Presence Of Staphylococcal Enterotoxin Research Articles

Antibiotic sensitivity patterns and molecular detection of Enterotoxin genes in Staphylococcus aureus isolated from frozen muscle foods

Stapylococcus aureus is considered to be the most common pathogen in commonly consumed and improperly stored frozen foods. Hence, this study investigated the antibiotic sensitivity patterns and the occurrence of enterotoxin genes (sea and seb) in S. aureus isolated from frozen poultry meat of chicken, turkey and fish, namely Trachurus trachurus, Scomber scrombus and Merluccious merluccious. A total of 500 samples of raw meat from chicken (n = 130), turkey (n =130), and different fish (n =240) were randomly purchased from various markets and shopping malls in Akure between September 2020 and December 2021. The antibiotic sensitivity of isolated S. aureus was determined by disk diffusion method. S. aureus was analyzed for enterotoxin genes by multiplex polymerase chain reaction (PCR). The highest staphylococcal count (7.70 ×105 CFU/g) was observed in T. trachurus. One hundred and twenty five S. aureus were isolated from meat and fish with the highest occurrence (40.80%) in turkey meat. S. aureus is highly resistant (33.3 to 100%) to the tested antibiotics. The PCR assay, respectively, showed sea and seb coded for staphylococcal enterotoxin A and B. The presence of staphylococcal enterotoxin A and B in frozen muscle foods could be attributed to unhygienic conditions during processing and storage. The occurrence of antibiotic resistant and enterotoxigenic S. aureus in muscle foods could pose a serious threat to people when consumed. Hence, urgent attention is required to avert the incidences of staphylococcal food poisoning after consuming frozen meat and fish.

Abstract 2874: The tetravalent structure of FS118, a bispecific antibody targeting LAG-3 and PD-L1, is required for its novel mechanism of LAG-3 shedding

Abstract FS118 is a tetravalent bispecific antibody targeting LAG-3 and PD-L1 that can overcome immune suppressive signals with greater preclinical activity than a combination of monoclonal antibodies. In vivo, FS118 downregulates LAG-3 on tumor-infiltrating lymphocytes (TILs) and increases soluble LAG-3 (sLAG-3) in the serum of mice. In a Phase 1 trial, FS118 demonstrated a dose-dependent increase in sLAG-3 in the serum of patients with advanced malignancies. Here, we demonstrate that the tetravalent structure of FS118 is required to mediate this novel LAG-3 shedding mechanism. Human ex vivo assays were performed by co-culturing expanded CD4+ T cells with iDCs in the presence of Staphylococcal enterotoxin B and FS118, or controls. sLAG-3 was measured by ECLIA. The binding valency of FS118 was determined using chemically-crosslinked mass spectrometry mapping (XL/MS). Variants of FS118 and FS118 surrogate molecules were generated with differing valency for LAG-3 and PD-L1. MC38 tumor-bearing C57BL/6 mice were dosed once interperitoneally with FS118 surrogate or valency variants. TILs were analyzed by flow cytometry, and sLAG-3 was measured in the serum by ECLIA. In patients receiving FS118 treatment, a dose-dependent increase in sLAG-3 was detected in the blood. In an ex vivo human T cell assay, the FS118-mediated increase in sLAG-3 was greater than with the combination of the individual bispecific components. sLAG-3 increase by FS118 was mediated by ADAM10 and ADAM17. FS118 demonstrated tetravalent binding to its target antigens using XL/MS. All four binding sites simultaneously bound to target antigens, with preferential binding to PD-L1 in the presence of equal amounts of both LAG-3 and PD-L1. Variants of FS118 with reduced binding valency for target antigens mediated lower levels of LAG-3 release than tetravalent FS118. Compared to FS118, monovalent PD-L1 binding reduced sLAG-3 release and importantly, a variant of FS118 with monovalent LAG-3 binding generated the lowest level of sLAG-3. Data will be presented showing the role of binding valency on LAG-3 downregulation by TILs and T cell activation in ex vivo murine samples. FS118 is tetravalent and can bind to two LAG-3 and two PD-L1 molecules simultaneously. Bivalent LAG-3 and PD-L1 binding is required for enhanced shedding of cell surface LAG-3 in vitro. Removing LAG-3 from the surface of exhausted TILs is a novel mechanism attributed to the tetravalent structure of FS118, which may be important to overcome compensatory upregulation of LAG-3 induced by blockade of PD-L1 in patients. Citation Format: Claire S. Reader, Wenjia Liao, Beatrice J. Potter-Landau, Christel Séguy Veyssier, Martyn C. Rhoades, Claire J. Seal, Michelle Morrow, Neil Brewis. The tetravalent structure of FS118, a bispecific antibody targeting LAG-3 and PD-L1, is required for its novel mechanism of LAG-3 shedding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2874.

Isolation of Methicillin Resistant (MR) Staphylococci from Chicken Meat Samples

The aim of this study was to determine the presence of methicillin resistant staphylococci (MRS) in retail raw chicken meat samples sold in Hatay. The antimicrobial susceptibility of the isolates was tested for 13 different antimicrobials by disc diffusion method and investigated for resistance genes encoding methicillin (mecA), tetracycline (tetM, tetK), penicillin (blaZ), macrolide (ermA, ermC), lincosamide (lnuA) and aminoglycoside [aac(6′)/aph(2′′), aph(3′)‐IIIa, ant(4′)‐Ia] resistance via the use of polymerase chain reaction (PCR). In addition, the presence of staphylococcal enterotoxin (SE) genes was also searched by PCR. Out of 50 collected chicken meat samples, 11 (22%) MRS was isolated and the following species were determined: S. sciuri (72.7%, 8/11) and S. epidermidis (27.3%, 3/11). While all isolates were resistant to oxacillin, penicillin and ampicillin, various rates of resistance were observed for tetracycline (8, 72.7%), clindamycin (3, 27.3%), trimethoprim‐sulfamethoxazole (2, 18.2%), erythromycin (2, 18.2%) and rifampicin (1, 9.1%). All MRS harbored mecA gene together with blaZ. The tetM gene responsible for ribosomal protection was detected in all phenotypically tetracycline resistant isolates. ermC gene in erythromycin resistant isolates and lnuA gene in clindamycin resistant isolates were detected. None of the isolates was found to be positive for SE genes. The results of this study indicated that contamination of retail raw chicken meat samples with MRS poses a risk to public health due to transmission of these bacteria to humans. Additionally, this study also highlights the importance of monitoring antimicrobial resistant bacteria in animal originated foods.

Abstract P105: Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures

Abstract VSIG4 (V-set immunoglobulin-domain-containing 4) is a B7 family related protein with known roles as a complement receptor involved in pathogen clearance, via interactions with C3 fragments, as well as a negative regulator of T cell activation by an undetermined mechanism. VSIG4 is expressed in tumor associated macrophages with exquisite specificity. In cancer, increased expression of VSIG4 has been associated with worse survival in multiple indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, stomach cancer and glioma, suggesting an important role in tumor immune evasion. Based upon computational analysis of transcript data across thousands of primary cancer and normal tissue samples, we hypothesized that VSIG4 has an important regulatory role in promoting M2-like immune suppressive macrophages in the tumor microenvironment, and that targeting VSIG4 via a monoclonal antibody could relieve VSIG4-mediated macrophage suppression by repolarizing tumor associated macrophages (TAMs) to an inflammatory phenotype capable of coordinating an anti-tumor immune response. Through a series of in vitro and ex vivo assays we demonstrate that anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Anti-VSIG4 antibodies upregulate pro-inflammatory cytokines in M-CSF plus IL-10 driven monocyte-derived M2c macrophages, as well as in TAM-like macrophages in vitro derived from monocytes cultured in the presence of ascites fluid from ovarian cancer patients. To determine whether anti-VSIG4-induced macrophage repolarization can activate T cells, monocyte-derived M2c macrophages were co-cultured with autologous T cells in the presence of staphylococcal enterotoxin B (SEB) activation and anti-VSIG4 antibodies. Here, anti-VSIG4 antibodies upregulate both pro-inflammatory myeloid-derived cytokines (GM-CSF) and T cell-derived cytokines (IFNγ). To extend these observations to a relevant translational model, we treated fresh, patient-derived tumor samples with anti-VSIG4 antibodies and relevant controls ex vivo. Across numerous patient-derived samples, which included multiple tumor types, anti-VSIG4 treatment resulted in a significant upregulation of cytokines involved in TAM repolarization and T cell activation, and chemokines involved in immune cell recruitment, at levels greater than observed by treatment with anti-PD-1 or a clinical macrophage repolarizing agent (anti-ILT-4). Taken together, these data suggest that VSIG4 represents a promising new target capable of stimulating an anti-cancer response via multiple key immune mechanisms. Citation Format: Steve Sazinsky, Ani Nguyen, Mohammad Zafari, Ryan Phennicie, Joe Wahle, Veronica Komoroski, Kathryn Rooney, Craig Mizzoni, Boris Klebanov, Jessica Ritter, Denise Manfra, Igor Feldman, Tatiana Novobrantseva. Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P105.

A comparative characterization of nasal and clinical isolates of Staphylococcus aureus from west of Iran.

Background and Objectives:Recently, the rise of methicillin-resistant Staphylococcus aureus (MRSA) isolated from hospital healthcare workers (HCWs) and various infectious samples has become one of the main concerns in hospital settings. Therefore, epidemiological studies are necessary to monitor antibiotic resistance patterns in each region and to study the pathogenesis of this strain to control infections.Materials and Methods:In this cross-sectional study, a total of 100 S. aureus isolates, including 50 isolates obtained from the anterior nares of healthcare workers, as well as 50 other isolates cultured from the various clinical specimens from the referral hospitals in Khorramabad (West of Iran) were tested. All isolates were examined to determine antibiotic resistance pattern, and the presence of staphylococcal enterotoxin A (sea), staphylococcal enterotoxin B (seb) and mecA genes.Results:The mecA gene was found among 36% (18/50) of the clinical S. aureus isolates (CSIs) and 14% (7/50) of nasal S. aureus isolates (NSIs), with statistically significant difference (X2 = 6.53; p = 0.011). The difference between the frequency rate of sea gene among MRSA strains isolated from clinical specimens (46.6%, 7/15) was significant compared to strains isolated from nostrils (14.3%, 1/7) (X2 = 3.85; p = 0.049).Conclusion:The frequency of mecA, sea, and seb genes among the clinical samples was more than strains isolated from the nostrils of healthcare personnel.

Enterotoxigenic Potential of Coagulase-Negative Staphylococci from Ready-to-Eat Food.

Although coagulase-positive staphylococci are considered to be the main factor responsible for food poisoning, an increasing role for the coagulase-negative staphylococci in the production of enterotoxins has been observed in recent years. This study was conducted to assess the occurrence of genes responsible for the production of staphylococcal enterotoxins (SE), enterotoxin-like toxins (SEI) and toxic shock syndrome toxin-1 (TSST-1) in coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food from bars and restaurants. One hundred and eighteen CoNS strains were tested using polymerase chain reaction (PCR) to five superantigenic toxin genes, including five different types of classical enterotoxins (sea, seb, sec, sed and see) and the toxic shock syndrome toxin-1 (tsst-1) as well as to supertoxin-like genes. PCR-positive isolates were then tested using immunoenzymatic methods (SET-RPLA, Vidas SET 2) for toxin expression. Out of 118 CoNS strains, the presence of staphylococcal enterotoxins was confirmed in 72% of them. The most frequently found enterotoxin-like genotype was ser, selu. Two of the tested strains had up to ten different enterotoxin genes in the genome at the same time. Although no production of enterotoxins was detected in the CoNS, which means that their possible role in the epidemiology of food-borne diseases is minimal, the data demonstrated that the toxigenic capacity of the CoNS should not be ignored, and that this group of microorganisms should be continuously monitored in food.

COAGULASE POSITIVE STAPHYLOCOCCI ENUMERATION AND ENTEROTOXINS DETECTION IN MILK AND DAIRY PRODUCTS FROM CENTRAL ITALY

This study aims at enumerating coagulase positive staphylococci (CPS) in 404 samples of milk and dairy products collected in own-checks or during the official controls from different dairy industries located in Central Italy. These microorganisms were enumerated using ISO 6888-2:1999/Amd. 1:2003 and only when they exceeded 105CFU/g, the presence of any of the seven more common staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED and SEE) was also investigated. Own-checks samples resulted always below the detection limit, whereas among those collected by the competent authorities in the framework of official controls, provola (100%) and mozzarella (22.9%) samples were positive to CPS, with mean values of 1.8x102 and 2.8x105 CFU/g respectively. Such values exceeded the maximum limits set by Commission Regulation (EC) No. 2073/2005, resulting in a request of hygiene improvements in the first case; in the second case, the presence of staphylococcal enterotoxins in 8 (2.7%) mozzarella samples out of 298 investigated cheese products was also observed, resulting in their withdrawn from the market. Therefore, this study aims at highlighting that monitoring of CPS incidence in dairy products and subsequent testing of cheeses for enterotoxins when appropriate represent an important tool for public health in order to avoid the occurrence of foodborne outbreaks.

Myeloid Dendritic Cells Induce HIV Latency in Proliferating CD4+ T Cells.

HIV latency occurs predominantly in long-lived resting CD4+ T cells; however, latent infection also occurs in T cell subsets, including proliferating CD4+ T cells. We compared the establishment and maintenance of latent infection in nonproliferating and proliferating human CD4+ T cells cocultured with syngeneic myeloid dendritic cells (mDC). Resting CD4+ T cells were labeled with the proliferation dye eFluor 670 and cultured alone or with mDC, plasmacytoid dendritic cells, or monocytes in the presence of staphylococcal enterotoxin B (SEB). Cells were cultured for 24 h and infected with CCR5-tropic enhanced GFP (EGFP) reporter HIV. Five days postinfection, nonproductively infected EGFP- CD4+ T cells that were either nonproliferating (eFluor 670hi) or proliferating (eFluor 670lo) were sorted and cultured for an additional 7 d (day 12) with IL-7 and antiretrovirals. At day 5 postinfection, sorted, nonproductively infected T cells were stimulated with anti-CD3/CD28, and induced expression of EGFP was measured to determine the frequency of latent infection. Integrated HIV in these cells was confirmed using quantitative PCR. By these criteria, latent infection was detected at day 5 and 12 in proliferating T cells cocultured with mDC and monocytes but not plasmacytoid dendritic cells, where CD4+ T cells at day 12 were poor. At day 5 postinfection, nonproliferating T cells expressing SEB-specific TCR Vβ-17 were enriched in latent infection compared with non-SEB-specific TCR Vβ-8.1. Together, these data show that both nonproliferating and proliferating CD4+ T cells can harbor latent infection during SEB-stimulated T cell proliferation and that the establishment of HIV latency in nonproliferating T cells is linked to expression of specific TCR that respond to SEB.

Bone marrow - mesenchymal stem cells impact on the U937 cells in the presence of staphylococcal enterotoxin B (SEB).

The growing resistance against conventional chemotherapy in acute myeloid leukemia (AML) is a noticeable clinical concern. Therefore, many researchers are looking for novel substances to overcome drug resistance in cancer. Staphylococcal enterotoxin B (SEB) is a superantigen (SAg) and a promising compound which has lethal effects on malignant cells. In this unprecedented study, SEB was used against U937 cells in a co-culture system in the presence of human bone marrow-mesenchymal stem cells (hBM-MSCs). The effects of hBM-MSCs on the proliferation and survival of U937 cell line with SEB was assessed using MTT assay and AnnexinV/PI flowcytometry, respectively. Moreover, the expression of IL-6, IL-10, TGF-β, and inhibitor of nuclear factor kappa-B kinase (IKKb) was evaluated by real-time PCR technique. The same experiments were also carried out using hBM-MSCs-conditioned medium (hBM-MSCs-CM). The results showed that SEB reduced the proliferation and survival of U937 cell line, but hBM-MSCs or hBM-MSCs-CM suppressed the effects of SEB. Furthermore, real-timePCR demonstrated that SEB could decrease the expression of IL-6, IL-10, and TGF-β in hBM-MSCs (P<.05), while the production of IKKb was increased in comparison with the control group. These findings help us to have a broader understanding ofthe usage of SEB in the treatment of haematological malignancies, especially if it is targeted against hBM-MSCs to disrupt their supportive effects on malignant cells.

Establishment of engineered cell-based assays mediating LAG3 and PD1 immune suppression enables potency measurement of blocking antibodies and assessment of signal transduction

LAG3 is an important regulator of T cell homeostasis and studies in mouse tumor models have demonstrated that simultaneously antagonizing LAG3 and PD1 can augment tumor-specific T cell responses and induce tumor rejection. The combined use of LAG3 antagonist antibodies with established anti-PD1 therapies is currently being evaluated in human clinical trials. A functional assay for human LAG3 was developed by co-culture of a Jurkat T-cell lymphoma line overexpressing LAG3 with a Raji B-cell lymphoma line in the presence of staphylococcal enterotoxins. Reversal of LAG3 repression was measured as an increase in IL-2 production or NFAT activation in response to treatment with MK-4280, an anti-human LAG3 antagonist antibody. Changes in cytokines, chemokines, and other mRNA transcripts were in agreement with published in vitro and in vivo models for LAG3 biology which highlights the physiological relevance of the Jurkat functional assay. Additional engineering of PD1 and PDL1 components into the LAG3 assay resulted in a bi-functional assay that is capable of inducing a 10-fold response to individual antibodies blocking either PD1 or LAG3. Importantly, when MK-4280 and pembrolizumab were combined to block both pathways, a synergistic 50-fold increase in response was observed.

Effects of CTLA4-Ig on human monocytes

BackgroundAbatacept, a CTLA4-Ig fusion protein attenuates T cell activation by inhibiting the CD80/86-CD28 costimulatory pathway that is required for the proper T cell activation and thus displays beneficial effects in the treatment of rheumatoid arthritis (RA). Although some studies have disclosed the in vitro effects of this biological agent on the immune-competent cells, the precise mechanisms of action in RA still remain unclear. The current studies were therefore undertaken to explore the effects of abatacept on monocytes in detail.MethodsMonocytes from healthy donors were cultured in the presence of staphylococcal enterotoxin B (SEB) with pharmacologically attainable concentrations of abatacept or control IgG-Fc. The expression of CD80 and CD86 and the induction of apoptosis of monocytes were measured by flow cytometry. The expression of CD80 and CD86 messenger RNA (mRNA) was determined by quantitative RT-PCR.ResultsAbatacept promoted apoptosis of SEB-stimulated monocytes. The induction of apoptosis of monocytes by these biological agents was reversed by the addition of IgG, but not IgG-F(ab′)2 fragments. Furthermore, abatacept significantly suppressed the expression of CD80, but not that of CD86 at protein levels. Finally, abatacept significantly suppressed the expression of mRNA for CD80 of monocytes stimulated with SEB, but not that of CD86.ConclusionsThese results demonstrate that one of the mechanisms of action of abatacept involves the induction of apoptosis of monocytes, which involves interaction with Fc receptor on monocytes. Moreover, the data also demonstrate that abatacept selectively suppresses the expression of CD80 at mRNA levels.

Prevalence of enterotoxin genes in Staphylococcus aureus colonising food handlers: does nasal carriage status matter?

This study investigated the association between the presence of staphylococcal enterotoxin (SE) genes and nasal carriage status, and determined temporal changes in the prevalence of these genes in Staphylococcus aureus strains isolated from healthy carriers between 2002 and 2011. Three large samples of food handlers recruited in 2002, 2003 and 2011 were nasally sampled on two occasions to determine S. aureus colonisation status. Those carrying the same spa type on both occasions were defined as persistent carriers. Genes for SEs SEA-SEU were amplified and associations between carriage status and presence of SE genes were investigated. Although 80% of nasal isolates harboured at least one SE gene over the sampling period, persistent carriers were significantly more likely to harbour enterotoxigenic S. aureus than transiently colonised subjects [odds ratio (OR) 2.52-3.06]. Strains from persistent carriers more commonly harboured sea, seb and sem. The prevalence of classical SE genes and sej, sem, sen, seo, seq and ses was stable over time, but seh, sel, sep, ser, set and selu increased significantly. Increased toxigenicity of isolates from persistent carriers is consistent with the elevated antibody levels to classical SEs previously reported in persistent carriers, supporting the hypothesis that superantigen production in the nasal cavity may enhance colonisation.

Effects of anti-IL-6 receptor antibody on human monocytes

Objective. To explore the effects of anti-IL-6 receptor antibody, tocilizumab on function of human monocytes.Methods. Monocytes from healthy donors were cultured in the presence of staphylococcal enterotoxin B (SEB) with pharmacologically attainable concentrations of tocilizumab or control IgG. The expression of IL-6 mRNA was determined using quantitative RT-PCR. The expression of CD80 and CD86 and the induction of apoptosis of monocytes were measured using flow cytometry.Results. Tocilizumab promoted apoptosis of SEB-stimulated monocytes. The induction of apoptosis of monocytes by tocilizumab were reversed by addition of IgG, but not IgG F(ab’)2 fragments. Tocilizumab significantly suppressed the expression of CD80, but not that of CD86, on SEB- stimulated monocytes. Finally, tocilizumab significantly suppressed the expression of mRNA for IL-6 of monocytes stimulated with SEB.Conclusions. These results demonstrate that one of the mechanism of action of tocilizumab involves the induction of apoptosis of monocytes, which requires interaction with Fc receptor on monocytes. Moreover, the data also indicate that tocilizumab inhibit IL-6 production of monocytes at mRNA levels.

Incidence of Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and Staphylococcal Enterotoxin in Two Types of Mexican Fresh Cheeses

Handcrafted fresh cheeses are popular among consumers in Mexico. However, unsafe raw materials and inadequate food safety practices during cheese manufacture and preservation make them a potential public health risk. The incidence of Salmonella, Listeria, Escherichia coli O157:H7, and staphylococcal enterotoxin was analyzed in two types of fresh cheese (panela and adobera) commonly marketed in Mexico. A total of 200 samples, 100 panela and 100 adobera, were acquired from 100 wholesale milk product distributors who supply small retailers in the Guadalajara metropolitan area, Jalisco State, Mexico. Pathogens were identified using culture and immunoassay (miniVidas) methods. The presence of staphylococcal enterotoxin was determined by an immunoassay method. Of the 200 analyzed samples, 92 were positive for at least one of the pathogens. The incidence in the panela samples was 56%: 34% Salmonella, 16% E. coli O157:H7, and 6% L. monocytogenes. In the adobera samples, incidence was 36%: 20% Salmonella, 4% E. coli O157:H7, and 12% L. monocytogenes. Staphylococcal enterotoxin was not detected in any of the 200 samples. Choice of technique had no effect on detection of pathogen incidence, although the immunoassay method identified more Salmonella serotypes than the culture method. Handcrafted panela and adobera fresh cheeses in Mexico frequently contain pathogenic bacteria and therefore pose a public health risk.

Extracellular acidosis promotes neutrophil transdifferentiation to MHC class II-expressing cells

Inflammation in peripheral tissues is usually associated with local acidosis. In the present study, we demonstrate that extracellular acidification enhances GM-CSF- and IFN- γ-induced expression of HLA-DR, CD80 and CD86 in human neutrophils (neutrophil transdifferentiation), and potentiates antigen-capturing capacities (both endocytosis and phagocytosis) of the transdifferentiated cells. Furthermore, in acidic conditions the transdifferentiated neutrophils have stronger antigen-presenting capacity, inducing more intense proliferation of autologous T lymphocytes in the presence of staphylococcal enterotoxin A. Thus, extracellular acidosis can represent a factor that promotes neutrophil transdifferentiation and potentiates the functional abilities of the transdifferentiated cells in inflammatory foci in vivo.

The effects of staphylococcal bacteriophage lysates on cancer cells in vitro

Bacteriophages constitute a serious alternative to antibiotic therapy of bacterial infections. They are also extremely numerous entities: phages can be found in almost all places on Earth and are constantly present in human and animal bodies. Observations of the effect of therapeutic staphylococcal phages and their bacterial hosts on melanoma migration in vitro are reported in this article. Together with bacteriophage preparations, disrupted Staphylococci (host strains) were investigated to compare the effects of bacteria with those of bacteriophages. Migration was decreased by all the investigated preparations in various ways and this was rather due to the activity of the bacterial components. Importantly, none of the investigated bacteriophage or bacterial preparations induced an increase in the migration activity of melanoma cells, which is important from the perspective of the therapeutic use of phage lysates. The possible presence of staphylococcal enterotoxins in the therapeutic bacteriophage preparations was also verified. All the studied therapeutic bacteriophage preparations were negative for the Staphylococcal enterotoxins A, B, C, D, and E (i.e., the enterotoxin content was less than 0.2-0.5 ng/ml).

Human Regulatory CD8+ T Cells

Administration of a humanized monoclonal anti-CD3 antibody (mAb) to patients with type 1 diabetes (T1D) increases their C-peptide responses and the CD8/CD4 ratio. Incubation of human peripheral blood mononuclear cells (PBMC) with mAb in vitro has been shown to induce CD8(+) regulatory T cells (Tregs) capable of inhibiting proliferation of CD4(+) T cells. We hypothesized that CD8(+) Tregs function through secretion of cytokines. To test that possibility, we generated CD8(+) Tregs, sorted them by FACS, incubated them with syngeneic CD8-depleted PBMC in the presence of staphylococcal enterotoxin B (SEB), and measured proliferation of T cells and cytokines. Using neutralizing anti-cytokine mAbs, we show that the inhibitory effect of CD8(+) Tregs could be partially alleviated by anti-CCL-4, anti-TNF, and to a lesser extent anti-IL2, suggesting that these cytokines contribute to CD8(+) Treg function.

Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France

Enterotoxins produced by Staphylococcus aureus are responsible for staphylococcal food-poisoning outbreaks (SFPO). In France, SFPO are the second cause of food-borne diseases after Salmonella. However, very little is known about the strains involved. The objective of this study was to characterize the staphylococcal strains related to these SFPO through phenotypic and genotypic analyses. A total of 178 coagulase-positive staphylococcal isolates recovered from 31 SFPO (1981–2002) were screened through biotyping. Thirty-three strains representative of the different biotypes in each SFPO were further examined for SmaI macrorestriction-type, phage-type, resistance to various antimicrobial drugs, presence of staphylococcal enterotoxin ( se) genes sea to sei, and production of enterotoxins SEA to SED. All these 33 strains were identified as S. aureus species: 27 were of human biotypes and six ovine or non-host-specific biotypes. Most (74.1%) strains reacted with group III phages. Eleven strains were resistant to at least two classes of antibiotics and among them, two were resistant to methicillin. Twenty-nine strains carried one or several of the eight se genes tested; the gene sea was most common ( n = 23), and often linked to sed ( n = 12) or seh ( n = 5). The novel se genes seg-i were in all cases associated with se genes sea to sed except for one strain which carried only seg and sei. Pulsed-Field Gel Electrophoresis (PFGE) of SmaI macrorestriction digests of the 33 strains discriminated 32 PFGE patterns grouped into nine biotype-specific clusters. All five strains carrying sea and seh were grouped together into the same sub-cluster. Three of the four se-gene-negative strains were in one PFGE cluster: all four should be tested for se genes not included in this study and, if negative, be further investigated for the presence of unidentified SEs.

Detection of enterotoxin genes seg, seh, sei, and sej and of a novel aroA genotype in Jordanian clinical isolates of Staphylococcus aureus

The presence of staphylococcal enterotoxin (SE) genes ( seg, seh, sei, and sej) and the correlation of their prevalence with the genotypes were studied in 100 clinical isolates of Staphylococcus aureus. Polymerase chain reaction (PCR) of SE genes indicated that 39% of the isolates were enterotoxigenic. Thirty-seven percent of the total isolates were seg positive, whereas 24% and 4% were sei and seh positive, respectively. All isolates containing sei were positive for seg, whereas sej gene was not detected. Genotyping by PCR-restriction fragment length polymorphism analysis of the aroA gene revealed that 39% of the isolates were type A and 11% were type B, and 50% displayed a novel (N) genotype. The presence of the enterotoxin genes was independent ( P < 0.05) of the genotypes of the tested S. aureus isolates. This study has demonstrated that the seg was the most dominant enterotoxin gene and that the enterotoxigenic Jordanian S. aureus isolates belong to different genotypes, and N genotype was predominant.

Human airway and peripheral blood eosinophils enhance Th1 and Th2 cytokine secretion

The effector function of eosinophils involves their release of toxic granule proteins, reactive oxygen species, cytokines, and lipid mediators. Murine studies have demonstrated that eosinophils can also enhance T cell function. Whether human eosinophils, in particular, airway eosinophils, have similar immunoregulatory activity has not been fully investigated. The aim of this study was to determine whether human blood and airway eosinophils can contribute to Th1 and Th2 cytokine generation from CD4+ T cells stimulated with superantigen. Eosinophils were obtained from blood or bronchoalveolar lavage fluid 48 h after segmental allergen bronchoprovocation. Purified eosinophils were co-cultured with autologous CD4+ blood T cells in the presence of staphylococcal enterotoxin B (SEB). Cytokine levels in the supernatant fluid were determined by enzyme-linked immunosorbent assay (ELISA). Eosinophil expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules was assessed by flow cytometry before culture, 24 h after granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation, and 24 h after co-culture with CD4+ T cells and SEB. Interleukin (IL)-5, IL-13, and interferon (IFN)-gamma generation increased when CD4+ T cells were co-cultured with either blood or airway eosinophils in the presence of SEB. The ability of eosinophils to enhance cytokine generation was independent of their source (blood vs airway), activation by GM-CSF, or detectable expression of human leukocyte antigen (HLA)-DR, CD80, or CD86. Our data demonstrate that SEB-induced generation of Th1 and Th2 cytokines is increased in the presence of human blood and airway eosinophils. Thus, eosinophils can have an immunoregulatory function in pathogen-associated allergic diseases such as atopic dermatitis, chronic sinusitis, and asthma exacerbations.