Corynebacterium Research Articles

Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform Bacterium Strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities

Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal ion-independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide and a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant. Previous studies identified Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) as critical residues in these activities. In terms of pairwise sequence, MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) is 38% identical with the Pseudomonas enzyme, including Pro-1 and Asp-37. However, Gln-73 replaces Arg-73, and the second arginine is shifted to Arg-76 by the insertion of a glycine. To determine how these changes relate to the activities of FG41 MSAD, the gene was cloned and the enzyme expressed and characterized. The enzyme has a comparable decarboxylase activity but a significantly reduced hydratase activity. Mutagenesis along with crystal structures of the native enzyme (2.0 Å resolution) and the enzyme modified by a 3-oxopropanoate moiety (resulting from the incubation of the enzyme and 3-bromopropiolate) (2.2 Å resolution) provided a structural basis. The roles of Pro-1 and Asp-37 are likely the same as those proposed for Pp MSAD. However, the side chains of Thr-72, Gln-73, and Tyr-123 replace those of Arg-73 and Arg-75 in the mechanism and play a role in binding and catalysis. The structures also show that Arg-76 is likely too distant to play a direct role in the mechanism. FG41 MSAD is the second functionally annotated homologue in the MSAD family of the tautomerase superfamily and could represent a new subfamily.

Surveillance of the condition of bacteria and Amaranthus viridis in a gasoline contaminated soil

The condition of bacteria and Amaranthus viridis in gasoline-contaminated soil was surveyed. The soil was treated with different concentrations of petrol oil: 0 ml (control), 18, 56, 112, 168 and 224 ml. Each of the concentrations was in triplicates. Seventeen (17) bacterial isolates were recovered from the control soil while twenty (20) bacterial isolates were recovered from the polluted soil. A total of twenty four (24) different genera were recovered from the soil. The organisms areAcinetobacter ceticus, Actinomyces sp, Aerococcus viridians, Alcaligenes paradoxus, Arthrobacter, Bacillus cereus, Bacillus polymxa, Bacillus subtilis, Chromobacterium violaceum, Clostridium sp., Corynebacterium hydrocarbodastus, Corynebacterium kutsceri, Corynebacterium xerosis, Cytophaga sp., Erwinia sp., Flavobacterium sp., Micrococcus luteus, Proteus vulgaris, Pseudomonas fluorescens, Pseudomonas putida, Sarcina ventricule, Serratia sp., Staphylococcus aureus, and Streptococcus sp. The pH, temperature, organic matter content, moisture content and water holding capacity of the soil were determined. A. viridis had 100% germination in both the control (0 ml) and 18 ml gasoline-treated soils while it had delayed germination in the other treatments (56, 112, 168 and 224 ml). The results obtained in this study showed that gasoline spillage posses a great threat to the survival and development of plants. It also revealed that certain bacteria could survive the gasoline-contaminated soil and thus may be useful in biodegradation and/or bioremediation of gasoline contaminated soils. Key words: Surveillance, bacteria, Amaranthus viridis, gasoline, contaminated soil.

Identification and characterization of the channel-forming protein in the cell wall of Corynebacterium amycolatum

The mycolic-acid layer of certain gram-positive bacteria, the mycolata, represents an additional permeability barrier for the permeation of small water-soluble solutes. Consequently, it was shown in recent years that the mycolic acid layer of individual bacteria of the group mycolata contains pores, called porins, for the passage of hydrophilic solutes. Corynebacterium amycolatum, a pathogenic Corynebacterium species, belongs to the Corynebacteriaceae family but it lacks corynomycolic acids in its cell wall. Despite the absence of corynomycolic acids the cell wall of C. amycolatum contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of C. amycolatum. Based on partial sequencing of the protein responsible for channel formation derived from C. amycolatum ATCC 49368 we were able to identify the gene coram0001_1986 within the known genome sequence of C. amycolatum SK46 that codes for the cell wall channel. The corresponding gene of C. amycolatum ATCC 49368 was cloned into the plasmid pXHis for its expression in Corynebacterium glutamicum ∆porA∆porH. Biophysical characterization of the purified protein (PorAcoram) suggested that coram0001_1986 is indeed the gene coding for the pore-forming protein PorAcoram in C. amycolatum ATCC 49368. The protein belongs to the DUF (Domains of Unknown Function) 3068 superfamily of proteins, mainly found in bacteria from the family Corynebacteriaceae. The nearest relative to PorAcoram within this family is an ORF which codes for PorAcres, which was also recognized in reconstitution experiments as a channel-forming protein in Corynebacterium resistens.

Pitted keratolysis, erythromycin, and hyperhidrosis

Pitted keratolysis (PK) is a plantar skin disorder mainly caused by coryneform bacteria. A common treatment consists of the topical use of erythromycin. Hyperhidrosis is considered a predisposing factor for bacterial proliferation and, consequently, for the onset of PK. The aim of this study was to evaluate the relationship between PK erythromycin and hyperhidrosis. All patients with PK seen in Sant'Andrea Hospital, between January 2009 and December 2011, were collected. PK was clinically and microscopically diagnosed. All patients underwent only topical treatment with erythromycin 3% gel twice daily. At the beginning of the study and after 5 and 10 days of treatment, a clinical evaluation and a gravimetric measurement of plantar sweating were assessed. A total of 97 patients were diagnosed as PK and were included in the study. Gravimetric measurements showed that in 94 of 97 examined patients (96.90%) at the time of the diagnosis, there was a bilateral excessive sweating occurring specifically in the areas affected by PK. After 10 days of antibiotic therapy, hyperhidrosis regressed together with the clinical manifestations. According to these data, we hypothesize that hyperhidrosis is due to an eccrine sweat gland hyperfunction, probably secondary to bacterial infection.

The first case report of peritoneal dialysis related peritonitis caused by Microbacterium paraoxydans

Peritonitis is still the major complication associated with peritoneal dialysis (PD). Microbacterium spp., a type of coryneform bacteria, is an environmental bacterium isolated from soil, waste water and animals. Human infection is rare, and only few cases have so far been reported in immunocompromised hosts, such as PD patients. Microbacterium paraoxydans, one type of Microbacterium spp. was identified for the first time in 2003. Only two cases of infection of Microbacterium paraoxydans have so far been reported. We herein report the first case of PD-related peritonitis caused by Microbacterium paraoxydans, which was identified by a sequence determination of the 16S rRNA gene. Based on the results of antibiotic sensitivity, the intravenous administration of erythromycin (EM) and oral administration of sulfamethoxazole/trimethoprim (ST) were selected, and PD was interrupted. EM administration was stopped after a total of 14 days. ST was administered for a total of 21 days, and later PD was resumed. Thereafter, no recurrence or relapse of peritonitis without removal of the PD catheter was observed. Microbacterium spp. exhibits multidrug resistance and such an infection is refractory in many cases. We assume that both accurate species identification and the use of antibiotic sensitivity tests are essential to effectively treat this kind of infection.

Emergence of coryneform bacteria as pathogens in nosocomial surgical site infections in a tertiary care hospital of North India

A prospective study was conducted to assess the role of coryneform bacteria in surgical site infections among obstetric and gynecological patients undergoing surgery. The surgery was graded according to the degree of contamination, and surgical site infections (SSIs) were classified as superficial or deep. Pus samples were collected from SSIs according to rigorous aseptic precautions, and the quality of specimens was assessed by Q-score. A detailed clinical and treatment history was elicited from all patients. The samples were processed using standard protocols. Coryneform bacteria were considered significant pathogens only if they fulfilled rigorous clinical and microbiological criteria. Antibiotic susceptibility testing was performed using the Kirby-Bauer method according to the CLSI guidelines. In total, 127 patients developed SSIs among 882 postoperative patients. Of these, 89 (70.1%) were culture positive: 40 (44.9%) were Gram-positive cocci, 27 (30.3%) were coryneform, and 22 (24.7%) were Gram-negative bacilli. All coryneform-infected patients had fever and post-operative wound dehiscence leading to a prolonged hospital stay. The most commonly isolated organism was Staphylococcus aureus (33.7%), followed by Corynebacterium amycolatum (11.2%), Escherichia coli (8.9%), Citrobacter spp. (7.8%) and coagulase-negative Staphylococci (6.7%). In our study, 45.5% were ESBL producers, 18.2% were Amp C producers, and 40% were MRSA. All the coryneform bacteria were multidrug resistant, and 51.8% of isolates were sensitive to only gatifloxacin and vancomycin. Symptomatic improvement was observed in all coryneform-infected patients after the administration of appropriate therapy. Coryneform bacteria appear to be emerging as significant nosocomial surgical site pathogens. The high level of multidrug resistance observed in coryneform bacteria in our study is cause for alarm.

The Role of ARGR Repressor Regulation on L-arginine Production in Corynebacterium crenatum

Corynebacterium crenatum SYA is an aerobic, Gram-positive, non-sporulating coryneform bacterium, and the mutant C. crenatum strain SYPA 5-5 can produce 30 g/l L-arginine under optimal culture conditions. In this study, the evolution of the cluster argCJBDFRGH (argC~H) involved in arginine biosynthesis in C. crenatum SYA, and SYPA 5-5 was investigated. Compared to the argR of its wild type C. crenatum SYA, a nucleotide substitution (C→T) within the argR gene of the mutant C. crenatum strain SYPA 5-5 was found. The inactivation of ARGR resulted in increased enzyme activities involved in L-arginine biosynthesis and increased L-arginine production in C. crenatum. In contrast, constructing an overexpressing argR C. crenatum/pTR, a complete and functional ARGR decreased the expression of enzymes, depressed transcriptional level of the argC~H cluster, and reduced the production of L-arginine in C. crenatum. It was thus evident that the inactivation of an ARGR suppressor could relieve a bottleneck in downstream steps of the L-arginine biosynthetic pathway, providing a good strategy for improving L-arginine production.

PM 7/42 (2) Clavibacter michiganensis subsp. michiganensis

Specific scopeThis standard describes a diagnostic protocol for Clavibacter michiganensis subsp. michiganensis.Specific approval and amendmentApproved in 2004‐09.Revision adopted in 2012‐09.

Study on anti-inflammatory, eliminating phlegm and bacteriostatic action of Yuxuan granule

Objective To reveal the mechanism of Yuxuan granule(YXG) in treating pharyngitis from anti-inflammatory,expectorant,and antibacterial actions.Methods Xylene-induced ear edema and cotton pellet induced granuloma mice models were adopted to observe the anti-inflammatory effects of YXG Phenol red secretion in mice test was adopted to observe the expectorant activity of YXG Five bacteria by tube dilution method was adopted to observe antimicrobial action of YXG Besides,the changes of routine indexes in blood and tumor necrosis factor (TNF),interleukin-1 (IL-1) in serum were measured in acute pharyngitis models.Results YXG has shown a dose-dependent of its anti-inflammatory action.In the test of xylene induced mice ear swelling and cotton ball induced granuloma,the swelling degree of the control group,positive control groupand YXG high (6.14g/kg),medium (3.07g/kg) and low (1.53g/kg) concentration group were (9.00± 1.50) mg,(3.50±0.10)mg,(4.25±1.08)mg,(5.54±0.97)mg,(8.05±0.08)mg and (1.22±1.50)mg,(0.50±0.10)ag,(0.45± 0.08) mg,(0.75± 0.97) mg,(0.70 ± 0.08) mg respectively.YXG also had obviously expectorant effect to the golden staphylocoecus ameus,hemolytic streptococcus,streptococcus pneumoniae,salmonella typhi and corynebacterium diphtheriae,with the minimum inhibition concentration of 55,100,68 and 35 mg/ml respectively; YXG also reduced leukocyte count [percentage of leukocyte was (9.88± 1.22) × 109/L,(7.50± 1.58) ×109/L,(6.02± 1.05) × 109/L,(8.38± 1.13) × 109/L,(8.75 ± 1.08) × 109/L in the five groups respectively],neutrophils [the value was (35.62±5.00)×109/L,(28.49±3.40)×109/L,(28.89±5.22)× 109/L,(29.34±4.83)×109/L,(33.43 ± 4.27) × 109/L in the five groups respectively],lymphocytes [the value was (70.72± 5.04) ％,(67.89 ±4.37)％,(60.14±3.88)％,(65.38±4.45)％,(65.65±4.28)％ in the five groups respectively],TNF in blood serum [the value was (90.25±2.29)ng/ml,(80.23 ± 0.60) ng/ml,(76.83±0.33)ng/ml,(78.00 ± 0.34) ng/ml,(80.52±1.14)ng/ml in the five groups respectively] and IL-1 [the value was (222.06±50.22)pg/ml,(149.55±50.02)pg/ml,(130.37 ± 30.61)pg/ml,(158.68 ± 40.00)pg/ml,(178.76 ± 39.26)pg/ml in the five groups respectively] in the acute pharyngitis rat.Conclusion Through the anti-inflammatory,expectorant,antimicrobial activity and reducing levels of TNF,IL-1 YXG achieves the goal of treating pharyngitis. Key words: Yuxuan granule; Anti-inflammatory; Expelling phlegm; Anti-bacterial effect; TNF; IL-1

First Case of Pseudoclavibacter bifida Bacteremia in an Immunocompromised Host with Chronic Obstructive Pulmonary Disease (COPD)

Pseudoclavibacter spp. are Gram-positive, aerobic, catalase-positive, coryneform bacteria belonging to the family of Microbacteriaceae. Identification of these species with conventional biochemical assays is difficult. This case report of a Pseudoclavibacter bifida bacteremia occurring in an immunocompromised host diagnosed with an acute exacerbation of chronic obstructive pulmonary disease, with a lethal outcome, confirms that this organism may be a human pathogen.

Development of loop-mediated isothermal amplification for detection of Leifsonia xyli subsp. xyli in sugarcane.

Ratoon stunt, caused by the xylem-limited coryneform bacterium Leifsonia xyli subsp. xyli (Lxx), is a deep bacteriosis and prevalent in most of sugarcane-producing countries. Based on loop-mediated isothermal amplification (LAMP), we developed a method for detecting Lxx. The major advantages of the LAMP method are visual judgment by color and time saving with only 60 min for identification of Lxx and without the need for costly PCR apparatus and gel scanner. In the present study, positive and negative samples detected by the LAMP method were clearly distinguishable. When total DNA extracted from internode juice was used as the template, the sensitivity of LAMP was 10 times higher than that of the conventional PCR detection. The LAMP assay is a highly specific, rapid, and sensitive method for the diagnosis of ratoon stunt caused by Lxx in sugarcane. This is the first report of LAMP-based assay for the detection of Lxx in sugarcane.

Cheese surface microbiota complexity: RT-PCR-DGGE, a tool for a detailed picture?

In this work, a culture-independent approach, based on PCR-DGGE and RT-PCR-DGGE, has been used to study the succession of bacterial communities that are encountered in Fontina PDO cheese. As already found for other smear ripened cheeses, it appeared that coryneform bacteria were actively present and could therefore be considered determinant in rind formation. DGGE profiles, especially at the RNA level, have shown the presence of Brevibacterium, Corynebacterium and Arthrobacter genera. RT-PCR-DGGE gels have lead to a richer band profile than the one obtained on the basis of DNA analysis, thus indicating that RNA analysis can highlight bacterial species that DNA analysis is not able to show. Thus, the biodiversity of the Fontina PDO surface has been described better by means of RT-PCR-DGGE, and RNA molecules should be considered a more informative target than DNA.

Brevibacterium otitidis: an elusive cause of neurosurgical infection

Coryneform bacteria are usually considered as non-pathogenic when isolated from clinical specimens. We present a case of Brevibacterium otitidis neurosurgical infection in an immunocompetent patient, and highlight the difficulty with identification and interpretation of antimicrobial susceptibility results for this unusual pathogen.

Bacterial analysis of combined periodontal-endodontic lesions using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)

Objective To compare the bacterial community profiles present in periodontium and root canals of the same tooth diagnosed as combined periodontal-endodontic lesions by using denaturing gradient gel electrophoresis (DGGE).Methods Samples were collected from 13 extracted teeth with advanced periodontitis,endodontic samples from root tip 1/3 root canal,and periodontal samples from the corresponding neighboring periodontium.Genomic DNA was collected for the following universal bacterial primersPCR.The PCR products were then loaded on the DGGE gels to gain separate bands.The typical DGGE bands were excised,PCR-cloned and sequenced.Results The number of bands,which was indicative of the number of bacterial species,was compared intra-group (periodontal and pulpal specimen from the same tooth).The difference was statistically significant (P＜0.01),but there was no positive correlation between them.The similarity (Dice Coefficient) between them was 13.1％-62.5％.Taxa identified through BLAST (≥98％ identity) were Campylobacter,Fusobacterium,Neisseria,et al in the periodontium,and Mogibacterium,Corynebacterium,Actinomyces,et al in the root canals.Conclusion Common bacteria existed between them,but not all of the periodontal bacteria would appear in neighboring root canal; and the bacteria in the root canal are not completely from neighboring periodontal tissue.The original bacteria in the root canals may resuscitate and enrich the bacterial community.In combined periodontal-endodontic lesions (periodontal source),it is probable that new species existed to be confirmed either in the periodontium or in the root canal. Key words: Combined periodontal-endodontic lesions; Denaturing gradient gel electrophoresis; 16S rDNA; Bacteria

Retrospective studies of the trends of occurrence of ruminant mastitis pathogens in a veterinary teaching hospital in Northwest, Nigeria

A retrospective study to determine the pattern of isolation of mastitis causing organisms from milk samples of cattle, sheep and goats submitted to the Microbiology laboratory of the Department of Veterinary Pathology and Microbiology Ahmadu Bello University Zaria from 1980-1991 and 1999-2009 was conducted . The samples were subjected to cultural isolation and biochemical characterization. The results of the isolates were assembled, reviewed and summarised. A total of 227 milk samples were submitted from cattle (40), sheep (31) and goats (156) respectively, from the years 1980 to 1991 and 1999 to 2009 and subjected to cultural isolation and biochemical characterization. A total of 18 different organisms were isolated from 158 of the samples submitted. Six of the submitted samples were un-typeable while 63 were culture negative i.e showed no growth. The most commonly isolated mastitis causing organisms from all samples were: Staphylococcus aureus (26.51%), Escherichia coli (12.20%), Staphylococcus spp (10.24%), Streptococcus spp (6.63%) and Corynebacterium spp (7.32%). Other organisms are , Micrococcus (1.22%), α-hemolytic Streptococcus (1.83%), β- haemolytic Streptococcus (2.42%), Pseudomonas aeroginosa (4.88%), Enterobacter spp (1.83%), Proteus spp (4.89%), Lactobacillus spp (2.42%), Pasteurella spp (4.27%), Klebsiella spp (3.05%), Citrobacter spp (0.61%), Candida albican (0.61%), Flavobacterium spp (0.61%), Bacillus spp (3.67%), Acinetobacter spp (1.20%) and Sarcina spp (0.61%). Staphylococcus aureus was the most predominant organism isolated from cattle, sheep and goat milk. There was a significant reduction in occurrence of mastitis from 201 (88.55%) cases within 1980-1991 and 26 (11.45%) cases within 1999-2009.

Corynebacterium aquatimens sp. nov., a lipophilic Corynebacterium isolated from blood cultures of a patient with bacteremia

An unknown lipophilic coryneform bacterium isolated from the blood cultures of a patient with bacteremia was characterized by phenotypic and molecular genetic methods. Chemical analysis revealed the presence of short chain mycolic acids consistent with the genus Corynebacterium. The DNA G+C content was 60.8mol%. Comparative 16S rRNA gene sequence analysis demonstrated that the isolate represents a new subline within the genus Corynebacterium. The closely phylogenetic relative of the unknown bacterium was found to be C. tuscaniense (97.8% sequence similarity). Partial rpoB gene sequence revealed that strain IMMIB L-2475T exhibited 13.5% sequence divergence with C. tuscaniense. The unknown bacterium was distinguished from C. tuscaniense by, DNA–DNA hybridization, cellular fatty acid profiles, MALDI-TOF analyses of cell extracts and biochemical tests. Based on the phylogenetic and phenotypic criteria, it is proposed that this bacterium be classified as new species, Corynebacterium aquatimens sp. nov., and is represented by strain IMMIB L-2475T (=DSM 45632T=CCUG 61574T).

Analysis of clinical disinfection effect at different time during operation course after surgical handa antisepsis

Objective To observe the influence of hands-washing and non-hands-washing on clinical disinfection effect before surgical hands antisepsis.Methods Totally 40 surgcal staff wee divided into the handa-washing group and the non-hands-washing group.The sterilizing effect of handa at diggerent time points during operations and kinds of bacteria colony were compared between two groups.Results Bacteria colony of hands at different time points during operations showed no significant difference between two groups.Both groups were detected bacteris colony on their hands,mainly made up of coagulase negative staphylococcus,corynebacterium and eaterococcus and no difference was seen between two groups.Conchusions The non-hands-washing method can acquire the same disinfection effect as traditional hands-washing method.Its disinfection effect lasts three hours,easy to operate,little stimulation on the skin,and was worthy of clinical application. Key words: Hands disinfection; Different time points; Colony counts monitoring

Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate

Here, we show that Corynebacterium glutamicum ATCC 13032 co-metabolizes formate when it is grown with glucose as the carbon and energy source. CO(2) measurements during bioreactor cultivation and use of (13)C-labelled formate demonstrated that formate is almost completely oxidized to CO(2). The deletion of fdhF (cg0618), annotated as formate dehydrogenase (FDH) and located in a cluster of genes conserved in the family Corynebacteriaceae, prevented formate utilization. Similarly, deletion of fdhD (cg0616) resulted in the inability to metabolize formate and deletion of cg0617 markedly reduced formate utilization. These results illustrated that all three gene products are required for FDH activity. Growth studies with molybdate and tungstate indicated that the FDH from C. glutamicum ATCC 13032 is a molybdenum-dependent enzyme. The presence of 100 mM formate caused a 25 % lowered growth rate during cultivation of C. glutamicum ATCC 13032 wild-type in glucose minimal medium. This inhibitory effect was increased in the strains lacking FDH activity. Our data demonstrate that C. glutamicum ATCC 13032 possesses an FDH with a currently unknown electron acceptor. The presence of the FDH might help the soil bacterium C. glutamicum ATCC 13032 to alleviate growth retardation caused by formate, which is ubiquitously present in the environment.

Research on the relationship of oxygen humidifier bottles filter element and humiditier liquid pollution

Objective To explore the relationship between use time limit of and humidifier liquid pollution,to make sure the time of oxygen humidifier bottles filter element,to provide the evidence for preventing hospital respiratory tract infection so as to reduce the nunber of oxybiontic bacteria and decrease the incidence rate of hospital respiratory tract infection.Methods The humidifier liquid of different period which patients long - term used in hospital was collected under sterile operation to observe whether pathogenic bacteria grown in different period and the number of bacterial colonies exceed standard.Results There were ten strains were found,corynebacteria and Neisseria were main strains.There were significant positive correlation relationship between humidifier liquid germ - carrying rate and filter element use rime.Humidifier liquid use time extended at the same time germ - carrying rate incerased In the first day unqualified rate of humidifier liquid was 51.28％,seven days later it reached 100％.Conclusions Humidifier bottles filter element increases the humidifier liquid pollution.It should he changed once a day. Key words: Oxygen humidifier bottles; Filter element; Humidifier liquid; Bacterial contamination

Proteomics identifies virulence factors in surface expressed proteins of Corynebacterium pseudotuberculosis

Proteomics identifies virulence factors in surface expressed proteins of Corynebacterium pseudotuberculosis