Isogenic Mutant Strains Research Articles

Impact of fluoroquinolone resistance on bactericidal and sterilizing activity of a moxifloxacin-containing regimen in murine tuberculosis

It has been shown previously that fluoroquinolone resistance (defined by resistance to at least 2 mg/liter ofloxacin) has a different impact on moxifloxacin monotherapy depending on the mutation in the sole fluoroquinolone target in Mycobacterium tuberculosis, i.e., DNA gyrase. Since tuberculosis treatment relies on multidrug therapy, we wished to determine the impact of fluoroquinolone resistance on the bactericidal and sterilizing activity of a second-line antituberculous regimen containing moxifloxacin. A total of 280 mice were inoculated with the wild-type Mycobacterium tuberculosis H37Rv strain or one of 3 isogenic fluoroquinolone-resistant mutant strains with increasing moxifloxacin resistance (the GyrB D500N, GyrA A90V, and GyrA D94G strains) and then treated for 6 months with a second-line regimen containing moxifloxacin, pyrazinamide, and ethionamide supplemented with amikacin during the first 2 months. Mice were sacrificed during treatment for measurement of bactericidal activity and 3 months after treatment completion for measurement of relapse rates (sterilizing activity). The CFU counts decreased faster in mice inoculated with the wild type than in mice inoculated with the mutant strains. The relapse rate after treatment completion was different among mice inoculated with mutant strains in relation to the drug resistance level: wild type, 0%; GyrB D500N strain, 33%; GyrA A90V strain, 50%; and GyrA D94G strain, 86%. The relapse rate observed with the GyrB D500N strain was the only one not statistically different from that observed with the wild-type strain. We demonstrated that the impact on sterilizing activity of the most active second-line drug regimen containing moxifloxacin depends on the MIC of moxifloxacin. We suggest that the precise level of moxifloxacin resistance be determined for all strains resistant to 2 mg/liter ofloxacin.

Detection of Resistance to Beta-Lactamase Inhibitors in Strains with CTX-M Beta-Lactamases: a Multicenter External Proficiency Study Using a Well-Defined Collection of Escherichia coli Strains

Under the auspices of the Spanish Society for Infectious Diseases and Clinical Microbiology Quality Control program, 14 Escherichia coli strains masked as blood culture isolates were sent to 68 clinical microbiology laboratories for antimicrobial susceptibility testing to β-lactam antibiotics. This collection included three control strains (E. coli ATCC 25922, an IRT-2 producer, and a CMY-2 producer), six isogenic strains with or without the OmpF porin and expressing CTX-M β-lactamases (CTX-M-1, CTX-M-15, and CTX-M-14), one strain carrying a double mechanism for β-lactam resistance (i.e., carrying CTX-M-15 and OXA-1 enzymes), and four strains carrying CTX-M variants with different levels of resistance to β-lactams and β-lactam-β-lactamase inhibitor (BLBLI) combinations. The main objective of the study was to ascertain how these variants with reduced susceptibilities to BLBLIs are identified in clinical microbiology laboratories. CTX-M variants with high resistance to BLBLIs were mainly identified as inhibitor-resistant TEM (IRT) enzymes (68.0%); however, isogenic CTX-M mutant strains with reduced susceptibilities to BLBLIs and cephalosporins were mainly associated with extended-spectrum β-lactamase production alone (51 to 80%) or in combination with other mechanisms (14 to 31%). Concerning all β-lactams tested, the overall interpretative discrepancy rate was 11.5%, of which 38.1% were the consequence of postreading changes in the clinical categories when a resistance mechanism was inferred. Therefore, failure to recognize these complex phenotypes might contribute to an explanation of their apparent absence in the clinical setting and might lead to inadequate drug treatment selection. A proposal for improving recognition is to adhere strictly to the current CLSI or EUCAST guidelines for detecting reduced susceptibility to BLBLI combinations, without any interpretative modification.

Moraxella catarrhalis Expresses a Cardiolipin Synthase That Impacts Adherence to Human Epithelial Cells

The major phospholipid constituents of Moraxella catarrhalis membranes are phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin (CL). However, very little is known regarding the synthesis and function of these phospholipids in M. catarrhalis. In this study, we discovered that M. catarrhalis expresses a cardiolipin synthase (CLS), termed MclS, that is responsible for the synthesis of CL within the bacterium. The nucleotide sequence of mclS is highly conserved among M. catarrhalis isolates and is predicted to encode a protein with significant amino acid similarity to the recently characterized YmdC/ClsC protein of Escherichia coli. Isogenic mclS mutant strains were generated in M. catarrhalis isolates O35E, O12E, and McGHS1 and contained no observable levels of CL. Site-directed mutagenesis of a highly conserved HKD motif of MclS also resulted in a CL-deficient strain. Moraxella catarrhalis, which depends on adherence to epithelial cells for colonization of the human host, displays significantly reduced levels of adherence to HEp-2 and A549 cell lines in the mclS mutant strains compared to wild-type bacteria. The reduction in adherence appears to be attributed to the absence of CL. These findings mark the first instance in which a CLS has been related to a virulence-associated trait.

Relative Contributions of Lipooligosaccharide Inner and Outer Core Modifications to Nontypeable Haemophilus influenzae Pathogenesis

Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.

LPS Structure and PhoQ Activity Are Important for Salmonella Typhimurium Virulence in the Gallleria mellonella Infection Model

The larvae of the wax moth, Galleria mellonella , have been used experimentally to host a range of bacterial and fungal pathogens. In this study we evaluated the suitability of G . mellonella as an alternative animal model of Salmonella infection. Using a range of inoculum doses we established that the LD50 of Salmonella Typhimurium strain NCTC 12023 was 3.6 × 103 bacteria per larva. Further, a set of isogenic mutant strains depleted of known virulence factors was tested to identify determinants essential for S . Typhimurium pathogenesis. Mutants depleted of one or both of the type III secretion systems encoded by Salmonella Pathogenicity Islands 1 and 2 showed no virulence defect. In contrast, we observed reduced pathogenic potential of a phoQ mutant indicating an important role for the PhoPQ two-component signal transduction system. Lipopolysaccharide (LPS) structure was also shown to influence Salmonella virulence in G . mellonella . A waaL (rfaL) mutant, which lacks the entire O-antigen (OAg), was virtually avirulent, while a wzz ST/wzz fepE double mutant expressing only a very short OAg was highly attenuated for virulence. Furthermore, shortly after infection both LPS mutant strains showed decreased replication when compared to the wild type in a flow cytometry-based competitive index assay. In this study we successfully established a G . mellonella model of S . Typhimurium infection. By identifying PhoQ and LPS OAg length as key determinants of virulence in the wax moth larvae we proved that there is an overlap between this and other animal model systems, thus confirming that the G . mellonella infection model is suitable for assessing aspects of Salmonella virulence function.

A LacI-Family Regulator Activates Maltodextrin Metabolism of Enterococcus faecium

Enterococcus faecium is a gut commensal of humans and animals. In the intestinal tract, E. faecium will have access to a wide variety of carbohydrates, including maltodextrins and maltose, which are the sugars that result from the enzymatic digestion of starch by host-derived and microbial amylases. In this study, we identified the genetic determinants for maltodextrin utilization of E. faecium E1162. We generated a deletion mutant of the mdxABCD-pulA gene cluster that is homologous to maltodextrin uptake genes in other Gram-positive bacteria, and a deletion mutant of the mdxR gene, which is predicted to encode a LacI family regulator of mdxABCD-pulA. Both mutations impaired growth on maltodextrins but had no effect on the growth on maltose and glucose. Comparative transcriptome analysis showed that eight genes (including mdxABCD-pulA) were expressed at significantly lower levels in the isogenic ΔmdxR mutant strain compared to the parental strain when grown on maltose. Quantitative real-time RT-PCR confirmed the results of transcriptome analysis and showed that the transcription of a putative maltose utilization gene cluster is induced in a semi-defined medium supplemented with maltose but is not regulated by MdxR. Understanding the maltodextrin metabolism of E. faecium could yield novel insights into the underlying mechanisms that contribute to the gut commensal lifestyle of E. faecium.

Interspecies Communication among Commensal and Pathogenic Streptococci

ABSTRACTQuorum sensing (QS) regulates diverse and coordinated behaviors in bacteria, including the production of virulence factors, biofilm formation, sporulation, and competence development. It is now established that some streptococci utilize Rgg-type proteins in concert with short hydrophobic peptides (SHPs) to mediate QS, and sequence analysis reveals that several streptococcal species contain highly homologous Rgg/SHP pairs. In group A streptococcus (GAS), two SHPs (SHP2 and SHP3 [SHP2/3]) were previously identified to be important in GAS biofilm formation. SHP2/3 are detected by two antagonistic regulators, Rgg2 and Rgg3, which control expression of the shp genes. In group B streptococcus (GBS), RovS is a known virulence gene regulator and ortholog of Rgg2, whereas no apparent Rgg3 homolog exists. Adjacent to rovS is a gene (shp1520) encoding a peptide nearly identical to SHP2. Using isogenic mutant strains and transcriptional reporters, we confirmed that RovS/SHP1520 comprise a QS circuit in GBS. More important, we performed experiments demonstrating that production and secretion of SHP1520 by GBS can modulate Rgg2/3-regulated gene expression in GAS in trans; likewise, SHP2/3 production by GAS can stimulate RovS-mediated gene regulation in GBS. An isolate of Streptococcus dysgalactiae subsp. equisimilis also produced a secreted factor capable of simulating the QS circuits of both GAS and GBS, and sequencing confirms the presence of an orthologous Rgg2/SHP2 pair in this species as well. To our knowledge, this is the first documented case of bidirectional signaling between streptococcal species in coculture and suggests a role for orthologous Rgg/SHP systems in interspecies communication between important human pathogens.

Impact of Fluoroquinolone Resistance on Bactericidal and Sterilizing Activity of a Moxifloxacin-Containing Regimen in Murine Tuberculosis

It has been shown previously that fluoroquinolone resistance (defined by resistance to at least 2 mg/liter ofloxacin) has a different impact on moxifloxacin monotherapy depending on the mutation in the sole fluoroquinolone target in Mycobacterium tuberculosis, i.e., DNA gyrase. Since tuberculosis treatment relies on multidrug therapy, we wished to determine the impact of fluoroquinolone resistance on the bactericidal and sterilizing activity of a second-line antituberculous regimen containing moxifloxacin. A total of 280 mice were inoculated with the wild-type Mycobacterium tuberculosis H37Rv strain or one of 3 isogenic fluoroquinolone-resistant mutant strains with increasing moxifloxacin resistance (the GyrB D500N, GyrA A90V, and GyrA D94G strains) and then treated for 6 months with a second-line regimen containing moxifloxacin, pyrazinamide, and ethionamide supplemented with amikacin during the first 2 months. Mice were sacrificed during treatment for measurement of bactericidal activity and 3 months after treatment completion for measurement of relapse rates (sterilizing activity). The CFU counts decreased faster in mice inoculated with the wild type than in mice inoculated with the mutant strains. The relapse rate after treatment completion was different among mice inoculated with mutant strains in relation to the drug resistance level: wild type, 0%; GyrB D500N strain, 33%; GyrA A90V strain, 50%; and GyrA D94G strain, 86%. The relapse rate observed with the GyrB D500N strain was the only one not statistically different from that observed with the wild-type strain. We demonstrated that the impact on sterilizing activity of the most active second-line drug regimen containing moxifloxacin depends on the MIC of moxifloxacin. We suggest that the precise level of moxifloxacin resistance be determined for all strains resistant to 2 mg/liter ofloxacin.

Mutations in the quinolone resistance determining region in isogenic mutant Acinetobacter baumannii strains

The aim of the study is to explore the change or mutation of DNA gyrase (gyrA) and topoisomerase IV (parC) in Quinolone Resistance Determining Region (QRDR) inAcinetobacter baumannii isogenic mutant strains which were insensitivity or resistant to fluoroquinolones (FQs). A total of 11 strains were studied, one is isolated from clinical sample as a wide-type parent strain and the others isogenic mutant strains which were selected by ciprofloxacin in vitro environment. Three of the isogenic mutant strains have amino acid mutation in GyrA and ParC of QRDR, two of the three have the mutation Ser-97 to Thr and Asp-150 to His in GyrA, another one which is highly resistant to ciproi¬‚oxacin (MICs of ciproi¬‚oxacin, 64 μg/ml) has the mutations Ser-97 to Thr in GyrA and Ala-85 to Pro in ParC. Key words: Acinetobacter baumannii, quinolone resistance determining region, mutation, isogenic strain.

Competitive Fitness During Feast and Famine: How SOS DNA Polymerases Influence Physiology and Evolution inEscherichia coli

Escherichia coli DNA polymerases (Pol) II, IV, and V serve dual roles by facilitating efficient translesion DNA synthesis while simultaneously introducing genetic variation that can promote adaptive evolution. Here we show that these alternative polymerases are induced as cells transition from exponential to long-term stationary-phase growth in the absence of induction of the SOS regulon by external agents that damage DNA. By monitoring the relative fitness of isogenic mutant strains expressing only one alternative polymerase over time, spanning hours to weeks, we establish distinct growth phase-dependent hierarchies of polymerase mutant strain competitiveness. Pol II confers a significant physiological advantage by facilitating efficient replication and creating genetic diversity during periods of rapid growth. Pol IV and Pol V make the largest contributions to evolutionary fitness during long-term stationary phase. Consistent with their roles providing both a physiological and an adaptive advantage during stationary phase, the expression patterns of all three SOS polymerases change during the transition from log phase to long-term stationary phase. Compared to the alternative polymerases, Pol III transcription dominates during mid-exponential phase; however, its abundance decreases to <20% during long-term stationary phase. Pol IV transcription dominates as cells transition out of exponential phase into stationary phase and a burst of Pol V transcription is observed as cells transition from death phase to long-term stationary phase. These changes in alternative DNA polymerase transcription occur in the absence of SOS induction by exogenous agents and indicate that cell populations require appropriate expression of all three alternative DNA polymerases during exponential, stationary, and long-term stationary phases to attain optimal fitness and undergo adaptive evolution.

Importance of Inhibition of Binding of Complement Factor H for Serum Bactericidal Antibody Responses to Meningococcal Factor H-binding Protein Vaccines

Factor H (fH) binding protein (fHbp) is part of vaccines developed for prevention of meningococcal serogroup B disease. More than 610 fHbp amino acid sequence variants have been identified, which can be classified into 2 subfamilies. The extent of cross-protection within a subfamily has been difficult to assess because of strain variation in fHbp expression. Using isogenic mutant strains, we compared cross-protective serum antibody responses of mice immunized with 7 divergent fHbp variants in subfamily B, including identification numbers (ID) 1 and 55, which were chosen for vaccine development. In the presence of the human complement downregulator fH, the ability of the anti-fHbp antibodies to deposit sufficient complement C3b on the bacterial surface to elicit bactericidal activity required inhibition of binding of fH by the anti-fHbp antibodies. With less bound fH, the bacteria became more susceptible to complement-mediated bactericidal activity. Among the different fHbp sequence variants, those more central in a phylogenic network than ID 1 or 55 elicited anti-fHbp antibodies with broader inhibition of fH binding and broader bactericidal activity. Thus, the more central variants show promise of extending protection to strains with divergent fHbp sequences that are covered poorly by fHbp variants in clinical development.

Flagellar Localization of a Helicobacter pylori Autotransporter Protein

ABSTRACTHelicobacter pylori contains four genes that are predicted to encode proteins secreted by the autotransporter (type V) pathway. One of these, the pore-forming toxin VacA, has been studied in great detail, but thus far there has been very little investigation of three VacA-like proteins. We show here that all three VacA-like proteins are >250 kDa in mass and localized on the surface of H. pylori. The expression of the three vacA-like genes is upregulated during H. pylori colonization of the mouse stomach compared to H. pylori growth in vitro, and a wild-type H. pylori strain outcompeted each of the three corresponding isogenic mutant strains in its ability to colonize the mouse stomach. One of the VacA-like proteins localizes to a sheath that overlies the flagellar filament and bulb, and therefore, we designate it FaaA (flagella-associated autotransporter A). In comparison to a wild-type H. pylori strain, an isogenic faaA mutant strain exhibits decreased motility, decreased flagellar stability, and an increased proportion of flagella in a nonpolar site. The flagellar localization of FaaA differs markedly from the localization of other known autotransporters, and the current results reveal an important role of FaaA in flagellar localization and motility.

The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis*

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

Collagen-binding Streptococcal Surface Proteins Influence the Susceptibility of Biofilm Cells to Endodontic Antimicrobial Solutions

IntroductionPrevious studies have indicated that the antimicrobial efficacy of endodontic irrigants may be diminished in the presence of patient tissues and fluids. With Streptococcus gordonii as a model microorganism, we used a genetic approach to investigate the hypothesis that bacterial surface proteins with collagen-binding abilities may function to protect biofilm cells from antiseptics commonly used in root canal treatment. MethodsS. gordonii strain DL1 or isogenic mutant strains with deletions of genes encoding collagen-binding surface proteins were grown in microtiter plates to form 8-hour biofilms. Planktonic cells were aspirated, and the remaining biofilm cells were buffer-washed and then incubated with either pH-adjusted buffer or potentially protective solutions of type I collagen, serum, or saliva. Biofilms were rewashed, pulsed with sodium hypochlorite, chlorhexidine digluconate, or BioPure MTAD, and then rewashed. Fresh medium was added, and survivor cell growth was monitored for 24 hours. ResultsBuffer-treated biofilm cells of all 3 strains were similarly killed by sodium hypochlorite, chlorhexidine digluconate, and MTAD. Collagen, serum, and saliva significantly protected strain DL1 from all 3 antiseptics compared with buffer-treated cells (P ≤ .0004). However, preincubation with collagen, serum, or saliva left both mutant strain biofilms significantly more susceptible to all 3 antiseptics than were respectively treated strain DL1 biofilms (P ≤ .005). ConclusionsInteractions of S. gordonii surface proteins with collagen or similar components in serum and saliva may play roles in protecting biofilm cells from endodontic antiseptics. Elucidating molecular mechanisms underlying bacterial resistance to antimicrobials may facilitate the development of more effective treatments.

Surfactant Protein A Blocks Recognition of Pseudomonas aeruginosa by CKAP4/P63 on Airway Epithelial Cells

We used isogenic mutant strains that were deficient or over-expressed capsule to study the function of the alginate exopolysaccharide in the interaction of Pseudomonas aeruginosa with the human airway epithelial cells (AEC) in the presence or absence of surfactant protein A (SP-A). SP-A prevented the invasion of AEC by alginate-producing P. aeruginosa strains because of a direct effect on the AEC. Monoclonal antibodies to CKAP4/P63, the principal SP-A-binding receptor on AEC, or inhibition of its expression using specific siRNA reduced the invasion of both highly encapsulated and poorly encapsulated strains, but not the invasion of the acapsular mutant. Treatment of AEC with SP-A, monoclonal antibodies to CKAP4/P63, or CKAP4/P63-specific siRNA decreased the binding of purified alginate exopolysaccharide to AEC. Alginate binding to AEC reduced SP-A release by these cells. Because the alginate exopolysaccharide is surface-exposed, levels of SP-A may be crucial to modulate the interaction of P. aeruginosa with AEC.

Pseudomonas aeruginosainactivation mechanism is affected by capsular extracellular polymeric substances reactivity with chlorine and monochloramine

The reactivity of capsular extracellular polymeric substances (EPS) to chlorine and monochloramine was assessed and compared in this study. The impact of capsular EPS on Gram-negative bacteria Pseudomonas aeruginosa inactivation mechanisms was investigated both qualitatively and quantitatively using a combination of batch experiments, viability tests with LIVE/DEAD staining, and Fourier transform infrared spectroscopy (FTIR). Both wild-type and isogenic mutant strains with different alginate EPS production capabilities were used to evaluate their susceptibility to chlorine and monochloramine. The mucA22 mutant strain, which overproduces the EPS composed largely of acidic polysaccharide alginate, exhibited high resistance and prolonged inactivation time to both chlorine and monochloramine relative to PAO1 (wild-type) and algT(U) mutant strains (alginate EPS deficient). Multiple analyses were combined to better understand the mechanistic role of EPS against chlorine-based disinfectants. The extracted EPS exhibited high reactivity with chlorine and very low reactivity with monochloramine, suggesting different mechanism of protection against disinfectants. Moreover, capsular EPS on cell membrane appeared to reduce membrane permeabilization by disinfectants as suggested by deformation of key functional groups in EPS and cell membrane (the C-O-C stretching of carbohydrate and the C=O stretching of ester group). The combined results supported that capsular EPS, acting either as a disinfectant consumer (for chlorine inactivation) or limiting access to reactive sites on cell membrane (for monochloramine inactivation), provide a protective role for bacterial cells against regulatory residual disinfectants by reducing membrane permeabilization.

Role of Heat-Stable Enterotoxins in the Induction of Early Immune Responses in Piglets after Infection with Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat - labile (LT) enterotoxins are cause of post – weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac+, LT+ STa+ STb+ ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.

Detection of Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOF Mass Spectrometry

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization - Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01–10.24; p = 0.023; CI 95%: 1.20–12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

Streptococcal Surface Proteins Activate the Contact System and Control Its Antibacterial Activity

Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects.

The influence of capsular extracellular polymeric substances on the interaction between TiO2 nanoparticles and planktonic bacteria

The role of capsular extracellular polymeric substances (EPS) at the surface of planktonic microorganisms was investigated for possible toxicity mitigation from titanium dioxide (TiO2) nanoparticles, using variable EPS producing wild-type and isogenic mutant strains of Pseudomonas aeruginosa. Membrane integrity assays revealed that increased capsular EPS reduced cell membrane damage. Acting as a barrier to the cell membrane, capsular EPS permitted attachment of nanoparticles to the cell, while simultaneously delaying cellular damage caused by the production of reactive oxygen species (ROS). Modulations in ROS production were monitored in situ; while changes in the chemical composition of the microorganisms before and after exposure were examined with Fourier transform infrared spectroscopy (FTIR). The addition of methanol, a known radical scavenger, was shown to vastly reduce ROS production and membrane integrity losses, while not affecting physical interactions of nanoparticles with the microorganism. The results support that EPS provides an attachment site for nanoparticles, but more importantly act as a barrier to cell membrane oxidation from ROS. These observations provide better understanding of the overall importance of ROS in TiO2 microbial toxicity.