Hemolytic Species Research Articles

Engineering Therapeutics to Detoxify Hemoglobin, Heme, and Iron.

Hemolysis (i.e., red blood cell lysis) can increase circulatory levels of cell-free hemoglobin (Hb) and its degradation by-products, namely heme (h) and iron (Fe). Under homeostasis, minor increases in these three hemolytic by-products (Hb/h/Fe) are rapidly scavenged and cleared by natural plasma proteins. Under certain pathophysiological conditions, scavenging systems become overwhelmed, leading to the accumulation of Hb/h/Fe in the circulation. Unfortunately, these species cause various side effects such as vasoconstriction, hypertension, and oxidative organ damage. Therefore, various therapeutics strategies are in development, ranging from supplementation with depleted plasma scavenger proteins to engineered biomimetic protein constructs capable of scavenging multiple hemolytic species. In this review, we briefly describe hemolysis and the characteristics of the major plasma-derived protein scavengers of Hb/h/Fe. Finally, we present novel engineering approaches designed to address the toxicity of these hemolytic by-products.

Role of Streptococci as etiological agents of dental caries

Dental plaques are notorious and lead to dental caries responsible for dental decay. Streptococci are the leading microorganisms associated with dental plaques. These are Gram-positive, normal microbial flora, non-motile, non-spore forming, and facultative anaerobes. These include Alpha, Beta, and Gamma hemolytic species. Streptococcus sp. produces a high amount of lactic acid through the fermentation of sugars, causes lowering of the pH leading to the plaque formation around teeth, and serves as a biofilm. Microbial biofilm provides certain attachment sites for growth and colonization of other bacteria, and also causes resistance to the antimicrobial agents. These Streptococci can be transmitted to the infants through parents or caretakers' kiss. This mode of transmission is the key role for the contribution of S. mutans in dental caries. In Pakistan, a national survey was conducted in 1992 on tooth decay, which showed that two teeth per person were decayed, missing, or filled, thus becomes an alarming situation. Minimal knowledge related to tooth decay is observed in the clinical settings; however, no similar studies have been carried out in Pakistan.

Differential expression of hemolysin genes in weakly and strongly hemolytic Brachyspira hyodysenteriae strains

BackgroundSwine dysentery (SD) is a diarrheal disease in fattening pigs that is caused by the strongly hemolytic species Brachyspira (B.) hyodysenteriae, B. hampsonii and B. suanatina. As weakly hemolytic Brachyspira spp. are considered less virulent or even non-pathogenic, the hemolysin is regarded as an important factor in the pathogenesis of SD. Four hemolysin genes (tlyA, tlyB, tlyC, and hlyA) and four putative hemolysin genes (hemolysin, hemolysin activation protein, hemolysin III, and hemolysin channel protein) have been reported, but their role in strong hemolysis is not entirely clear. Our study aimed to assess the transcriptional activity of eight (putative) hemolysin genes in a strongly hemolytic (B204) and a weakly hemolytic (G423) B. hyodysenteriae strain during non-hemolytic and hemolytic growth stages.ResultsStrongly and weakly hemolytic B. hyodysenteriae strains caused hemolysis on blood agar at different growth stages, namely during log phase (B204) and stationary/death phase (G423). During the lag, early log, late log (stationary phase in G423) and death phase (time points 1–4) strains differed in their hemolysin gene transcription patterns. At time point 1, transcription of the putative hemolysin gene was higher in B204 than in G423. At time point 2, tlyA and tlyC were upregulated in B204 during hemolysis. TlyB and hlyA were upregulated in both strains at all time points, but higher transcription rates were observed in the weakly hemolytic strain G423. The transcription activity of the hemolysin channel protein gene was quite similar in both strains, whereas the hemolysin activation protein gene was upregulated in the non-hemolytic stage of B204 at time point 4. Sequence analysis revealed deletions, insertions and single nucleotide polymorphisms in the G423 hlyA promoter, although without altering the transcription activity of this gene.ConclusionOur data indicate a combined activity of TlyA and TlyC as the most probable underlying mechanism of strong hemolysis in B. hyodysenteriae. Further studies should verify if the expression of tlyA is upregulated by the putative hemolysin gene. Depending on their immunogenic potential TlyA and TlyC may serve as possible vaccine candidates, especially since vaccines for an effective control of swine dysentery are currently not available.

Distribution and transmission of aetiological agents of swine dysentery

Swine dysentery is a serious production-limiting disease of pigs, with a global distribution. The condition is characterised by variable, and often severe, mucohaemorrhagic colitis, mainly seen in grower and finisher...

How much do we know about hemolytic capability of pathogenic Candida species?

Hemolytic factor production by pathogenic Candida species is considered an important attribute in promoting survival within the mammal host through the ability to assimilate iron from the hemoglobin-heme group. Hemolytic capability has been evaluated for Candida species based on hemolysis zones on plate assay, analysis of hemolytic activity in liquid culture medium, and hemolysis from cell-free culture broth. The production of hemolytic factor is variable among Candida species, where C. parapsilosis is the less hemolytic species. In general, no intraspecies differences in beta-hemolytic activities are found among isolates belonging to C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The production of hemolytic factor by Candida species is affected by several factors such as glucose supplementation in the culture medium, blood source, presence of erythrocytes and hemoglobin, and presence of electrolytes. On the basis of existing achievements, more researches are still needed in order to extend our knowledge about the biochemical nature of hemolytic molecules produced by distinct Candida species, the mechanism of hemolysis, and the molecular basis of the hemolytic factor expression.

Haemolytic activity and reactive oxygen species production of four harmful algal bloom species

Based on haemolytic activity and reactive oxygen species (ROS) production of Chattonella marina, Chattonella antiqua, Heterocapsa circularisquama, Alexandrium tamiyavanichii and Karenia mikimotoi, the species were categorized into four types. (1) H. circularisquama: haemolytic activity was detected in both cell suspension and cell-free culture supernatant, but with greater activity in cell suspension than in the supernatant suggesting the presence of both cell surface and secreted haemolytic agents. (2) A. tamiyavanichii: equal haemolytic activities were detected in both the cell suspension and cell-free culture supernatant suggesting the presence of only secreted haemolytic agents. (3) K. mikimotoi: haemolytic activity was detected only in the cell suspension, indicating haemolytic agents occur only on the cell surface. (4) C. marina and C. antiqua: no significant haemolytic activity was detected in either cell suspension or cell-free culture supernatant, but high ROS were detected in the cell suspensions. Heterocapsa circularisquama and K. mikimotoi showed lethal effects on rotifers (Brachionus plicatilis), whereas A. tamiyavanichii, C. marina and C. antiqua had no effect. Our results suggest that H. circularisquama, K. mikimotoi and A. tamiyavanichii produce haemolytic agents with distinct characteristics, whereas C. marina and C. antiqua have an extremely potent ability to produce ROS.

Isolation and Antibiotic Susceptibilities of Beta Hemolytic Streptococcus Species from Various Body Site Infections with Cytologic Evidences in Thoroughbred and Arabian Racehorses in Turkey

Isolation and Antibiotic Susceptibilities of Beta Hemolytic Streptococcus Species from Various Body Site Infections with Cytologic Evidences in Thoroughbred and Arabian Racehorses in Turkey

Emergence of Brachyspira species and strains: reinforcing the need for surveillance.

This short review discusses the increasing complexity that has developed around the understanding of Brachyspira species that infect pigs, and their ability to cause disease. It describes the recognition of new weakly haemolytic Brachyspira species, and the growing appreciation that Brachyspira pilosicoli and some other weakly haemolytic species may be pathogenic in pigs. It discusses swine dysentery (SD) caused by the strongly haemolytic Brachyspira hyodysenteriae, particularly the cyclical nature of the disease whereby it can largely disappear as a clinical problem from a farm or region, and re-emerge years later. The review then describes the recent emergence of two newly described strongly haemolytic pathogenic species, “Brachyspira suanatina” and “Brachyspira hampsonii” both of which appear to have reservoirs in migratory waterbirds, and which may be transmitted to and between pigs. “B. suanatina” seems to be confined to Scandinavia, whereas “B. hampsonii” has been reported in North America and Europe, causes a disease indistinguishable from SD, and has required the development of new routine diagnostic tests. Besides the emergence of new species, strains of known Brachyspira species have emerged that vary in important biological properties, including antimicrobial susceptibility and virulence. Strains can be tracked locally and at the national and international levels by identifying them using multilocus sequence typing (MLST) and comparing them against sequence data for strains in the PubMLST databases. Using MLST in conjunction with data on antimicrobial susceptibility can form the basis for surveillance programs to track the movement of resistant clones. In addition some strains of B. hyodysenteriae have low virulence potential, and some of these have been found to lack the B. hyodysenteriae 36 kB plasmid or certain genes on the plasmid whose activity may be associated with colonization. Lack of the plasmid or the genes can be identified using PCR testing, and this information can be added to the MLST and resistance data to undertake detailed surveillance. Strains of low virulence are particularly important where they occur in high health status breeding herds without causing obvious disease: potentially they could be transmitted to production herds where they may colonize more effectively and cause disease under stressful commercial conditions.

Anaerobic spirochaetes and animals

Anaerobic spirochaetes colonise the large intestine of many avian and mammalian host species. The most well known pathogenic species is the strongly haemolytic Brachyspira hyodysenteriae that was first isolated from pigs with swine dysentery (SD) in the early 1970s. Classical SD is a severe mucohaemorrhagic colitis that occurs in growing pigs and is endemic in most pig-rearing areas of the world. The spirochaete acts in concert with other components of the colonic microbiota to disrupt the integrity of the colonic epithelium and induce inflammation. In recent years two new strongly haemolytic species, the proposed ‘Brachyspira suanatina' and ‘Brachyspira hampsonii', both with reservoirs in migratory water birds, have been described as new and emerging agents of SD in the northern hemisphere. Weakly haemolytic species also have been described, some of which have pathogenic potential. In particular Brachyspira pilosicoli causes a mild colitis and diarrhoea in many species, including human beings, whilst Brachyspira intermedia is a common pathogen in adult poultry. Infection with B. intermedia and/or B. pilosicoli can cause wet litter, faecal staining of eggshells and delays in reaching peak egg production. Options for control of these widespread and economically significant anaerobic infections are limited.

Description of Acinetobacter venetianus ex Di Cello et al. 1997 sp. nov.

The name 'Acinetobacter venetianus' has been used previously to designate three marine hydrocarbon-degrading Acinetobacter strains, of which strain RAG-1 (=ATCC 31012) has industrial applications for the production of the bioemulsifier emulsan. However, to date, the name of this taxon has not been validly published. In this study, five strains were examined to corroborate the delineation of this taxon by means of phenotypic characterization, DNA-DNA hybridization, selective restriction fragment amplification (AFLP), amplified rDNA restriction analysis (ARDRA), rpoB gene sequence analysis and tRNA intergenic spacer length polymorphism analysis (tDNA-PCR) and to emend the description of 'Acinetobacter venetianus' (ex Di Cello et al. 1997). AFLP analysis showed that the five strains formed a tight cluster at 56.8+/-5.0 % genomic relatedness that was separated from strains of other haemolytic species of the genus Acinetobacter and from the type and reference strains of other Acinetobacter species at <or=27 % relatedness, indicating the distinctiveness of the novel strains. The strains were haemolytic and able to grow on citrate (Simmons), l-histidine and malonate. The strains did not oxidize d-glucose or utilize dl-lactate or l-aspartate. The G+C contents of strains RAG-1 and of VE-C3 were 43.9 % and 43.6 mol%, respectively. The novel strains could be recognized by a characteristic ARDRA pattern (CfoI 1, AluI 3, MboI 2, RsaI 2, MspI 3). The consensus tDNA-PCR pattern for the five strains consisted of amplified fragments of 87.9, 100.2, 134.6 and 248.5 bp and was indistinguishable from that of strains of Acinetobacter genomic species 14BJ. The five strains represent a novel species for which the name Acinetobacter venetianus sp. nov. is proposed. The type strain is RAG-1(T) (=ATCC 31012(T)=CCUG 45561(T)=LMG 19082(T)=LUH 3904(T)=NIPH 1925(T)).

Comparative studies of the Acinetobacter genus and the species identification method based on the recA sequences

The recA gene is indispensable for a maintaining and diversification of the bacterial genetic material. Given its important role in ensuring cell viability, it is not surprising that the RecA protein is both ubiquitous and well conserved among a range of prokaryotes. Previously, we reported Acinetobacter genomic species identification method based on PCR amplification of an internal fragment of the recA gene with subsequent restriction analysis (RFLP) withHin fl and Mbo I enzymes.1,2 In present study, the PCR products containing the internal fragment of the recA gene, for 25 Acinetobacter strains belonging to all genomic species, were sequenced. Based on the nucleotide sequences the restriction maps and phylogenetic tree were prepared. The restriction maps revealed that Tsp 509I restriction enzyme is the most discriminating for RFLP. To verify the computer analysis, the amplified DNAs from all reference genomic species available (43 strains) and 34 clinical strains were digested with each of the three restriction endonucleases mentioned. The results of digestion confirmed the computer analysis. The reconstructed phylogenetic tree showed linkages between genomic species 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3, «between 1 and 3», TU13 and «close to TU13»; genomic species 4, 6, BJ13, BJ14, BJ15, BJ16 and BJ17; genomic species 7 (Acinetobacter johnsonii) and TU14; genomic species 10 and 11; genomic species 8 (Acinetobacter lwoffii), 9, 12 (Acinetobacter radioresistens) and TU15; and genomic species 5 (Acinetobacter junii). It is interesting that one branch in the phylogenetic tree contains haemolytic species—genomic species 4 (A. haemolyticus), BJ13, BJ14, BJ15, BJ16 and BJ17. The proposed genotypic method clearly revealed that the RFLP profiles obtained with Tsp 509I enzyme might be useful for species identification of Acinetobacter strains. In this context, recA/RFLP genotypic method should be seen as an ideal preliminary screening method for large numbers of isolates, with the ultimate confirmatory role reserved for DNA hybridization analysis.

Identification of porcine Serpulina strains in routine diagnostic bacteriology

Serpulina strains from pigs were identified using simple tests. The large size of S. hyodysenteriae in stainings from colonic contents and faeces was found useful for the presumptive differentiation of this major pathogenic species from the other Serpulinae. However, this morphological characteristic gets lost upon cultivation. The `ring phenomenon' aided to confirm the strong haemolysis typical for S. hyodysenteriae. The weakly haemolytic species S. innocens, S. pilosicoli, S. intermedia and S. hyodysenteriae could be differentiated with the help of the indole spot test and two or four other simple enzymatic tests. Nearly half of the S. hyodysenteriae strains isolated in Belgium were indole-negative. Such strains have only rarely been reported earlier, and were absent among the strains from other European countries examined.

Effect of wastewater stabilization ponds on antimicrobial susceptibility and haemolysin occurrence among motile Aeromonas strains

Aeromonas strains (total=953) isolated from raw wastewater, stabilization pond effluent and sediments were evaluated for their susceptibilities to 17 antibiotics and for their ability to produce haemolysins. Stabilization ponds did not seem to select highly resistant strains of aeromonads. There were no differences in the resistance patterns of isolates from raw sewage, stabilization pond effluent and sediments. All strains were found to possess multiple resistance, most commonly to ampicillin, amoxicillin and novobiocin. Almost 90% of the strains of A. hydrophila and A. caviae were resistant to cephalothin, whereas more than 80% of A. sobria isolates were found to be susceptible to this antibiotic. Resistance to trimethoprim, oxytetracycline, nalidixic acid, chloramphenicol, tetracycline, trimethoprim-sulphamethoxazol, polymyxin B, kanamycin or erythromycin among all isolates did not exceed 10%. Moreover, no strain was found to be resistant to gentamycin and only 9 of the 953 isolates exhibited resistance to cefotaxim. The percentage of haemolytic strains was significantly higher in the stabilization pond effluent than in raw sewage. This high incidence of haemolytic activity was connected with a high proportion of A. sobria whereas, in samples from the raw sewage or stabilization pond sediments a high proportion of A. caviae decreased the total amount of haemolytic aeromonads. The high incidence of haemolytic activity (α+β) was associated particularly with A. sobria (93.3%) and A. hydrophila (88.7%) whereas A. caviae was found to be the lowest haemolytic species (16.3%).

Virulence of Listeria monocytogenes, Listeria seeligeri, and Listeria innocua Assayed with In Vitro Murine Macrophagocytosis

The survival of virulent and avirulent Listeria species internalized in cells of a murine macrophage-like cell line, RAW264.7, was monitored. Mouse macrophage cells (ca. 5 × 105/ml) suspended in fresh RPMI medium 1640 containing fetal bovine serum were mixed with 5 × 107 to 5 × 108Listeria cells per ml and incubated 1 h at 37°C with CO2-enriched air. Gentamicin (10 μg/ml) was added to kill bacteria not internalized by the cells. At 2, 4, and 6 h postinfection, 10-μl amounts of the suspensions were lysed in microtiter plate wells during serial decimal dilution in water. Triplicate dilutions (10-μl each) were plated on trypticase soy agar, and colonies were counted after 48 h incubation at 35°C. About 0.1 to 1% of the added hemolytic pathogen L. monocytogenes Scott A and the avirulent nonhemolytic L. innocua were internalized at 2 h. The number of internal L. monocytogenes cells increased significantly by 6 h, but L. innocua cells showed no significant change. A strain of the hemolytic species L. seeligeri behaved like the nonhemolytic L. innocua. This distinction between the intracellular behavior of pathogenic and nonpathogenic species, if a general phenomenon, may be useful as an in vitro virulence assessment parameter.

Production and characterization of neutralizing and nonneutralizing monoclonal antibodies against listeriolysin O

Listeriolysin O (LLO) is a thiol-activated toxin secreted by the facultative intracellular pathogen Listeria monocytogenes. LLO is essential for the survival of the bacterium in the infected cell because it promotes lysis of the phagosome membrane and escape of the bacterium into the cytosol. LLO was used as an antigen for the production of nine monoclonal antibodies (MAbs) in mice. Three of these could inhibit the hemolytic activity of LLO. One of them inhibited binding of LLO to erythrocyte membranes. The two other antibodies blocked the activity of LLO at a step subsequent to membrane binding. Only two of the nine MAbs recognized three other purified SH-activated toxins, streptolysin O, alveolysin, and pneumolysin. Western blot (immunoblot) analysis of culture supernatants of Listeria ivanovii and Listeria seeligeri, two hemolytic species of the genus Listeria, revealed that two MAbs recognized ivanolysin and seeligerolysin. The latter was also recognized by two other MAbs, including one of the neutralizing antibodies. MAbs raised against a peptide, ECTG LAWEWWR, present in all thiol-activated toxins sequenced to date, recognized all toxins and were not neutralizing. Taken together, these results demonstrate the existence of regions important for hemolytic activity that are unique to hemolysins of the genus Listeria and show that regions outside the conserved peptide are important for activity of LLO.

Comparison of hemolytic activity in eight species of polychaetes

Hemolytic capacity was investigated in homogenates of eight species of polychaetes, belonging to five different orders.Nainereis laevigata, Orbinia cuvieri, Cirriformia tentaculata, Notomastus latericeus, Arenicola claparedii andPetaloproctus terricola were collected during February 1990 from the coastal lagoon of Acquatina (Puglia, Italy).Nereis sp. (from the Yellow Sea, Japan) andMarphysa sanguinea (from Venice lagoon, Italy) were both purchased from a retail shop in Lecce (Italy). Homogenates from only three species [Nainereis laevigata andO. cuvieri (family Orbinidae, order Orbinia) andP. terricola (family Maldanidae, order Capitellida)] induced hemolysis in sheep red blood cells. Important differences in hemolytic titers, Ca- or Mg-dependence and in timecourse responses were noted between the three hemolytic species. On the other hand, thermolability and proteinase-inhibitor resistance (with the exception ofO. cuvieri) were features common to all three. Electrophoretic analysis revealed that hemolysis was mediated by apparently only one protein inN. laevigata andP. terricola, with no correlation between them or with the proteins responsible for hemolysis in the oligochaeteEisenia fetida andrei (Lumbricidae). No lytic protein was localized in hemolyticO. cuvieri homogenate, which was also the only homogenate to be partially inhibited by benzamidine, but not by phenylmethylsulfonyl fluoride or soybean trypsin inhibitor. These results underline the extreme variety existing among the species constituting the polychaete group.

Sequences homologous to the listeriolysin O gene region of Listeria monocytogenes are present in virulent and avirulent haemolytic species of the genus Listeria

Various parts of the hlyA gene region of Listeria monocytogenes which encodes a major virulence factor, listeriolysin O, have been used to detect the presence of homologous sequences in other species of the genus Listeria. Under low-stringency hybridization conditions, sequences homologous to the hlyA gene and its 5' adjacent regions were detected in the haemolytic and pathogenic species L. ivanovii, and in the haemolytic but non-pathogenic species L. seeligeri. In contrast, the region located downstream from hlyA appeared specific to L. monocytogenes. None of the probes spanning the region revealed homologies betwen L. monocytogenes and the non-pathogenic and non-haemolytic members of the genus, L. innocua, L. murrayi and L. welshimeri. Among various strains of L. monocytogenes tested, the gene hlyA and its 3' adjacent region appeared well-conserved. In contrast, a restriction length polymorphism was detected in the region located upstream from hlyA with no bvious correlation with the haemolytic phenotype or the serovar of the strains tested.

Evaluation of API 20 STREP system for identifying Listeria species.

The API 20 STREP system was used to identify 146 known strains from seven species of the genus Listeria, including both pathogenic and environmental strains. The gallery was easy to use and tests, with the exception of leucine arylamidase (LAP) and starch fermentation (AMD), were simple to interpret. Identification to genus level was satisfactory but differentiation between species was poor. Using the API 20 STREP the haemolytic species L monocytogenes, seeligeri, and ivanovii could easily be differentiated from the non-haemolytic species L welshimeri, innocua, grayii and murrayi. Of the haemolytic species, L monocytogenes could not be distinguished from L seeligeri but L ivanovii could be separated from the two other haemolytic species because it fermented ribose. Non-haemolytic L welshimeri could not be differentiated from non-haemolytic L innocua, but mannitol and ribose fermenting non-haemolytic L grayi and L murrayi were easily differentiated from the other two non-haemolytic species. The API 20 STREP identified Listeria in four hours and therefore might be used for rapid identification of strains causing infection in man. It would, however, not be useful to identify environmental isolates when speciation is important.

Sequences homologous to the listeriolysin O gene region of Listeria monocytogenes are present in virulent and avirulent haemolytic species of the genus Listeria

Various parts of the hlyA gene region of Listeria monocytogenes which encodes a major virulence factor, listeriolysin O, have been used to detect the presence of homologous sequences in other species of the genus Listeria. Under low-stringency hybridization conditions, sequences homologous to the hlyA gene and its 5′ adjacent regions were detected in the haemolytic and pathogenic species L. ivanovii, and in the haemolytic but non-pathogenic species L. seeligeri. In contrast, the region located downstream from hlyA appeared specific to L. monocytogenes. None of the probes spanning the region revealed homologies between L. monocytogenes and the non-pathogenic and non-haemolytic members of the genus, L. innocua, L. murrayi and L. welshimeri. Among various strains of L. monocytogenes tested, the gene hlyA and its 3′ adjacent region appeared well-conserved. In contrast, a restriction length polymorphism was detected in the region located upstream from hlyA with no obvious correlation with the haemolytic phenotype or the serovar of the strains tested.

HEMOLYSIS, A SYNAPOMORPHIC DISCRIMINATOR OF AN EXPANDED GENUS MEDICAGO (LEGUMINOSAE)

SummaryHemolysis is a traditional test for the presence of certain saponins in plant tissues. The hemolytic capacities of Medicago and allied genera were examined. It was found that leaves of Medicago species are hemolytic, although there are considerable differences in strength of reaction among species and populations of species. The leaves of species of six related genera consistently proved not to be hemolytic. Thus hemolysis by leaves is a synapomorphic discriminator, and supports a recent, controversial expanded circumscription of Medicago. The hemolytic capacities of seeds of Medicago and its closest allies, Trigonella and Melilotus, were also examined. Only about a third of Medicago species had hemolytic seeds, and the distribution of these is more or less characteristic of certain infrageneric groups. Three interrelated species of Trigonella proved to be the only species of genera other than those of Medicago with hemolytic seeds. In Medicago, hemolysis is caused by glycosides of the saponin medicagenic acid; different but as yet unidentified hemolytic agents are present in the seeds of the three hemolytic species of Trigonella.