Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer. Reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.     

Experimental Infection with Parent and L-Phase Variants of Neisseria meningitidis

Thirty-six strains of Neisseria meningitidis, including groups A, B, and C, produced L forms in vitro in the presence of an osmotic stabilizer and high concentrations of horse serum using penicillin as the transforming agent. Transformation to L growth occurred most readily among strains recently isolated from patients, and an unusually high rate of transformation was observed in 7 of the 36 strains. Revertant L strains developed diplococcal colonies on blood-agar and L colonies on sucrose-serum-penicillin-agar-always in a ratio of approximately 10 to 100 diplococcal colonies to 1 L colony. Using mucin as a host depressant, comparison was made between parent and revertant L strains of their initial pathogenicity and development of virulence by serial mouse passage. In general, revertant L strains showed the same pathogenic characteristics as the parent. Heart blood cultures from mice dying of infection with revertant L strains retained their ability to grow as L forms on penicillin media. Three stable L strains were completely avirulent for mice, although persistence of L forms could be demonstrated in peritoneal exudate for 6 days after inoculation.     

Clinical importance of infective agent persistence in Salmonella infection in children

The persistence properties of 70 S. enteriditis cultures and 12 S. typhimurium cultures isolated from the feces of children of the earliest age groups with the gastrointestinal form of Salmonellosis infection were studied. The study revealed that the low level of persistence properties occurred in most cases of mild forms of the disease. Salmonellae with a high level of anticomplement and antilysozyme activity were more often isolated in cases of moderate and severe forms of salmonellosis. The presence of correlation between the expression of the factors under study and the severity of the symptoms of intoxication in combination with the duration of the diarrhea syndrome in moderate and severe forms of Salmonella infection in children of the earliest age groups was established. The suggestion was made that the evaluation of the persistence properties of bacteria may be used for the prognosis of the course of the infectious process.     

Persistence of Staphylococcus aureus L-form during experimental lung infection in rats

The course of pulmonary infection in rats infected by intranasal inoculation with a Staphylococcus aureus stable protoplast L-form was studied. Blood and bronchoalveolar samples were taken on days 3, 7, 14 and 30 after challenge and were investigated by microbiological, electron-microscopic, cytochemical and cytometric methods. The electron microscopic data and isolation of L-form cultures from bronchoalveolar samples at all experimental times demonstrated the ability of S. aureus L-form cells to internalize, replicate and persist in the lungs of infected rats to the end of the observation period, in contrast to the S. aureus parental form. It was found that persisting L-form evoked ineffectual phagocytose by alveolar macrophages and low but long-lasting inflammatory reaction in rats. The experimental model of pulmonary infection with S. aureus L-form suggests that the cell-wall-deficient bacterial forms may be involved in the pathogenesis of chronic and latent lung infections.     

Group A streptococcal cysteine protease degrades C3 (C3b) and contributes to evasion of innate immunity

A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic DeltaspeB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the DeltaspeB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the DeltaspeB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections.     

Proteolysis and its regulation at the surface of Streptococcus pyogenes

Pathogenic bacteria often produce proteinases that are believed to be involved in virulence. Moreover, several host defence systems depend on proteolysis, demonstrating that proteolysis and its regulation play an important role during bacterial infections. Here, we discuss how proteolytical events are regulated at the surface of Streptococcus pyogenes during infection with this important human pathogen. Streptococcus pyogenes produces proteinases, and host proteinases are produced and released as a result of the infection. Streptococcus pyogenes also recruits host proteinase inhibitors to its surface, suggesting that proteolysis is tightly regulated at the bacterial surface. We propose that the initial phase of a S. pyogenes infection is characterized by inhibition of proteolysis and complement activity at the bacterial surface. This is achieved mainly through binding of host proteinase inhibitors and complement regulatory proteins to bacterial surface proteins. In a later phase of the infection, massive proteolytic activity will release bacterial surface proteins and degrade human tissues, thus facilitating bacterial spread. These proteolytic events are regulated both temporally and spatially, and should influence virulence and the outcome of S. pyogenes infections.     

Specific binding of collagen type IV to Streptococcus pyogenes

Many strains of Streptococcus pyogenes are capable of binding type IV collagen. In the present study, all 50 S. pyogenes strains isolated from patients with acute glomerulonephritis showed high or moderate affinity for radiolabelled type IV collagen. A majority of strains of other sources, such as reference strains of various M-types and strains isolated from patients with pharyngeal infections also bound type IV collagen; however, a number of weak binders or non-binders were found among those. The collagen type IV binding component(s) on S. pyogenes were susceptible to proteinase K digestion, partially sensitive to trypsin but insensitive to pepsin treatment at pH 5.5. According to tests with three M-positive strains and their M-negative derivatives, the binding was not dependent on M-protein. The binding was saturable with time and inhibited by unlabelled type IV collagen. Partially inhibition was found with type II collagen, gelatin and fibrinogen but not with a number of other serum proteins.     

Survival and transmission of Salmonella enterica serovar typhimurium in an outdoor organic pig farming environment

It was investigated how organic rearing conditions influence the Salmonella enterica infection dynamics in pigs and whether Salmonella persists in the paddock environment. Pigs inoculated with S. enterica serovar Typhimurium were grouped with Salmonella-negative tracer pigs. Bacteriological and serological testing indicated that organic pigs were susceptible to Salmonella infections, as 26 of 46 (56%) tracer pigs turned culture positive. An intermittent and mainly low-level excretion of Salmonella (<100 cells g-1) partly explains why the bacteriological prevalence appeared lower than the seroprevalence. Salmonella persisted in the paddock environment, as Salmonella was isolated from 46% of soil and water samples (n=294). After removal of pigs, Salmonella was found in soil samples for up to 5 weeks and in shelter huts during the entire test period (7 weeks). Subsequent introduction of Salmonella-negative pigs into four naturally Salmonella-contaminated paddocks caused Salmonella infections of pigs in two paddocks. In one of these paddocks, all tracer pigs (n=10) became infected, coinciding with a previous high Salmonella infection rate and high Salmonella excretion level. Our results showed that pigs reared under organic conditions were susceptible to Salmonella infections (just like conventional pigs) and that Salmonella persisting in the paddock environment could pose an infection risk. A driving force for these infections seemed to be pigs with a high Salmonella excretion level, which caused substantial contamination of the environment. This suggests that isolation of animals as soon as a Salmonella infection is indicated by clinical symptoms of diarrhea could be a means of reducing and controlling the spread and persistence of Salmonella in outdoor organic pig production environments.     

Region specific and worldwide distribution of collagen-binding M proteins with PARF motifs among human pathogenic streptococcal isolates

Some of the variety of Streptococcus pyogenes and Streptococcus dysgalactiae ssp. equisimilis (SDSE) M proteins act as collagen-binding adhesins that facilitate acute infection. Moreover, their potential to trigger collagen autoimmunity has been implicated in the pathogenesis of acute rheumatic fever and attributed to a collagen-binding motif called PARF (peptide associated with rheumatic fever). For the first time we determine the rate of clinical isolates with collagen-binding M proteins that use a PARF motif (A/T/E)XYLXX(L/F)N in a defined geographic region, Vellore in South India. In this region both, incidence of streptococcal infections and prevalence of acute rheumatic fever are high. M proteins with PARF motif conferred collagen-binding activity to 3.9% of 153 S. pyogenes and 10.6% of 255 SDSE clinical isolates from Vellore. The PARF motif occurred in three S. pyogenes and 22 SDSE M protein types. In one of the S. pyogenes and five of the SDSE M proteins that contained the motif, collagen-binding was impaired, due to influences of other parts of the M protein molecule. The accumulated data on the collagen binding activity of certain M protein types allowed a reanalysis of published worldwide emm-typing data with the aim to estimate the rates of isolates that bind collagen via PARF. The results indicate that M proteins, which bind collagen via a PARF motif, are epidemiologically relevant in human infections, not only in Vellore. It is imperative to include the most relevant collagen-binding M types in vaccines. But when designing M protein based vaccines it should be considered that collagen binding motifs within the vaccine antigen remain potential risk factors.     

M protein, a classical bacterial virulence determinant, forms complexes with fibrinogen that induce vascular leakage

Increased vascular permeability is a key feature of inflammatory conditions. In severe infections, leakage of plasma from the vasculature induces a life-threatening hypotension. Streptococcus pyogenes, a major human bacterial pathogen, causes a toxic shock syndrome (STSS) characterized by excessive plasma leakage and multi-organ failure. Here we find that M protein, released from the streptococcal surface, forms complexes with fibrinogen, which by binding to beta2 integrins of neutrophils, activate these cells. As a result, neutrophils release heparin binding protein, an inflammatory mediator inducing vascular leakage. In mice, injection of M protein or subcutaneous infection with S. pyogenes causes severe pulmonary damage characterized by leakage of plasma and blood cells. These lesions were prevented by treatment with a beta2 integrin antagonist. In addition, M protein/fibrinogen complexes were identified in tissue biopsies from a patient with necrotizing fasciitis and STSS, further underlining the pathogenic significance of such complexes in severe streptococcal infections.     

Is plasminogen deployed as a Streptococcus pyogenes virulence factor?

Streptococcus pyogenes (group A streptococcus) causes human skin and throat infections as well as highly invasive diseases including necrotizing fasciitis. Group A streptococcal infections and invasive disease have made a resurgence in developed countries during the past two decades. S. pyogenes use multiple pathways for the acquisition and activation of human plasminogen, securing potent proteolytic activity on the bacterial cell surface. Recent experimental evidence using a humanized transgenic mouse model suggests a crucial role for human plasminogen in the dissemination of S. pyogenes in vivo.     

SIC,a secreted protein of Streptococcus pyogenes that inactivates antibacterial peptides

Some isolates of the significant human pathogen Streptococcus pyogenes, including virulent strains of the M1 serotype, secrete protein SIC. This molecule, secreted in large quantities, interferes with complement function. As a result of natural selection, SIC shows a high degree of variation. Here we provide a plausible explanation for this variation and the fact that strains of the M1 serotype are the most frequent cause of severe invasive S. pyogenes infections. Thus, protein SIC was found to inactivate human neutrophil alpha-defensin and LL-37, two major antibacterial peptides involved in bacterial clearance. This inactivation protected S. pyogenes against the antibacterial effect of the peptides. Moreover, SIC isolated from S. pyogenes of the M1 serotype was more powerful in this respect than SIC variants from strains of M serotypes 12 and 55, serotypes rarely connected with invasive infections.     

Role of α2-macroglobulin in phagocytosis of group A and C streptococci

Binding of alpha 2-macroglobulin (alpha 2M) to streptococci and its effects on phagocytosis were investigated. Two types of streptococcal binding sites for alpha 2M were observed: Streptococcus pyogenes from human infections interacted only with native alpha 2M whereas S. dysgalactiae from bovine and S. equi from equine infections bound only a complex of alpha 2M with trypsin (alpha 2M-T). Preincubation of S. pyogenes with native alpha 2M substantially enhanced their phagocytosis by human polymorphonuclear neutrophils (PMN) whereas preincubation with alpha 2M-T was without any effect. On the other hand, incubation of S. dysgalactiae and S. equi with alpha 2M-T markedly reduced their phagocytosis by PMN from the respective host species. Native alpha 2M did not affect the phagocytosis of these streptococci. Digestion of the streptococcal binding sites for alpha 2M and alpha 2M-T pronase abolished the enhancement of phagocytosis of S. pyogenes by native alpha 2M as well as the inhibition of phagocytosis of S. dysgalactiae and S. equi by alpha 2M-T. Thus, binding of alpha 2M or its complexes appeared to play a role in streptococcal pathogenicity.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

Abstract A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae, S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae. S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

趾间型足癣患者细菌菌种分布特征

目的了解趾间型足癣患者趾间和健康志愿者趾间细菌菌种分布特征。方法对37例经临床和真菌镜检确诊的趾间型足癣患者的趾间进行细菌培养,然后给予1%联苯苄唑霜1次/d外用治疗4周,在治疗后的第1、2、4周分别对趾间再进行细菌培养。33例健康志愿者作为对照。结果健康志愿者趾间细菌培养葡萄球菌属占92.5%,其中表皮葡萄球菌占32.5%,未分离到金黄色葡萄球菌。趾间型足癣患者细菌培养葡萄球菌属占74.4%,其中金黄色葡萄球菌占27.9%,表皮葡萄球菌仅占2.3%,还分离到化脓性链球菌、粪肠球菌、屎肠球菌、奇异变形杆菌、铜绿假单胞菌等。经过4周的外用联苯苄唑霜治疗,金黄色葡萄球菌消失,表皮葡萄球菌又成为优势菌（37.5%）。结论趾间型足癣患者趾间金黄色葡萄球菌的分离率明显高,此乃足癣继发细菌感染的主要致病菌,因此积极治疗足癣对预防足癣合并症有非常重要的意义。