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Purpose: Extracellular protein toxins contribute to the pathogenesis of Staphylococcus aureus infections. The present study compared the effects of iclaprim and trimethoprim - two folic acid synthesis inhibitors - with nafcillin and vancomycin on production of Panton-Valentine leukocidin (PVL), alpha haemolysin (AH) and toxic-shock syndrome toxin I (TSST-1) in methicillin-resistant and vancomycin-intermediate S. aureus (MRSA and VISA, respectively)., Methodology: Northern blotting and RT-PCR were used to assess gene transcription; toxin-specific bioassays were used to measure protein toxin production., Results: As shown previously, sub-inhibitory concentrations (sub-MIC) of nafcillin increased and prolonged MRSA toxin gene transcription and enhanced PVL, TSST-1 and AH production. Sub-inhibitory doses of iclaprim and trimethoprim delayed maximal AH gene (hla) transcription and suppressed AH production; both drugs delayed, but neither reduced, maximal TSST-1 production. Trimethoprim significantly increased lukF-PV expression and PVL production compared to both untreated and iclaprim-treated cultures. Higher concentrations of iclaprim and trimethoprim markedly suppressed MRSA growth, mRNA synthesis and toxin production. In VISA, iclaprim, vancomycin and nafcillin variably increased tst and hla expression, but only nafcillin increased toxin production. Despite its ability to increase hla expression, iclaprim was the most potent inhibitor of AH production., Conclusions: We conclude that, due to its ability to suppress toxin production, iclaprim should be effective against severe staphylococcal infections caused by toxin-producing MRSA and VISA strains, especially given its ability to concentrate at sites of infection such as skin and skin structures and the lung.

Purpose: Potent extracellular toxins including alpha-haemolysin, Panton-Valentine leukocidin (PVL) and toxic-shock syndrome toxin 1 (TSST-1) significantly contribute to Staphylococcus aureus pathogenesis, thus, toxin suppression is a primary focus in treatment of staphylococcal disease. S. aureus maintains complex strategies to regulate toxin expression and previous data have demonstrated that subinhibitory concentrations of beta-lactam antibiotics can adversely increase S. aureus exotoxin production. The current study evaluates the effects of subinhibitory concentrations of tedizolid, a second-generation oxazolidinone derivative, on expression of staphylococcal exotoxins in both methicillin-resistant and methicillin-sensitive S. aureus., Methodology: S. aureus exotoxin expression levels were compared at 12 and 24 h following treatment with tedizolid, linezolid, nafcillin or vehicle control., Results: Our findings show that the level of antibiotic required to alter toxin production was strain-dependent and corresponds with the quantity of toxin produced, but both tedizolid and linezolid could effectively reduce expression of alpha-haemolysin, PVL and TSST-1 toxin at subinhibitory concentrations. In contrast, nafcillin showed less attenuation and, in some S. aureus strains, led to an increase in toxin expression. Tedizolid consistently inhibited toxin production at a lower overall drug concentration than comparator agents., Conclusion: Together, our data support that tedizolid has the potential to improve outcomes of infection due to its superior ability to inhibit S. aureus growth and attenuate exotoxin production.

Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in many developed countries. Recent studies have suggested that mutations in CovRS, a two-component regulatory system in Streptococcus pyogenes, play important roles in the pathogenesis of STSS. However, in vivo evidence of the significance of CovRS in human infections has not been fully demonstrated. We investigated five S. pyogenes strains isolated simultaneously from the pharynx, sputum, knee joint, cerebrospinal fluid and blood of a single STSS patient. All were emm89-type strains, and multilocus sequence typing (MLST) analysis revealed that the strains of pharynx and blood were isogenic. The growth rates of the strains from pharynx and sputum were faster than those of the other strains. Protein profiles of the culture supernatants of strains from the pharynx and sputum were also different from those of the other strains. Sequence analyses revealed that strains from the knee joint, cerebrospinal fluid and blood contained a single nucleotide difference in the covS coding region, resulting in one amino acid change, compared with the other strains. Introduction of a plasmid containing the covS gene from the pharynx strain to the blood strain increased the production of SpeB protein. This suggests that the one amino acid alteration in CovS was relevant to pathogenesis. This report supports the idea that mutated CovS plays important roles in vivo in the dissemination of S. pyogenes from the upper respiratory tract of human to aseptic tissues such as blood and cerebrospinal fluid.

Several recent reports have suggested that community-associated meticillin-resistant Staphylococcus aureus (MRSA) clones, particularly those harbouring genes for Panton-Valentine leukocidin (PVL) or toxic shock syndrome toxin 1 (TSST-1), are increasingly responsible for infections in hospitals. Here, a retrospective study was carried out to investigate whether antimicrobial resistance patterns could be used to detect these pathogens in a French university hospital. Isolates were characterized by antimicrobial susceptibility testing, PCR profiling (PVL genes and tst), PFGE typing and multilocus sequence typing. Demographic and clinical data were collected from all patients. For PVL-positive MRSA, the typical antimicrobial resistance pattern (susceptible to fluoroquinolones, non-susceptible to fusidic acid, kanamycin resistant and susceptible to gentamicin and tobramycin) had a sensitivity of 77.8 % and a positive predictive value (PPV) of 100 %. For tst-positive MRSA, the antimicrobial resistance pattern (susceptible to fluoroquinolones and non-susceptible to fusidic acid) had a sensitivity of 100 % and a PPV of 72.4 %. These results suggest that phenotypic rules based on antimicrobial resistance patterns are potentially useful for the detection of PVL- and tst-positive MRSA isolates.

Fusobacterium necrophorum, a Gram-negative anaerobe, causes a variety of necrotic infections in humans and animals. There are two subspecies: subsp. necrophorum and subsp. funduliforme. In cattle, subsp. necrophorum is more prevalent and production of leukotoxin is a major virulence factor. The leukotoxin operon (lktBAC) consists of three genes, lktB, lktA and lktC, of which lktA is the structural toxin gene. The subspecies identity of human F. necrophorum is less certain and it is not known whether human strains possess the leukotoxin gene or leukotoxin activity. Therefore, the objective of this study was to identify the subspecies status of four human clinical strains of F. necrophorum and determine whether they have the leukotoxin gene or leukotoxin activity. Phenotypic and genotypic characteristics suggested that the four strains belonged to subsp. funduliforme, which was confirmed by sequencing the 16S rRNA gene. Analysis of the four strains by PCR revealed the presence of the leukotoxin operon. Partial DNA sequencing identified one human strain with full-length lktA, whereas the others exhibited considerable heterogeneity in size. All strains had a leukotoxin operon promoter-containing intergenic region similar to that of bovine subsp. funduliforme strains, which was confirmed by DNA sequencing and Southern blotting. Despite variations in the lktA gene, all strains secreted leukotoxin as demonstrated by Western blotting. Flow cytometry assays revealed that the leukotoxin was toxic to human white blood cells. In conclusion, the human strains examined contained a leukotoxin gene whose gene product was biologically active. The importance of leukotoxin as a virulence factor in human fusobacterial infections needs further evaluation.

A case of purpura fulminans (PF) in a child secondary to infection with meticillin-sensitive Staphylococcus aureus (MSSA) encoding the Panton-Valentine leukocidin (PVL) toxin genes is presented. Occasional cases of PF have been documented secondary to S. aureus infection in adults, but, to the authors' knowledge, not in children. Here the first UK case of MSSA-PVL leading to PF is presented.

An exotoxin lethal to cells in culture (cytolethal toxin, CLT) was identified in culture filtrates of Burkholderia pseudomallei, the causative organism of melioidosis. CLT could pass through a 10-kDa cut-off ultrafilter and its properties suggest that it is a peptide. Isolates from soil, animals and man showed differential cytolethality in vitro. The isolates were divided into low, medium and high CLT producers with soil isolates being low producers and isolates from patients with melioidosis encephalitis being high producers. CLT levels are subject to regulation, as a strain isolated from an infected goat was one of the highest producers whereas the same strain isolated from soil was a low producer. In addition to CLT, all isolates produced a protein with cell-elongating activity which was also present in culture filtrates.

Pasteurella haemolytica isolates from cattle and sheep, including representatives of different serotypes and untypable strains, were examined for leukotoxin (Lkt) production at the end of the log phase of growth in brain heart infusion broth. There were marked differences in leukotoxic activity in culture supernate samples, as measured by chemiluminescence-inhibition assays with bovine and ovine neutrophils, even between strains of the same serotype. There was also some variation in the amount and mol. wt of the Lkt protein produced by different strains, as judged by SDS-PAGE, immunoblotting and ELISA. Some strains produced normal amounts of Lkt protein which had only low leukotoxic activity. Most strains produced Lkt of 105 kDa whereas four strains produced a higher mol. wt form of c. 108 kDa, including two of the five serotype A2 strains examined. Thus, the P. haemolytica isolates showed considerable heterogeneity in terms of leukotoxin production, mol. wt and activity, even within a given serotype.

The effects of exotoxin A (EXA) from Pseudomonas aeruginosa on polymorphonuclear leucocytes (PMNLs) were studied in a mouse model and in vitro. P. aeruginosa PA103, which produced EXA, was 20 times more virulent for normal mice than was its EXA-deficient mutant, PA103-29. EXA was detected in the plasma of mice infected with P. aeruginosa PA103, and its presence correlated with increasing numbers of bacteria in the blood and internal organs. A monoclonal antibody (MAb) against EXA prevented the death of the mice if it was given simultaneously with, or 2 h before, infection with strain PA103. The number of PMNLs in murine blood decreased by 50% within 30 min of intravenous injection of EXA, but this decrease was prevented by simultaneous or prior injection of MAb to the toxin. EXA inhibited in-vitro phagocytosis and killing of P. aeruginosa by human and murine PMNLs and decreased the number of the PMNLs by between 60 and 68%. Collectively, these results not only confirm that EXA is toxic in vivo, but also suggest that this toxin accelerates the growth of virulent P. aeruginosa in mice.

Nineteen strains of Staphylococcus aureus were isolated from the throat or the tooth surfaces of 19 cases amongst 127 patients with Kawasaki syndrome (KS) during the acute phases and 11 S. aureus isolates were obtained from five of 17 diseased controls and six healthy controls. The production of exotoxins, particularly superantigenic toxic shock syndrome toxin-1 (TSST-1), coagulase serotype, pigment production, haemolytic activity and tryptophan auxotrophy of these isolates were compared. Among 10 KS S. aureus strains isolated in 1990-1991, five (50%) secreted TSST-1, a higher frequency than two (18%) of 11 control isolates. In contrast, none of the nine KS strains collected in 1984 produced TSST-1. Four of five TSST-1-secreting KS strains produced white or white to golden pigmentation, whereas the two control strains capable of TSST-1 production formed golden colonies. There were no noticeable differences between S. aureus strains from KS patients and control children in the production of staphylococcal exotoxins A-E, coagulase serotype, haemolysis of sheep erythrocytes and tryptophan auxotrophy. The pathological or aetiological role of a new TSST-1-secreting S. aureus clone in patients with KS was not confirmed.

Mice that had been treated with cyclophosphamide and ampicillin were fed with Pseudomonas aeruginosa. These procedures induced an endogenous septicaemia under conditions mimicking the pathophysiology of the disease in man. This model was used to compare the mortality rates in mice infected with P. aeruginosa isolates from various clinical sources. Mortality rates in mice given isolates from blood cultures had a broad range (0-100%), but the mean rate was significantly higher than with isolates from other infection sites. Moreover, blood isolates persisted in the intestines of mice after oral inoculation, whereas most isolates from other sources were gradually eliminated. Most P. aeruginosa isolates from blood culture produced significantly higher levels of exotoxin A and total proteases than isolates from other infection sites. Amongst the blood isolates, all but one of the lethal strains produced large quantities of exotoxin A or total proteases or both. Taken together, the results suggest that the ability of P. aeruginosa to adhere to the intestinal tract and to produce high levels of exo-enzymes may contribute to the development of fatal septicaemia.

Eighty-six clinical isolates of Aeromonas hydrophila were studied for their ability to produce four exotoxins: a haemolysin active against rabbit erythrocytes, cytotoxin and enterotoxin detectable with Vero cell cultures, and the cholera toxin-like factor detected by an enzyme-linked immunosorbent assay. At least one exotoxin was produced by 80% of enteric and 96% of non-enteric isolates. The exotoxin profiles of non-enteric isolates were more restricted than those of enteric isolates, with haemolysin and cytotoxin producers preponderant. Although haemolysin and cytotoxin were produced by isolates from all sources, the enterotoxin and cholera toxin-like factor were more common amongst enteric isolates. The production of haemolysin and cytotoxin were closely related but the association between the enterotoxin and the cholera toxin-like factor was not significant.

The development of monoclonal antibody and enzyme-linked immunosorbent assay (ELISA) techniques has made possible the detection of specific antigens at extremely low concentrations. Diagnosis of recalcitrant diseases such as melioidosis depends upon either early isolation and identification of the causative organism or the identification of a specific marker antigen, Pseudomonas pseudomallei exotoxin, in serum; the latter is better because it allows more rapid and simple diagnosis. A method of detecting exotoxin concentrations of greater than 16 ng/ml by an ELISA based on a monoclonal antitoxin is here described; it is significantly more sensitive than the mouse lethality test (lower threshold 30 micrograms/ml) currently in use and an in-vitro cytotoxicity test (lower threshold 10 micrograms/ml) that we have developed and describe here.

The virulence of a protease-producing strain of Psuedomonas aeruginosa was compared with that of mutants that had lost the ability to produce proteases and other extracellular enzymes. Lethal infections were produced by inoculating mice intraperitoneally with bacteria in mucin, or by inoculating mice intraperitoneally or intravenously with bacteria 4 days after treatment with cyclophosphamide, 200 mg per kg body weight. No significant difference in virulence between the wild-type parent strain and some of its protease-deficient mutants was found. Histopatholoical examination of different organs in the cyclophosphamide-treated and infected mice showed striking fatty infiltration and focal necrosis of liver, multiple necrotic foci in the spleen and haemorrhagic cystitis with necorsis. The cystitis was produced by cyclophosphamide alone but was aggravated by the infection. In conclusion, no correlation between the production of protease in broth culture and the ability to produce lethal septicaemia in mice was found, and extracellular proteases probably didnot contribute to the virulence of P. aeruginosa. However, the histopathological changes in the liver suggested a role for exotoxin A insystemic infections.