Phylogenomic analyses of multidrug resistant Corynebacterium striatum strains isolated from patients in a tertiary care hospital in the UK.

Corynebacterium striatum is an emerging nosocomial pathogen. This is the first report showing the presence of three distinct multidrug resistant lineages of C. striatum among patients in a UK hospital. The presence of ErmX, Tet(W), Bla and AmpC proteins, and mutations in gyrA gene are associated with the resistance to clindamycin, doxycycline, penicillin and moxifloxacin, respectively. These strains are equipped with several corynebacterial virulence genes including two SpaDEF-type and a novel pilus gene cluster, which needs further molecular characterisation. This study highlights a need of developing an active surveillance strategy for routine monitoring and preventing potential cross-transmission among susceptible patients.

Phosphoinositide 3-Kinase δ Inhibition Improves Neutrophil Bacterial Killing in Critically Ill Patients at High Risk of Infection.

Acquired neutrophil dysfunction frequently develops during critical illness, independently increasing the risk for intensive care unit-acquired infection. PI3Kδ is implicated in driving neutrophil dysfunction and can potentially be targeted pharmacologically. The aims of this study were to determine whether PI3Kδ inhibition reverses dysfunction in neutrophils from critically ill patients and to describe potential mechanisms. Neutrophils were isolated from blood taken from critically ill patients requiring intubation and mechanical ventilation, renal support, or blood pressure support. In separate validation experiments, neutrophil dysfunction was induced pharmacologically in neutrophils from healthy volunteers. Phagocytosis and bacterial killing assays were performed, and activity of RhoA and protein kinase A (PKA) was assessed. Inhibitors of PI3Kδ, 3-phosphoinositide-dependent protein kinase-1 (PDK1), and PKA were used to determine mechanisms of neutrophil dysfunction. Sixty-six patients were recruited. In the 27 patients (40.9%) with impaired neutrophil function, PI3Kδ inhibition consistently improved function and significantly increased bacterial killing. These findings were validated in neutrophils from healthy volunteers with salbutamol-induced dysfunction and extended to demonstrate that PI3Kδ inhibition restored killing of clinical isolates of nine pathogens commonly associated with intensive care unit-acquired infection. PI3Kδ activation was associated with PDK1 activation, which in turn phosphorylated PKA, which drove phosphorylation and inhibition of the key regulator of neutrophil phagocytosis, RhoA. These data indicate that, in a significant proportion of critically ill patients, PI3Kδ inhibition can improve neutrophil function through PDK1- and PKA-dependent processes, suggesting that therapeutic use of PI3Kδ inhibitors warrants investigation in this setting.

In vitro effects of combined iron chelation, antibiotics and matrix disruption on clinical isolates of Pseudomonas aeruginosa.

OBJECTIVES: Pseudomonas aeruginosa is an important pathogen in chronic suppurative respiratory diseases, with adverse effects on severity, healthcare utilization and quality of life. Aerosolized combined biofilm disruption and iron chelators offer novel proof-of-concept for improving airway antimicrobial efficacy. Our aim was to assess the activity of desferrioxamine, Dornase alfa (DNase) and antibiotics on biofilm formation and against mature preformed biofilms of P. aeruginosa. METHODS: Fifty-six isolates of P. aeruginosa were screened for biofilm production and seven isolates with varying capacity to form biofilms were referred for further study. Three antibiotics (colistin, tobramycin and ciprofloxacin) as well as desferrioxamine and DNase were assessed for their ability to prevent biofilm formation using the crystal violet assay. The same method was used to assess their impact on mature biofilms. Each agent, as well as combinations of these agents, was also assessed for its effect on the metabolic activity and viability of preformed P. aeruginosa biofilm by the resazurin reduction assay and by performing viable counts. RESULTS: Antibiotics alone prevented the development of biofilms and partly reduced the viability of mature biofilms. Desferrioxamine and DNase did not reduce biofilm formation. For most isolates, desferrioxamine and DNase did not offer any clear advantage over the use of antibiotics alone with respect to reducing the viability of Pseudomonas biofilms. CONCLUSIONS: Colistin, tobramycin and ciprofloxacin prevented biofilm formation by P. aeruginosa and reduced the viability of mature biofilms. For most isolates, there was no clear advantage of combining these antimicrobials with desferrioxamine or DNase.

Synthesis and Antimicrobial Activity of Phosphonopeptide Derivatives Incorporating Single and Dual Inhibitors.

In diagnostic microbiology, culture media are widely used for detection of pathogenic bacteria. Such media employ various ingredients to optimize detection of specific pathogens such as chromogenic enzyme substrates and selective inhibitors to reduce the presence of commensal bacteria. Despite this, it is rarely possible to inhibit the growth of all commensal bacteria, and thus pathogens can be overgrown and remain undetected. One approach to attempt to remedy this is the use of "suicide substrates" that can target specific bacterial enzymes and selectively inhibit unwanted bacterial species. With the purpose of identifying novel selective inhibitors, six novel phosphonopeptide derivatives based on d/l-fosfalin and ß-chloro-l-alanine were synthesized and tested on 19 different strains of clinically relevant bacteria. Several compounds show potential as useful selective agents that could be exploited in the recovery of several bacterial pathogens including Salmonella, Pseudomonas aeruginosa, and Listeria.

C-Terminal 1-Aminoethyltetrazole-Containing Oligopeptides as Novel Alanine Racemase Inhibitors.

In clinical culture media inoculated with patient samples, selective inhibition of commensal bacteria is essential for accurate diagnosis and effective treatment, as they can mask the presence of pathogenic bacteria. The alanine analogue, 1-aminoethyltetrazole was investigated as a potential alanine racemase inhibitor. For effective uptake and enhanced and selective antibacterial activity, a library of C-terminal 1-aminoethyltetrazole containing di- and oligopeptides were synthesized by solid phase peptide coupling techniques. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These enabled differentiation between the closely related bacteria, Salmonella and Escherichia coli, which can be difficult to discriminate between in a clinical setting. In addition, differentiation between enterococci and other Gram-positive cocci was also seen.

Phosphonopeptides Revisited, in an Era of Increasing Antimicrobial Resistance.

Given the increase in resistance to antibacterial agents, there is an urgent need for the development of new agents with novel modes of action. As an interim solution, it is also prudent to reinvestigate old or abandoned antibacterial compounds to assess their efficacy in the context of widespread resistance to conventional agents. In the 1970s, much work was performed on the development of peptide mimetics, exemplified by the phosphonopeptide, alafosfalin. We investigated the activity of alafosfalin, di-alanyl fosfalin and ß-chloro-L-alanyl-ß-chloro-L-alanine against 297 bacterial isolates, including carbapenemase-producing Enterobacterales (CPE) (n = 128), methicillin-resistant Staphylococcus aureus (MRSA) (n = 37) and glycopeptide-resistant enterococci (GRE) (n = 43). The interaction of alafosfalin with meropenem was also examined against 20 isolates of CPE. The MIC50 and MIC90 of alafosfalin for CPE were 1 mg/L and 4 mg/L, respectively and alafosfalin acted synergistically when combined with meropenem against 16 of 20 isolates of CPE. Di-alanyl fosfalin showed potent activity against glycopeptide-resistant isolates of Enterococcus faecalis (MIC90; 0.5 mg/L) and Enterococcus faecium (MIC90; 2 mg/L). Alafosfalin was only moderately active against MRSA (MIC90; 8 mg/L), whereas ß-chloro-L-alanyl-ß-chloro-L-alanine was slightly more active (MIC90; 4 mg/L). This study shows that phosphonopeptides, including alafosfalin, may have a therapeutic role to play in an era of increasing antibacterial resistance.

Performance of RGM Medium for Isolation of Nontuberculous Mycobacteria from Respiratory Specimens from Non-Cystic Fibrosis Patients.

A new selective medium for rapidly growing mycobacteria (RGM medium) was evaluated on respiratory specimens from non-cystic fibrosis patients and compared to the mycobacterial growth indicator tube (MGIT) system and Middlebrook 7H11 agar for the isolation of all nontuberculous mycobacteria (NTM). A total of 203 mucolyzed respiratory specimens collected from 163 patients were inoculated on RGM medium and incubated at both 30°C (RGM30) and 35°C (RGM35) over a 28-day period. N-Acetyl-l-cysteine-sodium hydroxide (NALC-NaOH)-decontaminated specimens were inoculated into MGIT and Middlebrook 7H11 agar and incubated at 35°C for 42 days. NTM were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) or gene sequencing. A total of 133 NTM isolates were recovered overall from 101 (49.8%) specimens collected from 85 (52.1%) patients by a combination of all culture methods. The sensitivity of RGM30 for the recovery of NTM was significantly higher than that of either the MGIT system (76.7% versus 59.4%; P = 0.01) or Middlebrook 7H11 agar (76.7% versus 47.4%; P = 0.0001) alone, but it was not significantly different from that of an acid-fast bacillus culture (AFC) which includes both MGIT and Middlebrook 7H11 agar (76.7% versus 63.9%; P = 0.0647). RGM35 had significantly lower sensitivity than the MGIT system (49.6% versus 59.4%; P = 0.0367) and AFC (49.6% versus 63.9%; P = 0.0023). RGM medium was highly effective at inhibiting the growth of nonmycobacterial organisms in the respiratory specimens, with breakthrough contamination rates of 5.4% and 4.4% for RGM30 and RGM35, respectively.

Fluorogenic l-alanylaminopeptidase substrates derived from 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins and their potential applications in diagnostic microbiology.

Six novel fluorogenic enzyme substrates for detecting l-alanylaminopeptidase activity in microorganisms have been prepared and evaluated in Columbia agar media. The substrates are l-alanyl derivatives of 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins. Both the quinoline and coumarin series of substrates produced fluorescence in the presence of Gram-negative microorganisms. In contrast, fluorescence generation in the presence of the Gram-positive microorganisms and yeasts was limited or absent.

Fluorogenic 7-azidocoumarin and 3/4-azidophthalimide derivatives as indicators of reductase activity in microorganisms.

A series of fluorogenic heterocyclic azides were prepared and assessed as reductase substrates across a selection of Gram-negative and Gram-positive microorganisms. The majority of these azides showed similar activity profiles to nitroreductase substrates. Microorganisms that do not produce hydrogen sulfide reduced the azides, indicating reductase activity was not linked to hydrogen sulfide production.

Elevated urine IL-10 concentrations associate with Escherichia coli persistence in older patients susceptible to recurrent urinary tract infections.

BACKGROUND: Age is a significant risk factor for recurrent urinary tract (rUTI) infections, but the clinical picture is often confused in older patients who also present with asymptomatic bacteriuria (ASB). Yet, how bacteriuria establishes in such patients and the factors underpinning and/or driving symptomatic UTI episodes are still not understood. To explore this further a pilot study was completed in which 30 male and female community based older patients (mean age 75y) presenting clinically with ASB / rUTIs and 15 control volunteers (72y) were recruited and monitored for up to 6 months. During this period symptomatic UTI episodes were recorded and urines collected for urinary cytokine and uropathogenic Escherichia coli (UPEC) analyses. RESULTS: Eighty-six per cent of patients carried E. coli (102 ≥ 105 CFU/ml urine) at some point throughout the study and molecular typing identified 26 different E. coli strains in total. Analyses of urine samples for ten different cytokines identified substantial patient variability. However, when examined longitudinally the pro-inflammatory markers, IL-1 and IL-8, and the anti-inflammatory markers, IL-5 and IL-10, were significantly different in the patient urines compared to those of the controls (P < 0.0001). Furthermore, analysing the cytokine data of the rUTI susceptible cohort in relation to E. coli carriage, showed the mean IL-10 concentration to be significantly elevated (P = 0.04), in patients displaying E. coli numbers ≥105 CFU/ml. CONCLUSIONS: These pilot study data suggest that bacteriuria, characteristic of older rUTI patients, is associated with an immune homeostasis in the urinary tract involving the synthesis and activities of the pro and anti-inflammatory cytokines IL-1, IL-5, IL-8 and IL-10. Data also suggests a role for IL-10 in regulating bacterial persistence.

Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients.

RATIONALE: Aspiration of infective subglottic secretions causes ventilator-associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP. OBJECTIVES: To characterize subglottic host defense dysfunction in mechanically ventilated patients in the ICU; to determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth. METHODS: Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 min); isolation and culture of primary subglottic epithelial cells from control patients; laboratory analysis of host innate immune defenses. MEASUREMENTS AND MAIN RESULTS: Twenty-four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in the ICU was characterized by neutrophilic inflammation, significantly increased proinflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic impairment was reversible on treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air-liquid interface model of primary human subglottic epithelium. CONCLUSIONS: Mechanical ventilation in the ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil function and promotes bacterial growth. Mucolytic agents reverse mucin-mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage.

A Selective, Dual Emission ß-Alanine Aminopeptidase Activated Fluorescent Probe for the Detection of Pseudomonas aeruginosa, Burkholderia cepacia, and Serratia marcescens.

Selective detection of ß-alanyl aminopeptidase (BAP)-producing Pseudomonas aeruginosa, Serratia marcescens, and Burkholderia cepacia was achieved by employing the blue-to-yellow fluorescent transition of a BAP-specific enzyme substrate, 3-hydroxy-2-(p-dimethylaminophenyl)flavone derivative, incorporating a self-immolative linker to ß-alanine. Upon cellular uptake and accumulation of the substrate by viable bacterial colonies, blue fluorescence was generated, while hydrolysis of the N-terminal peptide bond by BAP resulted in the elimination of the self-immolative linker and the restoration of the original fluorescence of the flavone derivative.

A Decade of Development of Chromogenic Culture Media for Clinical Microbiology in an Era of Molecular Diagnostics.

In the last 25 years, chromogenic culture media have found widespread application in diagnostic clinical microbiology. In the last decade, the range of media available to clinical laboratories has expanded greatly, allowing specific detection of additional pathogens, including Pseudomonas aeruginosa, group B streptococci, Clostridium difficile, Campylobacter spp., and Yersinia enterocolitica. New media have also been developed to screen for pathogens with acquired antimicrobial resistance, including vancomycin-resistant enterococci, carbapenem-resistant Acinetobacter spp., and Enterobacteriaceae with extended-spectrum ß-lactamases and carbapenemases. This review seeks to explore the utility of chromogenic media in clinical microbiology, with particular attention given to media that have been commercialized in the last decade. The impact of laboratory automation and complementary technologies such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is also assessed. Finally, the review also seeks to demarcate the role of chromogenic media in an era of molecular diagnostics.

Anti-bacterial antibody and T cell responses in bronchiectasis are differentially associated with lung colonization and disease.

BACKGROUND: As a way to determine markers of infection or disease informing disease management, and to reveal disease-associated immune mechanisms, this study sought to measure antibody and T cell responses against key lung pathogens and to relate these to patients' microbial colonization status, exacerbation history and lung function, in Bronchiectasis (BR) and Chronic Obstructive Pulmonary Disease (COPD). METHODS: One hundred nineteen patients with stable BR, 58 with COPD and 28 healthy volunteers were recruited and spirometry was performed. Bacterial lysates were used to measure specific antibody responses by ELISA and T cells by ELIspot. Cytokine secretion by lysate-stimulated T cells was measured by multiplex cytokine assay whilst activation phenotype was measured by flow cytometry. RESULTS: Typical colonization profiles were observed in BR and COPD, dominated by P.aeruginosa, H.influenzae, S.pneumoniae and M.catarrhalis. Colonization frequency was greater in BR, showing association with increased antibody responses against P.aeruginosa compared to COPD and HV, and with sensitivity of 73% and specificity of 95%. Interferon-gamma T cell responses against P.aeruginosa and S.pneumoniae were reduced in BR and COPD, whilst reactive T cells in BR had similar markers of homing and senescence compared to healthy volunteers. Exacerbation frequency in BR was associated with increased antibodies against P. aeruginosa, M.catarrhalis and S.maltophilia. T cell responses against H.influenzae showed positive correlation with FEV1% (r = 0.201, p = 0.033) and negative correlation with Bronchiectasis Severity Index (r = - 0.287, p = 0.0035). CONCLUSION: Our findings suggest a difference in antibody and T cell immunity in BR, with antibody being a marker of exposure and disease in BR for P.aeruginosa, M.catarrhalis and H.influenzae, and T cells a marker of reduced disease for H.influenzae.

The synthesis of novel chromogenic enzyme substrates for detection of bacterial glycosidases and their applications in diagnostic microbiology.

The preparation and evaluation of chromogenic substrates for detecting bacterial glycosidase enzymes is reported. These substrates are monoglycoside derivatives of the metal chelators catechol, 2,3-dihydroxynaphthalene (DHN) and 6,7-dibromo-2,3-dihydroxynaphthalene (6,7-dibromo-DHN). When hydrolysed by appropriate bacterial enzymes these substrates produced coloured chelates in the presence of ammonium iron(III) citrate, thus enabling bacterial detection. A ß-d-riboside of DHN and a ß-d-glucuronide derivative of 6,7-dibromo-DHN were particularly effective for the detection of S. aureus and E. coli respectively.

A latent green fluorescent styrylcoumarin probe for the selective growth and detection of Gram negative bacteria.

A novel, green fluorescent ß-alanylstyrylcoumarin derivative was synthesized and evaluated for its performance as a fluorogenic enzyme substrate on a range of clinically relevant microorganisms. The substrate was selectively hydrolysed by ß-alanyl aminopeptidase producing P. aeruginosa resulting in an on-to-off fluorescent signal. Growth inhibitory effect of the substrate was observed on Gram positive bacteria and yeasts. Meanwhile, Gram negative species, despite their extremely protective cell envelope, showed ready uptake and accumulation of the substrate within their healthy growing colonies displaying intense green fluorescence.

Methods for the detection and identification of pathogenic bacteria: past, present, and future.

In order to retard the rate of development of antibacterial resistance, the causative agent must be identified as rapidly as possible, so that directed patient treatment and/or contact precautions can be initiated. This review highlights the challenges associated with the detection and identification of pathogenic bacteria, by providing an introduction to the techniques currently used, as well as newer techniques that are in development. Focusing on the chemical basis for these techniques, the review also provides a comparison of their advantages and disadvantages.

Targeting the Bacterial Cytoskeleton of the Burkholderia cepacia Complex for Antimicrobial Development: A Cautionary Tale.

Burkholderia cepacia complex (BCC) bacteria are a group of opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult, due to the inherent and acquired multidrug resistance of BCC. There is a pressing need to find new bacterial targets for antimicrobials. Here, we demonstrate that the novel compound Q22, which is related to the bacterial cytoskeleton destabilising compound A22, can reduce the growth rate and inhibit growth of BCC bacteria. We further analysed the phenotypic effects of Q22 treatment on BCC virulence traits, to assess its feasibility as an antimicrobial. BCC bacteria were grown in the presence of Q22 with a broad phenotypic analysis, including resistance to H2O2-induced oxidative stress, changes in the inflammatory potential of cell surface components, and in-vivo drug toxicity studies. The influence of the Q22 treatment on inflammatory potential was measured by monitoring the cytokine responses of BCC whole cell lysates, purified lipopolysaccharide, and purified peptidoglycan extracted from bacterial cultures grown in the presence or absence of Q22 in differentiated THP-1 cells. BCC bacteria grown in the presence of Q22 displayed varying levels of resistance to H2O2-induced oxidative stress, with some strains showing increased resistance after treatment. There was strain-to-strain variation in the pro-inflammatory ability of bacterial lysates to elicit TNFα and IL-1ß from human myeloid cells. Despite minimal toxicity previously shown in vitro with primary CF cell lines, in-vivo studies demonstrated Q22 toxicity in both zebrafish and mouse infection models. In summary, destabilisation of the bacterial cytoskeleton in BCC, using compounds such as Q22, led to increased virulence-related traits in vitro. These changes appear to vary depending on strain and BCC species. Future development of antimicrobials targeting the BCC bacterial cytoskeleton may be hampered if such effects translate into the in-vivo environment of the CF infection.

A Carbocyclic Curcumin Inhibits Proliferation of Gram-Positive Bacteria by Targeting FtsZ.

Inhibition of FtsZ assembly has been found to stall bacterial cell division. Here, we report the identification of a potent carbocyclic curcumin analogue (2d) that inhibits Bacillus subtilis 168 cell proliferation by targeting the assembly of FtsZ. 2d also showed potent inhibitory activity (minimum inhibitory concentrations of 2-4 mg/L) against several clinically important species of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In addition, 2d displayed a significantly reduced inhibitory effect on human cervical cancer cells in comparison to its effect on bacterial cells. Using live cell imaging of GFP-FtsZ by confocal microscopy, 2d was found to rapidly perturb the cytokinetic FtsZ rings in Bacillus subtilis cells. The immunofluorescence imaging of FtsZ also showed that 2d destroyed the Z-ring in bacteria within 5 min. Prolonged treatment with 2d produced filamentous bacteria, but 2d had no detectable effect either on the nucleoids or on the membrane potential of bacteria. 2d inhibited FtsZ assembly in vitro, whereas it had minimal effects on tubulin assembly. Interestingly, 2d strongly enhanced the GTPase activity of FtsZ and reduced the GTPase activity of tubulin. Furthermore, 2d bound to purified FtsZ with a dissociation constant of 4.0 ± 1.1 µM, and the binding of 2d altered the secondary structures of FtsZ. The results together suggested that the non-natural curcumin analogue 2d possesses powerful antibacterial activity against important pathogenic bacteria, and the evidence indicates that 2d inhibits bacterial proliferation by targeting FtsZ.

Evaluation of RGM Medium for Isolation of Nontuberculous Mycobacteria from Respiratory Samples from Patients with Cystic Fibrosis in the United States.

A novel selective agar (RGM medium) has been advocated for the isolation of rapidly growing mycobacteria from the sputa of cystic fibrosis (CF) patients. The aim of this study was to compare RGM medium to Burkholderia cepacia selective agar (BCSA) and a standard acid-fast bacillus (AFB) culture method for the isolation of nontuberculous mycobacteria (NTM) from patients with CF. The applicability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of NTM isolated on RGM medium was also assessed. Respiratory samples (n = 869) were collected from 487 CF patients and inoculated directly onto RGM medium and BCSA. Cultures were incubated at 30°C and examined for up to 28 days. A subset of 212 samples (from 172 patients) was also cultured by using a mycobacterial growth indicator tube (MGIT) and on Lowenstein-Jensen medium following dual decontamination. By using a combination of all methods, 98 mycobacteria were isolated from 869 samples (11.3%). The sensitivity of RGM medium (96.9%) was significantly higher than that of BCSA (35.7%) for the isolation of mycobacteria (P < 0.0001). The sensitivity of RGM medium was also superior to that of standard AFB culture for the isolation of mycobacteria (92.2% versus 47.1%; P < 0.0001). MALDI-TOF MS was effective for the identification of mycobacteria in RGM medium. RGM medium offers a simple and highly effective tool for the isolation of NTM from patients with CF. Extended incubation of RGM medium for 28 days facilitates the isolation of slow-growing species, including members of the Mycobacterium avium complex (MAVC).