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Quorum sensing (QS) system is an important bacterial cell-to-cell signaling system controlling expression of various genes in response to cell densities. In vibrios, LuxR/AphA are two established master QS regulators (MQSRs), and VqsA is recently identified to be the third putative MQSR. As a novel LysR-type regulator, the regulon and the underlying regulation mechanisms of VqsA remains to be elucidated. Here our investigation indicated that the yields of alkaline serine protease (Asp), the exotoxin in Vibrio alginolyticus was dependent on both LuxR and VqsA in growth phase dependent manner. Various in vivo and in vitro analyses including electrophoretic mobility shift assays (EMSA) along with DNase I footprinting investigations demonstrated that VqsA positively controls asp expression through directly binding to the partially palindromic 29 bp binding motif in the promoter region of asp. Moreover, RNA-seq analysis validated the regulatory roles of VqsA in various processes in the organism. Collectively, our data showed that VqsA positively regulates the expression of exotoxin and other virulence-associated genes and is essential for the QS regulation in V. alginolyticus., (© FEMS 2019.)

Unlabelled: The insecticidal activity of Bacillus thuringiensis is owing to the action of Cry and Cyt proteins. In addition to the synthesis of insecticidal proteins, some strains are able to synthesize β-exotoxin, which is highly toxic to humans. In this regard, it is very important to have a simple method to detect β-exotoxin to avoid the commercial production of this type of strains. In this work, we developed a simple and fast method, using the nematode Caenorhabditis elegans to detect indirectly the synthesis of β-exotoxin by B. thuringiensis strain. Using this assay, we detected that ~60% of Mexican native strains (i.e. LBIT-471, 491, 492, 497, 507, 511, 515, 536 and 537) were toxic to the nematode (44-97% mortalities) and their β-exotoxin (βEx(+) ) production, including a positive control (NRD-12), was confirmed by HPLC. In addition, the negative controls (βEx(-) ) LBIT-436 (HD-1) and LBIT-438 and also the native strains LBIT-499, 500, 521, 522, 533 and 542, did not show a detrimental effect against nematodes larvae, neither the synthesis of β-exotoxin as determined by HPLC. Finally, we did not find a correlation between B. thuringiensis strains with similar plasmid patterns and the β-exotoxin production., Significance and Impact of the Study: In this work, we implemented a qualitative and fast bioassay using the nematode Caenorhabditis elegans to detect the production of β-exotoxin in different strains of Bacillus thuringiensis. We show that this assay is useful to detect β-exotoxin in B. thuringiensis with high reliability, helping to discriminate strains that could not be used as bioinsecticides because of their putative risk to humans. Data show that qualitative bioassay with nematodes is a potential alternative to fly larvae bioassays, and correlated with the determination of β-exotoxin by HPLC., (© 2015 The Society for Applied Microbiology.)

Objectives: To examine the effect of subinhibitory concentrations (sub-MICs) of antistaphylococcal drugs on Panton-Valentine leucocidin (PVL), α-haemolysin (Hla) and protein A (SpA) expression by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA)., Methods: Five clinical isolates representing the main worldwide CA-MRSA clones were grown with sub-MICs (1/8, 1/4 and 1/2 MIC) of five antibiotics (clindamycin, daptomycin, linezolid, tigecycline and vancomycin). After 4 and 6 h of incubation, culture pellets were used for relative quantitative RT-PCR with primers specific for pvl, hla, spa and gyrB. The PVL, Hla and SpA concentrations were measured in the supernatant (for PVL and Hla) and in the cell pellet (for SpA) using specific ELISAs., Results: For all strains tested, clindamycin and linezolid dramatically reduced mRNA levels of PVL and SpA. Tigecycline also decreased the PVL and SpA mRNA levels of 3/5 and 4/5 strains tested, respectively, whereas daptomycin and vancomycin had no significant effect. PVL and SpA quantification confirmed the concentration-dependent inhibition of PVL and SpA production by clindamycin and, to a lesser extent, by linezolid and tigecycline. Only clindamycin decreased Hla mRNA expression, whereas linezolid, tigecycline and daptomycin showed heterogeneous strain-dependent results, and vancomycin had no significant effect. Analysis of the Hla level revealed a stronger concentration-dependent inhibition of Hla release by clindamycin than by linezolid., Conclusions: The effect of sub-MICs on virulence expression depended on the antibiotic and the virulence factor. Clindamycin and linezolid consistently suppressed the expression of different virulence factors by CA-MRSA, whereas tigecycline specifically suppressed PVL expression. Daptomycin and vancomycin seem to have no significant effects at these concentrations.

Background: Therapeutic use of vancomycin is characterized by decreased susceptibilities and increasing reports of clinical failures. Few studies have examined the clinical outcomes of patients with methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia treated with vancomycin. The primary objective was to compare clinical outcomes of patients with MRSA bacteraemia treated according to standard of care practices., Methods: Patients were included if: (i) admitted to University of New Mexico Hospital between 2002 and 2009; (ii) ≥18 years of age; (iii) had one blood culture positive for MRSA; and (iv) received vancomycin. Clinical outcomes were defined as cure, failure (relapse of infection 30 days after completion of therapy, death or change in therapy) or unevaluable. Patient demographics, source of bacteraemia, treatment regimen, and microbiological characteristics were determined., Results: Two hundred patients with MRSA bacteraemia were included. Sixty-one patients were unevaluable, leaving 139 patients for the final analysis. Seventy-two (51.8%) patients were cured and 67 (48.2%) experienced vancomycin failure. Vancomycin MIC(90) was 2 mg/L for both groups by Etest. Patients with endocarditis (P = 0.02) or pneumonia (P = 0.02) were more likely to fail therapy. Panton-Valentine leucocidin, loss of agr functionality and strain type were not predictors of outcomes in this study., Conclusions: High failure rates were observed in patients with MRSA bacteraemia treated with vancomycin, despite high vancomycin troughs and low rates of nephrotoxicity. Predictors of vancomycin failure included endocarditis and pneumonia. In these situations, vancomycin provides suboptimal therapy.

Objectives: To investigate the effect of subinhibitory concentrations of cell wall active antibiotics on virulence gene expression and biofilm formation in Staphylococcus aureus Newman and in laboratory strains., Methods: Promoter regions of spa, lukE and agr RNAIII were cloned upstream of a modified luxABCDE reporter. Using disc diffusion assays, the effects of antibiotics were observed on gene expression and quantitative real-time PCR was employed to confirm the results. Assays were performed to measure biofilm formation in wild-type S. aureus and respective spa-deficient and small colony variant mutants in the presence of subinhibitory concentrations of antibiotics., Results: Expression of spa and lukE was stimulated by subinhibitory concentrations of penicillin and cefalotin, while agr RNAIII expression was not affected. Denser biofilms were formed by S. aureus Newman and its small colony variant in the presence of subinhibitory concentrations of cefalotin., Conclusions: Subinhibitory concentrations of certain antibiotics have been shown to stimulate virulence gene expression in S. aureus; this may alter the progression of infection and thus render antimicrobial therapy unreliable. The use of appropriate combinations of antibiotics might be an approach to avoiding this situation. Promoter-lux reporters are sensitive tools for studying the modulation of transcription by antibiotic inhibitors, and could be used to predict novel therapeutic combinations for the treatment of infection.

Aim: To determine the antimicrobial activity of costus (Saussurea lappa) oil against Staphylococcus aureus, and to evaluate the influence of subinhibitory concentrations of costus oil on virulence-related exoprotein production in staph. aureus., Methods and Results: Minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of costus oil against 32 Staph. aureus strains ranged from 0.15 to 0.6 μl ml(-1) . The MIC(50) and MIC(90) were 0.3 and 0.6 μl ml(-1) , respectively. Western blot, haemolytic, tumour necrosis factor (TNF) release and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of costus oil on virulence-associated exoprotein production in Staph. aureus. The data presented here show that costus oil dose dependently decreased the production of α-toxin, toxic shock syndrome toxin 1 (TSST-1) and enterotoxins A and B in both methicillin-sensitive Staph. aureus (MSSA) and methicillin-resistant Staph. aureus (MRSA)., Conclusion: Costus oil has potent antimicrobial activity against Staph. aureus, and the production of α-toxin, TSST-1 and enterotoxins A and B in Staph. aureus was decreased by costus oil., Significance and Impact of the Study: The data suggest that costus oil may deserve further investigation for its potential therapeutic value in treating Staph. aureus infections. Furthermore, costus oil could be rationally applied in food products as a novel food preservative both to inhibit the growth of Staph. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

The current pandemic of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) skin infections is caused by several genetically unrelated clones. Here, we analyzed virulence of globally occurring CA-MRSA strains in a rabbit skin infection model. We used rabbits because neutrophils from this animal species have relatively high sensitivity to Panton-Valentine leukocidin (PVL), a toxin epidemiologically correlated with many CA-MRSA infections. Virulence in the rabbit model correlated with in vitro neutrophil lysis and transcript levels of phenol-soluble modulin α and α-toxin, but not PVL genes. Furthermore, abscesses caused by USA300 and its PVL-negative progenitor USA500 were comparatively large and similar in size, suggesting that PVL has played a limited role in the evolution of USA300 virulence in the context of skin infections. Our study indicates a major but not exclusive impact of virulence on the epidemiological success of USA300 and other CA-MRSA strains and emphasizes the importance of core genome-encoded toxins in CA-MRSA skin infections.

Group A streptococci (GAS) may engage different sets of virulence strategies, depending on the site of infection and host context. We previously isolated 2 phenotypic variants of a globally disseminated M1T1 GAS clone: a virulent wild-type (WT) strain, characterized by a SpeB(+)/SpeA(-)/Sda1(low) phenotype, and a hypervirulent animal-passaged (AP) strain, better adapted to survive in vivo, with a SpeB(-)/SpeA(+)/Sda1(high) phenotype. This AP strain arises in vivo due to the selection of bacteria with mutations in covS, the sensor part of a key 2-component regulatory system, CovR/S. To determine whether covS mutations explain the hypervirulence of the AP strain, we deleted covS from WT bacteria (DeltaCovS) and were able to simulate the hypervirulence and gene expression phenotype of naturally selected AP bacteria. Correction of the covS mutation in AP bacteria reverted them back to the WT phenotype. Our data confirm that covS plays a direct role in regulating GAS virulence.

Background: Postinfluenza Staphylococcus aureus pneumonias are increasingly recognized as a major form of life-threatening infections., Methods: A mouse model of postinfluenza S. aureus pneumonia was developed. Mice were intranasally infected with bacteria alone or bacteria plus virus. Infection was assessed by mouse survival, lung histopathology, bacterial density in the lungs, and cellular response to infection., Results: Mice infected with both influenza virus and S. aureus showed higher mortality, greater lung parenchymal damage, and greater bacterial density at metastatic tissue sites than mice infected with only S. aureus. At 4 h, more polymorphonuclear leukocytes and fewer CD11c(+) cells were found in lung samples from mice infected with virus and bacteria than in those from mice infected with bacteria. alpha-Hemolysin and protein A were maximally expressed 4 h after infection, and Panton-Valentine leukocidin was maximally expressed 72 h after infection, with higher levels of alpha-hemolysin expression in mice infected with bacteria alone. Interferon gamma expression was higher in tissue collected from mice infected with virus plus bacteria than in those from bacteria-infected mice., Conclusions: The results from this model demonstrate diverse effects caused by antecedent influenza virus infection, which have a profound influence on the morbidity and mortality associated with S. aureus pneumonia.

Objectives: Community-associated methicillin-resistant Staphylococcus aureus (MRSA) including those encoding Panton-Valentine leucocidin (PVL) are often described as more susceptible to a range of antibiotics than their hospital-associated counterparts. Recent scattered reports of the emergence of multiresistant PVL-MRSA have highlighted the potential for resistance to emerge. Here we detail polyclonal multiply antibiotic-resistant PVL-MRSA occurring in England., Methods: PVL-MRSA from community-based and hospitalized patients located across England were identified by PCR. Isolates were characterized via MIC determinations, toxin gene profiling, PFGE, SCCmec, spa and agr typing. Multilocus sequence typing (MLST) was performed on selected isolates. Patient demographic and available disease data were retained for analysis., Results: Seventy-six PVL-MRSA isolates resistant to three further classes of antibiotic were identified between 2005 and 2008 from centres in each of the Health Protection Agency's geographic regions in England. Patient demographics were typical for PVL-MRSA, and some travel associations were identified along with clonal spread. One instance of familial transmission in the community was detected. PVL-MRSA belonging to MLST clonal complex (CC) 1 (sequence type 772) were consistently highly resistant; multiply antibiotic-resistant representatives of CCs 5, 8, 22, 59 and 80 were also identified. Ciprofloxacin resistance was common amongst the study isolates (51 of 76 isolates)., Conclusions: Genetically diverse multiply antibiotic-resistant PVL-MRSA were identified, and included representatives of a recently emerged multiresistant clone (dubbed the Bengal Bay clone). Risk factors and disease presentations were typical for PVL-MRSA infections. This work highlights the diminishing utility of ciprofloxacin susceptibility for putative identification of PVL-MRSA.

sThe role of Panton-Valentine leukocidin (PVL) in Staphylococcus aureus pathogenesis is controversial. Here, we show that an unintended point mutation in the agr P2 promoter of S. aureus caused the phenotypes in gene regulation and murine pneumonia attributed to PVL by earlier investigators. In agreement with other studies that failed to detect similar effects of PVL using community-associated methicillin-resistant S. aureus strains, we found no significant effect of PVL on gene expression or pathogenesis after we repaired the mutation. These findings provide further evidence that PVL does not have a major impact on S. aureus pathogenesis. Moreover, our results demonstrate that a single nucleotide polymorphism in an intergenic region can dramatically affect bacterial physiology and virulence. Finally, our work emphasizes the need to frequently evaluate the integrity of the S. aureus agr locus.

Objectives: The rapid emergence of methicillin-resistant Staphylococcus aureus from the community (CA-MRSA) presents difficulties in making treatment choices. We evaluated whether combining another orally available agent commonly used to treat CA-MRSA with gemifloxacin would enhance gemifloxacin activity against CA-MRSA., Methods: Fifty strains of SCCmec IV, agr group 1, Panton-Valentine leucocidin-positive CA-MRSA were evaluated for susceptibilities to gemifloxacin, trimethoprim/sulfamethoxazole, doxycycline, levofloxacin, rifampicin, clindamycin and erythromycin. Twenty of these strains were evaluated for the potential for synergy between gemifloxacin and trimethoprim/sulfamethoxazole, clindamycin and rifampicin by time-kill analysis. Two strains were further evaluated in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model., Results: In time-kill analyses, gemifloxacin combined with trimethoprim/sulfamethoxazole produced additivity (6/20) or synergy (11/20) in 85% of the isolates tested. The addition of clindamycin to gemifloxacin showed additivity (3/20) or synergy (2/20) in 25% of the isolates. All isolates displayed indifference to the combination of gemifloxacin and rifampicin. In the PK/PD model, combining gemifloxacin and trimethoprim/sulfamethoxazole provided potent and sustained bactericidal activity to detection limits of 2 log(10) cfu/mL by 48 h; gemifloxacin combined with clindamycin or with rifampicin killed to detection limits by 56 h or later. One isolate developed efflux-mediated resistance to gemifloxacin at 96 h with gemifloxacin monotherapy. All combinations prevented the emergence of this resistance., Conclusions: Synergy or additivity was demonstrated by time-kill analysis between gemifloxacin and trimethoprim/sulfamethoxazole in most isolates tested. In the PK/PD model, the addition of trimethoprim/sulfamethoxazole, clindamycin and rifampicin enhanced the activity of gemifloxacin against CA-MRSA and suppressed the emergence of resistance to gemifloxacin.

Objectives: Panton-Valentine leucocidin (PVL)-positive Staphylococcus aureus are responsible for causing skin and soft tissue infections, with the potential to cause severe invasive disease. Recently, methicillin-resistant Staphylococcus aureus (MRSA) strains that produce PVL have emerged in the community. As residents of care homes are a key group at risk of MRSA colonization and infection, we have examined the epidemiology of MRSA in three large cohorts of residents in urban care homes to establish whether PVL-positive MRSA strains are present in this setting., Methods: Nasal swabs (n = 3037) collected from consenting residents of 69 care homes in Leeds, UK, were screened for MRSA using chromogenic agar over three periods (June-August 2005, November-December 2006 and October-November 2007). PCR amplification was used to detect genes encoding PVL. Antibiogram profile and PFGE were also used to characterize MRSA isolates (n = 601)., Results: MRSA prevalence was 21%, 20% and 19% in each cohort, respectively. The majority of the isolates were related epidemiologically to the predominant local nosocomial epidemic MRSA strain, EMRSA-15 (78%). No isolate carried the genes encoding PVL. Twelve percent of the isolates (n = 74) had increased susceptibility to non-beta-lactam agents and were distributed across 31 care homes., Conclusions: MRSA strains that produced PVL were not found to be colonizing residents of care homes between 2005 and 2007. Continued surveillance is, however, necessary to understand the interaction between MRSA in care homes and hospitals, especially to reduce the chance that the former may amplify community-associated MRSA strains.

Background: The predominant genetic background of community-associated methicillin-resistant Staphylococcus aureus has transitioned from USA400 to USA300 in most US communities. The explanation for this shift is unclear. We hypothesized that USA300 must be more pathogenic--specifically, that USA300 would have increased virulence when compared with USA400 in an animal model., Methods: Rats were inoculated intratracheally with 1 of 6 S. aureus isolates from the USA300 and USA400 backgrounds. We assessed mortality, in vivo bacterial growth, and histopathology. We assessed the in vitro expression of capsule and of selected genes believed to be important in virulence in S. aureus, including agr, saeRS, sarA, alpha-toxin (hla), and Panton-Valentine leukocidin (pvl)., Results: USA300 isolates were more lethal, produced more severe pneumonia, and had higher in vivo bacterial density in the lung than did USA400 isolates. In vitro expression of agr, saeRS, sarA, hla, and pvl were greater in USA300 isolates. USA300 isolates were unencapsulated, whereas 2 of 3 USA400 isolates produced capsule., Conclusions: USA300 isolates were more virulent than USA400 isolates in a model of necrotizing pneumonia. The explanation for this is unclear, but it likely results from increased expression of S. aureus regulatory systems (e.g., agr, saeRS, and sarA) and the resultant upregulation of key virulence factors including alpha-toxin and PVL.

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66 g l(-1) in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88 mmol glucose g(-1) h(-1), respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15 g l(-1) and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.

Skin infections associated with visits to tropical countries are well known. In most of the cases, the infection is caused by Staphylococcus aureus. After a sufficient antibiotic treatment, the skin infections resolve without any sequelae. But several patients suffer from recurrent skin infections. Panton-Valentine leukocidin (PVL) is a cytotoxin produced by S aureus, which is associated with severe necrotic skin lesions and with a high contagiosity.

Methicillin-resistant Staphylococcus aureus is increasingly responsible for staphylococcal outbreaks in prison. There is limited information on the source of the outbreak strains, risk factors for infection, and transmission of these strains within a prison. We conducted a survey to determine the prevalence of nasal colonization with S. aureus in 2 New York State prisons. S. aureus isolates from clinical cultures collected from all New York State prisons during a 6-month period were compared with the colonizing strains. Analyses were conducted to determine whether prison-level characteristics were associated with colonization or infection with S. aureus. The colonization rate was 25.5% (124/487); 10.5% of the isolates were methicillin resistant, all were staphylococcal chromosomal cassette (SCC)mec type IV, and 61.5% were Panton Valentine leukocidin (PVL) positive. Surprisingly, 21.6% of the methicillin-susceptible isolates were also PVL positive. Of the clinical isolates, 48.3% were methicillin resistant, with 93.1% of the latter being SCCmec type IV and 48.3% being PVL positive. The predominant clone was USA 300. Prison-level risk factors for infection included the proportion of inmates with drug offenses, the length of inmate stay, and the jail from which inmates originated. This study suggests that both new and long-term inmates act as sources of S. aureus strains, with the more virulent of the latter preferentially being selected as pathogens.

Aims: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture., Methods and Results: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C., Conclusions: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese., Significance and Impact of the Study: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.

Extracellular protein toxins contribute to the pathogenesis of a wide variety of Staphylococcus aureus infections. The present study investigated the effects that cell-wall active antibiotics and protein-synthesis inhibitors have on transcription and translation of genes for Panton-Valentine leukocidin, alpha-hemolysin, and toxic-shock syndrome toxin 1, in both methicillin-sensitive and methicillin-resistant S. aureus. Subinhibitory concentrations of nafcillin induced and prolonged mRNA for Panton-Valentine leukocidin, alpha-toxin, and toxic-shock syndrome toxin 1 and increased toxin production. In contrast, clindamycin and linezolid markedly suppressed translation, but not transcription, of toxin genes. These results suggest (1) that protein-synthesis inhibition is an important consideration in the selection of antimicrobial agents to treat serious infections caused by toxin-producing gram-positive pathogens and (2) that, by inducing and enhancing toxin production, inadvertent use of beta-lactam antibiotics to treat methicillin-resistant S. aureus infections may contribute to worse outcomes.

We report the first case of transmission of Panton-Valentine leukocidin-producing Staphylococcus aureus to a physician during the resuscitation of an infant with fatal pneumonia. The physician exhibited numerous furuncles. This case highlights the necessity for health care workers to protect themselves against transmission of infectious diseases from patient to care giver.

The role played by host-pathogen interactions in regulation of expression of streptococcal virulence factors in vivo is beginning to become clear. We have reported that the expression of 2 streptococcal virulence factors, the streptococcal pyrogenic exotoxin (Spe) A and the cysteine protease SpeB, was reciprocally modulated during infection with Streptococcus pyogenes. To identify host signals mediating this reciprocal regulation, we cocultured clonal M1T1 isolates with human peripheral blood mononuclear cells (PBMCs). In accordance with our in vivo findings, when bacteria were in direct contact with human PBMCs or were separated in transwells, expression of speA was induced, whereas expression of speB was down-regulated. This phenomenon was mediated by transferrin and lactoferrin and was influenced by the iron-saturation status of these proteins. Iron chelation from media induced expression of speA, but to a much lesser degree than did that with apotransferrin and lactoferrin, suggesting additional effects of these ferrins on modulation of expression of speA and speB. Thus, ferrins may play an important role in host-pathogen interactions in skin and mucosal tissues.

As part of a nationwide surveillance in The Netherlands during 1994-1997, 53 patients with invasive group A streptococcal (GAS) infections were evaluated for medical history, symptoms, and outcome. Patients' isolates were tested for the production of pyrogenic exotoxins A (SPE-A) and B (SPE-B). Acute-phase sera from all patients and convalescent sera from 12 patients were investigated for the presence of antibodies against SPE-A and SPE-B. Twenty-three patients developed toxic shock-like syndrome and 16 died. Absence of antibodies against SPE-A and/or SPE-B was a risk factor for developing invasive streptococcal disease. Toxic shock and mortality were associated with a lack of anti-SPE-A antibodies (P<.025). Anti-SPE-A antibodies were found in convalescent sera from all patients infected by speA-positive isolates. Virtually all invasive speA-positive streptococci expressed SPE-A protein in vitro. Thus antibodies against SPE-A appeared vital for mediating the outcome of invasive GAS disease in this population.

Analysis of 132 group A streptococcal (GAS) isolates from 151 invasive episodes, including streptococcal toxic shock syndrome (STSS), from 1983 to 1995 showed great genetic variation by use of T serotyping in combination with restriction fragment length polymorphism. In contrast, genetically homogenous T1M1 isolates appeared in epidemic patterns with significantly increased risk of STSS. The speA gene, with the allelic variants speA2 and speA3 carried by the T1M1 and T3M3 serotypes, respectively, was strongly associated with STSS. Infection with a GAS isolate carrying speA, alcohol abuse, and malignancy recently treated with cytostatic drugs were factors independently related to STSS. Neutralization of SpeA lymphocyte mitogenicity was totally absent in sera from patients with STSS and low in sera from persons with uncomplicated bacteremia compared with levels in sera from uncomplicated erysipelas. Neutralization of SpeB was significantly lower in sera of patients with STSS than in sera from persons with bacteremia or erysipelas.

The effects of medium composition and induction timing on expression of a chimeric fusion protein TGF-alpha-PE40 (TP-40) in Escherichia coli strain RR1 were examined using a complex medium at several fermentor scales. Two distinctive phases in E. coli catabolism were identified during fermentation based on preferential utilization between protein hydrolysate and glycerol. Maximum specific and volumetric productivities were achieved by inducing the culture when the cells were switching substrate utilization from protein hydrolysate to glycerol. By increasing the yeast extract concentration in the production medium, initiation of the catabolic switch was delayed until high cell mass was achieved. The final titer of TP-40 at the 15-L fermentation scale was doubled from 400 mg L-1 to 850 mg L-1 by increasing the yeast extract concentration from 1% to 4% (w/v) and delaying the time of induction. This fermentation process was rapidly scaled up in 180-L and 800-L fermentors, achieving TP-40 titers of 740 and 950 mg L-1, respectively.

The relationship between sugar availability and RTX (repeats in toxin) cytotoxin (leukotoxin) production in the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was investigated using a chemostat. A actinomycetemcomitans 301-b produced significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures at a dilution rate of 0.15 h-1 and at pH 7.0. When the growth limitation was relieved by pulsing the cultures with 50 or 150 mM fructose (final concentrations), leukotoxin production immediately stopped and the amount of cellular leukotoxin decreased until the culture was returned to fructose-limited conditions. Leukotoxin synthesis was also repressed in the chemostat cultures by pulsing with glucose but not with the non-fermentable sugar analog, alpha-methyl-D-glucoside. Leukotoxin production was also repressed by fructose in chemostat cultures of ATCC 33384, which is generally recognized as a non-leukotoxin-producing or minimally leukotoxic strain.

The speA gene encoding streptococcal erythrogenic toxin A (SPE A) from Streptococcus pyogenes bacteriophage T12 was overexpressed in Escherichia coli under the control of the T7 promoter. Since most of the expressed protein was found in the periplasmic space, an osmotic shock extraction with 0.5 M sucrose resulted in a highly enriched preparation of SPE A. An additional two-step purification employing high pressure liquid chromatography resulted in a purified SPE A protein.

The V beta repertoire of T cells of patients with gram-positive group A streptococcal (GAS) and non-GAS infections was analyzed to seek evidence for the role of superantigens in streptococcal toxic shock syndrome. No evidence of V beta overexpression but a consistent pattern of depletion of V beta 1, V beta 5.1, and V beta 12 was observed in patients with severe GAS infections. This pattern of V beta depletion was not observed in patients with nonsevere GAS infections or with severe non-GAS gram-positive infections. T cells from patients with severe GAS infections showed evidence of apoptosis; no apoptosis was found when there was no evidence of V beta depletion. There was no correlation with streptococcal M or T serotype or known spe genes. The depletion of specific V beta-bearing T cells in patients with severe GAS infections supports the role of a superantigen in these infections. The in vivo pattern of V beta specificity implicates a novel superantigen(s) in this disease.

phi CTX is a temperate phage of Pseudomonas aeruginosa harbouring the ctx gene that encodes cytotoxin (CTX). We identified phi CTX as an R pyocin-related phage, by serological and molecular analysis, based on the findings that the infectivity of the phage was inhibited with the antisera directed R pyocins and R pyocin-related phages and that the phi CTX genome showed DNA homology to the genome of PS17 (a representative of the R pyocin-related phages) as well as to the pyocin R2 genes. Another new CTX-converting, R pyocin-related phage named PS21 was isolated from a CTX-producing strain of P. aeruginosa, suggesting the distribution of the ctx gene by certain members of R pyocin-related phage family.

The molecular population genetics and pathogenic potential of North American and European invasive strains of Streptococcus pyogenes were assessed. Isolates from recent invasive infections and from infections in the 1920s and 1930s were characterized for multilocus enzyme genotype and allelic variation in the gene (speA) that encodes streptococcal pyrogenic exotoxin (SPE) A (scarlet fever toxin). A subset of strains was studied for allelic variation in genes that encode SPE B and streptokinase. All contemporary strains assigned to electrophoretic types (ETs) 1 and 2 that synthesize SPE A have the speA2 and speA3 allelic variants, respectively, and their relative virulence in two mouse models is similar to that of strains of the same ET and M protein types recovered earlier. In contrast, ET 1 and 2 isolates from disease episodes in the 1920s and 1930s contain the speA1 allele. The data suggest there may be temporal and geographic variation in the occurrence of clone--virulence factor allele combinations, an observation that may in part explain fluctuations in disease frequency, severity, and character.

The neutralizing and protective effect of murine monoclonal antibody (MAb) 3C7 (IgG1) against Pseudomonas aeruginosa exotoxin A was examined in an experimental mouse model of infection with exotoxin A-producing strains. Treatment with MAb 3C7 blocked the reduction of functional elongation factor 2 (EF-2) in the liver of mice but could not clear the bacteria. Administration of gentamicin caused bacteria to be cleared but did not block reduction of hepatic EF-2 level. Treatment with either MAb 3C7 or gentamicin individually did not prolong time to death; however, the combined therapy with both MAb 3C7 and gentamicin cleared bacteria and blocked the reduction of hepatic EF-2 level, resulting in a significant increase in the survival rate of mice. These results suggest that anti-exotoxin A MAbs show effectiveness against pseudomonal infection caused by exotoxin A-producing strains.

We report the study of 53 clinical isolates of group A streptococci, all from patients with streptococcal toxic shock-like syndrome. The strains were analysed for the occurrence of the genes of erythrogenic toxins (pyrogenic exotoxins) types A, B and C and in vitro production of these toxins. In contrast to reports indicating that 85% of the toxic shock-like syndrome-associated isolates contained the erythrogenic toxin A gene, only 58.5% of our strains harboured this gene. The erythrogenic toxin C gene was detected in 22.6% of the isolates. Erythrogenic toxin A and erythrogenic toxin B were produced by 68.7% and 58.3% of the strains containing either gene. For all group A streptococci, irrespective of clinical association, the erythrogenic toxin B gene was detected in all the isolates tested. Thus, it is difficult to define a specific role for erythrogenic toxin B in toxic shock-like syndrome as there was no clear correlation between this disease and the presence of toxin genes. Our results suggest the existence of other pathogenic factor(s) produced by group A streptococci which may stimulate human peripheral T lymphocytes in a manner similar to that of erythrogenic toxins, thus explaining different observations in previous epidemiological genetic studies.

Serious infections due to group A beta-hemolytic streptococcus (GABHS) have been reported with increasing frequency in recent years. We report a case of toxic shock syndrome (TSS) due to GABHS pharyngitis in an otherwise healthy 14-year-old boy. The organism was found to produce toxin A. To our knowledge, this is the second reported case of streptococcal TSS associated with the production of toxin A that is not associated with an invasive disease and the first case associated with a documented rise in the level of antibody to the streptococcal toxin itself. Clinicians must be especially vigilant for this entity in patients who have streptococcal pharyngitis because early recognition and institution of aggressive supportive therapy can be lifesaving.

Because of reported differences in the production of streptococcal pyrogenic exotoxins by group A strains associated with severe streptococcal infections, the stability of exotoxin production by specific strains was examined by passing group A streptococci on blood agar culture plates daily for 20 days. No changes were detected in either exotoxin genes or in exotoxin production during this time, suggesting that these reported differences are due to other explanations such as differences in the strains collected from various geographic areas or to laboratory methodologic differences.

Hamster tracheal organ cultures were used to evaluate the ability of two aminoglycoside and two beta-lactam antibiotics to protect the epithelium from damage due to Pseudomonas aeruginosa infection. Hamster tracheal explants were infected with strains of P. aeruginosa for 4 h and washed to remove nonadherent organisms. The explants were incubated for an additional 18 h in fresh minimal essential medium containing inhibitory or subinhibitory concentrations of antibiotics. The explants were examined by scanning electron microscopy and bacterial elastase and exotoxin A production was detected by ELISA and a western blot assay respectively. Concentrations of aminoglycosides below the MIC for the infecting strain protected the epithelium from damage and inhibited the production of exotoxin and elastase. The beta-lactam antibiotics were not protective and epithelial damage was observed at antibiotic levels equal to or higher than the MIC for that strain. The beta-lactam treated cultures continued to release elastase and exotoxin A at antibiotic concentrations equivalent to or higher than the MIC for that strain. Thus subinhibitory levels of aminoglycoside antibiotics could protect the infected epithelium from damage by inhibiting the release of toxic substances from the invading bacteria. In contrast, bacteria exposed to beta-lactam antibiotics may continue to release extracellular toxins which can damage the tissue.

Several groups of streptococci were tested for production of pyrogenic exotoxins (SPE) with Ouchterlony immunodiffusion, a newly developed passive hemagglutination inhibition assay, and an assay for pyrogenicity and capacity to enhance lethal endotoxin shock. With use of these assays, 68 (91%) of 75 group A streptococcal strains were positive for one or more of SPE types A, B, and C; seven were negative for both the known SPE types and antigenically unrelated pyrogenic exotoxins. Group A strains producing both SPE B and C were the most common, and strains producing A alone or AB and AC together were the least common. All of 11 rheumatogenic group A streptococci elaborated SPE C either alone or together with one or both of SPE types A and B. The 10 nephritogenic strains tested were positive for SPE B; five were positive for B alone. In contrast to group A streptococci, non-group A strains (41 tested) did not produce the known SPE types, and 19 of 19 tested were negative for antigenically unrelated pyrogenic exotoxins. Group A strains from Holland, India, and Japan also elaborated SPE. Several group A streptococci used widely in laboratory experiments were tested for SPE types produced.