Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with ␤-lactam antibiotics. The ␤-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed ␤-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by ␤-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to ␤-lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. KTG). Toho-1 was about 83% homologous to the ␤-lactamase mediated by the chromosome of K. oxytoca D488 and the ␤-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated ␤-lactamase Toho-1 produced by E. coli TUH12191 is similar to ␤-lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes. Toho-1 has shown the highest degree of similarity to K. oxytoca class A ␤-lactamase. Detailed comparison of Toho-1 with other ␤-lactamases implied that replacement of Asn-276 by Arg with the concomitant substitution of Thr for Arg-244 is an important mutation in the extension of the substrate specificity.Expanded-spectrum cephalosporins have chemical structures which confer stability to many ␤-lactamases from gram-negative bacteria. However, many members of the family Enterobacteriaceae other than Escherichia coli developed resistance to the expanded-spectrum cephems (40). The primary mechanism of this resistance was demonstrated to be excessive production of a chromosomal ␤-lactamase (AmpC) (23). However, bacteria that show resistance mediated by other ␤-lactamases appeared in 1984 (8). Species of the Enterobacteriaceae such as Klebsiella pneumoniae, Klebsiella oxytoca, and E. coli acquired resistance against expanded-spectrum cephem antibiotics by producing extended-spectrum ␤-lactamase. The extended spectrum of the ␤-lactamase was often acquired by the variation of ␤-lactamase genes on transmissible plasmids (43,44). Under the influence of antimicrobial agents, bacteria producing primarily TEM-type or SHV-type ␤-lactamases developed point mutations in structural genes which served to extend the substrate specificity of the enzymes (44). These TEM-type and SHV-type ␤-lactamases show about 65% amino acid sequence homology, with isoelectric points of 5.5 to 6.3 and 7.0 to 8.2, respectively (8, 9).

The factors involved in the survival of Legionella pneumophila in the microcosms of both hot spring water and tap water were studied by examining cultivability and metabolic activity. L. pneumophila could survive by maintaining metabolic activity but was noncultivable in all microcosms at 42°C, except for one microcosm with a pH of <2.0. Lower temperatures supported survival without loss of cultivability. The cultivability declined with increasing temperature, although metabolic activity was observed at temperatures of up to 45°C. The optimal range of pH for survival was between 6.0 and 8. The metabolic activity could be maintained for long periods even in microcosms with high concentrations of salt. The cultivability of organisms in the postexponential phase in a tap water microcosm with a low inoculum size was more rapidly reduced than that of organisms in the exponential phase. In contrast, the loss of cultivability in microcosms of a high inoculum size was significant in the exponential phase. Random(ly) amplified polymorphic DNA analysis of microcosms where cultivability was lost but metabolic activity was retained showed no change compared to cells grown freshly, although an effect on the amplified DNA band pattern by production of stress proteins was expected.Resuscitation by the addition of Acanthamoeba castellanii to the microcosm in which cultivability was completely lost but metabolic activity was maintained was observed only in part of the cell population. Our results suggest that L. pneumophila cell populations can potentially survive as free organisms for long periods by maintaining metabolic activity but temporarily losing cultivability under strict environments and requiring resuscitation by ingestion by amoebas.Legionella pneumophila, the organism that causes Legionnaires' disease and Pontiac fever, is a ubiquitous bacterium in natural or man-made aqueous environments and requires an intracellular environment of free-living amoebas for its replication (19). However, this pathogen is also known to survive as a free organism for long periods in low-nutrient environments under appropriate conditions (14, 15). Bacterial populations released in an aqueous environment are frequently exposed to stresses due to limitations and changes in nutrient availability, temperature, salinity, oxygen, and pH. To adapt to such a stressful environment, bacteria often enter a "temporarily noncultivable state," in which they regulate cell differentiation to adapt to such stresses and then resuscitate when environmental conditions become favorable for growth. This physical change is generally referred to as "viable but noncultivable," and some bacteria use this strategy (12). L. pneumophila cell populations that entered a noncultivable state in tap water were also reportedly resuscitated by injection into embryonated eggs (9) or by passage through Acanthamoeba castellanii (18).Japan is an eminently volcanic country, and hot springs are widespread. Japanese people enjoy a culture of bathing in hot springs, and recently ...

The effect of preexistent coronary collateral perfusion on the prevention of left ventricular aneurysm formation was examined in 47 patients undergoing an intracoronary thrombolysis within 6 hours after the onset of a first acute anterior myocardial infarction. Left ventricular aneurysm formation and wall motion were analyzed with cineventriculography. A left ventricular aneurysm was determined as well-defined demarcation of the infarcted segment from normally contracting myocardium. In 25 patients with successful thrombolysis (group A), a left ventricular aneurysm was observed in one patient (4%) during the chronic stage of infarction. In 10 patients who had a significant collateral circulation to the infarct-related coronary artery and unsuccessful reperfusion (group B), the left ventricular aneurysm was observed in only one patient (10%). In the remaining 12 patients with unsuccessful recanalization in the absence of a significant collateral perfusion (group C), there was a higher incidence (seven of 12, 58%) of left ventricular aneurysm formation than in groups A and B (p <0.05). In group A, both the global ejection fraction and regional wall motion in the infarct areas improved significantly (p< 0.05) between the acute and chronic stages of infarction. By contrast, in groups B and C, these indexes on the ventricular function did not change significantly during the convalescent period. Thus, although the collateral perfusion existing at the onset of acute myocardial infarction may not improve ventricular function, it exerts a beneficial effect on the prevention of left ventricular aneurysm formation. (Circulation 1989;79:791-796) It is generally accepted that early recanalization of an infarct-related coronary artery in the presence of residual flow resulting from either a subtotal obstruction of infarct-related coronary artery or collateral perfusion exerts a beneficial effect on the regional and global left ventricular wall motion evaluated during the chronic stage of infarction.1-3 However, it is still controversial whether a preexistent coronary collateral circulation contributes to the preservation of jeopardized myocardial function in the case of unsuccessful recanalization. The purpose of our study was to evaluate the relation of significant collateral perfusion to regional myocardial function and left ventricular aneurysm formation in patients with acute myocardial infarction who were treated with intracoronary thrombolysis. Methods Study PatientsOver a 4-year period, 47 consecutive patients with a first acute anterior myocardial infarction who had complete occlusion of the proximal part of the left anterior descending coronary artery were referred for intracoronary thrombolysis during the first 6 hours after the onset of symptoms. There were 39 men and eight women with a mean age of 60 years (range, 35-79). The diagnosis of acute myocardial infarction included the presence of persistent ST segment elevation of 2 mm or greater in two or more leads on the standard 12-lead electrocardiogram and seve...

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We analyzed the effects of temperature on the interaction of Legionella pneumophila with Acanthamoeba castellanii. At <20°C, overexpression of type 1 metacaspase, a stimulator of A. castellanii encystation, was associated with a reduced number of bacteria within amoeba. At low temperatures, A. castellanii seems to eliminate L. pneumophila by encystation and digestion.The intracellular pathogen Legionella pneumophila causes Legionnaires' disease and exploits aquatic protozoa for replication. L. pneumophila is more frequently isolated from man-made water systems with high water temperature (9, 17) than from relatively cold freshwater environments (4,16). This fact suggests that thermal conditions affect the relationship between L. pneumophila and protozoa, a notion supported by some reports (1, 3, 7, 11). The trophozoite of protozoa transforms into a cyst under harmful environments such as starvation, cold, and certain chemicals used in medical treatment. The effect of protozoal encystation on Legionella infection is not well understood although it seems that encystation is enhanced in a freshwater environment at low temperature. With these considerations in mind, we investigated whether the host-parasite relationship between L. pneumophila and protozoa is temperature dependent.Two strains of L. pneumophila serogroup 1, Suzuki and Lp01, and Acanthamoeba castellanii ATCC 30234 were used in the present study. Intracellular growth kinetics assays using A. castellanii were performed as described previously (15 Total RNA was purified using an RNeasy minikit (Qiagen, Valencia, CA) according to the instructions provided by the manufacturer. Total RNA was reverse transcribed into cDNA using an RNA PCR kit (Takara Bio Inc., Shiga, Japan). Real-time PCR with Sybr greenER (Invitrogen Life Sciences) was performed using the ABI Prism 7000 system (Applied Biosystems, Foster City, CA). Table 1 lists the primer pairs for target and internal control genes. Data were analyzed by Student's t test, and a two-tailed P value of Ͻ0