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Mannheimia haemolytica is an important cause of pneumonia in bighorn sheep (BHS; Ovis canadensis). Leukotoxin (Lkt), the primary virulence determinant of M. haemolytica, induces cytolysis of all subsets of leukocytes. Previously, we have shown that CD18, the beta subunit of beta2-integrins, mediates Lkt-induced cytolysis. However, it is not clear whether CD18 of all three beta2-integrins, LFA-1, Mac-1, and CR4, mediates Lkt-induced cytolysis. The objective of this study was to determine whether BHS LFA-1 (CD11a/CD18) serves as a receptor for Lkt. Plasmids encoding cDNA for BHS CD11a and CD18 were cotransfected into Lkt-resistant HEK-293 cells. Flow cytometric analysis of transfectants confirmed cell surface expression of BHS LFA- 1, Lkt-LFA-1 binding and Lkt-induced intra-cellular calcium elevation. More importantly, the transfectants were efficiently lysed by Lkt in a concentration-dependent manner. Collectively, these results indicate that BHS LFA-1 serves as a functional receptor for M. haemolytica Lkt.

This prospective study assessed the epidemiology of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) among patients with purulent skin and soft tissue infections (SSTIs) in Hong Kong. Among 298 patients with SSTIs, 10.4% (13/125) of all S. aureus isolates and 5% (12/241) of all abscesses were attributed to pvl-positive CA-MRSA. Overall, 77% and 69.9% of CA-MRSA and methicillin-sensitive S. aureus (MSSA) were susceptible to erythromycin, 77% and 74.8% to clindamycin, 100% and 97.1% to minocycline, and 100% and 98.1% to rifampin, respectively. Filipino ethnicity was the only clinical and epidemiologic factor significantly associated with CA-MRSA infection (odds ratio, 14.8; 95% confidence interval, 3.3-70.0; P < 0.001). Pulsed-field gel electrophoresis analysis showed that 6 CA-MRSA isolates belonged to the ST30-HKU100 clone, 5 belonged to the ST59-HKU200 clone, and 1 was singleton. Features of HKU100 isolates include SCCmec type IV, agr3, spa t019, and pan-susceptibility to non-beta-lactam antibiotics. In contrast, HKU200 isolates are characterized by having SCCmec type IV or V, agr4, spa t437, and variable non-beta-lactam susceptibility profiles. The major CA-MRSA spa types were shared by a minority of the MSSA.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates producing the Panton-Valentine leukocidin (PVL) have been reported worldwide. We describe the molecular characteristics of PVL-positive CA-MRSA strains isolated in Madrid, Spain, and analyze the clinical features of patients infected with these isolates. From 2004 to 2007, we collected 13 PVL-positive MRSA isolates from patients attending to the emergency department. The isolates were genotyped by pulsed-field gel electrophoresis, SCCmec typing, agr polymorphism, and multilocus sequence typing. Susceptibility to 29 antimicrobials was determined by the broth microdilution and by the E-test methods. The isolates belonged to 3 genotypes: ST8-SCCmec IVc (n = 11), ST5-SCCmec IVa (n = 1), and ST80-SCCmec IVc (n = 1). The corresponding agr types were I, II, and III, respectively. Five isolates were resistant to tetracycline and doxycycline, and 1 was resistant to fusidic acid (ST80). The isolates were from children (n = 9) and adults (n = 4), and were associated with skin and soft tissue infections (n = 9), otitis (n = 1), and bacteremia (n = 1). Nine patients were from South America. Our results indicate the transcontinental importation and recent emergence in Spain of PVL-positive CA-MRSA strains belonging to 3 distinct lineages, including 1 predominant (ST8-SCCmec IVc).

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.

Epidemiological surveillance for community-associated methicillin-resistant Staphylococcus aureus revealed prevalences of 33% and 13% in pediatric and adult patients, respectively, in Cordoba, Argentina, in 2005. This study describes for the first time the emergence and dissemination of the sequence type 5 (ST5) lineage as the most prevalent clone (89%) (pulsed-field gel electrophoresis type I-ST5-staphylococcal cassette chromosome type IVa-spa type 311) harboring the Panton-Valentine leukocidin and enterotoxin A genes.

A series of epidemics of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have occurred in Australia, starting in Western Australia in the early 1990s, in the Northern Territory soon thereafter and in eastern states in the mid 1990s. The Western Australian epidemic has been due mainly to Panton-Valentine leukocidin (PVL)-negative clones, whilst PVL-positive clones have predominated in the east. More recently, the major epidemic clones have spread throughout the country, whilst multiple new minor clones have emerged, mainly in Western Australia. A total of 45 clones of CA-MRSA have been detected in Australia to date: 30 of these carry SCCmec IV, 6 carry SCCmec V and 9 carry novel SCCmec types. Overall, CA-MRSA clones have been associated predominantly with skin and soft-tissue infections. PVL-positive clones have been associated with furunculosis, necrotising pneumonia and osteomyelitis and have caused fatalities in otherwise healthy children and young adults. Initial treatment of these infections remains problematic, as it is frequently inappropriate. Of particular concern, healthcare-associated acquisition of CA-MRSA clones is now increasing, although major hospital outbreaks have not occurred yet.

A 30-year-old man presented with community-acquired pneumonia (CAP), directly following influenza. Sputum Gram stain confirmed Staphylococcus aureus pneumonia. Initial empirical antimicrobial therapy did not cover S. aureus. The isolated S. aureus strain contained genes encoding exotoxins, such as Panton-Valentine leukocidin (PVL). This exotoxin is associated with high mortality and methicillin resistance, but in this patient the strain was susceptible to methicillin. The patient died. In the Netherlands the risk of methicillin resistance in PVL-positive S. aureus CAP is low but real. This should be taken into account when selecting empirical treatment, which can include the combination of flucloxacillin and rifampicin. This case report illustrates the difficulty in predicting the causative agent in CAP and highlights the usefulness of the sputum Gram stain. Moreover, clinical awareness and recognition of S. aureus CAP remains essential to the early initiation of directed therapy.

The capacity of Staphylococcus aureus strain LUG855 to release Panton-Valentine leukocidin (PVL) in the presence of sub-inhibitory concentrations of anti-staphylococcal drugs was examined. Oxacillin enhanced PVL release 2.5-fold, while clindamycin, linezolid, fusidic acid and rifampicin were inhibitory, and vancomycin, pristinamycin, tetracycline, ofloxacin and co-trimoxazole had no effect. In combination with oxacillin, sub-inhibitory concentrations of clindamycin or rifampicin inhibited PVL induction significantly, linezolid was less inhibitory, and fusidic acid did not inhibit PVL induction by oxacillin. These data support the use of oxacillin in combination with clindamycin, rifampicin or linezolid for the treatment of PVL-positive S. aureus infections.

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.

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.

Background: Panton-Valentine leukocidin (PVL-)producing Staphylococcus aureus is emerging as a serious problem worldwide. There has been an increase in the incidence of necrotizing lung infections in otherwise healthy young people with a very high mortality associated with these strains. Sporadic severe infectious complications after incision of Bartholin's abcesses have been described but involvement of S. aureus is rare., Case Report: We present a 23-year-old apparently healthy female patient without any typical predisposing findings who developed severe sepsis with necrotizing pneumonia and multiple abscesses following incision of a Bartholin's abscess. Methicillin-sensitive S. aureus harbouring Panton-Valentine leucocidin genes were cultured from the abscess fluid, multiple blood cultures and a postoperative wound swab. Aggressive antibiotic therapy with flucloxacillin, rifampicin and clindamycin, drainage and intensive supportive care lead finally to recovery., Conclusions: S. aureus, in particular PVL-positive strains, should be considered when a young, immunocompetent person develops a fulminant necrotizing pneumonia. Minor infections--such as Bartholin's abscess--can precede this life-threating syndrome. Bactericidal antistaphylococcal antibiotics are recommended for treatment, and surgical procedures may become necessary.

Objectives: To evaluate Staphylococcus aureus isolates from infected skin lesions for their potential to produce immune system-modulating toxins and to correlate these with white blood cell (WBC) counts associated with these lesions., Design: Specimens were obtained for bacterial culture and gram staining from 105 infected skin lesions, and the number of WBCs per low-power field (LPF) was determined. Chromosomal DNA was prepared from 84 bacterial isolates and subjected to real-time polymerase chain reaction analysis to determine the presence of genes encoding potential immunomodulating toxins. Bacterial populations were divided into 2 groups: those associated with low WBC counts (0-5 WBCs/LPF) and those with high WBC counts (> 5 WBCs/LPF). We applied chi(2) statistical analyses to compare the toxin gene profiles associated with WBC counts on initial swab for culture., Patients: Samples were obtained from patients at a single geographic location., Results: A higher than expected percentage of bacteria capable of producing the exfoliative toxins A and/or B (ETA and/or ETB) and Panton-Valentine leukocidin (PVL) was seen in all skin lesions infected with S aureus without regard to WBC count with initial cultures. Comparison of the toxins associated with the low WBC group vs the high WBC group showed that low WBC counts were associated with ETA and ETB, while high WBC counts were associated with PVL and toxic shock syndrome toxin. There were no differences in the clinical appearance of the lesions between groups., Conclusions: Staphylococcus aureus virulence factors ETA, ETB, and PVL are associated with WBC counts from infected skin lesions. The exact role they play in affecting the WBC counts remains to be determined.

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.

We report the largest documented healthcare-associated outbreak of Panton-Valentine leucocidin-positive meticillin-resistant Staphylococcus aureus (PVL(+) MRSA) in Europe. Six index patients from three long-term care facilities (LTCFs) were screened positive for PVL(+) MRSA in 2004 on admission to a community hospital in Germany. The purpose of this prospective study was to describe the prevalence of PVL(+) MRSA in the LTCFs before and after infection control interventions. Screening for MRSA with or without PVL was performed in all three LTCFs in 2004 [453 residents, 240 healthcare workers (HCWs)] and 2005 (440 residents, 192 HCWs). Swabs from anterior nares and wounds, if applicable, were collected. Colonised residents and staff were treated with mupirocin nasal ointment and topical antiseptics, and staff were provided with hygiene education. Total MRSA carrier rate of residents and HCWs in 2004 was 11.3% (PVL(+) MRSA 9.1%, PVL(-) MRSA 2.2%). There were comparable carrier rates between residents and HCWs in each LTCF. All PVL(+) MRSA isolates were of clonal origin (MLST 22) representing a novel spa sequence type t310. A decrease in total MRSA prevalence (from 11.3 to 5.5%) and PVL(+) MRSA (from 9.1 to 3.3%) was observed in 2005. The rate of PVL(-) MRSA remained unaffected. No symptomatic skin infections were noted among residents or HCWs. In this outbreak incomplete control of PVL(+) MRSA presumably resulted from difficult and delayed detection and decolonisation of carriers, incomplete compliance with control measures and lack of enforcement by public health authorities.

Osteo-articular infections represent the majority of invasive Staphylococcus aureus infections in children. It is demonstrated that, independently of the resistance or susceptibility to methicillin, the production of Panton-Valentine leukocidin (PVL) by S. aureus enhanced the severity of the disease. However, an increase of the rate of community acquired methicillin-resistant S. aureus (CA-MRSA) has been reported in osteo-articular infections. These CA-MRSA are the most often susceptible to the other antibiotics. SCCmec elements of type IV or V associated with the PVL coding genes seem to be molecular markers of CA-MRSA. The risk factors of acquisition of these strains are the young age of children and the overcrowding. It is now established that CA-MRSA are pathogens present all over the world. It thus became necessary to look after the epidemiology of these virulent strains.

Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus is emerging as a serious problem worldwide. Whilst usually causing skin and soft-tissue infections, particularly recurrent abscesses, there has in the last 10 years been an increase in the incidence of an associated devastating pneumonia affecting previously healthy young people and associated with a very high mortality. There are no evidence-based guidelines to consult for the management of PVL-associated staphylococcal pneumonia. The literature contains less than 100 cases, with widely differing antimicrobial therapies and the occasional use of other adjunctive therapies such as intravenous immunoglobulin, activated protein C and extracorporeal membrane oxygenation. This literature review focuses on the salient features of diagnosis and management, with particular attention to the choice of antimicrobials.

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.

Previously, it has been shown that the polysaccharide chitosan inhibits the growth of gram-positive bacteria. In this study, chitosan malate was evaluated in broth and thin-film cultures for its effect on the growth and exotoxin production of toxic shock syndrome (TSS)-inducing Staphylococcus aureus (five strains, three producing TSS toxin 1 and one each producing enterotoxin B or C) and group A streptococci (three strains producing streptococcal pyrogenic exotoxin A). Also, the compound was evaluated in a rabbit subcutaneous Wiffle ball model for its ability to prevent S. aureus and group A streptococcal induction of TSS. Finally, chitosan malate was evaluated for its ability to prevent TSS and necrotizing fasciitis in rabbits after subcutaneous inoculation with microbes. Chitosan malate inhibited both bacterial growth and, at sub-growth-inhibitory concentrations, the production of exotoxins, in both broth and thin-film cultures. Rabbits treated with chitosan malate in implanted Wiffle balls were protected from prior challenge with TSS-inducing S. aureus compared to animals not receiving chitosan malate (P < 0.001) and group A streptococci (P < 0.005). Chitosan malate protected rabbits from the development of streptococcal TSS with necrotizing fasciitis (P < 0.01). The data suggest that use of this growth- and toxin-inhibitory compound may be able to reduce the severity of S. aureus and group A streptococcal mucous membrane and trauma-associated skin infections.

Panton-Valentine leukocidin-producing Staphylococcus aureus is an emerging pathogen world-wide, causing necrotizing lung infections in otherwise healthy individuals. We describe 2 episodes of patient-to-patient transmission of Panton-Valentine leukocidin-producing S. aureus, resulting in acute, life-threatening pulmonary complications in patients with cystic fibrosis. Appropriate infection control measures may be warranted to prevent similar episodes.

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.

Panton-Valentine leukocidin-producing (PVL) Staphylococcus aureus is responsible for a highly lethal necrotizing pneumonia, which occurs predominantly in young immunocompetent patients. Hemoptysis and leucopenia often occur but are not always present. Detection of PVL gene on S. aureus strains responsible for pneumonia should help us to a better understanding of this disease, to improve its treatment with antibiotics capable of lower the toxin production and to prevent its diffusion to others persons by detection and elimination of a nasal S. aureus carriage.

Background: Toxic shock syndrome (TSS) is caused by Staphylococcus aureus infection. The disease entity manifests clinically as fever, hypotension, diffuse macular erythema that progresses to desquamation, and dysfunction of > 3 organ systems. Toxic shock-like syndrome (TSLS) has the same clinical manifestation as TSS but is caused by Streptococcus, usually group A, C or G. Here we report on a healthy woman who experienced group B Streptococcus (GBS)-related TSLS, possibly related to tampon use., Case: A 37-year-old woman, gravida 1, para 1, met the diagnostic criteria for TSS/TSLS 5 days after her last tampon use. Blood, urine and vaginal cultures were positive only for GBS. Analysis of the blood isolate suggested a novel GBS superantigen., Conclusion: This is the second reported case of GBS causing tampon-associated TSS/TSLS. Up to 40% of healthy menstruating women are vaginally colonized with GBS. Superantigens elaborated by staphylococci and streptococci induce an immunologic mediator storm that affects the circulatory and end-organ systems to produce the clinical picture. Prompt medical therapy involves large-volume isotonic fluid resuscitation and antibiotic coverage with vancomycin and an antistaphylococcal beta-lactam. Clindamycin may dampen the immunologic response and endotoxin production. Corticosteroids and intravenous immunoglobulin may be useful adjuncts; however, nonsteroidal antiinflammatories should be avoided.

We examined the capacity of Staphylococcus aureus strains to release Panton-Valentine leukocidin (PVL) in the presence of antibiotics. No PVL was detected when S. aureus was incubated at inhibitory concentrations, while subinhibitory concentrations of oxacillin enhanced the PVL level; clindamycin, linezolid, and fusidic acid were inhibitory; and vancomycin had roughly no effect.

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.

Pneumonia caused by Mannheimia (Pasteurella) haemolytica is a highly fatal disease of bighorn sheep (Ovis canadensis). Leukotoxin (Lkt), secreted by M. haemolytica, is an important virulence factor of this organism, and is cytolytic to bighorn sheep leukocytes. Previously, we have shown that CD18, the beta subunit of beta2 integrins, serves as the receptor for Lkt on bovine leukocytes. Furthermore, anti-CD18 antibodies inhibit Lkt-induced cytotoxicity of bighorn sheep leukocytes. Therefore, we hypothesized that Lkt utilizes CD18 as its receptor on bighorn sheep leukocytes. Confirmation of bighorn sheep CD18 as a receptor for Lkt requires the demonstration that the recombinant expression of bighorn sheep CD18 in Lkt-nonsusceptible cells renders them susceptible to Lkt. Therefore, we transfected cDNA encoding CD18 of bighorn sheep into a Lkt-nonsusceptible murine cell line. Cell surface expression of bighorn sheep CD18 on the transfectants was tested by flow cytometry with anti-CD18 antibodies. Transfectants stably expressing bighorn sheep CD18 on their surface were subjected to flow cytometric analysis for detection of Lkt binding, and cytotoxicity assays for detection of Lkt-induced cytotoxicity. Leukotoxin bound to the transfectants. More importantly, the transfectants were effectively lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. These results clearly indicate that M. haemolytica Lkt utilizes CD18 as a receptor on bighorn sheep leukocytes. Identification of CD18 as a receptor for Lkt on bighorn sheep leukocytes should enhance our understanding of the pathogenesis of pneumonia, which in turn should help in the development of control measures against this fatal disease of bighorn sheep.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.

Severe, invasive group A streptococcal infections have reemerged worldwide, and extracellular toxins, including streptococcal pyrogenic exotoxin B (SpeB), have been implicated in pathogenesis. The genetic regulation of SpeB is not fully understood, and the mechanisms involved in the processing of the protoxin to its enzymatically active form have not been definitively established. The present work demonstrated that the genes encoding SpeB (speB) and a peptidyl-prolyl isomerase (prsA) constitute an operon with transcription initiated from two promoters upstream of speB. Further, the speB-prsA operon was transcribed as a bicistronic mRNA. This finding is in contrast to the generally accepted notion that speB is transcribed only as a monocistronic gene. In addition, prsA has its own promoter, and transcription from this promoter starts in early log phase, prior to the transcription of speB. Genomic disruption of prsA decreased the production of enzymatically active SpeB but not the level of the pro-SpeB zymogen. Taken together, these results demonstrate that prsA is required for production of fully mature, enzymatically active SpeB.

Staphylococcal necrotizing pneumonia producing the Panton Valentine leukotoxin (PVL) has been described for many years. The french reference center for staphylococcal toxaemia defined it with precision in 1999. A 10-year-old child, died in 36 hours from respiratory distress and shock. Staphylococcal pneumonia was suspected then confirmed: S. Aureus producing PVL was isolated in lung, blood and articulations.

The M protein and streptococcus pyrogenic exotoxin (SPE A) are important virulence factors in group A streptococci (GAS) infections. The emm types of GAS strains isolated from patients with sepsis were determined by sequencing the 5' N-terminus of the emm gene, encoding the M protein, and clonality analysis using pulsed-field gel electrophoresis. The presence of speA and production of SPE A were also examined. There were no predominant GAS clones. The emm genotypes were variable, and the most common genotype was emm13 (17.9%). The production prevalence of SPE A was 21.4%. The low mortality rate (7.1%) of GAS sepsis might be attributable to the low incidence of virulent strains such as emm1 (10.7%) and emm3 (7.1%), as well as to low production rate of SPE A.

Actinobacillus actinomycetemcomitans is a Gram negative pathogen that is the etiologic agent of localized aggressive periodontitis (LAP), a rapidly progressing and severe disease of the oral cavity that affects predominantly adolescents. A. actinomycetemcomitans is also found in extraoral infections including infective endocarditis. As one of its many virulence determinants, A. actinomycetemcomitans produces the RTX (repeats in toxin) exotoxin, leukotoxin (LtxA). LtxA specifically kills leukocytes of humans and Old World Monkeys. All of our current knowledge of A. actinomycetemcomitans LtxA is based on the protein from strain JP2, a nonadherent laboratory isolate. Because laboratory isolates can lose virulence properties, we wished to examine LtxA from a clinical isolate, NJ4500. We show that localization patterns of LtxA do not differ between the strains. Subcellular localization studies with NJ4500 revealed that LtxA localizes to the outer membrane and that the interaction between LtxA and the surface of cells is specific. Surface localized LtxA was not removed with NaCl treatment and protease protection experiments revealed that approximately 10 kDa of LtxA is exposed. We purified secreted LtxA from NJ4500 and found that the specific activity of this toxin was greater than that of secreted LtxA from JP2. For other RTX toxins, fatty acid modification affects toxin activity, and A. actinomycetemcomitans LtxA is predicted to be modified. We show by two-dimensional gel electrophoresis that NJ4500 LtxA is more highly modified than JP2 LtxA, suggesting that the difference in activities could be due to differential modification. Studies of A. actinomycetemcomitans pathogenesis should therefore consider LtxA from clinical isolates.

Neutrophils are initially the predominant cells involved in the host defence of bacterial infections, including periodontal disease. Aggressive periodontitis is associated with Actinobacillus actinomycetemcomitans, a Gram-negative capnophilic microorganism. Infections caused by A. actinomycetemcomitans are not resolved by the host immune response despite the accumulation of neutrophils at the site of inflammation. To better understand the role of natural host defence mechanisms in A. actinomycetemcomitans infections, the interaction of phenotypically diverse strains of this pathogen with human neutrophils was assessed directly using techniques such as genetic labelling with the gene for green fluorescent protein, fluorescence-activated cell sorting and fluorescence imaging. The study included clinical isolates of A. actinomycetemcomitans represented by self-aggregating, biofilm-associated and isogenic planktonic variants. Data obtained showed that complement-mediated phagocytosis of A. actinomycetemcomitans was generally inefficient regardless of strain-specific serotype or leukotoxin production. Furthermore, the majority of ingested bacteria remained viable after exposure to neutrophils for 1 h. Interestingly, uptake of antibody-opsonized bacteria resulted in the rapid cell death of neutrophils. This was in contrast to ingestion of complement-opsonized bacteria, which did not affect neutrophil viability. The methods used in this study provided reliable and reproducible results with respect to adherence, phagocytosis and killing of A. actinomycetemcomitans when encountering human neutrophils.

We report 4 cases of community-acquired infections due to Staphylococcus aureus producing Panton-Valentin leukocidin (SA-PVL) with uncommon multivisceral localizations. These cases highlight the need to screen for PVL in patients with serious staphylococcal infections. All patients were cured. Two of them received intravenous immunoglobulins in addition to antibiotics.

The objective of the experiment is to explore the purification and production of immunotoxin. The chimeric toxin, which is composed of 40 peptides of interleukin 10 (from amino acids 18 to 57) fused to a mutant form of Pseudomonas extoxin (PE) devoid of its native cell recognition domain. Two kinds of prokaryotic expression vector containing the chimeric toxin IL-10(18-57)-PE40 were constructed respectively. After induction of IPTG for 3 hours, IL-10(18-57)-PE40 was expressed highly in cytoplasmic fraction in Rosettablue(DE3), and was directed to periplasmic space as soluble form in E. coli BL21(DE3)pLysS . Western -blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PE40. With the application of salting out of (NH4)2SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography, the purity of IL-10(18-57)-PE40 was about 96%. The cytotoxicity assay, Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10(18-57) based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.

Here we identify, using nonelectrophoretic proteomics, effects of subminimum inhibitory concentrations (subMIC) of two antibiotic preparations, chlortetracycline (CTC), and chlortetracycline-sulfamethazine (CTC + SMZ), on protein expression in the bovine respiratory pathogen Mannheimia haemolytica. The M. haemolytica genome is currently in draft form, and annotation is incomplete. Relying on the principle of gene sequence conservation across species, we used annotated genomes from closely related species to identify, confirm, and functionally annotate 495 M. haemolytica proteins. To conduct quantitative comparative proteomics, we developed a protein quantitation method based on the cross correlation function of the SEQUEST algorithm. When M. haemolytica was cultivated in the presence of 1/4 MIC of CTC and CTC + SMZ, expression of proteins involved in energy production, nucleotide metabolism, translation, and the bacterial stress response (chaperones) were affected. The most notable subMIC effect was a significant decrease in the expression of leukotoxin A, which is an important M. haemolytica virulence factor. Reduction in leukotoxin expression could be one of the molecular mechanisms responsible for the efficacy of these antibiotics against bovine respiratory disease.

Bacillus cereus is mainly known as a human food-borne opportunistic pathogen. Here, we used biological assays and HPLC to investigate the ability of B. cereus to produce insecticidal exotoxins during the stationary growth phase. None of the 575 B. cereus strains screened produced detectable levels of beta-exotoxin I, a small, heat-stable insecticidal nucleotide analogue. However, six out of a subset of 270 B. cereus strains produced several small, nonproteinaceous insecticidal exotoxins different from beta-exotoxin I. Thus, B. cereus can secrete a large array of proteinaceous and nonproteinaceous toxins acting on insects and mammals.

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals were examined by polymerase chain reaction. LukS and lukF genes were detected in all 48 avian and 72 porcine isolates of S. aureus. LukE and lukD genes, located in a putative staphylococcal pathogenicity island (Sapln3/Saplm3), were recognized in 44 (91.7%) of 48 avian isolates, but these genes were not detected in porcine isolates. In 297 bovine isolates collected from mastitic cow's milk and bulk milk from dairy farms in two regions, lukM and lukF-PV(P83) genes in addition to lukS-lukF and lukE-lukD genes were detected in 100 (62.5%) of the 160 isolates from Ishikawa and in118 (86.1%) of the 137 isolates from Hokkaido. When the lysogeny of S. aureus bovine isolates was examined by treatment with mitomycin C, clearing of the culture due to cell lysis was observed in 34 (91.9%) of 37 lukM-lukF-PV(P83) genes--positive isolates. In addition, we isolated a novel lukM-lukF-PV(P83)-carrying (designated phiLukM), and revealed that the lukM-lukF-PV(P83) genes were located very close to an amidase gene on the temperate phage genomes. These results suggest horizontal transmission of lukM-lukF-PV(P83) genes by temperate bacteriophages in S. aureus of bovine origin.

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.

Testing the supernatants of ctx(+) strains of V. cholerae eltor and V. cholerae O139 on cell subcultures confirmed the possibility of the synthesis of hemolysin by V. cholerae under the condition of growing them in tripton medium lacking FeCl3. At the same time ctx(+) strains of V. cholerae of both serogroups retained, simultaneously with hemolysin production, their capacity for the synthesis of cholera toxin.

Within certain infection sites, such as the lung of cystic fibrosis patients, Pseudomonas aeruginosa grows statically under either decreased oxygen tension or anaerobic conditions, a situation that is likely to influence the production of virulence factors. The goal of this study was to determine the effect of static growth under microaerobic (decreased oxygen) and anaerobic conditions on the expression of the P. aeruginosa exotoxin A (ETA) gene toxA and its positive regulator ptxR. Using toxA-lacZ and ptxR-lacZ fusion plasmids, the level of toxA and ptxR expression was measured throughout the growth cycle of strain PAO1, which was grown in either iron-deficient or iron-sufficient medium under four different conditions: 20%-SH (aerobic, shaking), 20%-ST (aerobic, static), 10%-ST (microaerobic, static) and 0%-ST (anaerobic, static). In iron-deficient medium, toxA expression was higher under 20%-ST and 10%-ST than under 20%-SH. However, the highest level of toxA expression occurred under 0%-ST. Analysis of ETA protein using sandwich ELISA revealed that at time points between 8 and 24 h of the growth curve, PAO1 produced higher levels of ETA under 0%-ST than under 20%-SH. In iron-sufficient medium, toxA expression was significantly repressed under all conditions. Additional analyses using PAO1 strains that carry lacZ fusions with the toxA regulatory genes regA and pvdS revealed that the expression of regA and pvdS is reduced rather than increased at 0%-ST. ptxR expression under different conditions paralleled that of toxA expression, except that it was repressed by iron under 20 %-SH only. Between 6 and 24 h of growth, and under all conditions, the level of dissolved oxygen (DO) within the PAO1 cultures was sharply reduced. These results suggest that (1) the combined effect of static growth and anaerobic conditions produce a significant increase in toxA and ptxR expression in PAO1; (2) this effect appears to be unique to toxA and ptxR, since the level of regA and pvdS expression was reduced under the same conditions; (3) neither static growth nor anaerobic conditions interfere with the repression of toxA expression by iron, although static growth deregulates ptxR expression with respect to iron; and (4) the enhanced expression of toxA and ptxR is not related to the reduced levels of DO in PAO1 cultures.

Objective: To construct the interleukin-18-PE38 fusion gene expression vector and explore the expression of the fusion gene in the chondrocyte., Methods: The recombinant eukaryotic expression vector PsecTag2B-IL-18-PE38 was constructed by inserting interleukin-18-PE38 fusion gene into eukaryotic expression vector PsecTag2B with molecular cloning technique. It was confirmed by restrictive enzymes (EcoR I) digestion assay and PCR. The vector was transfected into primary chrondrocyte by liposome protocol, and the transient expression was identified by fluorescence immunocytochemical assay., Results: Restrictive enzymes digestion analysis and PCR revealed that the interleukin-18-PE38 fusion gene was cloned into the eukaryotic expression vector PsecTag2B successfully. Immunofluorescence photograph of fluorescence immunocytochemical method confirmed that the fusion gene can be expressed in the cytomembrane and cytoplasm., Conclusion: The results confirmed that PsecTag2B-IL-18-PE38 fusion gene can be expressed in the chondrocyte, which could serve as a foundation for the study on rheumatoid arthritis therapy.

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.

Previously, we have performed T typing of Streptococcus pyogenes strains isolated from patients with streptococcal toxic shock syndrome (STSS) in Japan, and streptococcal pyrogenic exotoxin (SPE) typing for epidemiological examination. In this study, we conducted a drug sensitivity test using these strains, and investigated the results of gene analysis by pulse-field gel electrophoresis (PFGE) of S. pyogenes strains derived from patients with STSS, the patient's family, and patients other than those with STSS. To clarify the relationship between the host and bacterial factors, we investigated the association between clinical symptoms and T typing of the isolated strains/production of streptococcal pyrogenic exotoxin. There were no strains resistant to beta-lactams, and only 1 strain was resistant to multiple agents other than beta-lactams. The PFGE pattern of T1 type strains was classified into 2 ; the pattern was consistent between the strains derived from patients with STSS and those derived from the patient's family. The PFGE pattern of T3 type strains was classified into 5 (IV) ; Pattern I, which was most frequently observed, was detected in both the strains derived from patients with STSS/non-STSS. However, Patterns II and III were detected only in the strains derived from patients with non-STSS. Patterns IV and V were detected only in the strains derived from patients with STSS. When examining the association between clinical symptoms and bacterial factors, disseminated intravascular coagulation (DIC) was associated with T1-SPE B-producing strains, and pharyngitis was associated with T3-SPE A-producing strains. In the future, the relationship between the host and bacterial factors should be further investigated.

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.

IL-15 and IL-15 receptors (IL-15R) play a crucial role in the pathogenesis of adult T-cell leukemia (ATL), multiple myeloma and inflammatory autoimmune diseases. To develop a novel therapeutic agent capable of eliminating IL-15R-over-expressing abnormal cells, the gene coding for human IL-15 antagonist (IL-15M) was fused with a DNA fragment coding for the mutated form of Pseudomonas exotoxin, PEdelta293. The resulting gene fusion was cloned into pET16b under the control of T7 promoter, giving rise to the expression plasmid pET-IL15M-PEdelta293. Using Ni2+ -NTA affinity chromatography, IL15M-PEdelta293 was purified from E. coli BL21 (DE3) pLysS transformed with pET-IL15M-PEdelta293. The fusion toxin showed cytotoxicity to IL-15R-bearing myelogenous leukemia cell line K562 and K562-derived multidrug resistant cell line K562/AO2. However, IL-15R negative cell line Jurkat was insensitive to IL15M-PEdelta293. In addition, the toxic effect of IL15M-PEdelta293 on K562 was completely blocked by excessive amount of recombinant human IL-15. These results demonstrated that the selective cytotoxicity of IL15M-PEdelta293 correlated with the appropriate IL-15R expression on target cells. The present data suggest that the chimeric toxin constructed in this report may have therapeutic potential in the treatment of diseases associated with abnormal expression of IL-15/IL-15R, even in the treatment of chemotherapy refractory tumors.

High-dose clindamycin (CLDM) and benzylpenicillin (PCG) are the recommended chemotherapeutic remedies for toxic shock-like syndrome caused by group A streptococci. One reason for this is that it has been shown that CLDM suppresses the expression of some exoproteins, e.g., SpeB, SpeA, and streptolysin O (Slo). We analyzed the effects of antibiotics on the production of whole exoproteins by two-dimensional gel electrophoresis. Unexpectedly, we found that the levels of several exoproteins, Slo, NAD(+)-glycohydrolase (Nga), M protein, and Sic, were increased by CLDM treatment, although we also confirmed previous findings that the levels of various exoproteins, including SpeB, were decreased. The increases in exoprotein levels were also detected by using other protein synthesis inhibitor antibiotics: erythromycin, kanamycin, tetracycline, chloramphenicol, and linezolid. Peptidoglycan synthesis inhibitors (such as PCG, cefazolin, and imipenem), DNA replication inhibitors (such as gatifloxacin), and an RNA polymerase inhibitor (rifampin) did not have significant effects on exoprotein production. The combination of CLDM and PCG had no advantageous effects with regard to exoprotein production compared to the effect achieved with CLDM alone. We also analyzed the transcriptional levels of slo and nga by reverse transcription-PCR and found that this change was also detected at the transcriptional level. Furthermore, the phenomenon was seen not only in strains of the M1 serotype but also in strains of the other M serotypes. Our study suggests that the clinical effectiveness of CLDM might be due to the inhibition of the production of a limited number of exoproteins.

Aim: To investigate the inhibitory effect of translocating peptide/granzyme B fusion protein on cell growth., Methods: The translocating peptide gene of Pseudomonas exotoxin A (PE) was fused with active granzyme B gene by recombinant PCR to construct PE II-GrBa fusion protein gene. PE II-mGrBa with a mutation of serine to cystein at active center of GrB was used as negative control. The resulting PE II-GrBa and PE II-mGrBa genes were transiently transfected into mammalian cells via lipofectamine mediation. The effects of expression of PE II-GrBa gene on morphology and growth of transfected cells were detected by MTT colorimetry, TUNEL assay and indirect immunofluorescence staining., Results: Transient expression of PE II-GrBa resulted in cytoskeleton abnormality, cell growth inhibition, and apoptosis in some cells., Conclusion: Expression of PE II-GrBa fusion protein can inhibit cell growth.

In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.