Patricia Colque-Navarro

Identification and Characterization of Pathogenic Aeromonas veronii Biovar Sobria Associated with Epizootic Ulcerative Syndrome in Fish in Bangladesh

ABSTRACT Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans. In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate [PhP] typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors. The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting. Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates. The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines. All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes. Our results suggest that a clonal group of A. veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.

Antibody Responses in Patients with Staphylococcal Septicemia against Two Staphylococcus aureus Fibrinogen Binding Proteins: Clumping Factor and an Extracellular Fibrinogen Binding Protein

ABSTRACT We analyzed the serum antibody responses against twoStaphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureussepticemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens, and cutoff levels (upper 95th percentile) against these antigens were determined. No correlation was seen between serum antibody levels against Clf and Efb. In acute-phase samples 27% of patients showed positive antibody levels against Clf and 10% showed positive levels against Efb, while in convalescent-phase samples 63% (26 of 41) showed a positive serology against Clf and 49% (20 of 41) showed a positive serology against Efb. Antibody levels against Efb were significantly lower in the acute-phase sera than in sera from healthy individuals (P = 0.002). An antibody response against Clf was most frequent in patients suffering from osteitis plus septic arthritis and from endocarditis (80% positive). The antibody response against Efb appeared to develop later in the course of disease. A possible biological effect of measured antibodies was demonstrated with the help of an inhibition ELISA, in which both high-titer and low-titer sera inhibited the binding of bacteria to fibrinogen. In conclusion, we have demonstrated in vivo production ofS. aureus fibrinogen binding proteins during deep S. aureus infections and a possible diagnostic and prophylactic role of the corresponding serum antibodies in such infections.

Phenotypic and genotypic characterisation of blood isolates of coagulase‐negative staphylococci in the newborn

Coagulase‐negative staphylococci (CNS) are the leading cause of late‐onset sepsis in newborns (>72 h of age). Our aim was to determine whether phenotypic and/or genotypic differences existed between blood isolates of CNS regarded as inducers of sepsis or as contaminants. Ninety‐seven bloodisolates of CNS recovered from newborns at the neonatal intensive care unit, Örebro, Sweden in 1983–1997 were analysed. Twenty‐nine of them (30%) were classified as sepsis isolates and 68 (70%) as contaminants. The most prevalent species was Staphylococcus epidermidis (n=59). Staphylococcus haemolyticus (n=16) was most often isolated from newborns with the lowest gestational age and birth weight. Biochemical typing using the Phene Plate system (PhP) and genotyping using pulsed‐field gel electrophoresis (PFGE) showed that the S. epidermidis isolates regarded as inducers of sepsis (n=16) were more homogeneous than isolates considered contaminants (n=37). One main genotypic group, representing seven (44%) isolates, was identified among the sepsis isolates. Phenotypically the S. epidermidis sepsis isolates comprised three major clusters. In contrast, among the S. epidermidis contaminants, eight genotypic groups and two phenotypic clusters were identified. The dominating genotypic group among the sepsis isolates of S. epidermidis may represent strains with higher invasive capacity.

Microplate-based microbial assay for risk assessment and (eco)toxic fingerprinting of chemicals

Abstract We have developed a multi-species microbial assay, MARA, for assessing the (eco)toxic risks of chemical compounds and for the determination of their toxic fingerprints. The main advantages with MARA are (1) the simultaneous testing on several microbial strains; (2) the concept of toxic fingerprinting; (3) the simple and inexpensive handling and reading of the test. The toxic activity is measured in parallel on 11 different micro-organisms lyophilised in a microplate. A concentration gradient of the chemical to be tested is added and growth is indicated through the reduction of tetrazolium red (TTC). The microplates are read by a common flatbed scanner or a microplate spectrophotometer. The array of the 11 different inhibition values constitute a toxic fingerprint, characteristic for each type of chemical compound, and it is shown that the assay can distinguish between 12 standard chemicals. Both the reproducibility (CV≈20%) and the sensitivity are similar to other toxicity tests based on micro-organisms.

Antibody responses in patients with invasive Staphylococcus aureus infections.

Correlation between antibody response and clinical outcome in Staphylococcus aureus bacteremia has yielded conflicting results. Immunization schedules have failed in clinical trials. Is the humoral response toward S. aureus of protective nature? A prospective study was performed in patients with invasive S. aureus (ISA) infections during the period 2003–2005. The antibody levels were determined at the beginning and at the end of treatment and one month later (n = 96, n = 71, and n = 51, respectively). As controls, 115 healthy individuals were used. A quantitative enzyme-linked immunosorbent assay (ELISA) against eight purified antigens was performed. Bacterial isolates were grouped as to the production of alpha-toxin, agr type, and pulsed-field gel electrophoresis (PFGE) type. Large variations were seen in the antibody levels. The levels in the second sample were the highest. A correlation between agr group, PFGE group, alpha-toxin production, and initial antibody levels was observed. Patients with fatal outcome displayed lower initial antibody levels to all antigens and significantly so in regard to teichoic acid, lipase, enterotoxin A, and scalded skin syndrome toxin. In episodes with complicated bacteremia, initial significantly low levels to teichoic acid and lipase were registered. Low initial antibody levels against several antigens were associated with increased mortality and complicated bacteremia in invasive S. aureus infections. Bacterial properties, strain, and toxin production affected the antibody response.

Levels of Antibody against 11 Staphylococcus aureus Antigens in a Healthy Population

ABSTRACT Serum samples from 151 healthy individuals aged from 15 to 89 years were investigated by enzyme-linked immunosorbent assay (ELISA) for IgG levels against 11 different purified antigens from Staphylococcus aureus. Surface antigens, such as teichoic acid, clumping factors A and B, and bone sialoprotein binding protein, and extracellular proteins, such as alpha-toxin, lipase, enterotoxin A, toxic shock syndrome toxin, scalded-skin syndrome toxin, fibrinogen binding protein, and extracellular adherence protein, were used. The IgG values were analyzed in relation to the state of nasal carriage at the time of sampling. There was great individual variation in antibody levels in both young and elderly healthy subjects. Occurrence of S. aureus in the nares at the time of sampling was correlated with higher antibody levels, while elderly individuals over 65 years of age showed slightly lower levels than younger adults. More individuals than was expected from random probability calculations showed high antibody levels against several antigens, and more individuals than would be expected showed low levels against several antigens. Certain extracellular proteins had more often induced IgG levels of the same magnitude in the same individuals, indicating that among these individuals, there was a tendency to respond to certain antigens in the same way. Most individuals had circulating IgG antibodies to the 11 tested antigens, and some individuals had the tendency to be “good responders” to several antigens, while others were “poor responders.” These findings constitute basic knowledge for the development of improved serological diagnostics, immune prophylaxis, individual prognosis tools, and therapy against invasive Staphylococcus aureus infections.

The Staphylococcus aureus Alpha-Toxin Perturbs the Barrier Function in Caco-2 Epithelial Cell Monolayers by Altering Junctional Integrity

ABSTRACT Increased microvascular permeability is a hallmark of sepsis and septic shock. Intestinal mucosal dysfunction may allow translocation of bacteria and their products, thereby promoting sepsis and inflammation. Although Staphylococcus aureus alpha-toxin significantly contributes to sepsis and perturbs the endothelial barrier function, little is known about possible effects of S. aureus alpha-toxin on human epithelial barrier functions. We hypothesize that S. aureus alpha-toxin in the blood can impair the intestinal epithelial barrier and thereby facilitate the translocation of luminal bacteria into the blood, which may in turn aggravate a septic condition. Here, we showed that staphylococcal alpha-toxin disrupts the barrier integrity of human intestinal epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER) and reduced cellular levels of junctional proteins, such as ZO-1, ZO-3, and E-cadherin. The Caco-2 cells also responded to alpha-toxin with an elevated cytosolic calcium ion concentration ([Ca2+]i), elicited primarily by calcium influx from the extracellular environment, as well as with a significant reduction in TER, which was modulated by intracellular calcium chelation. Moreover, a significantly larger reduction in TER and amounts of the junctional proteins, viz., ZO-3 and occludin, was achieved by basolateral than by apical application of the alpha-toxin. These experimental findings thus support the hypothesis that free staphylococcal alpha-toxin in the bloodstream may cause intestinal epithelial barrier dysfunction and further aggravate the septic condition by promoting the release of intestinal bacteria into the underlying tissues and the blood.

staphylococcal α-toxin in septicaemic patients; detection in serum, antibody response and production in isolated strains

Serum samples from 41 patients with septicaemia due to Staphylococcus aureus were analysed by a sandwich enzyme immunoassay (EIA) based on a monoclonal antibody against α-toxin. The detection limit of α-toxin was approximately 1 ng ml−1. Alpha-toxin was detected in 22% (627) of the sera taken on admission from patients whose symptoms had been present for <7 days. In all, α-toxin was found in eight (19.5%) of 41 serum samples taken on admission. In seven of these eight samples low antibody titres were also noted. Quantitation of α-toxin production from the isolated S. aureus strains was also achieved by the EIA. Alpha-toxin was demonstrated in the supernatants of all the cultured strains, at concentrations ranging from 0.25–2.4 μg ml−1 (mean 1.14 μg ml−1). The mean α-toxin concentrations from the strains of the antigen-positive and -negative patients were 1.4 and 1.07 μg ml−1, respectively. Twenty (57%) of 35 patients showed a positive antibody response to α-toxin when followed up for ≥ 14 days from the onset of illness.

Lipase versus teichoic acid and alpha-toxin as antigen in an enzyme immunoassay for serological diagnosis ofStaphylococcus aureus infections

Titres of IgG antibodies toStaphylococcus aureus lipase were analysed in 448 sera from patients suspected of havingStaphylococcus aureus infections and the results compared to those for the routinely used staphylococcal antigens teichoic acid and alpha-toxin. The results indicated that determination of serum antibodies to lipase is a sensitive assay for serological diagnosis of staphylococcal infections and increased sensitivity may be achieved by selection of optimal antigen combinations.

Antibody response in patients with osteomyelitis of the mandible

Patients with mandibular osteomyelitis had quantification of 10 antibodies against certain bacterial proteins and polysaccharides. Sera from 31 patients with acute or chronic osteomyelitis of the mandible and from 17 healthy controls were analyzed. Some patients showed low levels of investigated antibodies in general and a lack of specific antiteichoic acid antibodies, as well as of different antipneumococcal antibodies particularly. Two patients with therapy-resistant osteomyelitis showed IgG2 and IgG3 subclass deficiency. They had replacement therapy with intravenous 10 or 15 gm immunoglobulin every 3 weeks for 6 months. Both patients showed considerable improvement in their clinical symptoms after treatment with immunoglobulin. This study indicates that impaired humoral immune response may be of importance in subgroups of patients with osteomyelitis of the mandible.