes involved in metabolism and virulence.We found that norepinephrine was a potent stimulator of growth, via a mechanism involving norepinephrine-delivery of transferrin-iron and internalisation of the inotrope. Stress hormone exposure also markedly increased biofilm formation. Importantly, gene profiling showed that norepinephrine significantly enhanced expression of genes involved in central metabolism and host colonisation. Analysis of the response of the pneumococcal pspA and pspC mutants to the stress hormone showed them to have a central involvement in the catecholamine response mechanism. CONCLUSIONS: Collectively, our evidence suggests that the pneumococcus has mechanisms to recognise and process host stress hormones to augment its virulence properties. The ability to respond to host stress signals may be important for the pneumococcal transition from colonization to invasion mode, which is key to its capacity to cause life-threatening pneumonia, septicaemia and meningitis.

Respiratory syncytial virus increases the virulence of Streptococcus pneumoniae by binding to penicillin binding protein 1a. A new paradigm in respiratory infection.

RATIONALE: Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are major respiratory pathogens. Coinfection with RSV and S. pneumoniae is associated with severe and often fatal pneumonia but the molecular basis for this remains unclear. OBJECTIVES: To determine if interaction between RSV and pneumococci enhances pneumococcal virulence. METHODS: We used confocal microscopy and Western blot to identify the receptors involved in direct binding of RSV and pneumococci, the effects of which were studied in both in vivo and in vitro models of infection. Human ciliated respiratory epithelial cell cultures were infected with RSV for 72 hours and then challenged with pneumococci. Pneumococci were collected after 2 hours exposure and changes in gene expression determined using quantitative real-time polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Following incubation with RSV or purified G protein, pneumococci demonstrated a significant increase in the inflammatory response and bacterial adherence to human ciliated epithelial cultures and markedly increased virulence in a pneumonia model in mice. This was associated with extensive changes in the pneumococcal transcriptome and significant up-regulation in the expression of key pneumococcal virulence genes, including the gene for the pneumococcal toxin, pneumolysin. We show that mechanistically this is caused by RSV G glycoprotein binding penicillin binding protein 1a. CONCLUSIONS: The direct interaction between a respiratory virus protein and the pneumococcus resulting in increased bacterial virulence and worsening disease outcome is a new paradigm in respiratory infection.

Role of porin proteins in acquisition of transferrin iron by enteropathogens.

Acquisition of iron from key innate immune defence proteins such as transferrin (Tf) and lactoferrin is an important mechanism by which pathogenic bacteria obtain essential iron for growth within their host. Bacterial species that do not produce siderophores often use specific Tf-binding proteins, the best characterized being the Neisseriaceae-type Tf-binding proteins TbpA and TbpB. Previous work from our laboratory has shown that siderophore-producing enteric species such as Escherichia coli also readily bind Tf, although no genomic evidence exists for Tbp-like Tf-binding proteins. Application of proteomic analyses and molecular mutagenesis strategies to an enteropathogenic E. coli identified the OmpA and OmpC porins as Tf-binding proteins. Mutagenesis of the ompA or ompC genes affected E. coli Tf binding and, furthermore, compromised the ability of the ompA mutant to respond to growth promotion by certain catecholamine stress hormones. Evidence was also found to implicate the OmpA porin as an entry point for catecholamine stress hormones. Further proteomic analyses in other bacterial pathogens revealed wide-scale involvement of porins in Tf binding: Salmonella typhimurium (OmpC), and Shigella sonnei, Shigella flexneri and Shigella boydii (OmpC and/or OmpA). This study shows that in addition to their existing housekeeping functions, the Gram-negative porin family of proteins can also act as Tf-capture proteins for those bacteria that lack the classical Neisseriaceae-type Tf-binding proteins.

Mutation design and strain background influence the phenotype of Escherichia coli luxS mutants.

Previous analyses of luxS in Escherichia coli have used different strain backgrounds and design formats to produce the luxS mutation, resulting in luxS mutants with confusingly dissimilar phenotypes. This study therefore investigates the roles that strain background and mutational design strategy have upon the phenotype of the pathogenic E. coli luxS mutant. We inactivated luxS in three E. coli backgrounds: enteropathogenic E. coli E2348-69, and enterohaemorrhagic strains Sakai and NCTC12900. To investigate the influence of mutational design strategy, four mutation formats were used: antibiotic resistance insertion methodologies as previously employed, using tetracycline and chloramphenicol resistance cassettes, and non-polar strategies creating deletion and premature termination mutations. Our study showed that the E. coli luxS phenotype was markedly dependent on strain background: in some strains disruption of luxS caused significant metabolic stress or no stress at all. How the luxS mutation was constructed also shaped its phenotype: non-polar mutants were very similar to wild type, while mutations made using the antibiotic insertion methodologies produced phenotypes defective in growth and virulence. Proteomic profiling of our luxS mutants showed only a few proteins were differentially expressed and those that were altered suggested a metabolic rather than communication role for the E. coli luxS gene product.

Communication between Bacteria and Their Hosts.

It is clear that a dialogue is occurring between microbes and their hosts and that chemical signals are the language of this interkingdom communication. Microbial endocrinology shows that, through their long coexistence with animals and plants, microorganisms have evolved sensors for detecting eukaryotic hormones, which the microbe uses to determine that they are within proximity of a suitable host and to optimally time the expression of genes needed for host colonisation. It has also been shown that some prokaryotic chemical communication signals are recognized by eukaryotes. Deciphering what is being said during the cross-talk between microbe and host is therefore important, as it could lead to new strategies for preventing or treating bacterial infections.

Pseudomonas aeruginosa-catecholamine inotrope interactions: a contributory factor in the development of ventilator-associated pneumonia?

BACKGROUND: Ventilated patients receiving intensive care are at significant risk of acquiring a ventilator-associated pneumonia that is associated with significant morbidity and mortality. Despite intensive research, it is still unclear why Pseudomonas aeruginosa, a microbe that rarely causes pneumonia outside of intensive care, is responsible for so many of these infections. METHODS: We investigated whether medications frequently prescribed to patients in the ICU, the catecholamine inotropes, were affecting the growth and virulence of P aeruginosa . Effects of clinically attainable concentrations of inotropes on P aeruginosa pathogenicity were explored using in vitro growth and virulence assays and an ex vivo model of infection using ciliated human respiratory epithelium. RESULTS: We found that inotropes were potent stimulators of P aeruginosa growth, producing upto 50-fold increases in bacterial numbers via a mechanism involving inotrope delivery of transferrin-ron,internalization of the inotrope, and upregulation of the key pseudomonal siderophore pyoverdine.Inotropes also markedly increased biofilm formation on endotracheal tubing and enhanced the biofilm production and toxicity of P aeruginosa in its interaction with respiratory epithelium.Importantly, catecholamine inotropes also facilitated the rapid recovery of P aeruginosa from tobramycin antibiotic challenge. We also tested out the effect of the inotropes vasopressin and phenylephrine on the growth and virulence of P aeruginosa and found that, in contrast to the catecholamines,these drugs had no stimulatory effect. CONCLUSIONS: Collectively, our results suggest that catecholamine inotrope-bacterial interactions may be an unexpected contributory factor to the development of P aeruginosa -ventilator-associated pneumonia.

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.

Norepinephrine augments Salmonella enterica-induced enteritis in a manner associated with increased net replication but independent of the putative adrenergic sensor kinases QseC and QseE.

Stress has long been correlated with susceptibility to microbial infection. One explanation for this phenomenon is the ability of pathogens to sense and respond to host stress-related catecholamines, such as norepinephrine (NE). In Gram-negative enteric pathogens, it has been proposed that NE may facilitate growth by mediating iron supply, or it may alter gene expression by activating adrenergic sensor kinases. The aim of this work was to investigate the relative importance of these processes in a model in which NE alters the outcome of Salmonella enterica serovar Typhimurium infection. A bovine ligated ileal loop model was used to study the effect of NE on enteritis induced by S. Typhimurium and on the bacterial in vivo replication rate. Mutants lacking putative adrenergic receptor genes were assessed in the loop model, in a calf intestinal colonization model, and in vitro. S. Typhimurium-induced enteritis was significantly enhanced by addition of 5 mM NE. This effect was associated with increased net bacterial replication in the same model. Exogenous ferric iron also stimulated bacterial replication in the medium used but not transcription of enteritis-associated loci. The putative adrenergic sensors QseC and QseE were not required for NE-enhanced enteritis, intestinal colonization of calves, or NE-dependent growth in iron-restricted medium and did not influence expression or secretion of enteritis-associated virulence factors. Our findings support a role for stress-related catecholamines in modulating the virulence of enteric bacterial pathogens in vivo but suggest that bacterial adrenergic sensors may not be the vital link in such interkingdom signaling in Salmonella.

Norepinephrine represses the expression of toxA and the siderophore genes in Pseudomonas aeruginosa.

Among the different extracellular virulence factors produced by Pseudomonas aeruginosa are exotoxin A (ETA) and the pyoverdine and pyochelin siderophores. Production of ETA and the siderophores requires the function of the iron-starvation sigma factor PvdS, the transcriptional activator RegA, and the AraC-activator PchR. Iron represses the production of ETA and the siderophores by repressing the expression of pvdS, regA, and pchR. PvdS regulates the expression of the ETA gene, toxA, regA, and the pyoverdine synthesis genes. The catecholamine norepinephrine enhances the growth of pathogenic bacteria by transferring iron from host-binding proteins. In this study, we elucidated the mechanism by which norepinephrine and other catecholamines induce P. aeruginosa growth. We also investigated whether norepinephrine regulates the expression of toxA and the siderophore genes, and the mechanism of this regulation. Norepinephrine enhanced the growth of P. aeruginosa by supplying iron from transferrin. This provision of iron repressed the expression of toxA, the pyoverdine genes pvdD and pvdE, and their regulators, pvdS, regA, and pchR, suggesting that norepinephrine accomplishes this repression through PvdS and PchR. Additionally, norepinephrine bypassed PvdS and supported the growth of a pvdS deletion mutant, indicating that norepinephrine transfers iron to P. aeruginosa independent of pyoverdine. Thus, norepinephrine apparently influences the pathogenesis of P. aeruginosa by affecting its pattern of growth and the production of virulence factors.

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.