María del Carmen Díaz Fernández

Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

ABSTRACT Multidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence in Enterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates of E. cloacae were used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). The acrA and tolC genes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrA and EcΔtolC and JcΔacrA and JcΔtolC knockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance of E. cloacae to several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that the acrA and tolC genes both affect the fitness of E. cloacae, as fitness was clearly reduced in the acrA and tolC KO strains. The median CI values obtained in vitro and in vivo were, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in both E. cloacae clinical strains when either the acrA or tolC gene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates of E. cloacae.

Comparative analysis of the in vitro cytotoxicity of the dietary biogenic amines tyramine and histamine

Tyramine and histamine, the most toxic biogenic amines (BA), are often found in high concentrations in certain foods. Prompted by the limited knowledge of BA toxicity, and increasing awareness of the risks associated with high intakes of dietary BA, the in vitro cytotoxicity of tyramine and histamine was investigated. Tyramine and histamine were toxic for HT29 intestinal cell cultures at concentrations commonly found in BA-rich food, as determined by real-time cell analysis. Surprisingly, tyramine had a stronger and more rapid cytotoxic effect than histamine. Their mode of action was also different, while tyramine caused cell necrosis, histamine induced apoptosis. To avoid health risks, the BA content of foods should be reduced and legal limits established for tyramine.

Impact on Human Health of Microorganisms Present in Fermented Dairy Products: An Overview

Fermented dairy products provide nutrients in our diet, some of which are produced by the action of microorganisms during fermentation. These products can be populated by a diverse microbiota that impacts the organoleptic and physicochemical characteristics foods as well as human health. Acidification is carried out by starter lactic acid bacteria (LAB) whereas other LAB, moulds, and yeasts become dominant during ripening and contribute to the development of aroma and texture in dairy products. Probiotics are generally part of the nonstarter microbiota, and their use has been extended in recent years. Fermented dairy products can contain beneficial compounds, which are produced by the metabolic activity of their microbiota (vitamins, conjugated linoleic acid, bioactive peptides, and gamma-aminobutyric acid, among others). Some microorganisms can also release toxic compounds, the most notorious being biogenic amines and aflatoxins. Though generally considered safe, fermented dairy products can be contaminated by pathogens. If proliferation occurs during manufacture or storage, they can cause sporadic cases or outbreaks of disease. This paper provides an overview on the current state of different aspects of the research on microorganisms present in dairy products in the light of their positive or negative impact on human health.

A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis

BackgroundAntibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity.ResultsCells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the β-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after β-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different β-lactams and vancomycin, confirming the applicability of the methodology.ConclusionThe technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis.

Distant and new mutations in CTX-M-1 beta-lactamase affect cefotaxime hydrolysis.

ABSTRACT The CTX-M β-lactamases are an increasingly prevalent group of extended-spectrum β-lactamases (ESBL). Point mutations in CTX-M β-lactamases are considered critical for enhanced hydrolysis of cefotaxime. In order to clarify the structural determinants of the activity against cefotaxime in CTX-M β-lactamases, screening for random mutations was carried out to search for decreased activity against cefotaxime, with the CTX-M-1 gene as a model. Thirteen single mutants with a considerable reduction in cefotaxime MICs were selected for biochemical and stability studies. The 13 mutated genes of the CTX-M-1 β-lactamase were expressed, and the proteins were purified for kinetic studies against cephalothin and cefotaxime (as the main antibiotics). Some of the positions, such as Val103Asp, Asn104Asp, Asn106Lys, and Pro107Ser, are located in the 103VNYN106 loop, which had been described as important in cefotaxime hydrolysis, although this has not been experimentally confirmed. There are four mutations located close to catalytic residues—Thr71Ile, Met135Ile, Arg164His, and Asn244Asp—that may affect the positioning of these residues. We show here that some distant mutations, such as Ala219Val, are critical for cefotaxime hydrolysis and highlight the role of this loop at the top of the active site. Other distant substitutions, such as Val80Ala, Arg191, Ala247Ser, and Val260Leu, are in hydrophobic cores and may affect the dynamics and flexibility of the enzyme. We describe here, in conclusion, new residues involved in cefotaxime hydrolysis in CTX-M β-lactamases, five of which are in positions distant from the catalytic center.

Fast Assessment of Resistance to Carbapenems and Ciprofloxacin of Clinical Strains of Acinetobacter baumannii

ABSTRACT Infections caused by multidrug-resistant Acinetobacter baumannii constitute a major life-threatening problem worldwide, and early adequate antibiotic therapy is decisive for success. For these reasons, rapid detection of antibiotic susceptibility in this pathogen is a clinical challenge. Two variants of the Micromax kit were evaluated for a rapid detection in situ of susceptibility or resistance to meropenem or ciprofloxacin, separately, in 322 clinical isolates. Release of the nucleoid is the criterion of susceptibility to the beta-lactams (carbapenems), whereas diffusion of DNA fragments emerging from the nucleoid characterizes the quinolone activity. All the susceptible and resistant strains were correctly categorized in 100 min according to the MIC results and CLSI criteria. Thus, our technology is a promising tool for rapid identification of carbapenem and quinolone resistance of A. baumannii strains in hospital settings.

Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase

Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.

Ultrastructural evidence of endoplasmic reticulum changes during the maturation of the olive pollen grain (Olea europaea L., Oleaceae).

Characteristic features of rough endoplasmic reticulum (rER) distribution and proliferation were noted during olive pollen (Olea europaea L.) development, suggesting the physiological significance of this organelle. Initially scarce in the young microspore, ER increases as cytoplasmic vacuoles form. At the vacuolated microspore stage the cytoplasm contains numberous polysomes and elongated rER cisternae arranged preferentially in stacks, with an average intracisternal width of 0.07 µm. Stacks persist in the bicellular pollen grain but consist of fewer, shorter, dilated cisternae (mean intracisternal width 0.1 µm) containing a considerable electron-dense matrix. Cisternae in the mature grain are fragmented, leaving behind an ER of swollen pockets. Pockets of ER containing a material of greater electron density are evenly deposited along the plasmalemma, in close relation with it. A dense material is seen in the tubules of the apertural region, which was lacking in earlier stages. Our results show that ER may be involved in protein transport to the intine.

A review of the terminology applied to apertural thickenings of the pollen grain: Zwischenkörper or oncus?

Abstract The various terms employed to designate a particular structure in the pollen grain aperture or pore are reviewed along with the equivocal use of such terms to describe two apparently identical structures which actually differ in moment and place of origin. The factors which may have led to this confusion are analysed as well as the problems encountered in the search for an appropriate term which would avoid misinterpretation of results, given the need to adopt an international terminology to be used by all authors. The term “oncus”, originally proposed by Hyde in 1955, is considered the most appropriate in this sense, as it has the advantage of reflecting the origin of this structure in the form of a swelling or tumor, as long as it is clearly indicated whether the swelling is located in the intine or the exine.

Aperture structure, development and function in Lycopersicum esculentum Miller (Solanaceae) pollen grain

Abstract The pollen grain of Lycopersicum esculentum Miller is trizonocolporate. The endoapertures are covered by the ectexine layer, which shows a central suture line. This covering is similar in appearance to a bridge, hence the ectexine in the form of a bridges was chosen to denote these structures. Observations in L. esculentum pollen grains suggest that their harmomegathic function may be limited to the colpal zone (ectoaperture), although the characteristics of the interapertural zones in the wall, with small gaps in the tectum, a discontinuous and fragmented foot layer and numerous white lines in the endexine (typical tectate collumellate structure), indicate that the interapertural zones may also play a role in harmomegathy. Pollen tube germination occurs through the endoaperture, located upon the intinous oncus (IO) in the equatorial zones of the apertures.