Non-exponential growth of Mycobacterium leprae Thai-53 strain cultured in vitro.

In this study, attempts were made to culture this bacterium in media supplemented with a variety of biological materials to determine why cultivation of Mycobacterium leprae in vitro has not this far been successful. A slight increase in the number of cells in medium supplemented with human blood plasma and an extract of nude mouse tissue as observed after more than 3 months of cultivation at 30 °C. To ascertain whether this increase was real growth, the growth was analyzed by droplet digital PCR, which showed a slow increase in the copy number of cell-associated DNA and the release of a large amount of DNA into the culture medium from bacterial cells during cultivation. These results were supported by electron microscopic examination of M. leprae in infected mouse tissues, which showed that most of the replicated bacteria had degenerated and only a few cells survived. Based on these results, it was postulated that many of the replicated cells degenerate during M. leprae growth and that only a few cells remain to participate in the next growth stage. This means that, unlike other cultivable bacteria, the growth of M. leprae is not exponential and the number of cells therefore increase extremely slowly. Thus, accurate judging of the success of M. leprae cultivation requires observation of growth over a long period of time and careful measurement of the increase in number of viable cells.

Legionella dumoffii Tex-KL Mutated in an Operon Homologous to traC-traD is Defective in Epithelial Cell Invasion / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To understand the mechanism of invasion by Legionella dumoffii.</p><p><b>METHODS</b>The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903dIIlacZ. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in HeLa and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR.</p><p><b>RESULTS</b>The transposon insertion was in a gene homologous to Salmonella typhi traC, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the traC gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a traC deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain..</p><p><b>CONCLUSION</b>Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.</p>

Filament formation of Salmonella Paratyphi A accompanied by FtsZ assembly impairment and low level ppGpp.

Previously, we reported that Salmonella enterica serovar Paratyphi A strain S602 grew into multinuclear, nonseptate, and nonlethal filaments on agar plates containing nitrogenous salts. Strain S602 was more sensitive to osmotic and oxidative stress than the reference strain 3P243 of nonfilamentous Salmonella Paratyphi A. Strain S602 had an amber mutation (C154T) in rpoS. The revertant of this mutant, SR603, was repressed to form filaments under conditions with abundant nitrogenous salts. However, 3PR244, an rpoS mutant of 3P243 (C154T), did not form filaments, which implies that the rpoS mutation is not the sole cause of filamentation in strain S602. Next, we examined whether the level of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in S602 strain is involved in filament formation. The intracellular ppGpp level in filamentous cells was lower than that in nonfilamentous cells. Furthermore, cells belonging to strain RE606, a derivative of S602 where the intracellular concentration of ppGpp was increased by overexpression of the relA gene, exhibited normal Z-ring formation and cell division. In the S602 strain, the decrease in the ppGpp level induced by the presence of nitrogenous salt and the rpoS mutation led to the inhibition of Z-ring formation and the subsequent filamentation of cells.

Elevated guanosine 5'-diphosphate 3'-diphosphate level inhibits bacterial growth and interferes with FtsZ assembly.

FtsZ, a protein essential for prokaryotic cell division, forms a ring structure known as the Z-ring at the division site. FtsZ has a GTP binding site and is assembled into linear structures in a GTP-dependent manner in vitro. We assessed whether guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a global regulator of gene expression in starved bacteria, affects cell division in Salmonella Paratyphi A. Elevation of intracellular ppGpp levels by using the relA expression vector induced repression of bacterial growth and incorrect FtsZ assembly. We found that FtsZ forms helical structures in the presence of ppGpp by using the GTP binding site; however, ppGpp levels required to form helical structures were at least 20-fold higher than the required GTP levels in vitro. Furthermore, once formed, helical structures did not change to the straight form even after GTP addition. Our data indicate that elevation of the ppGpp level leads to inhibition of bacterial growth and interferes with FtsZ assembly.

Hydrogen peroxide causes Vibrio vulnificus bacteriolysis accelerated by sulfonyl fluoride compounds.

Induction of bacteriolysis of Vibrio vulnificus cells by 10 mM hydrogen peroxide (H(2)O(2)) was analyzed. All Vibrio species examined, except for Vibrio hollisae, were lysed by 10 mM H(2)O(2). Bacteriophage induction was not the cause of H(2)O(2)-induced bacteriolysis. Autolysis is also known to cause bacteriolysis. VvpS protein is a serine protease of V. vulnificus essential for autolysis. vvpS mutant underwent H(2)O(2)-induced bacteriolysis in the same manner as the wild type. Protease inhibitors including serine protease inhibitors did not inhibit H(2)O(2)-induced bacteriolysis, which means that bacteriolysis is not due to autolysis. Unexpectedly, H(2)O(2)-induced bacteriolysis was accelerated by adding 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and phenylmethylsulfonyl fluoride which are serine protease inhibitors. The hydroxyl radical was generated by H(2)O(2)-AEBSF interaction. It was considered that H(2)O(2)-induced bacteriolysis was caused by the hydroxyl radical which was generated by Fenton reaction, and possibly mediated by AEBSF. Deferoxamine, an agent chelating ferric ion and Fenton reaction inhibitor, suppressed both H(2)O(2)-induced bacteriolysis and its acceleration by AEBSF. This suggests that both phenomena were Fenton reaction dependent, and hydroxyl radical generated by Fenton reaction caused bacteriolysis of V. vulnificus though the reason for high susceptibility of Vibrio species to hydroxyl radical is not known.

The usefulness of semi-solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka, Japan.

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi-solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira-positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi-solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi-solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.

Leptospira idonii sp. nov., isolated from environmental water.

Strain Eri-1(T) was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of members of the genus Leptospira, but the spiral structure of the isolate was sharper under dark-field microscopy. Cells were 10.6 ± 1.3 µm long and 0.2 µm in diameter, with a wavelength of 0.9 µm and an amplitude of 0.4 µm. Strain Eri-1(T) grew in Korthof's medium at both 13 and 30 °C, and also in the presence of 8-azaguanine. 16S rRNA gene-based phylogenetic analysis placed strain Eri-1(T) within the radiation of the genus Leptospira where it formed a unique lineage within the clade of the known saprophytic species of the genus Leptospira. The strain was not pathogenic to hamsters. Strain Eri-1(T) exhibited low levels (11.2-12.6 %) of similarity by DNA-DNA hybridization to the three most closely related species of the genus Leptospira. The DNA G+C content of the genome of strain Eri-1(T) was 42.5 ± 0.1 mol%. These results suggest that strain Eri-1(T) represents a novel species of the genus Leptospira, for which the name Leptospira idonii sp. nov. is proposed. The type strain is Eri-1(T) ( = DSM 26084(T) = JCM 18486(T)).

Effect of hyperbaric oxygen on Vibrio vulnificus and murine infection caused by it.

Vibrio vulnificus is a bacterium known to cause fatal necrotizing soft tissue infection in humans. Here, a remarkable therapeutic effect of hyperbaric oxygen (HBO) on V. vulnificus infection provoked by its injection into mouse footpads is described. HBO was shown to be bactericidal to this bacterium in vitro as well as in the infected tissue. The bactericidal activity of HBO was shown to be due to reactive oxygen species (ROS), the efficacy of HBO against V. vulnificus infection being accounted for by the high sensitivity of this bacterium to ROS. Besides being somewhat weak in ROS-inactivating enzyme activities, this bacterium is also unusually sensitive to ultraviolet light and other DNA-damaging agents. It seems likely that the sensitivity of V. vulnificus to HBO is mainly due to its poor ability to repair oxidative damage to DNA. These findings encourage clinical application of HBO against potentially fatal V. vulnificus infection in humans.

Importance of the icmN gene in the growth of Legionella pneumophila in amoebic cells at low temperature.

Legionella pneumophila grows in amoebae and has achieved the ability to grow at various temperatures, although the mechanisms controlling this ability remain poorly understood. The Icm/Dot type IVB secretion system is composed of more than 25 proteins and is known to be essential for intracellular growth. The role of the icmN gene in intracellular multiplication and the effects of culture temperatures on it are not precisely understood. We conducted our investigation using an icmN mutant made by gene replacement mutagenesis. Intracellular growth of the mutant was impaired both in mammalian macrophages and amoeba at 37 °C. In particular, intracellular growth in amoebae was completely impaired at 25 °C. It was found that genes from icmN to icmC formed an operon, i.e., icmN, -M, -L, -E, -G, -C,, and the promoter activity of the icmN operon was stronger at 25 than at 37 °C. It was suggested that icmM and its downstream genes had a secondary promoter that enables icmN mutant grow in amoebae at lower temperatures and macrophages at 37 °C. These results show that the icmN promoter has a low temperature inducible nature, and gene products of the icmN operon require high expression for bacterial proliferation at low temperatures within amoeba.

Systemic cytokine response in moribund mice of streptococcal toxic shock syndrome model.

Streptococcus pyogenes causes severe invasive disease in humans, including streptococcal toxic shock syndrome (STSS). We previously reported a mouse model that is similar to human STSS. When mice were infected intramuscularly with 10(7) CFU of S. pyogenes, all of them survived acute phase of infection. After 20 or more days of infection, a number of them died suddenly accompanied by S. pyogenes bacteremia. We call this phenomenon "delayed death". We analyzed the serum cytokine levels of mice with delayed death, and compared them with those of mice who died in the acute phase of intravenous S. pyogenes infection. The serum levels of TNF-α and IFN-γ in mice of delayed death were more than 100 times higher than those in acute death mice. IL-10 and IL-12, which were not detected in acute death, were also significantly higher in mice of delayed death. IL-6 and MCP-1 (CCL-2) were elevated in both groups of mice. It was noteworthy that not only pro-inflammatory cytokines but also anti-inflammatory cytokines were elevated in delayed death. We also found that intravenous TNF-α injection accelerated delayed death, suggesting that an increase of serum TNF-α induced S. pyogenes bacteremia in our mouse model.

Glucose metabolism in Legionella pneumophila: dependence on the Entner-Doudoroff pathway and connection with intracellular bacterial growth.

Glucose metabolism in Legionella pneumophila was studied by focusing on the Entner-Doudoroff (ED) pathway with a combined genetic and biochemical approach. The bacterium utilized exogenous glucose for synthesis of acid-insoluble cell components but manifested no discernible increase in the growth rate. Assays with permeabilized cell preparations revealed the activities of three enzymes involved in the pathway, i.e., glucokinase, phosphogluconate dehydratase, and 2-dehydro-3-deoxy-phosphogluconate aldolase, presumed to be encoded by the glk, edd, and eda genes, respectively. Gene-disrupted mutants for the three genes and the ywtG gene encoding a putative sugar transporter were devoid of the ability to metabolize exogenous glucose, indicating that the pathway is almost exclusively responsible for glucose metabolism and that the ywtG gene product is the glucose transporter. It was also established that these four genes formed part of an operon in which the gene order was edd-glk-eda-ywtG, as predicted by genomic information. Intriguingly, while the mutants exhibited no appreciable change in growth characteristics in vitro, they were defective in multiplication within eukaryotic cells, strongly indicating that the ED pathway must be functional for the intracellular growth of the bacterium to occur. Curiously, while the deficient glucose metabolism of the ywtG mutant was successfully complemented by the ywtG(+) gene supplied in trans via plasmid, its defect in intracellular growth was not. However, the latter defect was also manifested in wild-type cells when a plasmid carrying the mutant ywtG gene was introduced. This phenomenon, resembling so-called dominant negativity, awaits further investigation.

Discrimination of live, anti-tuberculosis agent-injured, and dead Mycobacterium tuberculosis using flow cytometry.

Flow cytometry (FCM) using propidium iodide (PI)/bis-oxonol (BOX) staining can distinguish live, dead, and sublethally injured Escherichia coli by detecting intact vs. nonintact membranes (PI) and membrane potential (BOX). However, live bacteria, especially Mycobacterium tuberculosis, are not likely to be successfully discriminated from injured bacterium by FCM when utilizing the live/dead staining agents currently on the market. As injured cell membranes have integrity like that of live cells and are regarded as such by FCM, the distinction between live and injured cells has depended on the culture method, where injured bacteria cannot grow in general. We have previously shown that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro. In this study, we found that the chromosomal DNA of antibiotic-injured, but not live, M. tuberculosis could be cleaved within 2 h by EMA, and that the resultant decrease in the spaces of DNA base pairs could greatly inhibit the intercalation of SYTO9 in FCM. The percentage value of SYTO9(+)/PI(-) quadrant from antibiotic-injured M. tuberculosis after EMA treatment decreased by at least 80%, compared with that before EMA, but such a phenomenon did not take place in live cells. FCM (SYTO9/PI) following EMA treatment is a very rapid, simple, and effective method for discriminating live, antibiotic-injured, and dead M. tuberculosis without culture.

Conjugative plasmid pLD-TEX-KL promotes growth of host bacterium Legionella dumoffii at low temperatures.

Legionella (Fluoribacter) dumoffii is a resident of various aquatic environments and occasionally causes pneumonia in humans. We found that L. dumoffii strain TEX-KL carries a 66-kb circular plasmid. As predicted by the presence of tra genes similar to those of other transferable plasmids, we showed that pLD-TEX-KL was actually capable of transferring itself to a plasmid-cured derivative of the original strain. Unexpectedly, this plasmid-free derivative turned out to be partially defective in terms of growth at temperatures 30 degrees C or lower. Subsequent works revealed that the growth defect was attributable to the loss of the plasmid gene traA(Ti) homologous to the traA gene of Ti plasmid from Agrobacterium tumefaciens, and that the growth was restored by the introduction of the mobA/repB gene of plasmid pMMB207. Since the existence of a DNA nickase domain is the only feature common to the traA(Ti) and mobA/repB gene products, we hypothesized that this growth defect at low temperature is related to insufficient DNA transactions, which can somehow be alleviated by the nickase activity of those plasmid-encoded proteins. It was also noted that the above features of growth defect at low temperatures were seen in L. dumoffii cells parasitizing the amebic host Acanthamoeba culbertsoni.

Method To Detect Only Live Bacteria during PCR Amplification.

Ethidium monoazide (EMA) is a DNA cross-linking agent and eukaryotic topoisomerase II poison. We previously reported that the treatment of EMA with visible light irradiation (EMA + Light) directly cleaved chromosomal DNA of Escherichia coli (T. Soejima, K. Iida, T. Qin, H. Taniai, M. Seki, A. Takade, and S. Yoshida, Microbiol. Immunol. 51:763-775, 2007). Herein, we report that EMA + Light randomly cleaved chromosomal DNA of heat-treated, but not live, Listeria monocytogenes cells within 10 min of treatment. When PCR amplified DNA that was 894 bp in size, PCR final products from 10(8) heat-treated L. monocytogenes were completely suppressed by EMA + Light. When target DNA was short (113 bp), like the hly gene of L. monocytogenes, DNA amplification was not completely suppressed by EMA + Light only. Thus, we used DNA gyrase/topoisomerase IV and mammalian topoisomerase poisons (here abbreviated as T-poisons) together with EMA + Light. T-poisons could penetrate heat-treated, but not live, L. monocytogenes cells within 30 min to cleave chromosomal DNA by poisoning activity. The PCR product of the hly gene from 10(8) heat-treated L. monocytogenes cells was inhibited by a combination of EMA + Light and T-poisons (EMA + Light + T-poisons), but those from live bacteria were not suppressed. As a model for clinical application to bacteremia, we tried to discriminate live and antibiotic-treated L. monocytogenes cells present in human blood. EMA + Light + T-poisons completely suppressed the PCR product from 10(3) to 10(7) antibiotic-treated L. monocytogenes cells but could detect 10(2) live bacteria. Considering the prevention and control of food poisoning, this method was applied to discriminate live and heat-treated L. monocytogenes cells spiked into pasteurized milk. EMA + Light + T-poisons inhibited the PCR product from 10(3) to 10(7) heat-treated cells but could detect 10(1) live L. monocytogenes cells. Our method is useful in clinical as well as food hygiene tests.

Concerted action of lactate oxidase and pyruvate oxidase in aerobic growth of Streptococcus pneumoniae: role of lactate as an energy source.

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both H(2)O(2)-forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.

Onset of streptococcal toxic shock syndrome is accelerated by bruising in a mouse model.

Streptococcal toxic shock syndrome (STSS) is the severest form of human infections caused by Streptococcus pyogenes. In our animal model of STSS [Saito M, Kajiwara H, Ishikawa T, et al. Delayed onset of systemic bacterial dissemination and subsequent death in mice injected intramuscularly with Streptococcus pyogenes. Microbiol Immunol 2001;45:777-86], mice inoculated intramuscularly with S. pyogenes strains initially suffer from a short illness, then recover and remain healthy for about 20 days, and finally become sick and incur a sudden death. Here we report that the death during the convalescent period was facilitated by artificially bruising an extremity remote from the site of the initial inoculation. Bacterial burden was found to be higher in the bruised site than in a non-bruised control extremity of each mouse examined. Bacteremia started to occur approximately 20 days after infection. These findings imply that a fresh bruise serves as a focus for bacterial multiplication in the presence of bacteremia, thereby facilitating the development of STSS.

Complete nucleotide sequence of pLD-TEX-KL, a 66-kb plasmid of Legionella dumoffii TEX-KL strain.

The complete nucleotide sequence of a large (66 kb) plasmid pLD-TEX-KL of Legionella dumoffii TEX-KL strain was determined. Of the 57 predicted open reading frames (ORFs), 39 (68%) encoded proteins similar to previously known proteins, five (9%) were assigned with putative functions, three (5%) encoded conserved hypothetical proteins, and 10 (18%) had no homology to any genes present in the current open databases. The ORFs with similar functions were organized in a modular structure; thus, transfer region was identified, as well as a putative heavy-metal ion transporter system (hel). The transfer region encoded homologs of the Salmonella entrica serovar Typhi conjugative system components involved in conjugation. In addition, we also found a potential protein that was analogous to the DNA polymerase III epsilon subunit. It is rarely found that plasmid encode the DNA polymerase.

A novel agar medium to detect hydrogen peroxide-producing bacteria based on the prussian blue-forming reaction.

The classic method for H(2)O(2) detection involving Prussian blue formation was adapted to formulate a novel agar medium that makes possible in situ detection of H(2)O(2) produced by bacteria. Using this medium, colonies of H(2)O(2)-producing species including Streptococcus pyogenes were easily identified by the appearance of blue halos. The utility of the medium was further illustrated by its successful application to the isolation of H(2)O(2)-producing mutants from a non-H(2)O(2)-producing stain of S. pyogenes.

Photoactivated ethidium monoazide directly cleaves bacterial DNA and is applied to PCR for discrimination of live and dead bacteria.

Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 microg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 microg/ml) led to breaks of double-stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single-stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 microg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca. When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.