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TEM-1 hyperproduction in two ampicillin-sulbactam-resistant Shigella flexneri strains was studied. In both strains the blaTEM gene was encoded as a single copy on a large conjugatively transferable plasmid. A single G-->T transversion at position 1 of the -10 consensus sequence was identified to be the mechanism of TEM-1 hyperproduction.
We have identified the gene for transcription termination factor Rho in Staphylococcus aureus. Deletion of rho in S. aureus reveals that it is not essential for viability or virulence. We also searched the available bacterial genomic sequences for homologs of Rho and found that it is broadly distributed and highly conserved. Exceptions include Streptococcus pneumoniae, Streptococcus pyogenes, Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum, and Synechocystis sp. strain PCC6803, all of which appear not to possess a Rho homolog. Complementation studies indicate that S. aureus Rho possesses the same activity as Escherichia coli Rho and that the Rho inhibitor bicyclomycin is active against S. aureus Rho. Our results explain the lack of activity of bicyclomycin against many gram-positive bacteria and raise the possibility that the essentiality of rho may be the exception rather than the rule.
Pediococcus species isolated from forage crops were characterized, and their application to silage preparation was studied. Most isolates were distributed on forage crops at low frequency. These isolates could be divided into three (A, B, and C) groups by their sugar fermentation patterns. Strains LA 3, LA 35, and LS 5 are representative isolates from groups A, B, and C, respectively. Strains LA 3 and LA 35 had intragroup DNA homology values above 93.6%, showing that they belong to the species Pediococcus acidilactici. Strain LS 5 belonged to Pediococcus pentosaceus on the basis of DNA-DNA relatedness. All three of these strains and strain SL 1 (Lactobacillus casei, isolated from a commercial inoculant) were used as additives to alfalfa and Italian ryegrass silage preparation at two temperatures (25 and 48 degrees C). When stored at 25 degrees C, all of the inoculated silages were well preserved and exhibited significantly (P < 0.05) reduced fermentation losses compared to that of their control in alfalfa and Italian ryegrass silages. When stored at 48 degrees C, silages inoculated with strains LA 3 and LA 35 were also well preserved, with a significantly (P < 0.05) lower pH, butyric acid and ammonia-nitrogen content, gas production, and dry matter loss and significantly (P < 0.05) higher lactate content than the control, but silages inoculated with LS 5 and SL 1 were of poor quality. P. acidilactici LA 3 and LA 35 are considered suitable as potential silage inoculants.
Archaeal community structures in microhabitats in a deep-sea hydrothermal vent chimney structure were evaluated through the combined use of culture-independent molecular analyses and enrichment culture methods. A black smoker chimney was obtained from the PACMANUS site in the Manus Basin near Papua New Guinea, and subsamples were obtained from vertical and horizontal sections. The elemental composition of the chimney was analyzed in different subsamples by scanning electron microscopy and energy-dispersive X-ray spectroscopy, indicating that zinc and sulfur were major components while an increased amount of elemental oxygen in exterior materials represented the presence of oxidized materials on the outer surface of the chimney. Terminal restriction fragment length polymorphism analysis revealed that a shift in archaeal ribotype structure occurred in the chimney structure. Through sequencing of ribosomal DNA (rDNA) clones from archaeal rDNA clone libraries, it was demonstrated that the archaeal communities in the chimney structure consisted for the most part of hyperthermophilic members and extreme halophiles and that the distribution of such extremophiles in different microhabitats of the chimney varied. The results of the culture-dependent analysis supported in part the view that changes in archaeal community structures in these microhabitats are associated with the geochemical and physical dynamics in the black smoker chimney.
Outbreaks of listeriosis and febrile gastroenteritis have been linked to produce contamination by Listeria monocytogenes. In order to begin to understand the physiology of the organism in a produce habitat, the ability of L. monocytogenes to attach to freshly cut radish tissue was examined. All strains tested had the capacity to attach sufficiently well such that they could not be removed during washing of the radish slices. A screen was developed to identify Tn917-LTV3 mutants that were defective in attachment to radish tissue, and three were characterized. Two of the three mutations were in genes with unknown functions. Both of the unknown genes mapped to a region predicted to contain genes necessary for flagellar export; however, only one of the two insertions caused a motility defect. The third insertion was found to be in an operon encoding a phosphoenolpyruvate-sugar phosphotransferase system. All three mutants were defective in attachment when tested at 30 degrees C; the motility mutant had the most severe phenotype. However, not all of the mutants were defective when tested at other temperatures. These results indicate that L. monocytogenes may use different attachment factors at different temperatures and that temperature should be considered an important variable in studies of the molecular mechanisms of Listeria fitness in complex environments.
The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.
The need for protozoa for the proliferation of Legionella pneumophila in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of L. pneumophila in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous L. pneumophila originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-microm pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable L. pneumophila, and for their concentrations of protozoa. L. pneumophila grew to high numbers (6.3 log CFU/cm2) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of Hartmannella vermiformis in all flasks in which L. pneumophila multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of L. pneumophila in trophozoites of H. vermiformis, with 25.9% +/- 10.5% of the trophozoites containing L. pneumophila on day 10 and >90% containing L. pneumophila on day 14. Calculations confirmed that intracellular growth was most likely the only way for L. pneumophila to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher L. pneumophila concentrations, and therefore the growth of L. pneumophila within engineered water systems can be limited by controlling biofilm formation.
We studied the impact of grazing and substrate supply on the size structure of a freshwater bacterial strain (Flectobacillus sp.) which showed pronounced morphological plasticity. The cell length varied from 2 to >40 microm and encompassed rods, curved cells, and long filaments. Without grazers and with a sufficient substrate supply, bacteria grew mainly in the form of medium-sized rods (4 to 7 microm), with a smaller proportion (<10%) of filamentous forms. Grazing experiments with the bacterivorous flagellate Ochromonas sp. showed that freely suspended cells of <7 microm were highly vulnerable to grazers, whereas filamentous cells were resistant to grazing and became enriched during predation. A comparison of long-term growth in carbon-limited chemostats with and without grazers revealed that strikingly different bacterial populations developed: treatments with flagellates were composed of >80% filamentous cells. These attained a biomass comparable to that of populations in chemostats without grazers, which were composed of medium-sized rods and c-shaped cells. Carbon starvation resulted in a fast decrease in cell length and a shift towards small rods, which were highly vulnerable to grazing. Dialysis bag experiments in combination with continuous cultivation revealed that filament formation was significantly enhanced even without direct contact of bacteria with bacterivores and was thus probably stimulated by grazer excretory products.
Yersinia enterocolitica is an enteric pathogen capable of causing systemic disease in a murine model. We have identified a novel protein, systemic factor protein A (SfpA), conserved in other pathogenic bacteria that is involved in systemic disease. Analysis of bacterial colonization revealed that a DeltasfpA strain is defective in mesenteric lymph node colonization. Bioinformatics and functional studies suggest that SfpA is a porin.
The importance of individual amino acids in mediating the broad-spectrum bactericidal action of a 20-mer amphipathic, cationic peptide (CG 117-136) of human lysosomal cathepsin G was determined by using a single amino acid replacement strategy. This strategy revealed an important role for arginine because loss of any of the four arginine residues in CG 117-136 due to substitution with alanine, citrulline, or lysine residues resulted in a reduction of its bactericidal activity against both Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 33593. However, the replacement of a single alanine residue in CG 117-136 with arginine, but not glutamic acid, enhanced the activity of CG 117-136 against both P. aeruginosa and S. aureus. The importance of certain bulky, nonpolar amino acids for the bactericidal activity of CG 117-136 was also evident, since their substitutions by alanine diminished bactericidal activity. Accordingly, contributions of hydrophobic amino acids and structural considerations of the guanidinium side chain of arginine are major determinants in the broad-spectrum antimicrobial action of CG 117-136.
We have compared the sensitivity and specificity of quantitative mycobacterial culture against results obtained by using the polymerase chain reaction for the detection of DNA from organisms of the Mycobacterium tuberculosis complex in 82 clinical specimens from patients suspected of having tuberculosis. Two amplification protocols were used, a standard amplification protocol, which amplifies a segment of the gene coding for the 65-kDa antigen, and a protocol in which the initial amplification products are reamplified with a second set of nested oligonucleotide primers. Although the standard amplification protocol gave positive results for 18 of 18 samples which grew greater than 100 CFU/ml and gave positive results in 4 of 35 specimens from patients with tuberculosis which were negative by culture, only 1 of 6 samples which grew less than 100 CFU/ml was positive. This lack of sensitivity could not be explained by the presence of inhibitors of Taq polymerase present in the original samples. In contrast, the reamplification protocol gave positive results for 24 of 24 samples which were positive by culture as well as for 13 of 35 samples from patients with tuberculosis which were negative by culture (overall sensitivity, 63%, P less than 0.02, compared with the standard amplification protocol and routine culture). Two of 23 samples from patients not diagnosed as having tuberculosis gave positive results when the standard amplification protocol was used, but no additional false-positive results were seen with the reamplification protocol (overall specificity, 91%). We conclude that the use of a reamplification protocol improves the sensitivity of detection of mycobacterial DNA in clinical samples without sacrificing specificity. The sensitivity of this approach appears to be superior to that of standard culture techniques.
The dominant faecal flora of the rat was determined using randomly cloned 16S rDNA comparative sequence analysis. A total of 109 near full-length 16S rDNA clones were sequenced, representing 69 unique 16S rRNA phylotypes or operational taxonomic units (OTUs). Estimates of species richness indicated that approximately 338 species were present in the faeces, suggesting that only 20% of species were identified. Only two of 39 Gram-negative clones aligned with previously cultured species, the remainder fell into a separate lineage within the Bacteroides-Cytophaga phylum. Several clones within this new group were related to 16S rDNA sequences previously identified from mouse faeces. Lactobacilli were the most abundant Gram-positive species, representing 23% of the total clones but only 7% of OTUs. The remaining Gram-positive clones were distributed among the Clostridium coccoides group (9%), the Clostridium leptum subgroup (18%), and throughout the low GC Gram-positive bacteria (13%). The majority of OTUs (63/69 or 91%) were less than 97% homologous to previously cultured bacteria. Faecal samples were also cultured using a variety of anaerobic media. With the exception of the lactobacilli, the cultured isolates demonstrated low species diversity and poorly reflected the population, as defined through comparative sequence analysis.
It has been suggested that the induction of lipocortin-1, a phospholipase A2-inhibitory protein, may mediate the anti-inflammatory action of glucocorticoids. We assessed the production of prostaglandin E2, thromboxane B2, and leukotriene B4 and the expression of lipocortin-1 in different populations of blood leukocytes and in alveolar macrophages (obtained by bronchoalveolar lavage) from patients with inflammatory lung diseases (bronchial asthma, n = 21; interstitial lung disease, n = 6) undergoing glucocorticoid treatment at clinically effective doses. No inhibition of eicosanoid production was observed in either whole blood or single populations of blood leukocytes (granulocytes and monocytes) stimulated with ionophore A-23187, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, or zymosan. Conversely, eicosanoid production from alveolar macrophages (assessed in 9 cases) was significantly inhibited by glucocorticoids. After ionophore stimulation, eicosanoid production was as follows (in ng/ml): prostaglandin E2, 0.19 +/- 0.06 and 0.06 +/- 0.01; thromboxane B2, 2.9 +/- 0.9 and 0.5 +/- 0.1; leukotriene B4, 6.6 +/- 1.1 and 3.6 +/- 1.0, before and after treatment, respectively (P < 0.05 for all differences). Lipocortin-1 expression, determined by Western blot and enzyme immunoassay, was significantly (P < 0.05) stimulated in alveolar macrophages, but not in blood leukocytes, by glucocorticoid treatment. These results indicate that alveolar macrophages, at variance from blood leukocytes, are the most likely cell target for glucocorticoid-induced eicosanoid inhibition and lipocortin expression. We suggest that cell responsiveness to glucocorticoids is acquired during differentiation from monocyte to tissue macrophage.
OBJECTIVE: In the present study we investigated the cytologic and colposcopic characteristics of a cohort of HIV-infected women, with the aim to determine a relationship between immunologic status and frequency and/or severity of cervical abnormalities. MATERIALS AND METHODS: 21 women, who tested positive for the HIV antibody and who were admitted as outpatients because of various gynecologic complications or because of an HIV infection that was under regular clinical surveillance. A pelvic examination was performed and Papanicolaou smears were obtained from endocervix and ectocervix before colposcopic examination. Cytologic samples for human papillomavirus (HPV) detection by polymerase chain reaction were also collected. Results obtained in the group of HIV-infected women were compared with findings in a group of 473 seronegative women recruited consecutively from our outpatient population. Serum samples for T lymphocytes were drawn within 2 weeks of cytologic and colposcopic examination. CD4 and CD8 monoclonal antibodies were purchased from Becton Dickinson (Mountain View, Calif., USA). RESULTS: HIV-infected women had a significantly higher percentage of HPV DNA positivity with respect to the outpatient population (67 vs. 7%, respectively, p < 0.001). Analysis of cytologic specimens revealed 9 women (43%) with cytologic evidence of cervical dysplasia in the HIV-seropositive group vs. 23 (5%) of 473 in the outpatient population (p < 0.001). All the HIV-seropositive women with cervical dysplasia presented an associated HPV DNA positivity; in particular, the percentage of associated HPV DNA type 16 in cervical dysplasia was 78% (7/9 cases). In HIV-infected women, the evaluation of T lymphocyte subset distribution suggested a significant relationship between CD4+ cell decrease and severity of cytologic findings (p = 0.03). DISCUSSION: The HIV-infected women had a tenfold higher prevalence of both HPV infection and cervical dysplasia than the outpatient population; this increased risk seems to be limited mainly to those who also had genital HPV infection. The analysis of immunologic status confirmed previous observations that an impaired immune system results in increased cervical disease.
BACKGROUND: Short tandem repeats (STRs) are repetitive DNA sequences with 1–6 nucleotide repeat units, exhibiting high polymorphism due to varying repeat counts. STRs are more variable than SNPs and can cause genetic disorders. With population-scale cattle whole-genome sequencing data available, whole-genome STR identification has attracted new interest, but challenges remain due to the lack of standardized methods, sequencing data limitations, and the diversity of STR-calling tools. This study compared six STR-calling tools: HipSTR, GangSTR, and ExpansionHunter for short-read data, and Straglr, RepeatHMM, and LongTR for Oxford Nanopore (ONT) long-read data—using sequences from five Holstein cattle (two parent–offspring trios with a shared sire). This is the first cattle study to evaluate short- and long-read STR callers using both data types from the same animals. RESULTS: In short-read data, ExpansionHunter identified the highest number of polymorphic STRs (pSTRs) (327,690), followed by HipSTR (205,900) and GangSTR (110,680), with 93,023 loci detected by all three tools. In long-read data, LongTR detected 470,250 pSTRs, RepeatHMM 224,185, and Straglr 90,275, with only 33,253 loci shared among them. Mendelian consistency of STR genotypes in the trio offspring was high (> 0.8) for all short-read tools, with HipSTR and GangSTR highest at 0.98. LongTR was the only long-read tool with high consistency (0.88). Short-read tools also showed higher concordance in STR genotypes among themselves than was observed among long-read tools. However, long-read tools had a clear advantage in detecting large STRs. Relative to computational efficiency, HipSTR and GangSTR (short-reads), and LongTR (long-reads) required less memory and shorter runtimes than the other tools. CONCLUSIONS: Tool selection is critical for accurate whole-genome STR identification in cattle. For short-read data, HipSTR showed relatively high Mendelian consistency and concordance compared to the other tools, while ExpansionHunter was able to detect longer STRs but with lower Mendelian consistency. For long-read data, LongTR demonstrated higher consistency and computational efficiency relative to the other tools. Based on these results, HipSTR and LongTR are suggested as preferred options for short-read and ONT long-read datasets, respectively, in cattle STR analysis. These recommendations are based on the metrics observed in this study, and confirmatory analyses across additional breeds, larger sample sizes, and validated truth sets are encouraged.
PURPOSE: We have investigated whether the extent of residual leukemia in the marrows obtained at the completion of chemotherapy can predict subsequent relapse in children with B-lineage acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Marrow samples of 24 patients were examined for residual disease at the end of treatment using a quantitative method based on the polymerase chain reaction (PCR) amplification of the complementarity determining region-3 of the immunoglobulin heavy chain. RESULTS: Of the 15 patients who remain in continuous bone marrow remission (range, 41 to 98 months), 14 had no detectable leukemic cells; one patient had a very low level (one in approximately 335,000 marrow cells) of residual leukemic cells that underwent clonal evolution. Among the nine patients who had a marrow relapse after the completion of treatment, eight patients whose relapses occurred 4 to 54 months from the end of therapy had no detectable leukemic cells, whereas only the one patient who relapsed 2 months after the completion of therapy had detectable residual disease. CONCLUSION: These observations indicate that the absence of detectable residual leukemia by PCR at the end of chemotherapy is not sufficient to assure that the patient is cured and suggest that frequent serial monitoring is required for the early prediction of relapse off therapy.
The proteose-peptone fraction was studied to measure proteolysis in 86 samples of quarter milk from 31 cows with subclinical mastitis. The relative content of component PP3 decreased significantly in the total of proteose-peptone as plasmin activity increased, but the content of this component in milk was not correctly correlated with the plasmin activity (r = .52). Electrophoresis, amino acid composition, and sequence analysis showed that the component beta-CN-1P (f29-105/7) of the proteose-peptone fraction was a terminal product of plasmin-like activity and was located correctly electrophoretically. Correlation factors between the plasmin activity and content in milk of component beta-CN-1P (f29-105/7) was very high (r = .87). This component could be used as indicator of the endogenous proteolysis in milk from cows with subclinical mastitis.
CTLA-4, a homologue of CD28, is a negative regulator of T cell activation in the periphery and is transiently expressed on the cell surface after T cell activation. However, the role of CTLA-4 in T cell activation in the thymus is not clear. This investigation was initiated to determine the role of CTLA-4 in the activation of CD4(+)CD8(+) double-positive (DP) and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes using fetal thymic organ cultures (FTOC) of MHC class II-restricted, OVA(323-339)-restricted TCR transgenic mice (DO11.10). We found that treatment of the FTOC with anti-CTLA-4-blocking Ab during activation with OVA(323-339) increased the proportion and number of DP thymocytes, but decreased the proportion and number of SP thymocytes compared with OVA(323-339)-stimulated FTOC without anti-CTLA-4 Ab treatment. In addition, anti-CTLA-4 Ab treatment inhibited OVA(323-339)-induced expression of the early activation marker, CD69, in DP thymocytes, but increased CD69 in SP thymocytes. Similarly, CTLA-4 blockage decreased phosphorylation of ERK in DP thymocytes by Ag-specific TCR engagement, but increased phosphorylation of ERK in SP thymocytes. CTLA-4 blockage inhibited deletion of DP thymocytes treated with a high dose of OVA(323-339), whereas CTLA-4 blockage did not inhibit deletion of DP thymocytes treated with a low dose of OVA(323-339). We conclude that CTLA-4 positively regulates the activation of DP thymocytes, resulting in their deletion, whereas blocking CTLA-4 suppresses the activation of DP thymocytes, leading to inhibition of DP thymocyte deletion. In contrast, CTLA-4 negatively regulates the activation of SP thymocytes.