Studies on the antibacterial effects of statins--in vitro and in vivo

Background

Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections.

Aim

The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract.

Methods

MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert).

Findings

The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria.

Conclusions

Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1–15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.     

Expression of bacteriocin divercin AS7 in Escherichia coli and its functional analysis

Bacteriocins are small peptides with antimicrobial activity, that are produced by bacteria. Four classes of bacteriocins produced by lactic acid bacteria have been defined. Class IIa bacteriocins are promising candidates for industrial applications due to their high biological activity and their physicochemical properties. Divercin AS7 is a class IIa bacteriocin produced by Carnobacterium divergens AS7. It shows antibacterial activity against pathogens and food spoilage flora including Listeria spp. Little is known about the impact of class IIa bacteriocins upon eukaryotic cells. The safe use of bacteriocins as food biopreservatives requires the absence of cytotoxicity to human cells. To analyze the impact of divercin AS7 on human enterocytes, we expressed the recombinant divercin AS7 in the Escherichia coli BL21DE3pLys strain and conducted in vitro studies to evaluate the safety of recombinant divercin AS7. No cytotoxic effect on differentiated monolayer Caco-2 cells and no apoptotic appearance were observed when recombinant divercin AS7 was used at a concentration of 2 μg ml^?1. In our study, divercin AS7 also did not interfere with differentiated Caco-2 cells monolayer integrity. The obtained results suggest that divercin AS7 is a promising peptide for the food industry.     

The incidence of Streptococcus pneumoniae otitis media is affected by the polymicrobial environment particularly Moraxella catarrhalis in a mouse nasal colonisation model

Otitis media (OM) is a highly prevalent paediatric disease with both bacterial and viral triggers of infection. This study has investigated how combinations of bacteria associated with nasal colonisation and the occurrence and absence of viral infection (Sendai virus) induce OM in a mouse nasal colonisation model. The respiratory virus significantly contributed to bacterial OM for all bacterial combinations (p < 0.001). Streptococcus pneumoniae consistently dominated as the causative bacterium of OM and when co-infected with S. pneumoniae, Moraxella catarrhalis more significantly affected pneumococcal OM than did non-typeable Haemophilus influenzae (p < 0.001) by increasing the incidence rate, infection bacterial load and duration of infection. Nitric oxide levels in the middle ear, an indicator of inflammation, peaked at day 3 in single bacterium groups, but at day 1 in mixed bacterial groups and was produced in all bacteria inoculated groups even in the absence of viable bacterial recovery. Phagocytic cells were recruited rapidly to the ear following nasal inoculation but over time their numbers did not correlate with persistence of bacterial infection. The study has shown that the composition of bacteria in the nasal cavity and respiratory viral infection significantly affected the OM incidence rate, duration of infection and bacterial load (severity).     

Incidence, risk factors and mortality of nosocomial pneumonia in Intensive Care Units: A prospective study

Background

Increasing antimicrobial resistance among the key pathogens responsible for community-acquired respiratory tract infections has the potential to limit the effectiveness of antibiotics available to treat these infections. Since there are regional differences in the susceptibility patterns observed and treatment is frequently empirical, the selection of antibiotic therapy may be challenging. PROTEKT, a global, longitudinal multicentre surveillance study, tracks the activity of telithromycin and comparator antibacterial agents against key respiratory tract pathogens.

Methods

In this analysis, we examine the prevalence of antibacterial resistance in 1,336 bacterial pathogens, isolated from adult and paediatric patients clinically diagnosed with acute bacterial sinusitis (ABS).

Results and discussion

In total, 58.0%, 66.1%, and 55.8% of S. pneumoniae isolates were susceptible to penicillin, cefuroxime, and clarithromycin respectively. Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of S. pneumoniae isolates (128 isolates were resistant to penicillin [MIC >= 2 mg/L], 72 intermediate [MIC 0.12–1 mg/L]) while 99.5% and 95.5% of isolates were susceptible to telithromycin and amoxicillin-clavulanate, respectively. In total, 88.2%, 87.5%, 99.4%, 100%, and 100% of H. influenzae isolates were susceptible to ampicillin, clarithromycin, cefuroxime, telithromycin, and amoxicillin-clavulanate, respectively. In vitro, telithromycin demonstrated the highest activity against M. catarrhalis (MIC₅₀ = 0.06 mg/L, MIC₉₀ = 0.12 mg/L).

Conclusion

The high in vitro activity of against pathogens commonly isolated in ABS, together with a once daily dosing regimen and clinical efficacy with 5-day course of therapy, suggest that telithromycin may play a role in the empiric treatment of ABS.     

Release of Enterococcus mundtii Bacteriocin ST4SA from Self-Setting Brushite Bone Cement

Maxillofacial and craniofacial surgery is on the increase, which exposes more patients at risk of acquiring microbial infections. The use of antibiotic-loaded calcium phosphate bone cements has been shown to reduce the incidence of infection. A marked increase in antibiotic-resistant pathogens, including multidrug-resistant pathogens, has been reported. This has led to the investigation of various compounds as alternatives to conventional treatments. In this paper, we report on the incorporation and release of a broad-spectrum class II antimicrobial peptide, bacteriocin ST4SA produced by Enterococcus mundtii, into a calcium orthophosphate-based bone cement. Our results suggest class II bacteriocins may be incorporated into self-setting bone cements to produce implants with antimicrobial activity over extended periods of time.     

Genetic requirement for pneumococcal ear infection

Background

Ear infection or otitis media (OM) accounts for most bacterial respiratory infections in children in both developed and developing nations. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the major OM pathogens. However, little is known about the genetic basis of bacterial OM largely due to practical difficulties in conducting research in ear infection models and genetically manipulating clinical isolates. Here, we report the first genome-scale in vivo screen for bacterial genes required for ear infection in a chinchilla model by signature tagged mutagenesis (STM), a high throughput mutant screen technique.

Methodology/Principal Findings

STM strains were constructed with a multi-drug resistant OM isolate ST556 (serotype 19F) and screened in a chinchilla OM model. Out of 5,280 mutants tested, 248 mutants were substantially underrepresented in the mutant pools recovered from the middle ear fluids of the infected chinchillas, indicating the impaired ability to survive and replicate in the middle ears due to genetic disruptions in the chromosome of strain ST556. Further DNA sequencing analysis mapped the mutations to 169 pneumococcal genes. Surprisingly, only 52 of these genes were required for pneumococcal nasopharyngeal colonization in a murine model. This infection site-specific gene requirement was verified by targeted mutagenesis in the selected genes.

Conclusions/Significance

These findings suggest that there are a subset of pneumococcal genes required for ear infection and that these may be distinct from those required for nasal colonization. Our data thus provide comprehensive gene targets for mechanistic understanding of pneumococcal ear infection. Finally, this study has also developed a model for future genome-scale search for virulence determinants in other pathogens associated with ear infections.     

Gassericin A: a circular bacteriocin produced by Lactic acid bacteria Lactobacillus gasseri

During the recent years extensive efforts have been made to find out bacteriocins from lactic acid bacteria (LAB) active against various food spoilage and pathogenic bacteria, and superior stabilities against heat treatments and pH variations. Bacteriocins isolated from LAB have been grouped into four classes. Circular bacteriocins which were earlier grouped among the four groups of bacteriocins, have recently been proposed to be classified into a different class, making it class V bacteriocins. Circular bacteriocins are special molecules, whose precursors must be post translationally modified to join the N to C termini with a head-to-tail peptide bond. Cyclization appears to make them less susceptible to proteolytic cleavage, high temperature and pH, and, therefore, provides enhanced stability as compared to linear bacteriocins. The advantages of circularization are also reflected by the fact that a significant number of macrocyclic natural products have found pharmaceutical applications. Circular bacteriocins were unknown two decades ago, and even to date, only a few circular bacteriocins from a diverse group of Gram positive organisms have been reported. The first example of a circular bacteriocin was enterocin AS-48, produced by Enterococcus faecalis AS-48. Gassereccin A, produced by Lactobacillus gasseri LA39, Reutericin 6 produced by Lactobacillus reuteri LA6 and Circularin A, produced by Clostridium beijerinickii ATCC 25,752, are further examples of this group of antimicrobial peptides. In the present scenario, Gassericin A can be an important tool in the food preservation owing to its properties of high pH and temperature tolerance and the fact that it is produced by LAB L. gasseri, whose many strains are proven probiotic.     

Swab transport in Amies gel followed by frozen storage in skim milk tryptone glucose glycerol broth (STGGB) for studies of respiratory bacterial pathogens

Nasopharyngeal carriage studies are needed to monitor changes in important bacterial pathogens in response to vaccination and antibiotics. Commercial swab transport followed by transfer to skim milk tryptone glucose glycerol broth for frozen storage is an option for studies of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.     

Wheeze in preschool age is associated with pulmonary bacterial infection and resolves after antibiotic therapy

Background

Neonates with airways colonized by Haemophilus influenzae, Streptococcus pneumoniae or Moraxella catarrhalis are at increased risk for recurrent wheeze which may resemble asthma early in life. It is not clear whether chronic colonization by these pathogens is causative for severe persistent wheeze in some preschool children and whether these children might benefit from antibiotic treatment. We assessed the relevance of bacterial colonization and chronic airway infection in preschool children with severe persistent wheezing and evaluated the outcome of long-time antibiotic treatment on the clinical course in such children.

Methodology/Principal Findings

Preschool children (n = 42) with severe persistent wheeze but no symptoms of acute pulmonary infection were investigated by bronchoscopy and bronchoalveolar lavage (BAL). Differential cell counts and microbiological and virological analyses were performed on BAL samples. Patients diagnosed with bacterial infection were treated with antibiotics for 2–16 weeks (n = 29). A modified ISAAC questionnaire was used for follow-up assessment of children at least 6 months after bronchoscopy. Of the 42 children with severe wheezing, 34 (81%) showed a neutrophilic inflammation and 20 (59%) of this subgroup had elevated bacterial counts (≥10⁴ colony forming units per milliliter) suggesting infection. Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were the most frequently isolated species. After treatment with appropriate antibiotics 92% of patients showed a marked improvement of symptoms upon follow-up examination.

Conclusions/Significance

Chronic bacterial infections are relevant in a subgroup of preschool children with persistent wheezing and such children benefit significantly from antibiotic therapy.     

Leuconostoc mesenteroides SJRP55: A Bacteriocinogenic Strain Isolated from Brazilian Water Buffalo Mozzarella Cheese

The production of bacteriocins by Leuconostoc mesenteroides represents an important opportunity for exploration of their potential use for industrial purpose. The antimicrobial compounds produced by L. mesenteroides subsp. mesenteroides SJRP55 strain were characterized and purified. Cell-free supernatant of Leuc. mesenteroides subsp. mesenteroides SJRP55 produced antibacterial compounds against Listeria spp. strains and not inhibiting against Lactobacillus spp. The antimicrobial substances were stable at high temperatures (100 °C for 2 h and 121 °C for 20 min) and low pH (pH 2–4) values, but sensitive to proteolytic enzymes and resistant to α-amylase, lipase and catalase enzymes. The optimal temperature for active peptides production was 25 °C. The antimicrobial compounds were purified by ammonium sulfate precipitation, affinity column and reverse-phase chromatography. Mass spectrometry and amino acids analyses showed that the bacteriocins were identical to mesentericin Y105 and B105. The producer strain’s DNA analysis revealed presence of open reading frames possibly coding for virulence factors, such as enterococcal surface protein (esp), collagen adhesion (ace) and intrinsic vancomycin resistance (vanA); however, biogenic amines encoding genes were not observed. Leuc. mesenteroides subsp. mesenteroides SJRP55 is a promising biopreservative culture in fermented milk, and the purified bacteriocins can also be applied in food preservation.     

Circular Bacteriocins: Biosynthesis and Mode of Action

Circular bacteriocins are a group of N-to-C-terminally linked antimicrobial peptides, produced by Gram-positive bacteria of the phylum Firmicutes. Circular bacteriocins generally exhibit broad-spectrum antimicrobial activity, including against common food-borne pathogens, such as Clostridium and Listeria spp. These peptides are further known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make this group of bacteriocins highly promising for potential industrial applications and their biosynthesis of particular interest as a possible model system for the synthesis of highly stable bioactive peptides. In this review, we summarize the current knowledge on this group of bacteriocins, with emphasis on the recent progress in understanding circular bacteriocin genetics, biosynthesis, and mode of action; in addition, we highlight the current challenges and future perspectives for the application of these peptides.     

Antimicrobial Activity,Inhibition of Urogenital Pathogens,and Synergistic Interactions Between <Emphasis Type="Italic">Lactobacillus</Emphasis> Strains

Lactobacillus fermentum strain L23 and L. rhamnosus strain L60 were selected as an alternative treatment to prevent or treat urogenital infections based on their probiotic properties and production of bacteriocins. The objectives of the present work were to study the inhibitory activities of these two bacteriocin-producing strains, and to analyze the interactions between pairs of bacteriocins that inhibit urogenital pathogens. Antimicrobial activity tests of L23 and L60 were performed by a diffusion method with 207 bacterial strains, isolated from female patients presenting a urogenital infection. Inhibitory substances interaction tests were carried out by using a streak-diffusion method on agar plates. One hundred percent of the clinical isolates showed sensitivity to the antimicrobial substances produced by L23 and L60. The selected lactobacilli produced larger inhibition halos when compared to several antibiotics commonly used for treating these infections. Synergistic interactions and indifferent interactions were recorded in 68.6% and 31.4% of the cases, respectively. No antagonistic interactions were observed. In conclusion, the bacteriocin-producing strains L23 and L60 are potential candidates for probiotic prophylaxis and treatment of urogenital disorders in women.     

Bacteriocin production by Bifidobacterium spp. A review

Bacteriocins are ribosomally-synthesized antibacterial peptides. These compounds are produced by a broad variety of different bacteria belonging mainly to the genus Bifidobacterium, to which health promoting properties have frequently been attributed. However, despite the fact that the identification of Bifidobacterium-associated bacteriocins was first reported in 1980 and that they exhibit antimicrobial activity against pathogenic microorganisms such as Listeria monocytogenes, Clostridium perfringens, and Escherichia coli, relatively little information is still available about the antimicrobial compounds produced by strains of this genus. More detailed understanding of the action mechanisms of these antimicrobials could allow us to determine the extent to which their production contributes to the probiotic properties of specific bifidobacteria strains and, potentially, be of crucial significance for ultimate preservation of functional foods or pharmaceutical applications. Here we review what is already known about their structure, classification, mode of action, functionality, immunity, production and purification.     

Peptide deformylase inhibitors with retro-amide scaffold: Synthesis and structure–activity relationships

Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins. Thus inhibition of PDF activity is considered to be one of the most effective antibiotic strategies. Reported herein are the synthesis and structure–activity relationship studies of retro-amide inhibitors based on actinonin, a naturally occurring PDF inhibitor. Analysis of the structure–activity relationships led to the discovery of 7a, which exhibits potent enzyme inhibition and antibacterial activity against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.     

BACTIBASE: a new web-accessible database for bacteriocin characterization

Background  

Bacteriocins are very diverse group of antimicrobial peptides produced by a wide range of bacteria and known for their inhibitory activity against various human and animal pathogens. Although many bacteriocins are now well characterized, much information is still missing or is unavailable to potential users. The assembly of such information in one central resource such as a database would therefore be of great benefit to the exploitation of these bioactive molecules in the present context of increasing antibiotic resistance and natural bio-preservation need.     

Inhibitory effect of plantaricin peptides (Pln E/F and J/K) against Escherichia coli

Plantaricins are small bioactive peptides produced by Lactobacillus plantarum strains that exhibit significant antimicrobial activity against closely-related Gram-positive bacteria, including food spoilage organisms. In comparison, bacteriocins including plantaricins, are usually less effective against Gram-negative organisms. In this study, we demonstrate that heterologously expressed and purified plantaricins, Pln E, -F, -J, and -K when tested against Gram negative model organism Escherichia coli K-12 were highly effective under certain conditions. The apparent tolerance of Gram-negative members to these peptides has been explained on the basis of the presence of the outer membrane (OM) that acts as a protective barrier. We have shown that agents and/or conditions that destabilize OM of E. coli K-12, make it susceptible to plantaricin peptides. In order to further strengthen this conclusion, an OM lipoprotein-defective lpp mutant strain of E. coli K-12 was also studied and compared. A significant loss of cell viability both in terms of CFU/ml as well as with live–dead dual staining combined with flow cytometry, could be demonstrated with the lpp mutant in comparison to the wild type strain. The results indicate that plantaricins can inhibit Gram-negative bacteria if the outer-membrane is weakened and it can be used in preservation of food with the help of some food-grade chelating agents.     

NKLP27: A Teleost NK-Lysin Peptide that Modulates Immune Response,Induces Degradation of Bacterial DNA,and Inhibits Bacterial and Viral Infection

NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.     

Production,purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1

Background  

The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances.     

Otopathogens Detected in Middle Ear Fluid Obtained during Tympanostomy Tube Insertion: Contrasting Purulent and Non-Purulent Effusions

Otitis media is a prominent disease among children. Previous literature indicates that otitis media is a polymicrobial disease, with Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis and Moraxella catarrhalis being the most commonly associated bacterial pathogens. Recent literature suggests that introduction of pneumococcal conjugate vaccines has had an effect on the etiology of otitis media. Using a multiplex PCR procedure, we sought to investigate the presence of the aforementioned bacterial pathogens in middle ear fluid collected from children undergoing routine tympanostomy tube placement at Wake Forest Baptist Medical Center during the period between January 2011 and March 2014. In purulent effusions, one or more bacterial organisms were detected in ~90% of samples. Most often the presence of H. influenzae alone was detected in purulent effusions (32%; 10 of 31). In non-purulent effusions, the most prevalent organism detected was A. otitidis (26%; 63 of 245). Half of the non-purulent effusions had none of these otopathogens detected. In purulent and non-purulent effusions, the overall presence of S. pneumoniae was lower (19%; 6 of 31, and 4%; 9 of 245, respectively) than that of the other pathogens being identified. The ratio of the percentage of each otopathogen identified in purulent vs. non-purulent effusions was >1 for the classic otopathogens but not for A. otitidis.     

Characterization of Mundticin L,a Class IIa Anti-Listeria Bacteriocin from Enterococcus mundtii CUGF08

Enterococcus mundtii CUGF08, a lactic acid bacterium isolated from alfalfa sprouts, was found to produce mundticin L, a new class IIa bacteriocin that has a high level of inhibitory activity against the genus Listeria. The plasmid-associated operons containing genes for the mundticin L precursor, the ATP binding cassette (ABC) transporter, and immunity were cloned and sequenced. The fifth residue of the conservative consensus sequence YGNGX in the mature bacteriocin is leucine instead of valine in the sequences of the homologous molecules mundticin KS (ATO6) and enterocin CRL35. The primary structures of the ABC transporter and the immunity protein are homologous but unique.Bacteriocins are ribosomally synthesized proteinaceous compounds that inhibit closely related bacteria (19). Due to consumer concerns with chemical and irradiation preservation methods and due to the rising demand for minimally processed food products, alternative methods for shelf life extension and enhanced safety are needed. Bacteriocins are considered “natural” antimicrobials since many bacteriocins are produced by food grade lactic acid bacteria, which are generally recognized as safe. Bacteriocins can be divided into three main classes: the class I lanthionine-containing lantibiotics, exemplified by nisin; the class II non-lanthionine-containing bacteriocins; and the class III heat-labile, large proteins (6). Class III bacteriocins have limited application due to their thermal instability and cytolytic activity against eukaryotic cells. Class II can be further divided into class IIa containing pediocin-like bacteriocins, class IIb containing two-peptide bacteriocins, and class IIc containing other bacteriocins (8). Class IIa bacteriocins have been extensively studied since pediocin PA-1 was first discovered (12) and characterized (20). Currently, only nisin in class I has been approved by the FDA as a natural food additive. Bacteriocins belonging to class IIa are promising alternative antimicrobials since they are more stable over a broader range of heating regimens and pH conditions. In addition, these bacteriocins exhibit stronger antimicrobial activity against the genus Listeria than nisin (17) but have a narrower antimicrobial spectrum.The potential applications of class IIa bacteriocins in both meat and plant-based foods as a means to provide protection against potential food-borne pathogens and extend shelf life continue to expand. In an attempt to use biological methods for controlling food-borne pathogens on fresh sprouts, a number of food grade lactic acid bacteria were isolated from the indigenous microbiota on alfalfa sprouts. Some of these isolates were found to be bacteriocinogenic. This study describes a new class IIa bacteriocin, mundticin L produced by Enterococcus mundtii CUGF08 isolated from alfalfa sprouts.