Beta-Lactam Sensitive Bacteria Can Acquire ESBL-Resistance via Conjugation after Long-Term Exposure to Lethal Antibiotic Concentration

Beta-lactams are commonly used antibiotics that prevent cell-wall biosynthesis. Beta-lactam sensitive bacteria can acquire conjugative resistance elements and hence become resistant even after being exposed to lethal (above minimum inhibitory) antibiotic concentrations. Here we show that neither the length of antibiotic exposure (1 to 16 h) nor the beta-lactam type (penam or cephem) have a major impact on the rescue of sensitive bacteria. We demonstrate that an evolutionary rescue can occur between different clinically relevant bacterial species (Klebsiella pneumoniae and Escherichia coli) by plasmids that are commonly associated with extended-spectrum beta-lactamase (ESBL) positive hospital isolates. As such, it is possible that this resistance dynamic may play a role in failing antibiotic therapies in those cases where resistant bacteria may readily migrate into the proximity of sensitive pathogens. Furthermore, we engineered a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-plasmid to encode a guiding CRISPR-RNA against the migrating ESBL-plasmid. By introducing this plasmid into the sensitive bacterium, the frequency of the evolutionarily rescued bacteria decreased by several orders of magnitude. As such, engineering pathogens during antibiotic treatment may provide ways to prevent ESBL-plasmid dispersal and hence resistance evolution.

Essential Oil of Artemisia judaica L. (ssp. Sahariensis) from Algerian Sahara: Antimicrobial Effects and Mechanisms of Action

The aim of this work was to investigate the essential oil of Artemisia judaica L. (ssp. Sahariensis) originated from south of Algeria by evaluating its antimicrobial activity as well as its mechanisms of action against bacterial and fungal microorganisms. The antimicrobial activity was evaluated against 17 microbial strains. The mechanisms of action of Artemisia judaica essential oil (AJEO) have been studied by the evaluation of the release of cellular material (260 nm), the cell viability, the activity of lipase, and the salt tolerance. The AJEO yield was 1.27 ± 0.24% (w/w). The chemical composition of AJEO was characterized by a high content of piperitone (71.1%). The most sensitive bacterium was Staphylococcus aureus with an MIC of 2.73 mg/mL. AJEO was found to be enough to kill strains tested at least 40% after thirty seconds of incubation and to induce release of cellular material. On the other hand, AJEO showed complete inhibition of lipase activity of Candida albicans cells as well as the ability of cells to form colonies on saline medium. AJEO has a powerful antimicrobial activity against different microbial organisms with different mechanisms of action. AJEO seem to be useful in pharmaceutical, food, and cosmetic application.

In vitro Antimicrobial Activity of Methanolic Extract from Varthemia iphionoides Leaves

<p>In the Mediterranean region, <em>Varthemia iphionoides</em> is commonly used in folk medicine for the treatment of gastrointestinal disorders. The present study described the antimicrobial activity of the methanolic extract of <em>V.</em> <em>iphionoides</em> leaves. The extract was assayed against a panel of pathogenic bacteria and fungi using agar well diffusion method. The antibacterial activity of the methanolic extract was investigated against six standard bacterial species and was found to exhibit high antibacterial activity. The most sensitive bacterium was <em>Klebsiella pneumoniae </em>ATCC 13883 followed by <em>Proteus vulgaris</em> ATCC 13315 and methicillin-resistant <em>Staphylococcus aureus</em> ATCC 95047. The least sensitive bacterium to <em>V.</em> <em>iphionoides</em> methanolic extract was <em>Escherichia coli</em> O157:H7 ATCC43895. Antifungal susceptibility of 13 fungal species was tested against <em>V.</em> <em>iphionoides</em> methanolic extract. Among the fungal species studied, <em>Fusarium lini</em> was the most sensitive and <em>Beauveria bassiana</em> was the most resistant to the extract. Good antifungal activity has been displayed by the methanolic extract of <em>V.</em> <em>iphionoides</em> against <em>Aspergillus brasiliensis</em>,<em> Aspergillus niger</em>, and <em>Aspergillus alliaceus</em>,<em> Aspergillus flavus</em>, <em>Cunninghamella echinulata</em>, <em>Gibberella fujikuroi</em>, <em>Macrophomina phaseolina</em>, <em>Cephalosporum aphidicola</em>, <em>Rhizoupus stolonifer</em>, <em>Curvularia lunata</em> and <em>Cunninghamella elegans</em>. The observed antimicrobial potential of <em>V.</em> <em>iphionoides</em> indicated that this plant possesses bioactive compounds that are able to combat pathogenic microorganisms and support its traditional use in the treatment of pathogen infection.</p>

Immunity to the Bacteriocin Sublancin 168 Is Determined by the SunI (YolF) Protein of Bacillus subtilis

ABSTRACT Bacillus subtilis strain 168 produces the extremely stable lantibiotic sublancin 168, which has a broad spectrum of bactericidal activity. Both sublancin 168 production and producer immunity are determined by the SPβ prophage. While the sunA and sunT genes for sublancin 168 production have been known for several years, the genetic basis for sublancin 168 producer immunity has remained elusive. Therefore, the present studies were aimed at identifying an SPβ gene(s) for sublancin 168 immunity. By systematic deletion analysis, we were able to pinpoint one gene, named yolF, as the sublancin 168 producer immunity gene. Growth inhibition assays performed using plates and liquid cultures revealed that YolF is both required and sufficient for sublancin 168 immunity even when heterologously produced in the sublancin-sensitive bacterium Staphylococcus aureus. Accordingly, we propose to rename yolF to sunI (for sublancin immunity). Subcellular localization studies indicate that the SunI protein is anchored to the membrane with a single N-terminal membrane-spanning domain that has an Nout-Cin topology. Thus, the bulk of the protein faces the cytoplasm of B. subtilis. This topology has not yet been reported for known bacteriocin producer immunity proteins, which implies that SunI belongs to a novel class of bacteriocin antagonists.

The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

ABSTRACT Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5′-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa d-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, k cat/Km = 1.7 10−2 μM−1 s−1; riboflavin, k cat/Km = 7.5 10−3 μM−1 s−1). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, k cat/Km = 1.3 10−2 μM−1 s−1; riboflavin, k cat/Km = 1.3 10−2 μM−1 s−1). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.

Antimicrobial Effects of Alginate-Based Films Containing Essential Oils on Listeria monocytogenes and Salmonella Typhimurium Present in Bologna and Ham

Bologna and ham slices (300 of each) were inoculated with Salmonella Typhimurium or Listeria monocytogenes at 103 CFU/cm2. Alginate-based edible films that had been immersed in a 2 or 20% (wt/vol) CaCl2 solution and contained 1% (wt/vol) essential oils of Spanish oregano (O; Corydothymus capitatus), Chinese cinnamon (C; Cinnamomum cassia), or winter savory (S; Satureja montana) were then applied to slices to control pathogen growth. On bologna, C-based films pretreated with 20% CaCl2 were the most effective against the growth of Salmonella Typhimurium and L. monocytogenes. L. monocytogenes was the more sensitive bacterium to O-, C-, and S-based films. L. monocytogenes concentrations were below the detection level (&lt;10 CFU/ml) after 5 days of storage on bologna coated with O-, C-, or S-based films pretreated with 20% CaCl2. On ham, a 1.85 log CFU/cm2 reduction of Salmonella Typhimurium (P ≤ 0.05) was found after 5 days of storage with C-based films regardless of the type of pretreatment used (2 or 20% CaCl2) or when coated with O-based films pretreated with 20% CaCl2. L. monocytogenes was highly resistant in ham, even in the presence of O-, C-, or S-based films. However, C-based films pretreated with 20% CaCl2 were the most effective against the growth of L. monocytogenes. Evaluation of the availability of active compounds in films revealed a significantly higher release of active compounds in C-based films (P ≤ 0.05) regardless of pretreatment or meat tested (bologna or ham). O-based films had the lowest release level of active compounds. The release of active compounds from O- and S-based films pretreated with 20% CaCl2 was faster than that in the same respective films pretreated with 2% CaCl2 regardless of the meat type. C-based film pretreated by immersion in a 20% CaCl2 solution was most efficient against both pathogens, and migration of active compounds was higher in C-based films than in O- and S-based films.

Evidence for Distinct l-Methionine Catabolic Pathways in the Yeast Geotrichum candidum and the Bacterium Brevibacterium linens

ABSTRACT Tracing experiments were carried out to identify volatile and nonvolatile l-methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the l-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and α-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of l-methionine catabolism. However, the l-methionine degradation pathways differ in the first steps of l-methionine degradation. l-Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum. Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G.candidum could also be used as precursors for VSC production by B. linens.

Increased inflammation in lysozyme M–deficient mice in response to Micrococcus luteus and its peptidoglycan

More than 70 years ago, Alexander Fleming discovered lysozyme and proposed that nonpathogenic bacteria fail to cause disease because they are very susceptible to destruction by lysozyme, an enzyme that is one of the principal proteins of phagocytes. Although much has been learned about the effects of lysozyme in vitro, its biological role in vivo has not been determined. We examined transgenic mice deficient in lysozyme M after challenge by the normally nonpathogenic and highly lysozyme-sensitive bacterium Micrococcus luteus. Despite partial compensation by newly expressed lysozyme P in macrophages, lysozyme M–deficient mice developed much more severe lesions than wild-type mice. The tissue injury was due to the failure of lysozyme M–deficient mice to inactivate peptidoglycan, resulting in an intense and prolonged inflammatory response. Our data indicate that tissue injury is normally limited by prompt degradation of bacterial macromolecules that trigger innate immunity and inflammation.