Streptococcal necrotizing fasciitis: development of an animal model to study its pathogenesis

Necrotizing fasciitis is a serious and increasingly common human disease which can be caused by an infection with beta-haemolytic streptococci (BHS) of Lancefield groups A, C or G, spreading rapidly in the loose connective tissue over the muscle fascia. To facilitate study of its pathogenesis, we have developed an animal model for the production of a spreading infection with BHS in the loose connective tissue over the muscle layer in the skin of New Zealand White rabbits. Intradermal injection of group A BHS alone into the flank was unsatisfactory in that a spreading lesion occurred on only 12% of occasions. When the group A BHS were co-injected with cultures of Staphylococcus aureus, the results depended on the strain of S. aureus used: an abscess-producing strain isolated from pigs gave rise to a spreading lesion on 50% of occasions. When BHS were injected with the alpha-lysin of S. aureus at a titre which produced inflammation without necrosis, spreading lesions occurred on 75% of occasions. However, both inoculated and uninoculated broth acted synergistically with the alpha-lysin in potentiating the spread of the streptococci. This demonstration of synergy between BHS and alpha-lysin of S. aureus may reflect the clinical situation in the human, as both organisms have been found to occur together at sites where spreading streptococcal infections have originated.     

Effect of phenethyl alcohol on Staphylococcus aureus alpha-lysin production.

Phenethyl alcohol, at the maximum concentration which did not inhibit growth (0.3% [vol/vol]), inhibited the production of alpha-lysin and exoproteases but not that of delta-lysin in Staphylococcus aureus Wood 46. The inhibition of alpha-lysin was reversible, and transient accumulation of cell-associated alpha-lysin occurred in the presence of PEA. A precursor of alpha-lysin ca. 3,000 daltons larger than extracellular alpha-lysin was immunologically detected in the sodium dodecyl sulfate extracts of membranes and whole cells of phenethyl alcohol-treated S. aureus cultures. Also, a degraded form of alpha-lysin was detected in membranes prepared from cells lysed by lysostaphin but not in membranes from cells lysed with an X-press.     

Bactericidal Effect of Oleic Acid on Group A Streptococci: Mechanism of Action

In contrast to Staphylococcus aureus and coagulase-negative staphylococci, group A streptococci are infrequently present on normal human skin, except in certain populations with endemic impetigo. This has been attributed to differences in susceptibility to the bactericidal effect of skin surface lipids, particularly unsaturated fatty acids. When an M type 6 strain group A streptococcus was exposed to 500 mug of oleic acid per ml, viable counts decreased by 4 logs in 5 min. The rank order of killing was 35 > 20 > 4 degrees C. Oleic acid did not kill a strain of S. aureus, a strain of coagulase-negative staphylococcus, or a strain of Escherichia coli, but bound rapidly to these bacteria as well as to the group A streptococcus. The loss of [(3)H]uridine from labeled oleic acid-treated group A streptococcal cells was greater than 100 times that of controls. There was no loss of [(3)H]-thymidine from group A streptococci or of [(3)H]uridine or [(3)H]thymidine from identically exposed coagulase-negative staphylococci. When [(3)H]uridine was added to group A streptococci during mid-log-phase growth, cessation of uptake occurred within 5 min of addition of 50 mug of oleic acid per ml. Electron microscopic changes seen within 5 min included condensation of the nucleoid and distortion of the streptococcal surface by numerous clumps and blebs. Coagulase-negative staphylococci, S. aureus, and E. coli similarly exposed showed no comparable electron microscopic changes. We propose that oleic acid kills group A streptococci by altering the integrity of the cell membrane with resulting loss of ribonucleic acid but not deoxyribonucleic acid.     

Connective tissue responses to some heavy metals. I. Sodium loaded ion exchange beads as a control: histology and ultrastructure.

Ion exchange resin beads (Amberlite IR-120) were implanted into loose connective tissue as the vehicle for cations in both control and experimental studies. Beads were loaded with sodium for control purposes or with the metal of interest for subsequent experimental studies. When single control beads were implanted in the loose connective tissue of the rat pinna, they were well accepted by tissue and permitted rapid healing. The bead implantation initially produced minimal inflammatory disruption and subsequent repair at the bead-tissue interface which rapidly matured leaving no fibro-collagenous capsule. Some minor geometric rearrangement occurred in the alignment of the connective tissue to compensate for the physical presence of the bead. This system provides a valid control for studies of the effects of metal cations on tissue inflammation and repair--free from anionic complications, surface phenomena or systemic interference.     

Connective tissue responses to some heavy metals. I. Sodium loaded ion exchange beads as a control: histology and ultrastructure

Ion exchange resin beads (Amberlite IR-120) were implanted into loose connective tissue as the vehicle for cations in both control and experimental studies. Beads were loaded with sodium for control purposes or with the metal of interest for subsequent experimental studies. When single control beads were implanted in the loose connective tissue of the rat pinna, they were well accepted by tissue and permitted rapid healing. The bead implantation initially produced minimal inflammatory disruption and subsequent repair at the bead-tissue interface which rapidly matured leaving no fibro-collagenous capsule. Some minor geometric rearrangement occurred in the alignment of the connective tissue to compensate for the physical presence of the bead. This system provides a valid control for studies of the effects of metal cations on tissue inflammation and repair--free from anionic complications, surface phenomena or systemic interference.     

Tissue colonization from implantable biomaterials with low numbers of bacteria.

This study was undertaken to evaluate the risk of infection (defined as the recovery of the relevant organism from the implant site) in a mouse model when low numbers of bacteria were present on an implanted biomaterial. Segments of different types of suture with adherent bacteria were implanted subcutaneously into mice. The infection risk with Staphylococcus aureus was greater than with Staphylococcus epidermidis RP62A or Candida albicans. The infection risk with the implantation of multifilament sutures was significantly greater than with monofilament sutures. When <10 colony forming units (cfu) of S. aureus were present on monofilament suture material, the infection rate was 3%. When <10 cfu of S. aureus were present on multifilament suture material, the infection rate was 7%. An infection rate of 15% occurred with <10 cfu of S. aureus on multifilament nylon sutures. When >10 but <20 cfu of S. aureus were present, the infection rates were 4 and 51%, respectively. These data confirm that the infection rate with multifilament sutures (or porous materials) is greater than with monofilament sutures (or solid materials) when the organisms are encountered at implantation (acute model) and indicate that a significant risk of infection may occur when only a few organisms are on a device at implantation.     

Prevalence and molecular diversity of invasive <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> in a German tertiary care medical centre

Prevalence of invasive ß-haemolytic streptococci (BHS) at a tertiary care hospital and molecular diversity of S. pyogenes and S. dysgalactiae was studied. Between 2012 and 2016, all blood culture sets (n?=?55,839), CSF (n?=?8413) and soft tissue (n = 20,926) samples were analysed for BHS positivity using HYBASE software. Molecular profiles of 99 S. pyogenes and S. dysgalactiae were identified by sequencing of M protein genes (emm types) and multiplex PCR typing of 20 other virulence determinants. Streptococci contributed to 6.2% of blood, 10.7% of CSF and 14.5% of soft tissue isolates, being among the most common invasive isolates. The overall rates of invasive S. pyogenes, S. agalactiae, S. dysgalactiae and S. pneumoniae were 2.4, 4.4, 2.1, and 5.3%. Whereas S. pneumoniae was 1.5% more common in CSF samples, BHS isolates were 2-fold and 11-fold higher in bacteraemia and invasive soft tissue infections. Genetic BHS typing revealed wide molecular diversity of invasive and noninvasive group A and group G BHS, whereas one emm-type (stG62647.0) and no other virulence determinants except scpA were detected in invasive group C BHS. BHS were important invasive pathogens, outpacing S. pneumoniae in bacteraemia and invasive soft tissue infections. The incidence of S. dysgalactiae infections was comparable to that of S. pyogenes even with less diversity of molecular virulence. The results of this study emphasise the need for awareness of BHS invasiveness in humans and the need to develop BHS prevention strategies.     

Bacteraemia caused by beta-haemolytic streptococci in North Queensland: changing trends over a 14-year period

Group A streptococci (GAS) are usually the predominant species in cases of bacteraemia caused by β haemolytic streptococci (BHS). An increasing worldwide incidence of invasive disease from non-group A BHS has been reported. Little is known about the changing trends in invasive disease caused by BHS in Australia. North Queensland has a relatively large indigenous population, who experience significantly higher rates of group A-related disease than the non-indigenous population. This prospective study examined changing trends of disease from large colony BHS that group with A, B, C and G antisera over a 14-year period at the single large tertiary referral hospital in the area. We identified 392 bacteraemic episodes caused by BHS. GAS were most commonly isolated (49%), with adjusted rates remaining stable over the period. There was a significant increase in the incidence of non-neonatal bacteraemia caused by group B streptococci (GBS) over the study period (r = 0.58; p 0.030), largely driven by infection in older, non-indigenous women. Rates of bacteraemia caused by group C streptococci also experienced a modest, but significant, increase over time (r = 0.67; p 0.009). GAS, which had no predominant emm type, were seen most commonly in indigenous subjects (52%). Mortality rates ranged from 3.2% (group G) to 10.3% (group C), with a rate of 7.9% associated with group A disease. The marked rise in GBS disease has been noted worldwide, but the relatively low incidence in indigenous Australian patients has not been described before, despite the burden of wellrecognized risk factors for GBS disease within this group.     

Immunohistochemical evidence of Clostridium sp, Staphylococcus aureus, and group A Streptococcus in severe soft tissue infections related to injection drug use

Severe soft tissue infections are caused by either single or multiple microorganisms. We performed a retrospective immunohistochemical (IHC) study on formalin-fixed, paraffin-embedded soft tissue samples from 20 injection drug users who were part of a cluster of severe illness and death after skin and soft tissue infections in Scotland and Ireland in 2000. The IHC assays used antibodies against Clostridium sp, Staphylococcus aureus, group A streptococci, and Bacillus anthracis. Intact bacilli and granular Clostridium antigen staining in areas with necrosis, edema, and inflammation were observed in skin, fascia, or muscle samples of 12 (60%) patients. A variety of clostridia were isolated from affected soft tissues in 10 IHC-positive cases. Staphylococcus aureus antigens were observed in 3 cases including 1 where S aureus was isolated, 1 with negative cultures, and 1 where mixed cultures were obtained. Group A streptococcal antigens were observed in 1 case in which Streptococcus pyogenes and S aureus were isolated. By using IHC, we detected different bacteria in archival soft tissue samples from patients with severe skin and soft tissue infections. Immunohistochemical assays can be of great diagnostic value, particularly for bacteria such as Clostridium sp, which are difficult to isolate because of their anaerobic fastidious growth requirements.     

Stenotic intimal thickening of the external iliac artery in competition cyclists

Twenty-three cases of an arterial disease that affects competition cyclists are reported. Patients complained of intermittent acute claudication appearing on one lower limb only at the time of a maximal strain while cycling. Doppler hemodynamic investigation on an ergometric bicycle revealed a collapse of the ankle systolic pressure. Arteriography showed a sinuous lengthening and moderate stenosis of the external iliac artery. Pathologic examination of the artery disclosed a stenotic intimal thickening due to moderately cellular loose connective tissue with a variable distribution of collagen and elastic fibers. The cells in the affected zone were readily labeled with anti-actin and anti-myosin antibodies, and electron microscopy revealed features of synthetic smooth muscle cells. The lesion observed differs from intimal fibrodysplasia and from artherosclerosis. Abnormal local hemodynamic conditions may lead to this type of lesion. Thus, stenotic intimal thickening of the external iliac artery appears to be a new arterial disease defined by clinical, arteriographic, and pathologic features.     

Production of gamma-hemolysin and lack of production of alpha-hemolysin by Staphylococcus aureus strains associated with toxic shock syndrome.

The hemolytic activity of toxic shock syndrome isolates of Staphylococcus aureus is enhanced when agarose is substituted for agar in blood plates or when strains are grown in liquid culture in the presence of 20% (vol/vol) CO2 in air. Hemolytic activity of a representative panel of toxic shock syndrome isolates was rigorously assessed both on blood agar and in liquid culture to unequivocally identify the predominant hemolysins produced. As determined by isoelectric focusing and Western immunoblotting, 15 of 15 TSS isolates produced gamma-lysin and 10 of 15 produced delta-lysin. None produced beta-lysin, and only 2 of 15 produced alpha-lysin. The low rate of alpha-lysin production was a most striking characteristic, since all strains were found to have the alpha-lysin gene by Southern blot hybridization.     

Antiserum agar method for identification of Smith type exopolysaccharides in clinical isolates of Staphylococcus aureus.

We used an antiserum agar method to identify clinical Staphylococcus aureus strains producing an exopolysaccharide antigenically identical to the S. aureus Smith diffuse strain. S. aureus blood isolates were obtained from 137 patients, and three additional isolates were obtained from bone debridement. The 140 patients were clinically divided into the following groups: endocarditis (7 patients); pneumonia, empyema, or both (33 patients); intravascular device (34 patients); superficial or wound infection or both (35 patients); deep tissue infections (18 patients); and 6, unknown bacteremias (13 patients). Ninety (64.3%) of the total 140 S. aureus isolates were found to produce precipitin halos on the antiserum agar. The percentage was greatest in the isolates from the endocarditis group (100%) and least in deep tissue infections (55.5%). The presence of clinical S. aureus strains producing exopolysaccharides antigenically identical to the Smith diffuse strain exopolysaccharide appears to be a common phenomenon.     

Secondary infections with beta-hemolytic streptococci in skin lesions

Secondary infections (SI) in skin lesions are common. In the present study 40 beta-hemolytic streptococci were isolated from 36 patients suffering from SI due to various skin diseases. Staphylococcus aureus coexisted with beta-hemolytic streptococci in 29 of these cases (81%), and beta-hemolytic streptococci were often associated with coagulase-negative staphylococci and gram-positive rods. Eighteen patients (50%) carried beta-hemolytic streptococci predominantly. In most cases of SI due to atopic dermatitis (AD), the predominant species was S. aureus, while in other skin diseases, S. aureus and beta-haemolytica streptococci were predominant in approximately 50% of the patients, except for SI due to tumors and viral diseases. The mean age of patients with SI and beta-hemolytic streptococci was 37 years and that of patients with SI and predominant S. aureus was 32 years. The lower mean age found for S. aureus was due to SI found in patients with AD. This study emphasizes the polymicrobial microbiology of SI.     

Nationwide laboratory-based surveillance of invasive beta-haemolytic streptococci in Denmark from 2005 to 2011

The aim of this work was to describe national surveillance of invasive beta-haemolytic streptococci (BHS) in Denmark and to report overall trends and major findings by groups and types of BHS causing laboratory-confirmed disease from 2005 to 2011. A total of 3063 BHS isolates were received from 2872 patients. Based on confirmed cases the overall annual incidence increased from 6.2 to 8.9 per 100 000 persons between 2005 and 2011. In 2011 the incidences of group A, B, C and G streptococci were 3.1, 2.3, 0.9 and 2.6 per 100 000 persons, respectively. An increase was observed for all groups of BHS, but in particular for group G in men above 65 years of age. Among group A streptococci (GAS), five T-types (1, 28,12, 3,13,B3264 and B3264) represented 71% and five emm-types (1, 28, 3, 89 and 12) 76% of all isolates. Among group B streptococci (GBS) four types (III, Ia, V, Ib) represented 79% of the isolates. Potential coverage for future vaccines against GAS and GBS disease was 76% compared with the 26-valent GAS vaccine and 89% based on GBS serotypes Ia, Ib, II, III and V. The number of reported cases of invasive BHS disease increased in Denmark from 2005 to 2011. Nationwide laboratory-based surveillance of BHS is required to monitor epidemiological changes, explore potential outbreaks and determine potential vaccine coverage.     

Macrolide resistance in beta-hemolytic streptococci: changes after the implementation of the separation of prescribing and dispensing of medications policy in Korea

This study evaluated the antimicrobial susceptibilities and macrolide resistance mechanisms of beta-hemolytic streptococci (BHS), and an additional objective was to assess the effects of 'the separation of prescribing and dispensing (SPD) of medications' on bacterial resistance rate and distribution of phenotypes and genotypes of erythromycin-resistant BHS by comparing the antimicrobial susceptibility data before (1990- 2000) and after the implementation of SPD at one tertiary care hospital in South Korea. Between the period of January 2001 and December 2002, the minimal inhibitory concentrations of six antimicrobials were determined for 249 clinical isolates of BHS. Resistance mechanisms of erythromycin-resistant (intermediate and resistant) isolates were studied by using the double disk test and PCR. Overall, the resistance rates to tetracycline, erythromycin, and clindamycin were 75.5%, 32.9%, and 32.5%, respectively. Sixty-seven (81.7%) of 82 erythromycin- resistant isolates expressed constitutive resistance to macrolide- lincosamide-streptogramin B antibiotics (a constitutive MLSB phenotype); 11 isolates (13.4%) expressed an M phenotype; and four isolates (4.9%) had an inducible MLSB resistance phenotype. erm(A) was found in isolates with constitutive/ inducible MLSB phenotypes, erm(B) with the constitutive/ inducible MLSB phenotype, and mef(A) with the M phenotype. We found that resistance rates to erythromycin and clindamycin among S. agalactiae, S. pyogenes, and group C streptococci isolates were still high after the implementation of the SPD policy in Korea, and that the constitutive MLSB resistance phenotype was dominant among erythromycin- resistant BHS in this Korean hospital.     

Bacteremia due to beta haemolytic streptococci

The Beta haemloytic streptococci (BHS) are well recognised human pathogens causing a variety of infections, including septicemia. It is important to ensure their isolation from clinical specimens by using optimum media. Moreover, since the different groups have different pathogenic potential, it is equally important to routinely serogroup them; this is emphasized here. Since, BHS are uniformly will greatly decrease morbidity and mortality due to BHS infection.     

Protection of Chicken Embryos by Viridans Streptococci Against the Lethal Effect of Staphylococcus aureus

Chicken embryos were used to investigate the mechanism by which viridans streptococci inhibit the growth of pathogenic staphylococci. Ten-day-old embryonated eggs were infected allantoically. At a concentration of 1.8 x 10(2) colony-forming units (CFU) of viridans streptococci, the percentage of fatalities was less than 10%. There was 80% fatality with 8 x 10(1) CFU of Staphylococcus aureus strain 502A and 100% when a 100-fold increase in concentration was used. An inoculum size of 10(2) to 10(3) CFU of viridans streptococci was chosen to protect the embryos against the lethal effect of strain 502A when challenged 24 h later. The survival after challenging at 4 days was 93% in protected eggs and 37% in unprotected eggs. Chicken embryos receiving heat-killed viridans and challenged with strain 502A when examined after 4 days did not demonstrate a protective effect. This protection of embryonated eggs could not be transferred by administration of sterile filtrate of allantoic fluid in which protecting strain was grown. The experimental infection of embryonated eggs has demonstrated that prior allantoic infection with viridans streptococci affords significant protection against subsequent challenge with virulent staphylococci.     

Murine model of cutaneous infection with gram-positive cocci.

Staphylococcus aureus has remained an important cause of nosocomial wound infections, but standardized or reproducible systems for analyzing cutaneous infections caused by S. aureus do not exist. A variety of foreign materials, variable inocula, and skin traumas have been used to promote infection. To minimize these variables and ensure reproducibility, we chose a model using subcutaneous injections of a fixed quantity of dextran microbeads (Cytodex) as the foreign material added to standardized broth suspensions of S. aureus. Suspensions (0.2 ml) injected into an outbred strain of immunocompetent hairless mice generated reproducible, measurable lesions. With S. aureus Smith Diffuse, fluctuant, erythematous lesions with a peak diameter of 15 mm were observed; these lesions yielded purulent material containing gram-positive cocci and neutrophils and yielded growth of S. aureus on culture. Lesion size was proportional to the bacterial inoculum size. Histologic examination of excised lesions revealed typical abscesses. A second strain of S. aureus (SLC3) produced dermonecrosis instead of abscesses at an inoculum size of 10(7) CFU. Control injections with a sterile Cytodex suspension regularly produced nondraining, nonerythematous nodules with maximum diameters of less than or equal to 5 mm. Streptococcus pyogenes produced late-onset necrotic lesions and abscesses. Using a foreign substance, this model generates easily observed and reproducible cutaneous infection with S. aureus and streptococci that can potentially discriminate between inter- and intrastrain differences. Such a model could be used to test the pathogenicity of isogeneic strains of these bacterial species and to evaluate the efficacy of antimicrobial agents.     

Virulence and immunity of Staphylococcus aureus BB and certain deficient mutants.

Coagulase-negative and deoxyribonuclease-negative mutants were isolated from Staphylococcus aureus BB by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Comparison of virulence (50% lethal dose) to mice of these six mutant strains and S. aureus BB was determined by both intravenous and intraperitoneal routes. The ratios of the 50% lethal dose of coagulase-negative mutants to that of the parental strain S. aureus BB ranged from 201 to 403 for intravenous infection and 30.7 to 52.7 for intraperitoneal infection. The virulence of deoxyribonuclease-negative mutants was essentially the same as that of S. aureus BB. When mice were immunized subcutaneously with live S. aureus BB or its deoxyribonuclease-negative mutants, the resulting protection against the intravenous challenge of S. aureus BB was remarkable. The ratios of the 50% lethal dose for the mice that were immunized by these strains to that for the untreated mice extended from 40.1 to 60.6 for intravenous infection and 6.61 to 11.5 for the intraperitoneal route. However, no effect against S. aureus BB challenge was shown in the mice that were immunized with coagulase-negative mutants.     

Evaluation of Hemofast MRSA for identification of Staphylococcus aureus and detection of methicillin-resistance staphylococci, directly in blood culture broths]

The newly developed Hemofast MRSA system for Staphylococcus aureus identification and methicillin-resistant staphylococci (MRS) detection in blood culture broths was evaluated in 106 Bactec broths containing grapelike clusters of Gram-positive cocci. All 26 S. aureus positive broths were correctly identified by Hemofast MRSA within two hours, and 81% were identified within one hour. Sensitivity and specificity were both 100%. Accuracy of MRS detection was 100%, i.e., no discrepancies versus the agar diffusion method were found. When the 28 broths containing coagulase negative staphylococci (CNS) were tested using an inoculum ten fold larger than for S. aureus testing, a number of discrepancy were recorded. For 49 other broths containing CNS, accuracy was 98.5% when the test was interpreted based on the growth rate of the strain, according to the manufacturer's instructions. One broth containing S. epidermidis strain susceptible yielded a false result with Hemofast MRSA, indicating that it probably contained a contaminant. The other discrepancies occurred with specimens containing mixed populations with CNS or a Micrococcus strain. Most (85%) results were available within 4 hrs 30 min, irrespective of the S. aureus or CNS strains. Avoiding the isolation step on agar, Hemofast MRSA saves 24 to 48 hours, thus allowing earlier antistaphylococcal treatment.