Detection of homology to the beta bacteriophage integration site in a wide variety of Corynebacterium spp.

In toxigenic conversion of Corynebacterium diphtheriae C7, beta bacteriophage DNA integrates into either of two chromosomal attachment sites, attB1 or attB2. These attB sites share a 96-base-pair sequence with the attP sites of beta-related phages. The distribution of attB-related sites in other species of Corynebacterium was assessed by hybridization with a DNA probe containing both attB sites of the C7 strain and a second DNA probe containing the attP site of a beta-related phage. All but one of the 15 C. diphtheriae strains tested, regardless of origin or colonial type, contained at least two BamHI fragments that hybridized strongly to both of these probes under conditions of high stringency. Strains of C. ulcerans and C. pseudotuberculosis, species in which conversion to toxinogeny has also been demonstrated, also had one or two hybridizing BamHI fragments. The functionality of these sites as integration sites was demonstrated by isolating lysogens of all three species following single infection with one or more beta-related phages. As predicted, following lysogenization one of the DNA fragments that had exhibited homology with the attB1-attB2 probe was replaced by two hybridizing fragments. Other species of Corynebacterium, including pathogens and nonpathogens from animals, plant pathogens, and soil isolates also carried at least one BamHI fragment that hybridized with the attB1-attB2 and attP probes. The data indicate that sequences homologous to the beta phage integration sites in C. diphtheriae have been conserved in members of the genus Corynebacterium.     

Diphtheria in Europe: current problems and new challenges

Diphtheria, caused by toxigenic strains of Corynebacterium diphtheriae, is an ancient disease with high incidence and mortality that has always been characterized by epidemic waves of occurrence. Whilst towards the beginning of the 1980s, many European countries were progressing towards the elimination of diphtheria, an epidemic re-emergence of diphtheria in the Russian Federation and the Newly Independent States of the former Soviet Union demonstrated a continuous threat of the disease into the 1990s. At present, the epidemic is under control and only sporadic cases are observed in Europe. However, the circulation of toxigenic strains is still observed in all parts of the world, posing a constant threat to the population with low levels of seroprotection. More recently, Corynebacterium ulcerans has been increasingly isolated as emerging zoonotic agent of diphtheria from companion animals such as cats or dogs, indicating the enduring threat of this thought-to-be controlled disease.     

Etiologic role of Corynebacterium non diphtheriae in patients with different pathology

Bacteriologic examination of 1589 patients showed that, aside from C. diphtheriae, 11% of acute upper respiratory tract infections were caused by other Corynebacterium species. Such bacteria can cause infections of various localizations (bronchitis, pyelonephritis, urethritis, colpitis, dermatitis, arthritis, etc.). C. pseudodiphtheriticum and C. xerosis were isolated from clinical specimens most frequently. Corynebacterium spp. have adhesive, hemolytic, hemagglutinating, and neuraminidase activity; some of them are highly pathogenic. The most virulent, were following species: C. diphtheriae, C. pseudotuberculosis, C. urealyticum, and C. ulcerans. Corynebacterium non diphtheriae were frequently isolated from clinical specimens in association with staphylococci and streptococci. In such cases, factors of pathogenicity and resistance to antibiotics were more pronounced. Strains isolated with association with other bacteria have lost susceptibility to tetracycline, oleandomycin, penicillin, and erythromycin. It is important to be vigilant about bacteria from Corynebacterium genus in clinical settings, and thoroughly study their biologic characteristics, especially in immunocompromised patients.     

Heavy metal effects on Proteus mirabilis Superoxide dismutase production

Abstract Levels of genomic DNA relatedness were determined using a SI nuclease procedure for reference bacteria identified as biotypes of Corynebacterium diphtheriae , biovars of Corynebacterium pseudotuberculosis , and ' Corynebacterium ulcerans '. These results showed that the three species are separate taxa at the genomospecies level whereas biotypes and biovars are closely related genomically within each species. Phylogenetic analyses of small-subunit rDNA sequences revealed that ' Corynebacterium ulcerans ' forms a tight cluster with Corynebacterium pseudotuberculosis within the robust branch that groups all Corynebacterium sequenced to date. Therefore, we propose that the species incertae sedis ' C. ulcerans ' should be conclusively recognized as a distinct species within the genus Corynebacterium with strain CCUG 2708 = NCTC 7910 as type strain. This species is characterized by urease production and fermentation of glycogen.     

DNA sequence homology between attB-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP side of γ-Cornephage

Chromosomal restriction fragments of Corynebacterium ulcerans and C. diphtheriae, containing an integration site for corynephages of the beta family, show homology on Southern blots. Homologous DNA in also found in the soil isolate C. glutamicum, although this strain is not susceptible to beta-corynephages. Three of these DNA fragments, one for each bacterial strain, and a fragment of gamma-corynephage DNA previously shown to contain the phage integration site, were cloned and sequenced. Alignment of the 3 bacterial sequences shows a very high degree of homology in a stretch of ca 120 nucleotides, whereas the rest of the sequences is generally non-homologous. Within this common bacterial portion, a segment of ca. 96 nucleotides (core sequence) is also highly homologous to the phage sequence. The first half (ca. 50 bp) of the core sequence is identical in all aligned sequences whereas the second half, which is largely occupied by a stem-and-loop structure, contains point mutations peculiar to each clone. The described sequences are likely to be involved in phage integration/excision processes.     

Construction and consequences of directed mutations affecting the hemin receptor in pathogenic Corynebacterium species

Genes encoding an ATP-binding cassette transporter system involved in hemin iron utilization from Corynebacterium ulcerans were cloned and characterized. The genes are homologous to a hemin transport system previously identified in Corynebacterium diphtheriae. Disruption of the hmuT gene, which encodes the putative hemin receptor, resulted in greatly reduced ability of C. ulcerans to use hemin or hemoglobin as an iron source. Inactivation of hmuT in C. diphtheriae by site-specific recombination had no effect on hemin utilization, which suggests that C. diphtheriae has an additional system for transporting hemin.     

Identification of monkey chromosomes by differential staining with Romanovsky--Giemza stain. III. Cercopithecus aethiops]

The distribution of the G-bands in chromosomes of bone marrow cells of Cercopithecus aethiops was studied by means of differentail staining with the Romanovsky - Gimsa dye. All the homologous chromosome pairs were identified and morphometric parameters were detected. The comparison of C. aethiops karyotypes with those of Macacca mulatta has shown that they are different in numbers and in the character of banding pattern of the most chromosomes. Both species revealed 12 pairs of chromosomes similar in their morphology and parameters.     

First detection of Corynebacterium ulcerans producing a diphtheria-like toxin in a case of human with pulmonary infection in the Rio de Janeiro metropolitan area, Brazil

The frequency and severity of human infections associated with Corynebacterium ulcerans appear to be increasing in different countries. Here, we describe the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro metropolitan area.     

Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets

The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.     

Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters

Corynebacterium diphtheriae and Corynebacterium ulcerans use haemin and haemoglobin as essential sources of iron during growth in iron-depleted medium. C. diphtheriae and C. ulcerans mutants defective in haemin iron utilization were isolated and characterized. Four clones from a C. diphtheriae genomic library complemented several of the Corynebacteria haemin utilization mutants. The complementing plasmids shared an approximately 3 kb region, and the nucleotide sequence of one of the plasmids revealed five open reading frames that appeared to be organized in a single operon. The first three genes, which we have termed hmuT, hmuU and hmuV, shared striking homology with genes that are known to be required for haemin transport in Gram-negative bacteria and are proposed to be part of an ABC (ATP-binding cassette) transport system. The hmuT gene encodes a 37 kDa lipoprotein that is associated with the cytoplasmic membrane when expressed in Escherichi coli and C. diphtheriae. HmuT binds in vitro to haemin- and haemoglobin-agarose, suggesting that it is capable of binding both haemin and haemoglobin and may function as the haemin receptor in C. diphtheriae. This study reports the first genetic characterization of a transport system that is involved in the utilization of haemin and haemoglobin as iron sources by a Gram-positive bacterium.     

龙须菜几株附生细菌对铜离子吸附性能的比较

重金属污染和富营养化是沿岸海域的主要污染形式, 造成沿海生态功能的退化。在重金属污染区域往往伴随着富营养化现象。本文对分离来自电镀废水中铜污染海域内的龙须菜体表的附生微生物对Cu2+的生物吸附进行试验, 分析和比较这些微生物对Cu2+的生物吸收性能。共分离到6株优势附生细菌：人苍白杆菌GF-S1、杀鲑气单胞菌GF-S2、莱拉微球菌GF-S3、纹带棒杆菌GF-S4、假交替单胞菌GF-S5和弗氏弧菌GF-S6。经对Cu2+的生物吸附试验发现, GF-S2效果最好, GF-S6最差, 其余间于这两者之间。对GF-S2、GF-S4和GF-S6进行了最佳吸附时间和pH筛选, 以及使用不同化学试剂对细菌Cu2+吸附的影响试验。结果显示：最佳吸附时间在40 min~60 min, pH介于4~5对GF-S2、GF-S4吸附Cu2+的效果较好; HCl处理导致GF-S2丧失吸附能力, NaOH和乙醇能提高其吸附性能; 3种试剂都能较大地提高GF-S4和GF-S6的吸附性能, 尤其对GF-S4经处理后, 对环境中的Cu2+的去除率得以大幅度提高, 去除较彻底。     

Novelty of laboratory diagnosis for diphtheria

Selected elements of simplified, bacteriological diagnosis of diphtheria were presented. The procedure of Corynebacterium strains isolation from diphtheria suspected persons and performing of toxin testing of potentially toxigenic isolates: C. diphtheriae, C. ulcerans and C. pseudotuberculosis were shortened. The role of selective tellurite media was underlined but Loeffler medium was rejected. Columbia blood agar plate was utilized for preliminary culture. Biochemical tests and toxin testing were performed from this medium. Presented diphtheria diagnosis scheme may have practical application for the laboratory work in Poland.     

Biologic data of Macaca mulatta, Macaca fascicularis, and Saimiri sciureus used for research at the Fiocruz primate center

Physiological parameters of laboratory animals used for biomedical research is crucial for following several experimental procedures. With the intent to establish baseline biologic parameters for non-human primates held in closed colonies, hematological and morphometric data of captive monkeys were determined. Data of clinically healthy rhesus macaques (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis), and squirrel monkeys (Saimiri sciureus) were collected over a period of five years. Animals were separated according to sex and divided into five age groups. Hematological data were compared with those in the literature by Student's t test. Discrepancies with significance levels of 0.1, 1 or 5% were found in the hematological studies. Growth curves showed that the sexual dimorphism of rhesus monkeys appeared at an age of four years. In earlier ages, the differences between sexes could not be distinguished (p < 0.05). Sexual dimorphism in both squirrel monkeys and cynomolgus monkeys occurred at an age of about 32 months. Data presented in this paper could be useful for comparative studies using primates under similar conditions.     

Isolation and characterization of Corynebacterium ulcerans from cephalic implants in macaques

To determine the prevalence of colonization by Corynebacterium ulcerans, we cultured samples from the cephalic implant-skin margin and pharynx of 26 rhesus macaques and one pig-tailed macaque. All but one of the samples from the cephalic implants yielded a mixed population of bacteria. C. ulcerans grew from the cephalic implants in 56% and from the pharynx in 3% of the implanted animals. We screened nine of these isolates for diphtheria toxin (DT) and phospholipase D (PLD). Polymerase chain reactions (PCR) failed to identify DT in any of the tested isolates, which also lacked DT activity in Elek tests. However, all nine isolates tested had PLD toxin activity as determined by conjoint hemolysis on sheep blood agar plates in the presence of equi factor (Rhodococcus equi). In addition, PCR assays and Southern blot hybridization confirmed the presence of pld in the isolates. The role of the PLD toxin in promoting colonization of cephalic implants by C. ulcerans is unknown. We found C. ulcerans to be a frequent contaminant of the cephalic implant-skin margin. Further studies are necessary to investigate the relative clinical importance of this organism and the efficacy of various implant maintenance protocols in preventing infection.     

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.     

限制修饰系统cglI赋予钝齿棒杆菌抗噬菌体功能活性

为解决氨基酸发酵工业中的噬菌体污染问题, 对cglI基因复合体在钝齿棒杆菌中的功能活性表达进行研究。通过PCR从谷氨酸棒杆菌基因组扩增cglI基因复合体, 构建重组质粒pJL23-cglI, 转化钝齿棒杆菌T6-13后得到重组菌株。定性和定量检测重组菌株的噬菌体抗性。实验结果表明, 携带cglI基因复合体的重组钝齿棒杆菌显示了明显的抗噬菌体功能活性和较广的抗噬菌体谱, 进而证实了cglI基因复合体用于构建钝齿棒杆菌抗噬菌体菌株的可行性, 为解决氨基酸发酵生产中的噬菌体污染问题提供了一种有效方法。     

限制修饰系统cglI赋予钝齿棒杆菌抗噬菌体功能活性

为解决氨基酸发酵工业中的噬菌体污染问题, 对cglI基因复合体在钝齿棒杆菌中的功能活性表达进行研究。通过PCR从谷氨酸棒杆菌基因组扩增cglI基因复合体, 构建重组质粒pJL23-cglI, 转化钝齿棒杆菌T6-13后得到重组菌株。定性和定量检测重组菌株的噬菌体抗性。实验结果表明, 携带cglI基因复合体的重组钝齿棒杆菌显示了明显的抗噬菌体功能活性和较广的抗噬菌体谱, 进而证实了cglI基因复合体用于构建钝齿棒杆菌抗噬菌体菌株的可行性, 为解决氨基酸发酵生产中的噬菌体污染问题提供了一种有效方法。     

Eosinophilic bronchitis-like lesion as the cause of death in a Macaca mulatta: a first case report

BACKGROUND: Eosinophilic bronchitis is a recently described, relatively benign condition in humans that is characterized by a corticosteroid-responsive chronic cough and sputum eosinophilia without the abnormalities of airway function seen in asthma. The exact cause of this condition is currently unknown, however has been associated with various occupational exposures in humans. It has also been reported to progress to irreversible airway obstruction. This disease has been reported in dogs and horses, but not in non-human primates. METHODS: Gross examination of an otherwise healthy 13-year-old, colony-born Macaca mulatta, which died of severe non-responsive respiratory distress revealed that the lungs were markedly inflated and moist. RESULTS: Hematoxylin and eosin-stained sections from the lungs contained widespread accumulation of eosinophils, sloughed epithelial cells, and mucus centered around bronchioles and adjacent airways. There was no evidence of mast cell infiltration of peribronchiolar smooth muscle, goblet cell hyperplasia, or basement membrane thickening. CONCLUSIONS: This ruled out recurrent episodes as would be expected in asthma, favoring the diagnosis of an eosinophilic bronchitis-like lesion. We report a first case of eosinophilic bronchitis-like features in a M. mulatta.     

Vaccine-elicited antibodies mediate antibody-dependent cellular cytotoxicity correlated with significantly reduced acute viremia in rhesus macaques challenged with SIVmac251

Effector cells armed with Abs can eliminate virus-infected target cells by Ab-dependent cellular cytotoxicity (ADCC), an immune mechanism that has been largely overlooked in HIV vaccine development. Here, we show that a prime/boost AIDS vaccine approach elicits potent ADCC activity correlating with protection against SIV in rhesus macaques (Macacca mulatta). Priming with replicating adenovirus type 5 host range mutant-SIV recombinants, followed by boosting with SIV gp120, elicited Abs with ADCC activity against SIV(mac251)-infected cells. In vitro ADCC activity correlated with in vivo reduced acute viremia after a mucosal challenge with pathogenic SIV. Our findings expose ADCC activity as an immune correlate that may be relevant in the rational design of an efficacious vaccine against HIV.