Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

Injury of Macrophages Induced by Clostridium perfringens Type C Exotoxins.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

Proof of concept for detection of staphylococcal enterotoxins in platelet concentrates as a novel safety mitigation strategy.

BACKGROUND AND OBJECTIVES: Staphylococcus aureus is a predominant contaminant of platelet concentrates (PCs) that can evade detection during screening with culture methods. Importantly, S. aureus produces staphylococcal enterotoxins (SEs) during PC storage, which are linked to slow growth and enhanced biofilm formation. This study investigated timing of SE production during PC storage and feasibility of SE detection as a PC safety strategy. MATERIALS AND METHODS: Genomic and transcriptomic data of transfusion-relevant S. aureus PS/BAC/169/17/W, PS/BAC/317/16/W, CI/BAC/25/13/W and CBS2016-05 were used to determine the presence and differential expression of exotoxin genes in PCs. Trypticase soy broth (TSB) and PCs were inoculated with 1.0E+06 cfu/mL of S. aureus PS/BAC/169/17/W and CBS2016-05. Expression of SEs at different growth phases was confirmed with Western blotting. PCs were inoculated with 30 cfu/unit of the same strains, and SE detection during PC storage was optimized with a sandwich dot-ELISA assay. RESULTS: S. aureus genomes contain multiple exotoxin genes including those encoding for SEs. Transcriptome data revealed significant upregulation (0.5-6.7-fold, p < 0.05) of SE genes in PCs versus TSB. Western blots demonstrated SE production at all growth phases. Notably, dot-ELISA detected clinically relevant concentrations of SEs (~0.2 µg/mL) at 32 h of PC storage when S. aureus PS/BAC/169/17/W and CBS2016-05 counts were 1.8E+04 and 1.4E+04 cfu/mL, respectively. CONCLUSION: Genomic analyses revealed that staphylococcal exotoxins are widely distributed and highly conserved among transfusion-relevant S. aureus isolates. Furthermore, SEs are significantly upregulated in PCs and detected at 30 h of PC storage. Therefore, bacterial toxin detection could supplement mitigation strategies to enhance PC safety.

T-cell receptor Vß8 for detection of biologically active streptococcal pyrogenic exotoxin type C.

Streptococcus pyogenes is an important human pathogen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPE). The current method for detection cannot distinguish between the biologically active form of SPE that has been reported to cause foodborne outbreaks and the inactivated toxin that poses no health risk. To measure the biological activity of SPE type C (SPE-C), one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vß8. With this finding, we used a T-cell line natively expressing Vß8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element in combination with a B-cell line to present the recombinant SPE-C (rSPE-C) toxin via major histocompatibility complex (MHC) class II to the Vß8 T-cell receptor (TCR) in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross-reactivity with SPE-B and significant loss of SPE-C biological activity in spiked phosphate-buffered saline while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment.

Invasive Streptococcus pyogenes disease in Spain: a microbiological and epidemiological study covering the period 2007-2019.

The aim of this study is to present the first nationwide microbiological and epidemiological study of invasive group A Streptococcus (iGAS) disease in Spain. One thousand eight hundred ninety-three iGAS isolates were analyzed over 2007-2019. emm typing was performed by sequencing the gene's variable 5' end, exotoxin genes were identified by PCR, and antimicrobial susceptibility explored via the E test and disk diffusion. Five hundred twenty-three isolates were associated with sepsis, 292 with cellulitis, 232 with scarlet fever, 153 with pneumonia, 141 with streptococcal toxic shock syndrome, and 94 with necrotizing fasciitis. The most prevalent emm types were emm1 (449/1893 isolates), emm89 (210/1893), emm3 (208/1893), emm4 (150/1893), emm12 (112/1893) emm6 (107/1893), emm87 (89/1893), emm28 (88/1893), emm75 (78/1893), emm77 (78/1893), emm11 (58/1893), and emm22 (35/1893). emm1, emm3, emm4, and emm6 were the predominant types affecting children (mostly respiratory infections), while emm11, emm77, and emm89 prevailed in the elderly (mostly skin infections). Each emm type was associated with one or more exotoxin gene (spe, sme, and ssa) profiles. speA was detected in 660 isolates, speB in 1829, speC in 1014, speF in 1826, speG in 1651, speJ in 716, speH in 331, smeZ in 720, and ssa in 512. Isolates with speA were associated with the most severe infections. Penicillin susceptibility was universal. Two hundred twenty-four isolates were resistant to tetracycline, 169 to erythromycin, and 81 to clindamycin. Tetracycline, erythromycin, and clindamycin resistance rates declined over the study period. The above information could serve as the basis for continued surveillance efforts designed to control disease cause by this bacterium.

Exotoxins from Staphylococcus aureus activate 5-lipoxygenase and induce leukotriene biosynthesis.

Massive neutrophil infiltration is an early key event in infectious inflammation, accompanied by chemotactic leukotriene (LT)B4 generation. LTB4 biosynthesis is mediated by 5-lipoxygenase (5-LOX), but which pathogenic factors cause 5-LOX activation during bacterial infections is elusive. Here, we reveal staphylococcal exotoxins as 5-LOX activators. Conditioned medium of wild-type Staphylococcus aureus but not of exotoxin-deficient strains induced 5-LOX activation in transfected HEK293 cells. Two different staphylococcal exotoxins mimicked the effects of S. aureus-conditioned medium: (1) the pore-forming toxin α-hemolysin and (2) amphipathic α-helical phenol-soluble modulin (PSM) peptides. Interestingly, in human neutrophils, 5-LOX activation was exclusively evoked by PSMs, which was prevented by the selective FPR2/ALX receptor antagonist WRW4. 5-LOX activation by PSMs was confirmed in vivo as LT formation in infected paws of mice was impaired in response to PSM-deficient S. aureus. Conclusively, exotoxins from S. aureus are potent pathogenic factors that activate 5-LOX and induce LT formation in neutrophils.

Molecular detection of type III secretory toxins in Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa has been known as a common unscrupulous pathogen that reasons cause nosocomial infections in patients with immunocompromise. Infection with multi-drug resistant Pseudomonas aeruginosa infection in many patients is a public health problem. The bacterium causes urinary tract infections, respiratory tract infections, skin inflammation and inflammation, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and various systemic infections, especially in patients with severe burns, cancer and AIDS, whose immune systems are suppressed. Among diverse virulence factors, the type III secretion system is known as a significant agent in virulence and development of antimicrobial resistance in P. aeruginosa. A total of 50 isolates of P. aeruginosa were gathered from burn wound and milk specimens. Documentation and antimicrobial susceptibility evidence were performed using the VITEK 2 system. Multiplex PCR was done to detect the secretion toxins-encoding genes. Out of 50 samples: 45/225 (20%) burn wound and 6/120 (5%) raw milk samples were found positive for P. aeruginosa. The multiplex PCR analysis of ExoT and ExoY genes showed that all P. aeruginosa 50 (100%) were positive. The occurrence of the ExoS and ExoU genes was 97.7% and 86.6% among clinical isolates while none of the raw milk isolates harbored the ExoU gene and 60% of them carried the ExoS gene. The results found 20 (40%) of isolates were multidrug resistance and the most effective antibiotics against clinical isolates were Ciprofloxacin and Meropenem. The aim of this study was to prevalence the exotoxin genes encoded type III secretion system and pattern of antimicrobial susceptibility of P. aeruginosa isolated from clinical and raw milk specimens.

Timing Is Everything: Impact of Naturally Occurring Staphylococcus aureus AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction.

Mutations in the polymorphic Staphylococcus aureusagr locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction, thus reducing virulence and maintaining S. aureus in an agr-downregulated "colonization" mode.IMPORTANCE Virulence factor expression in Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.

Evaluation of the ability of colistin, amoxicillin (components of Potencil® ), and fluoroquinolones to attenuate bacterial endotoxin- and Shiga exotoxin-mediated cytotoxicity-In vitro studies.

Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS- and Stx-mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS-mediated cytotoxicity in an experiment mimicking "natural infection," (c) the effect of antibiotics to decrease Stx2e-mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS- and Stx2e-induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS- and Stx2e-mediated cytotoxicity.

Genetic features of Pseudomonas aeruginosa isolates associated with eye infections referred to Farabi Hospital, Tehran, Iran.

BACKGROUND: Pseudomonas is the most common cause of microbial keratitis especially in people who use contact lens. The virulence of Pseudomonas aeruginosa in different eye infections is associated with different virulence factors . METHODS: In this study, 54 P. aeruginosa isolates including 39 isolates from keratitis and 15 isolates from conjunctivitis were evaluated for their ability to form biofilm, production of protease, elastase, alkaline protease and their antibiotic-resistant patterns. The distribution of the exoS and exoU genes in the test strains were determined using PCR assays. RESULTS: Most of the eye infections (90.74%) were seen in people who used contact lenses, and in most of patients (72.22%), the infection was presented as keratitis. None of the isolates were resistant to a single antibiotic as tested. Multidrug resistance (MDR) was detected in two isolates (3.5%) which were resistant to more than one category of antibiotics. The exoU+/exoS+ isolates were in majority although in total, compared to exoS, there were more exoU in a greater number of samples. Most of the strains produce elastase but among all of ocular isolates, only 5.8% of the strains showed alkaline protease activity. Most of the ocular isolates were not capable of producing biofilm. CONCLUSIONS: In our study, a high prevalence of virulence factors was observed in P. aeruginosa isolates from contact lens wearer with keratitis. As the P. aeruginosa isolates from different infection origins and different geographic region may have different virulence factors, having a better perception of these differences could help to improve development of clinical instructions for the control of keratitis.

Emergence of a Staphylococcus aureus Clone Resistant to Mupirocin and Fusidic Acid Carrying Exotoxin Genes and Causing Mainly Skin Infections.

Skin and soft tissue infections (SSTIs) caused by mupirocin-resistant Staphylococcus aureus strains have recently increased in number in our settings. We sought to evaluate the characteristics of these cases over a 43-month period. Data for all community-acquired staphylococcal infections caused by mupirocin-resistant strains were retrospectively reviewed. Genes encoding products producing high-level resistance (HLR) to mupirocin (mupA), fusidic acid resistance (fusB), resistance to macrolides and lincosamides (ermC and ermA), Panton-Valentine leukocidin (PVL) (lukS/lukF-PV), exfoliative toxins (eta and etb), and fibronectin binding protein A (fnbA) were investigated by PCRs in 102 selected preserved strains. Genotyping was performed by SCCmec and agr typing, whereas clonality was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 437 cases among 2,137 staphylococcal infections were recorded in 2013 to 2016; they were all SSTIs with the exception of 1 case of primary bacteremia. Impetigo was the predominant clinical entity (371 cases [84.9%]), followed by staphylococcal scalded skin syndrome (21 cases [4.8%]), and there were no abscesses. The number of infections detected annually increased during the study years. All except 3 isolates were methicillin susceptible. The rates of HLR to mupirocin and constitutive resistance to clindamycin were 99% and 20.1%, respectively. Among the 102 tested strains, 100 (98%) were mupA positive and 97 (95%) were fusB positive, 26/27 clindamycin-resistant strains (96.3%) were ermA positive, 83 strains (81.4%) were lukS/lukF positive, 95 (93%) carried both eta and etb genes, and 99 (97%) were fnbA positive. Genotyping of methicillin-sensitive S. aureus (MSSA) strains revealed that 96/99 (96.7%) belonged to one main pulsotype, pulsotype 1, classified as sequence type 121 (ST121). The emergence of a single MSSA clone (ST121) causing impetigo was documented. Resistance to topical antimicrobials and a rich toxinogenic profile confer to this clone adaptability for spread in the community.

IL-22/IL-22R1 signaling regulates the pathophysiology of chronic rhinosinusitis with nasal polyps via alteration of MUC1 expression.

BACKGROUND: IL-22 is an IL-10-family cytokine that regulates chronic inflammation. We investigated the role of IL-22 and its receptor, IL-22R1, in the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP). METHODS: IL-22 and IL-22R1 protein and mRNA expression in NP and in uncinate tissues (UT) from CRS and non-CRS patients was examined using immunohistochemistry and real-time PCR, respectively. Dispersed NP and UT cells were cultured with the Staphylococcus aureus exotoxins, staphylococcal enterotoxin B and alpha-toxin, following which exotoxin-induced IL-22 levels and their association with clinicopathological factors were analyzed. Effects of IL-22 on MUC1 expression and cytokine release in NP cells were also determined. RESULTS: IL-22 and IL-22R1 in NP were mainly expressed in infiltrating inflammatory cells and in epithelial cells, respectively. IL-22 mRNA levels in NP were significantly higher than those in UTs from non-CRS patients whereas IL-22R1 levels were conversely lower in NPs. NP cells produced substantial amounts of IL-22 in response to exotoxins. Exotoxin-induced IL-22 production by NP cells significantly and negatively correlated with the degree of local eosinophilia and postoperative computed tomography (CT) score, whereas conversely it positively correlated with the forced expiratory volume in 1s (FEV1)/forced vital capacity (FVC) ratio. IL-22 significantly enhanced MUC1 mRNA expression in NP cells. IL-22-induced MUC1 mRNA levels were significantly and positively correlated with IL-22R1 mRNA levels in NPs. CONCLUSIONS: These data suggest that imbalance of IL-22/IL-22R1 signaling regulates the pathogenesis of CRSwNP, including local eosinophilia, via alteration of MUC1 expression.

Cytotoxic effects of Aeromonas hydrophila culture supernatant on peripheral blood leukocytes of Nile tilapia (Oreochromis niloticus): Possible presence of a secreted cytotoxic lectin.

Number of exotoxins like haemolysin, leukocidin, aerolysin etc. were reported from Aeromonas hydrophila. In this study, we report the haemolytic and cytotoxic effect of A. hydrophila culture supernatant (CS) that is specifically inhibited by lactose and also by serum and mucus of Nile tilapia (Oreochromis niloticus). Hence, we assume the presence of a secreted lectin in the CS. CS is toxic to peripheral blood leukocytes (PBL) of O. niloticus as revealed by MTT assay and by flow cytometry. DNA laddering assay indicates that CS causes necrosis to PBL. As a result of necrosis, CS treated PBL showed increased production of reactive oxygen species as indicated by nitroblue tetrazolium and 2',7' -dichlorofluorescin diacetate assays. CS treated PBL showed reduced mRNA expression of TNF-α and IFN-γ genes. When CS was subjected to polyacrylamide gel electrophoresis, it showed a single band corresponding to the molecular weight of 45 kDa. However, upon concentrating the CS by ultrafiltration, many bands were visualized. Further studies at molecular level are required to unravel this macromolecular-leukocyte interaction which would ultimately benefit the aquaculture industry.

Effect of licorice extract on cell viability, biofilm formation and exotoxin production by Staphylococcus aureus.

Staphylococcus aureus is one of the most significant clinical pathogen, as it causes infections to humans and animals. Even though several antibiotics and other treatments have been used to control S. aureus infections and intoxication, bacterium is able to adapt, survive and produces exotoxins. Licorice (Glycyrrhiza glabra L.) has been used traditionally in various medicinal (antimicrobial) preparations, and Glycyrrhizic acid (GA) is the major active constituents present in it. In the present investigation the effect of licorice extract on methicillin susceptible S. aureus (FRI 722) and methicillin resistant S. aureus (ATCC 43300) growth and toxin production was studied. The MIC of licorice extract was found to be 0.25 and 2.5 mg GA ml(-1) against S. aureus FRI 722 and S. aureus ATCC 43300, respectively. Inhibition of biofilm formation was observed even at very low concentration (25 µg GA ml(-1)). Gradual decrease in expression and production of exotoxins such as α and ß hemolysins and enterotoxin B was observed with the increasing concentrations of licorice extract, however, suboptimal concentration induced the expression of some of the virulence genes. This study indicated efficacy of licorice extract in controlling growth and pathogenicity of both methicillin susceptible and methicillin resistant S. aureus, however, the mechanisms of survival and toxin production at suboptimal concentration needs further study.

Outbreak of Streptococcus pyogenes emm type 58 in a high dependency unit of a level-1 trauma center of India.

BACKGROUND AND AIMS: Group A Streptococcus (GAS) can cause illnesses ranging from self-limited to severe, life-threatening, invasive infections. The objective of the following study was to investigate a suspected Streptococcus pyogenes outbreak in a high dependency unit (HDU) of our trauma center. MATERIALS AND METHODS: All the isolates of beta hemolytic Streptococci were identified by standard microbiological methods, Vitek 2 system and latex agglutination tests. Antimicrobial susceptibility testing was performed as recommended by Clinical Laboratory Standards Institute. Exotoxin genes, including speA, speB, speC, speF, smeZ, ssa, speG, speH, speJ, speL, speM and speI were detected by polymerase chain reaction (PCR). The emm types of isolates of S. pyogenes were determined by sequencing the variable 5' end of emm gene after amplification by PCR. RESULTS: In a 28 bedded poly-trauma ward with a four bedded HDU three out of four patients developed S. pyogenes emm type 58 infection. The strain was macrolide and tetracycline resistant and produced the Streptococcal pyrogenic exotoxins speB, speC, speG, speF and smeZ. Surveillance sampling was done for investigation from patients, health-care workers and environmental samples. CONCLUSION: An outbreak of GAS infections was established caused by the uncommonly reported emm type 58. The outbreak was controlled by prompt treatment, intensive surveillance, feedback and training.

Changes in Streptococcus pyogenes causing invasive disease in Portugal: evidence for superantigen gene loss and acquisition.

The emergence of highly virulent and successful Streptococcus pyogenes (group A streptococci - GAS) clones has been attributed to the exchange of virulence factors by lateral gene transfer mechanisms, which strongly contribute to genomic diversity. We characterized a collection of 191 GAS isolates recovered from normally sterile sites in Portugal during 2006-2009 and compared them to invasive isolates obtained during 2000-2005. Antimicrobial resistance rates did not change significantly between the two periods and were generally low. In 2006-2009, emm1, emm89, emm3, and emm6 represented 60% of the isolates. The chromosomally encoded superantigen (SAg) genes speG and smeZ were present in the majority (>90%) of the isolates, while speJ was found in only 45%. The phage encoded SAgs varied greatly in prevalence (2-53%). The distribution of emm types, pulsed-field gel electrophoresis profiling (PFGE) clusters, and SAg profiles changed significantly between the periods, although there were no statistically supported changes in the prevalence of individual types. While the macrolide susceptible clone emm1-T1-ST28 remained dominant (28%), there was a significant decrease in clonal diversity as indicated by both PFGE profiling and emm typing. This was accompanied by intra-clonal divergence of SAg profiles, which was statistically confirmed for isolates representing emm1, emm28, and emm44. This diversification was associated with the loss and acquisition of SAg genes, carried by phages and of chromosomal origin. These data suggest an ongoing genomic diversification of GAS invasive isolates in Portugal that may contribute to the persistence of clones with improved fitness or virulence.

Evidence of exotoxin secretion of Piscirickettsia salmonis, the causative agent of piscirickettsiosis.

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial-free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE-214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.

Recurrent staphylococcal scalded skin syndrome in a 20-month old-A case report.

We present a case of a 20-month-old child with a history of atopic dermatitis that exhibited recurrent erythematous-bullous lesions consistent with Staphylococcal Scalded Skin syndrome (SSSS). SSSS is an exfoliative toxin-mediated skin disorder most commonly found in children. In this paper, we discuss the importance of recognizing the clinical symptomatology and progressive nature of SSSS, particularly in patients with a history of atopic dermatitis, to ensure prompt treatment and resolution of the syndrome.

Screening for Antimicrobial Resistance and Genes of Exotoxins in Pseudomonas aeruginosa Isolates from Infected Dogs and Cats in Poland.

Pseudomonas aeruginosa has assumed an increasingly prominent role as the aetiological agent in serious hard-to-treat infections in animals and humans. In this study, 271 P. aeruginosa strains collected from dogs and cats were investigated. The aim of the research was to screen these P. aeruginosa strains for antibiotic resistance and the presence of selected virulence factor genes. Antibiotic resistance was determined using the Kirby-Bauer method, while virulence genes were detected by polymerase chain reaction (PCR). The most frequently detected resistance was to fluoroquinolones, ranging in prevalence from 17.3% for ciprofloxacin up to 83% for enrofloxacin. The resistance to carbapenems was 14% and 4.8% for imipenem and meropenem, respectively. Almost all P. aeruginosa strains harboured the exoT (97.8%) and lasB (93.4%) genes, while the lowest prevalence was found for exoU (17.3%) and plcH (17.3%). P. aeruginosa strains isolated from dogs that harboured the toxA gene were more frequently resistant to ceftazidime (p = 0.012), while the presence of the exoU gene was found to be connected with resistance to marbofloxacin (p = 0.025) and amikacin (p = 0.056). In strains originating from cats, only the connection between the presence of the exoU gene and resistance to enrofloxacin (p = 0.054) was observed. The confirmation of associations between virulence-factor-encoding genes and antibiotic resistance indicates that problems of antibiotic resistance may not only cause complications at the level of antibiotic dosage but also lead to changes in the virulence of the bacteria; thus, further studies in this area are required.

Secretory proteins in the orchestration of microbial virulence: The curious case of Staphylococcus aureus.

Microbial virulence showcases an excellent model for adaptive changes that enable an organism to survive and proliferate in a hostile environment and exploit host resources to its own benefit. In Staphylococcus aureus, an opportunistic pathogen of the human host, known for the diversity of the disease conditions it inflicts and the rapid evolution of antibiotic resistance, virulence is a consequence of having a highly plastic genome that is amenable to quick reprogramming and the ability to express a diverse arsenal of virulence factors. Virulence factors that are secreted to the host milieu effectively manipulate the host conditions to favor bacterial survival and growth. They assist in colonization, nutrient acquisition, immune evasion, and systemic spread. The structural and functional characteristics of the secreted virulence proteins have been shaped to assist S. aureus in thriving and disseminating effectively within the host environment and exploiting the host resources to its best benefit. With the aim of highlighting the importance of secreted virulence proteins in bacterial virulence, the present chapter provides a comprehensive account of the role of the major secreted proteins of S. aureus in orchestrating its virulence in the human host.