Evaluation of a rapid immunochromatographic test for detection of KPC in clinical isolates of Enterobacteriaceae and Pseudomonas species.

The KPC K-SeT immunochromatographic test (Coris BioConcept®, Gembloux, Belgium) has been widely used for detection of KPC in Enterobacteriaceae with reported sensitivities and specificities of 100%. However, to our knowledge, there are no reports of its use in KPC-positive Pseudomonas species. We evaluated the KPC K-SeT test in 36 clinical isolates of Enterobacteriaceae (21 KPC-positive and 15 KPC-negative) and 20 Pseudomonas species (5 KPC-positive and 15 KPC-negative) using conventional PCR for carbapenemase genes as the reference method. The KPC K-SeT test detected 25 out of 26 KPC-positive isolates (96.1%). The undetected isolate was 1 P. aeruginosa bearing the mutation D179Y in the omega loop region of KPC-2 carbapenemase. This mutation was already reported in Enterobacteriaceae as conferring resistance to ceftazidime-avibactam. To our knowledge, this is the first report of evaluation of KPC K-SeT test in KPC-positive P. aeruginosa isolates.

Clinical and microbiological response of mice to intranasal inoculation with Lactococcus lactis expressing Group A Streptococcus antigens, to be used as an anti-streptococcal vaccine.

Protein subunit vaccines are often preferred because of their protective efficacy and safety. Lactic acid bacteria expressing heterologous antigens constitute a promising approach to vaccine development. However, their safety in terms of toxicity and bacterial clearance must be evaluated. Anti-Streptococcus pyogenes (S. pyogenes) vaccines face additional safety concerns because they may elicit autoimmune responses. The assessment of toxicity, clearance and autoimmunity of an anti-streptococcal vaccine based on Lactococcus lactis (L. lactis) expressing 10 different M protein fragments from S. pyogenes (L. lactis-Mx10) is here reported. Clearance of L. lactis from the oropharynges of immunocompetent mice and mice devoid of T/B lymphocytes mice was achieved without using antibiotics. The absence of autoimmune responses against human tissues was demonstrated with human brain, heart and kidney. Assessment of toxicity showed that leucocyte counts and selected serum biochemical factors were not affected in L. lactis-Mx10-immunized mice. In contrast, mice immunized with L. lactis wild type vector (L. lactis-WT) showed increased neutrophil and monocyte counts and altered histopathology of lymph nodes, lungs and nasal epithelium. Two days after immunization, L. lactis-Mx10-immunized and L. lactis-WT-immunized mice weighed significantly less than unimmunized mice. However, both groups of immunized mice recovered their body weights by Day 6. Our results demonstrate that L. lactis-WT, but not the vaccine L. lactis-Mx10, induces alterations in certain hematologic and histopathological variables. We consider these data a major contribution to data on L. lactis as a bacterial vector for vaccine delivery.

Protective immunity induced by an intranasal multivalent vaccine comprising 10 Lactococcus lactis strains expressing highly prevalent M-protein antigens derived from Group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N-terminal fragment of streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N- terminal fragment is hypervariable: there are more than 200 different M types. In this study, an intranasal live bacterial vaccine comprising 10 strains of Lactococcus lactis, each expressing one N-terminal fragment of M protein, has been developed. Live bacterial-vectored vaccines cost less to manufacture because the processes involved are less complex than those required for production of protein subunit vaccines. Moreover, intranasal administration does not require syringes or specialized personnel. Evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All 10 strains combined in a 10-valent vaccine (M×10) induced serum and bronchoalveolar lavage IgG titers that ranged from three- to 10-fold those of unimmunized mice. After intranasal challenge with M28 streptococci, survival of M×10-immunized mice was significantly higher than that of unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of M×10-immunized mice did not differ significantly from that of unimmunized mice. Mx-10 immunized mice had significantly less S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge than did unimmunized mice. Our L. lactis-based vaccine may provide an alternative solution to development of broadly protective group A streptococcal vaccines.

Cytochrome c peroxidase (CcP) is a molecular determinant of the oxidative stress response in the extreme acidophilic Leptospirillum sp. CF-1.

Bioleaching processes are used to recover metals from sulfidic ores. Biofilm formation on ores is important for bioleaching because the attached microorganisms start the leaching process by concentrating ferric ions in the extracellular matrix. It has been shown that hydrogen peroxide is spontaneously generated on the surface of ores and that it negatively influences the growth and activity of microorganisms. However, the mechanism by which bioleaching microorganisms tolerate exogenous H2O2 as an adaptive trait remains elusive. Herein, we demonstrate that the gene yhjA, encoding a predicted periplasmic cytochrome c peroxidase (CcP), is important for the response to exogenously generated oxidative stress in the iron-oxidizing acidophilic bacterium Leptospirillum sp. CF-1. Our results show that yhjA is co-transcribed with the genes encoding the peroxide-responsive transcription regulator PerR and peroxiredoxin AhpC. CcP activity, but not yhjA mRNA level, significantly increased in response to hydrogen peroxide and ferric ion exposure, suggesting a post-translational regulation. In agreement with these results, challenging planktonic cells with hydrogen peroxide significantly increased their attachment to pyrite surfaces. In summation, our results suggest that CcP is important to cope with exogenous H2O2, thus favoring the early steps of attachment to mineral substrates.

Cultivable psychrotolerant yeasts associated with Antarctic marine sponges.

Unlike filamentous fungi and bacteria, very little is known about cultivable yeasts associated with marine sponges, especially those from Antarctic seas. During an expedition to King George Island, in the Antarctica, samples of 11 marine sponges were collected by scuba-diving. From these sponges, 20 psychrotolerant yeast isolates were obtained. Phylogenetic analyses of D1/D2 and ITS rRNA gene sequences revealed that the marine ascomycetous yeast Metschnikowia australis is the predominant organism associated with these invertebrates. Other species found belonged to the Basidiomycota phylum: Cystofilobasidium infirmominiatum, Rhodotorula pinicola, Leucosporidiella creatinivora and a new yeast from the Leucosporidiella genus. None of these yeasts have been previously associated with marine sponges. A screening to estimate the ability of these yeasts as producers of extracellular enzymatic activities at several pH and temperature conditions was performed. Several yeast isolates demonstrated amylolytic, proteolytic, lipolytic or cellulolytic activity, but none of them showed xylanolytic activity under the conditions assayed. To our knowledge, this work is the first description of cultivable yeasts associated with marine sponges from the Antarctic sea.

Reduction of the monounsaturated fatty acid content of Escherichia coli results in increased resistance to oxidative damage.

Reactive oxygen species (ROSs) affect several macromolecules and cellular components in eukaryotic and prokaryotic cells. In this work, the effect of various ROS-generating compounds on the Escherichia coli membrane was studied. Membrane fatty acid profiles, oxidative damage levels and bacterial resistance to these toxicants were determined. Studies included wild-type cells as well as a strain exhibiting a modified monounsaturated fatty acid (MUFA) profile (accomplished by overexpressing the ß-hydroxyacyl acyl carrier protein dehydratase-encoding gene, fabA). Levels of membrane MUFAs and oxidative damage markers decreased slightly upon toxicant exposure with a concomitant increase in cell resistance to these ROS-generating compounds. A direct relationship between MUFAs and lipid peroxidation was observed. The lower the MUFA the lower the peroxide levels, suggesting that MUFAs are targets for membrane lipid oxidation.