In Yersinia pestis KIM there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2" response virulence plasmid pCD1. Only YopM and YopN are found in easily detectable amounts in the culture medium. In our previous work, we characterized the yopM gene. In the present study, we constructed a YopMmutant to elucidate the role of YopM in the virulence of Y. pestis. A lacZYA sequence was inserted 126 base pairs downstream from the start codon of the yopM gene in pCD1. The YopMmutant had the same growth properties as the parent, Y. pestis KIM5-3001. The inserted lacZ gene was regulated by the promoter of the yopM gene. Accordingly, it was expressed strongly at 37°C in the absence of Ca2' and was decreased in expression when Ca2+ was present. Northern blot (RNA blot) analysis revealed that the yopM gene was in a monocistronic operon, suggesting that the yopM insertion mutation was unlikely to have polar effects on other genes. The YopMmutant had strongly decreased virulence in mice, with a 50% lethal dose of 3.4 x 105 CFU. Virulence was restored by the cloned yopM-containing 5.5-kilobase Hindlll F fragment of pCDl. However, supplying a cloned 1.57-kilobase fragment containing little more than the yopM structural gene caused the yopM mutant to signfficantly overexpress YopM and failed to restore virulence. The infection kinetics of the YopMmutant revealed growth in both spleens and livers from days 2 to 4 after infection, followed by a precipitous clearance of the bacteria. YopM-containing supernatant proteins of Y. pestis inhibited thrombinor ristocetin-induced platelet aggregation, whereas there was no inhibition by supernatant proteins from the YopM-Y. pestis mutant. Accordingly, YopM may prevent platelet-mediated events and serve as an important strategy for the yersiniae in the initial stages of a plague infection.

In previous studies, Yersinia pestis YopM has been shown through mutational analysis to be necessary for virulence in mice and found to have homology with the thrombin-binding domain of the platelet receptor GPIba. In this study, YopM was purified and shown by dot blot and chemical cross-linking tests to bind to human ct-thrombin. No cross-linked product could be detected when human prothrombin was incubated with YopM. As a functional test of thrombin binding, it was shown that native but not boiled YopM inhibits thrombin-induced aggregation of human platelets. Control tests showed that YopM did not inactivate the platelets themselves, nor was its effect a nonspecific consequence of its very acidic isoelectric point. Microsequencing of YopM revealed an intact N terminus, indicating that functional YopM is not processed at the N terminus or secreted by a mechanism involving a cleavable signal sequence. Further characterization was made of an interesting effect on yopM expression that had been noticed in a previous study. A 1.5-kb HaeTI subclone overexpressed YopM in both Y. pestis and Escherichia coli compared with a larger clone containing the 5.3-kb HindHI-F fragment. To search for a possible regulator ofYopM expression, the Hindm-F fragment was sequenced, revealing several open reading frames and three large repeated sequences. Deletional analysis showed that these were not involved in regulation ofyopM. The data implicated a DNA structure 5' to yopM in moderating yopM expression.

Bafilomycin A(1), a selective inhibitor of V-type H(+)-translocating ATPase (V-ATPase), may be a useful adjunct in cancer chemotherapy (Altan et al. [1998] J Exp Med 187:1583-1598). Therapeutic uses of the enzyme inhibitor need to consider the agent's potential effects on normal (nontumor) cells. This study determined the effects of bafilomycin A(1) on resident alveolar macrophages (mphi). Treatment of alveolar mphi with bafilomycin A(1) (10 microM, 1 h) caused a significant decrement in cytosolic pH. This was accompanied by marked alteration of mphi bactericidal capabilities. The enzyme inhibitor caused a marginal reduction in the phagocytosis of opsonized Staphylococcus aureus and significantly suppressed intracellular killing of the phagocytosed bacteria. In keeping with the effects on intracellular killing, bafilomycin A(1) significantly reduced the production of reactive oxygen species (ROS). On the other hand, cell spreading was enhanced significantly by bafilomycin A(1). Comparable changes in ROS generation and mphi spreading were produced by altering cytosolic pH through changes in extracellular pH (pH(o)) in the absence of bafilomycin A(1). These findings suggest that the agent's effects on ROS production and mphi spreading were related to the accompanying changes in cytosolic pH. The enzyme inhibitor also altered mphi morphology, leading to the shortening of microvilli and focal loss of surface ruffles. These morphologic effects differed from those produced by altering cytosolic pH by changes in pH(o). The results demonstrate that V-ATPase activity is an important determinant of mphi functioning and structure. Therapeutic use of V-ATPase inhibitors might be expected to compromise the bactericidal activity of alveolar mphi.

The pattern of ampicillin resistance and possible association with virulence factors of 78 Escherichia coli isolates taken from 78 pregnant women with pyelonephritis were evaluated. The current incidence of ampicillin resistance among pyelonephritis isolates (46%) was significantly higher than that reported in 1985 (22%). Resistance was found more frequently during the first (60%) and third (53%) trimesters than during the second trimester (33%). Of all dra(+) E. coli isolates, 75% were ampicillin resistant, whereas dra(+) isolates of O75 serotype E. coli accounted for 87% of ampicillin-resistant strains. The significant increase of ampicillin resistance among gestational pyelonephritis E. coli and the association with the dra gene cluster encoding colonization and invasive capacity may warrant further study involving obstetric and neonate wards, with the latter being at the higher risk for potential problems.

A 7-day incubation protocol was instituted with the BACTEC 9240 system for a 1-year period to determine the times to detection of clinically relevant organisms. A total of 23,686 blood and 693 sterile body fluid cultures were received; some cultures were held longer by special request. Of 1,609 likely skin contaminants, 42 were recovered on day 5, 34 on day 6, 16 on day 7, and 5 on day 8. Of 2,803 usual pathogens, 34 were recovered on day 5, 24 on day 6, 15 on day 7 and 1 on day 8. Twenty-one of the latter organisms were considered significant laboratory isolates because they were the first isolates from the respective patients. Chart review showed that 10 of 21 were considered clinically significant, but only 3 (all yeasts) affected the treatment of the patient. Our data show that 4 days of incubation were sufficient to recover all clinically relevant bacteria and 6 days were required to recover all clinically relevant yeasts.