Open-label, phase II, multicenter study of lasofoxifene plus abemaciclib for treating women with metastatic ER+/HER2- breast cancer and an ESR1 mutation after disease progression on prior therapies: ELAINE 2.

BACKGROUND: Acquired ESR1 mutations in estrogen receptor-positive (ER+) metastatic breast cancer (mBC) drive treatment resistance and tumor progression; new treatment strategies are needed. Lasofoxifene, a next-generation, oral, endocrine therapy and tissue-specific ER antagonist, provided preclinical antitumor activity, alone or combined with a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) in ESR1-mutated mBC. PATIENTS AND METHODS: In the open-label, phase II, ELAINE 2 trial (NCT04432454), women with ESR1-mutated, ER+/human epidermal growth factor receptor 2-negative (HER2-) mBC who progressed on prior therapies (including CDK4/6i) received lasofoxifene 5 mg/day and abemaciclib 150 mg b.i.d until disease progression/toxicity. The primary endpoint was safety/tolerability. Secondary endpoints included progression-free survival (PFS), clinical benefit rate (CBR), and objective response rate (ORR). RESULTS: Twenty-nine women (median age 60 years) participated; all but one were previously treated with a CDK4/6i (median duration 2 years). The lasofoxifene-abemaciclib combination was well tolerated with primarily grade 1/2 treatment-emergent adverse events (TEAEs), most commonly diarrhea, nausea, fatigue, and vomiting. One patient (with no prior CDK4/6i) discontinued treatment due to grade 2 diarrhea. No deaths occurred during the study. Median PFS was 56.0 weeks [95% confidence interval (CI) 31.9 weeks-not estimable; ∼13 months]; PFS rates at 6, 12, and 18 months were 76.1%, 56.1%, and 38.8%, respectively. CBR at 24 weeks was 65.5% (95% CI 47.3% to 80.1%). In 18 patients with measurable lesions, ORR was 55.6% (95% CI 33.7% to 75.4%). ESR1-mutant circulating tumor DNA (ctDNA) allele fraction decreased from baseline to week 4 in 21/26 (80.8%) patients. CONCLUSIONS: Lasofoxifene plus abemaciclib had an acceptable safety profile, was well tolerated, and exhibited meaningful antitumor activity in women with ESR1-mutated, ER+/HER2- mBC after disease progression on prior CDK4/6i. Observed decreases in ESR1-mutant ctDNA with lasofoxifene concordant with clinical response suggest target engagement. If the ELAINE 2 findings are confirmed in the initiated, phase III, ELAINE 3 trial, these data could be practice-changing and help address a critical unmet need.

The ability of a host plant to associate with different symbiotic partners affects ectomycorrhizal functioning.

Some plants that associate with ectomycorrhizal (ECM) fungi are also able to simultaneously establish symbiosis with other types of partners. The presence of alternative partners that may provide similar benefits may affect ECM functioning. Here we compared potential leucine-aminopeptidase (LA) and acid phosphatase (AP) enzyme activity (involved in N and P cycling, respectively) in ECM fungi of three hosts planted under the same conditions but differing in the type of partners: Pinus (ECM fungi only), Eucalyptus (ECM and arbuscular mycorrhizal -AM- fungi) and Acacia (ECM, AM fungi and rhizobial bacteria). We found that the ECM community on Acacia and Eucalyptus had higher potential AP activity than the Pinus community. The ECM community in Acacia also showed increased potential LA activity compared to Pinus. Morphotypes present in more than one host showed higher potential AP and LA activity when colonizing Acacia than when colonizing another host. Our results suggest that competition with AM fungi and rhizobial bacteria could promote increased ECM activity in Eucalyptus and Acacia. Alternatively, other host-related differences such as ECM community composition could also play a role. We found evidence for ECM physiological plasticity when colonizing different hosts, which might be key for adaptation to future climate scenarios.

Intraspecific competition between ectomycorrhizal Pisolithus microcarpus isolates impacts plant and fungal performance under elevated CO2 and temperature.

Root systems are simultaneously colonized by multiple individuals of mycorrhizal fungi. Intraspecific competitive interactions between fungal isolates are likely to affect both fungal and plant performance and be influenced by abiotic factors. Here, we assessed the impact of intraspecific competition between three Pisolithus microcarpus isolates on the establishment of, and benefit derived from, symbioses with Eucalyptus grandis seedlings. We investigated the outcomes of competition under ambient and elevated temperature and CO2 concentration ([CO2]) in a factorial design. We observed a reduction in mycelium growth, mycorrhiza formation and seedling mass when two P. microcarpus isolates were co-inoculated on a single E. grandis seedling. Isolates invested more in mycelium than in mycorrhizas in the presence of a competitor. All isolates responded negatively to elevated [CO2] and positively to elevated temperature, which led to no changes on the outcomes of the interactions with changing conditions. However, the presence of a competitor hindered the positive response of P. microcarpus isolates to warming, which resulted in larger negative effects of competition under elevated temperature than under ambient conditions. Our study highlights the need to consider how competition affects individual fungal responses as well as plant performance when trying to predict the impacts of climate change.

FragMatch--a program for the analysis of DNA fragment data.

FragMatch is a user-friendly Java-supported program that automates the identification of taxa present in mixed samples by comparing community DNA fragment data against a database of reference patterns for known species. The program has a user-friendly Windows interface and was primarily designed for the analysis of fragment data derived from terminal restriction fragment length polymorphism analysis of ectomycorrhizal fungal communities, but may be adapted for other applications such as microsatellite analyses. The program uses a simple algorithm to check for the presence of reference fragments within sample files that can be directly imported, and the results appear in a clear summary table that also details the parameters that were used for the analysis. This program is significantly more flexible than earlier programs designed for matching RFLP patterns as it allows default or user-defined parameters to be used in the analysis and has an unlimited database size in terms of both the number of reference species/individuals and the number of diagnostic fragments per database entry. Although the program has been developed with mycorrhizal fungi in mind, it can be used to analyse any DNA fragment data regardless of biological origin. FragMatch, along with a full description and users guide, is freely available to download from the Aberdeen Mycorrhiza Group web page (http://www.aberdeenmycorrhizas.com).

Pine microsatellite markers allow roots and ectomycorrhizas to be linked to individual trees.

Linking roots and ectomycorrhizas (EcM) to individual host trees in the field is required to test whether individual trees support different ectomycorrhizal communities. Here we describe a method that identifies the source of EcM roots by PCR of polymorphic pine nuclear microsatellite loci using fluorescently labelled primers and high-throughput fragment analysis. ITS-PCR can also be performed on the same EcM DNA extract for fungal identification. The method was tested on five neighbouring Scots pine (Pinus sylvestris var scotica) trees in native woodland. Successful host tree identification from DNA extracts of EcM root tips was achieved for 93% of all root fragments recovered from soil cores. It was estimated that each individual mature pine sampled was colonised by between 15 and 19 EcM fungi. The most abundant fungal species were found on all five trees, and within the constraints of the sampling scheme, no differences between trees in EcM fungal community structure or composition were detected.

Nutrient cycling responses to fire frequency in the Kruger National Park (South Africa) as indicated by stable isotope analysis.

Fires, which are an intrinsic feature of southern African ecosystems, produce biogenic and pyrogenic losses of nitrogen (N) from plants and soils. Because of the long history of fires in these savannas, it was hypothesized that N2 fixation by legumes balances the N losses caused by fires. In this study, the N2 fixation activity of woody legumes was estimated by analyzing foliar delta15N and proportional basal area of N2 fixing species along experimental fire gradients in the Kruger National Park (South Africa). In addition, soil carbon (C) and N pools, foliar phosphorus (P) and gross N mineralization and nitrification rates were measured, to indicate the effects of fires on nutrient stocks and the possible N cycling processes modified by fires. Although observations of increased soil C/N and mineralization rates in frequently burned plots support previous reports of N losses caused by fires, soil %N did not decrease with increasing fire frequency (except in 1 plot), suggesting that N losses are replenished in burned areas. However, relative abundance and N2 fixation of woody legumes decreased with fire frequency in two of the three fire gradients analyzed, suggesting that woody legume N2 fixation is not the mechanism that balances N losses. The relatively constant %N along fire gradients suggests that these ecosystems have other mechanisms to balance the N lost by fires, which could include inputs by atmospheric deposition and N2 fixation by forbs, grasses and soil cyanobacteria. Soil isotopic signatures have been previously used to infer patterns of fire history. However, the lack of a relationship between soil delta15N and fire frequency found in this study indicates that the effects of fires on ecosystem delta15N are unpredictable. Similar soil delta15N along fire gradients may reflect the contrasting effects of increased N gaseous emissions (which increases delta15N) and N2 fixation other than that associated with woody legumes (which lowers delta15N) on isotopic signatures.

Stromelysin-3 suppresses tumor cell apoptosis in a murine model.

Stromelysin-3 (STR-3) is a matrix metalloproteinase with a unique pattern of expression and substrate specificity. During embryogenesis and remodeling of normal adult tissues, STR-3 is produced by stromal cells in direct contact with epithelial cells undergoing regional apoptosis and selective cell survival. STR-3 is also overexpressed by interdigitating stromal cells in primary epithelial malignancies. Although STR-3 does not degrade classic extracellular matrix components, the enzyme promotes the establishment of local tumors in nude mice by as yet undefined mechanisms. STR-3 is induced when malignant epithelial cells come into contact with surrounding stromal elements; the active stromal cell-derived 45 kDa enzyme is subsequently processed to a 35 kDa protein without enzymatic activity. We have generated MCF-7 transfectants expressing wild type or catalytically inactive 45 kDa STR-3 (STR-3wt and STR-3cat-) or secreted 35 kDa STR-3 (35 kDa STR-3sec) and evaluated their implantation and survival in nude mice. Tumors developed significantly more rapidly in animals receiving STR-3wt, rather than vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Most importantly, STR-3wt tumors had a significantly lower percentage of apoptotic cells than tumors derived from vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Taken together, these studies suggest that the active STR-3 enzyme may increase tumor take by suppressing tumor cell apoptosis and that 45 kDa to 35 kDa STR-3 processing limits STR-3 activity at the tumor/stromal interface. Because STR-3 is secreted as an active enzyme rather than a proform, subsequent 45 kDa to 35 kDa STR-3 processing may represent a novel mechanism for regulating enzymatic activity.

The angiogenic factor interleukin 8 is induced in non-small cell lung cancer/pulmonary fibroblast cocultures.

The interactions between tumor cells and surrounding stromal elements may promote the release of angiogenic factors. Although interleukin 8 (IL-8) is a major angiogenic factor in non-small cell lung cancer (NSCLC), the stromal contribution to IL-8 expression in primary NSCLC remains to be defined. To elucidate the role of stromal elements in NSCLC IL-8 production, normal pulmonary fibroblasts were cocultured with six representative NSCLC lines in direct and transwell assays. IL-8 transcripts and protein were consistently induced in fibroblasts and a subset of NSCLCs as a consequence of tumor/stromal coculture. In these cocultures, IL-8 was induced by IL-1alpha and an additional, as yet unidentified, soluble factor. These data underscore the importance of tumor/stromal interaction in the production of angiogenic peptides such as IL-8 in NSCLC.

Stromelysin-3 is induced in tumor/stroma cocultures and inactivated via a tumor-specific and basic fibroblast growth factor-dependent mechanism.

Stromelysin-3 (STR-3) is a recently characterized matrix metalloproteinase (MMP) with a unique pattern of expression and substrate specificity. Unlike other MMPs, STR-3 is consistently and dramatically overexpressed by multiple epithelial malignancies, including carcinomas of the breast, lung, colon, head and neck, and skin. Recent studies suggest that STR-3 promotes the local establishment of epithelial malignancies, contributing to tumor cell survival and implantation in host tissues; however, STR-3's mechanism of action remains undefined. STR-3 is a stromal cell product, prompting speculation that infiltrating stromal cells secrete STR-3 in response to tumor-derived factors. To explore this possibility, we developed a tumor/"stroma" coculture assay in which non-small cell lung cancer (NSCLC) cell lines were grown on confluent monolayers of normal pulmonary fibroblasts. In these tumor/stroma cocultures, NSCLCs stimulate normal pulmonary fibroblasts to secrete STR-3 and release extracellular basic fibroblast growth factor. Thereafter, STR-3 is processed at a unique internal sequence via a basic fibroblast growth factor- and MMP-dependent mechanism to a previously unidentified 35-kDa protein that lacks enzymatic activity. 35-kDa STR-3 is the most abundant STR-3 protein in tumor/stroma cocultures and is only detected when normal pulmonary fibroblasts are cultured with malignant bronchial epithelial cells. Therefore, the tumor-specific processing of STR-3 to the 35-kDa protein is likely to be an important regulatory mechanism.

Limited-stage small cell lung cancer: a case report.

Because small cell lung cancer (SCLC) is very responsive to chemotherapy, an attempt at treatment is warranted even in poor-prognosis patients with limited-stage disease. Concurrent thoracic radiotherapy and prophylactic cranial irradiation should be considered in such cases. A case report of an elderly, debilitated patient with limited-stage SCLC is presented, and his management is discussed.

Combination therapy including a gelatinase inhibitor and cytotoxic agent reduces local invasion and metastasis of murine Lewis lung carcinoma.

The efficacy of combination therapy including an oral gelatinase inhibitor (CT1746) and cytotoxic agent was analyzed using the murine Lewis lung carcinoma model. Primary tumors, pulmonary metastases, and sera from tumor-bearing animals had increased gelatinase B activity that was inhibited by CT1746 levels achievable in vivo. The combination of CT1746 and cyclophosphamide (CTX) was significantly more effective than either single agent in delaying local tumor growth (CT1746/CTX, 30.9 +/- 1.7 days; CT1746, 2.6 +/- 0.3 days; CTX, 19.5 +/- 1.1 days; P < .001) and reducing the number and size of pulmonary metastases [CT1746/CTX, 5 +/- 2 (15% metastases > 3 mm); CT1746, 15 +/- 4 (55% > 3 mm); CTX, 11 +/- 3 (63% > 3 mm); no treatment, 24 +/- 5 (62% > 3 mm); P < .001]. These data support the notion of combining matrix metalloproteinase inhibitors and cytotoxic agents to treat certain epithelial malignancies.

Stromelysin-3 is overexpressed by stromal elements in primary non-small cell lung cancers and regulated by retinoic acid in pulmonary fibroblasts.

Stromelysin-3 (STR-3) is a recently characterized matrix metalloproteinase (MMP) that was cloned on the basis of differential expression in benign and malignant breast tumors. This MMP has a unique processing mechanism and substrate specificity. Unlike previously characterized MMPs that are secreted as inactive zymogens, STR-3 is processed within the constitutive secretory pathway and secreted as an active enzyme. Although STR-3 has a characteristic MMP structure, the enzyme does not hydrolyze many of the extracellular matrix components that are substrates for other MMPs. However, STR-3 cleaves certain serine protease inhibitors (serpins), including the alpha 1 proteinase inhibitor (alpha 1 anti-trypsin). Because alpha 1 proteinase inhibitor deficiency has a known pathogenetic role in pulmonary disease, the role of STR-3 in non-small cell lung carcinomas (NSCLC) is of great interest. STR-3 transcripts and protein were significantly more abundant in primary NSCLC than in adjacent normal lung specimens in an extensive panel of stage I-III squamous cell and adenocarcinomas. The major form of STR-3 detectable in the primary NSCLC was the mature fully processed active enzyme. STR-3 transcripts and protein were primarily localized to NSCLC stromal elements, prompting analysis of STR-3 induction in normal pulmonary fibroblasts. Although STR-3 could be induced in normal pulmonary fibroblasts with growth factors (basic fibroblast growth factor and platelet-derived growth factor) and/or 12-O-tetradecanoylphorbol-13-acetate, STR-3 induction was inhibited by all-trans retinoic acid, a commonly used chemopreventive agent for aerodigestive tract malignancies. Taken together, these data suggest that STR-3 may be a novel marker and potential therapeutic target in NSCLC.

A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis.

Soil microorganisms are important sources of the nitrogen trace gases NO and N2O for the atmosphere. Present evidence suggests that autotrophic nitrifiers such as Nitrosomonas europaea are the primary producers of NO and N2O in aerobic soils, whereas denitrifiers such as Pseudomonas spp. or Alcaligenes spp. are responsible for most of the NO and N2O emissions from anaerobic soils. It has been shown that Alcaligenes faecalis, a bacterium common in both soil and water, is capable of concomitant heterotrophic nitrification and denitrification. This study was undertaken to determine whether heterotrophic nitrification might be as important a source of NO and N2O as autotrophic nitrification. We compared the responses of N. europaea and A. faecalis to changes in partial O2 pressure (pO2) and to the presence of typical nitrification inhibitors. Maximal production of NO and N2O occurred at low pO2 values in cultures of both N. europaea (pO2, 0.3 kPa) and A. faecalis (pO2, 2 to 4 kPa). With N. europaea most of the NH4+ oxidized was converted to NO2-, with NO and N2O accounting for 2.6 and 1% of the end product, respectively. With A. faecalis maximal production of NO occurred at a pO2 of 2 kPa, and maximal production of N2O occurred at a pO2 of 4 kPa. At these low pO2 values there was net nitrite consumption. Aerobically, A. faecalis produced approximately the same amount of NO but 10-fold more N2O per cell than N. europaea did. Typical nitrification inhibitors were far less effective for reducing emissions of NO and N2O by A. faecalis than for reducing emissions of NO and N2O by N. europaea.(ABSTRACT TRUNCATED AT 250 WORDS)

Elimination of malignant clonogenic breast cancer cells from human bone marrow.

Autologous bone marrow transplantation is a promising approach to the treatment of breast cancer but is at present limited to patients without bone marrow metastases. To eliminate malignant clonogenic breast cancer cells from normal human bone marrow, immunomagnetic separation has been combined with chemoseparation using 4-hydroperoxycyclophosphamide. Breast cancer cell lines have been mixed with a 10-fold excess of irradiated human bone marrow from normal donors. Mixtures have been incubated with a combination of five different monoclonal antibodies which bind to epithelial cell surface antigens of Mr 42,000, 55,000, 72,000, 200,000, and greater than 200,000. Antiglobulin coated microspheres which contained magnetite were added, and tumor cells were trapped in a magnetic field. Elimination of tumor cells from the decanted marrow was measured in a limiting dilution assay. Two treatments with antibody and microspheres permitted elimination of 2-4 logs of clonogenic breast cancer cells, depending upon the cell line studied. Similar treatment of nonirradiated normal marrow failed to affect levels of colony forming units-granulocyte-macrophage significantly. Use of immunomagnetic purging in combination with 4-hydroperoxycyclophosphamide eliminated up to 5 logs of tumor cells but reduced the recovery of colony forming units-granulocyte-macrophage. If prompt engraftment is observed following reinfusion of similarly treated marrow in phase I trials, these techniques should permit extension of autologous bone marrow transplantation to a larger population of breast cancer patients.

Relative rates of nitric oxide and nitrous oxide production by nitrifiers, denitrifiers, and nitrate respirers.

Biogenic emissions of nitric and nitrous oxides have important impacts on the photochemistry and chemistry of the atmosphere. Although biogenic production appears to be the overwhelming source of N(2)O, the magnitude of the biogenic emission of NO is very uncertain. In soils, possible sources of NO and N(2)O include nitrification by autotrophic and heterotrophic nitrifiers, denitrification by nitrifiers and denitrifiers, nitrate respiration by fermenters, and chemodenitrification. The availability of oxygen determines to a large extent the relative activities of these various groups of organisms. To better understand this influence, we investigated the effect of the partial pressure of oxygen (pO(2)) on the production of NO and N(2)O by a wide variety of common soil nitrifying, denitrifying, and nitrate-respiring bacteria under laboratory conditions. The production of NO per cell was highest by autotrophic nitrifiers and was independent of pO(2) in the range tested (0.5 to 10%), whereas N(2)O production was inversely proportional to pO(2). Nitrous oxide production was highest in the denitrifier Pseudomonas fluorescens, but only under anaerobic conditions. The molar ratio of NO/N(2)O produced was usually greater than unity for nitrifiers and much less than unity for denitrifiers. Chemodenitrification was the major source of both the NO and N(2)O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of NO and N(2)O in nitrifier cultures but only when high concentrations of nitrite had accumulated or were added to the medium. Although most of the denitrifiers produced NO and N(2)O only under anaerobic conditions, chemostat cultures of Alcaligenes faecalis continued to emit these gases even when the cultures were sparged with air. Based upon these results, we predict that aerobic soils are primary sources of NO and that N(2)O is produced only when there is sufficient soil moisture to provide the anaerobic microsites necessary for denitrification by either denitrifiers or nitrifiers.

Further Evidence that Cytoplasmic Acidosis Is a Determinant of Flooding Intolerance in Plants.

We present two pieces of evidence that regulation of cytoplasmic pH near neutrality is a prerequisite for survival of root tips during hypoxia. First, blackeye peas and navy beans show earlier cytoplasmic acidosis under hypoxia than soybeans or pumpkin or maize, and die earlier. Second, when cytoplasmic acidosis in maize root tips is greatly retarded by treatment with 25 millimolar Ca(NO(3))(2), they remain viable under hypoxia for a much longer period of time than untreated hypoxic root tips. We also show that viability of maize root tips is unaffected by the supply of exogenous sugar (and so on the rate of ethanolic fermentation) for at least 16 hours of hypoxia.

Auto-antibody to kidney tubular cells during retrograde chronic pyelonephritis in rats.

Retrograde pyelonephritis was induced in inbred Fischer rat kidneys with Proteus mirabilis (1 X 10(8) organisms/ml). Sera from pyelonephritic animals sacrificed at 4 and 6 weeks contained cytotoxic antibodies to cultured syngeneic 51Cr-labeled kidney tubular cells (p less than 0.02 and p less than 0.05, respectively) which could be absorbed with plasma membranes. Immune sera from 4- and 6-week pyelonephritic animals also displayed a granular fluorescent pattern along the surface of cultured kidney tubular cells. Quick-frozen syngeneic rat kidney sections stained positively with fluorescent antibody on the intraluminal side of kidney tubular cells. The results suggest that during chronic phases of pyelonephritis, auto-antibodies to kidney tubular cells are induced.

Tropospheric sources of NOx: lightning and biology.

Laboratory experiments to quantify the global production of NOx (NO + NO2) in the troposphere due to atmospheric lightning and biogenic activity in soil are presented. These laboratory experiments, as well as other studies, suggest that the global production of NOx by lightning probably ranges between 2 and 20 MT(N)y-1 of NO and is strongly dependent on the total energy deposited by lightning, a quantity not well-known. In our laboratory experiments, nitrifying micro-organisms is soil were found to be a significant source of both NO and nitrous oxide (N2O). The measured production ratio of NO to N2O averaged 2-3 for oxygen partial pressures of 0.5-10%. Extrapolating these laboratory measurements to the global scale, which is somewhat risky, suggests that nitrifying micro-organisms in soil may account for as much as 10 MT(N) y-1 of NO. Additional experiments with denitrifying micro-organisms gave an NO to N2O production ratio ranging from 2 to 4 for an oxygen partial pressure of 0.5% and a ratio of less than unity for oxygen partial pressures ranging from 1 to 20%. The production of NO and N2O, normalized with respect to micro-organism number indicates that the production of both NO and N2O by denitrifying micro-organisms is at least an order of magnitude less than production by nitrifying micro-organisms for the micro-organisms studied.

In situ development of sublethal stress in Escherichia coli: effects on enumeration.

Development of sublethal stress in Escherichia coli exposed in situ to estuarine waters was examined during various seasons. An electrochemical detection technique was utilized to derive a stress index based upon the difference between a predicted electrochemical response time in Trypticase soy broth or EC medium at 44.5 degrees C estimated from a standard curve for unstressed cells and an observed response time for cells exposed to seawater. This stress index was related to recovery efficiencies of seawater-exposed cells, using a variety of standard and resuscitative enumeration procedures. Stress was further studied by determination of the adenylate energy charge. Sublethal stress as measured by the electrochemical detection method was an inverse function of water temperature, with maximum stress occurring after exposure to temperatures below 10 degrees C. Total adenylates and ATP decreased dramatically at low temperatures, although energy charge remained relatively constant under various environmental conditions. Decreases in E. coli ATP suggest that ATP may not be an adequate measure of biomass for in situ stressed cells. Discrepancies in enumeration efficiency were most pronounced at temperatures below 10 degrees C. Resuscitative procedures for solid-media techniques increased the recovery of stressed cells under cold water conditions but were not as effective as the standard most-probable-number procedure.