Importance of Medium in Demonstrating Penicillin Tolerance by Group B Streptococci

A total of 30 clinical isolates of group B streptococci were studied for penicillin tolerance in vitro. Minimal inhibitory and bactericidal concentrations of penicillin were determined simultaneously in three test media which have been used for group B streptococci, tryptose phosphate, Mueller-Hinton, and Todd-Hewitt broths, using a logarithmic-phase inoculum of 10(5) colony-forming units per ml. Minimal inhibitory concentrations in the three media did not differ significantly. However, minimal bactericidal concentrations were significantly higher in tryptose phosphate broth (mean, 1.04 mug/ml) than in Mueller-Hinton broth (0.22 mug/ml) or Todd-Hewitt broth (0.15 mug/ml). Similarly, ratios of minimal bactericidal to minimal inhibitory concentrations were significantly greater in tryptose phosphate broth than in Mueller-Hinton or Todd-Hewitt broth. After incubation in tryptose phosphate broth for an additional 24 h, the minimal bactericidal concentration consistently fell to levels which were only twice or equal to the minimal inhibitory concentration. This study illustrates the importance of the medium in the demonstration of penicillin tolerance and of controlling laboratory variables in the susceptibility testing of group B streptococci with penicillin.     

Effects of erythromycin in combination with penicillin, ampicillin, or gentamicin on the growth of Listeria monocytogenes.

Since the optimal antimicrobial therapy for infections caused by Listeria monocytogenes, particularly in patients allergic to penicillin, is uncertain, we investigated the in vitro effects of erythromycin, alone and in combination with other antibiotics, on listeriae. Seven strains of listeriae were inhibited but not killed by erythromycin, penicillin G, or ampicillin when tested by a microtiter broth dilution method. Susceptibility to gentamicin decreased when tryptose phosphate broth was substituted for Mueller-Hinton broth, but was independent of their calcium and magnesium concentrations. Quantitative killing studies performed with erythromycin combined with either penicillin G or ampicillin yielded antagonism for all strains, in contrast to microtiter checkerboard determinations, which did not indicate antagonism in all instances. Antagonism occurred with strains in both the stationary and log phases of growth and was slightly reversed by a 120-min preincubation of the listeriae with penicillin before the addition of erythromycin. Erythromycin and gentamicin were antagonistic in quantitative killing studies. Based on these in vitro findings, we conclude that the addition of gentamicin to erythromycin offers no advantage in the treatment of listeriosis in the penicillin-allergic patient.     

CYTOTOXIC EFFECTS OF SOME MINERAL DUSTS ON SYRIAN HAMSTER PERITONEAL MACROPHAGES

Hamster peritoneal macrophages were grown in cell culture and their response to various conditions was examined. The cultures responded favorably to high concentrations of serum and to medium which had been preconditioned by contact with tumor cells. After 2–3 days of adaptation, they entered into a period of stability which lasted from the 4th to the 9th day. Macrophage cultures in this stable phase were treated with various samples of mineral dusts and their response determined by counting the number of viable macrophages/cm² at intervals over a period of 72 hr. Crystalline silica Snowit was found to be nontoxic. Amorphous silica Fransil caused a characteristic cytotoxic effect and a rapid decline in cell population at doses less than 150 µg/5 x 10⁵ cells. Of the three different kinds of asbestos used, chrysotile was toxic and amosite and crocidolite nontoxic at equivalent concentrations. A comparison of two preparations of chrysotile which differed in surface area showed that weight rather than surface area determines toxicity. Pretreatment of chrysotile with tryptose phosphate broth under drastic conditions accelerated but did not increase the final intensity of the cytotoxic effect.     

Utilization of phosphate rock as a sole source of phosphorus for uranium biomineralization mediated by Penicillium funiculosum

In this work, uranium(vi) biomineralization by soluble ortho-phosphate from decomposition of the phosphate rock powder, a cheap and readily available material, was studied in detail. Penicillium funiculosum was effective in solubilizing P from the phosphate rock powder, and the highest concentration of the dissolved phosphate reached 220 mg L⁻¹ (pH = 6). A yellow precipitate was immediately formed when solutions with different concentrations of uranium were treated with PO₄³⁻-containing fermentation broth, and the precipitate was identified as chernikovite by Fourier transform infrared spectroscopy, scanning electron microscope, and X-ray powder diffraction. Our study showed that the concentrations of uranium in solutions can be decreased to the level lower than maximum contaminant limit for water (50 μg L⁻¹) by the Environmental Protection Agency of China when Penicillium funiculosum was incubated for 22 days in the broth containing 5 g L⁻¹ phosphate rock powder.

In this work, uranium(vi) biomineralization by soluble ortho-phosphate from decomposition of the phosphate rock powder, a cheap and readily available material, was studied in detail.     

VISA–Daptomycin non-susceptible Staphylococcus aureus frequently demonstrate non-susceptibility to Telavancin

Telavancin was evaluated against S. aureus isolates with reduced susceptibility to other antimicrobial agents using two broth microdilution methods and Etest® strips. The three methods provided comparable results. Differences in telavancin susceptibility versus non-susceptibility were noted mainly in the VISA-daptomycin non-susceptible group of isolates. In this group the percent susceptibility was 38% for the Etest® method and 50% and 54% for the 2 broth microdilution methods. All differences in susceptibility were within one 2-fold dilution.     

<Emphasis Type="Italic">Rhizobium</Emphasis> Mutant Deficient in Mineral Phosphate Solubilization Activity Shows Reduced Nodulation and Plant Growth in Green Gram

The aim of the present study was to understand the mineral phosphate solubilization (MPS) in Rhizobium sp. MR-54 (MPS⁺) by using forward genetic approach and its effect on nodulation as well as nitrogen accumulation on green gram. Tn5 mutants (MPS^?) were generated from the MPS⁺ strain by Tn5 mutagenesis. Screening 1,603 colonies of MR-54::Tn5 resulted in identification of those mutants which failed to show any solubilization zone up to 72 h after incubation on Tri-calcium phosphate medium (TCP). pH drops and Pi release were highly correlated up to 15 days after incubation in TCP broth. Gluconic acid was detected in the culture supernatants of MPS⁺ isolates through thin layer chromatography indicating the presence of direct oxidation pathway in MR-54 strain. Detailed characterization of mutants was carried out for related properties like colony morphology, Pi release and pH change in TCP broth, organic acid production, surface characteristics and plant growth promoting ability etc. Mutants deficient in MPS showed reduced nodulation and nitrogen accumulation in green gram under green house conditions in leonard jar apparatus over the wild strain indicating the MPS by Rhizobium being involved in nodulation, nitrogen accumulation and plant growth.     

Medium-dependent properties of mycoplasmas

Without a cell wall, the morphology, growth rate, and composition of mycoplasmas are culture media-dependent with variable properties best desribed as environmentally related. The adaptation of mycoplasmas to either a tissue cell or cell-free culture media, with dependency upon specific animal or plant products for survival, has led to investigations of their human host-related properties. The influence of culture media on the antibiotic sensitivities of mycoplasmas was measured by use of three different broths in two different assay systems. The variable results indicate that the inhibition of mycoplasma protein synthesis or growth may also be host-tissue dependent. The addition of noninhibitory penicillins to different culture media was found to affect the composition and antigenicity of some mycoplasmas. Using the complement fixation test, we found some human sera that were more reactive than rabbit antisera to mycoplasmas cultured in human synovial broth or in myelin-enriched broth. Mycoplasmas cultured in human lung broth and pig lung broth had media-dependent antigenicity. The antigenicity and the growth of mycoplasmas were found to depend on the proteolytic enzymes used to provide the essential peptides in tissue broths. The media-affected mycoplasmas indicate the presence of species-, strain-, and tissue-specific antigen sites that may determine immunopathogenicity in the genetically susceptible host.     

Susceptibility testing of anaerobic bacteria

With the significant increase in resistance of anaerobic bacteria to antimicrobial agents in recent years, susceptibility testing of these organisms becomes very important. In addition to survey studies, hospitals should be determining their local patterns, and therapy of at least the more serious infections should be guided by susceptibility tests. Of the various tests available, those most suitable for smaller hospitals for guidance of patient management are the broth disc elution procedure and the micro broth dilution tray test.     

Preservation of acid phosphatase activity in medico-legal specimens

Vaginal acid phosphatase has been preserved with a protective broth containing, per liter, 50 of bovine albumin, 0.2 g of sodium azide, 10 mmol of phosphate (pH 7.4), and 9.0 g of NaCl. Samples may be maintained at ambient temperature for one month without loss of activity. Several other commonly used preservative methods are compared and are shown to be inadequate. With a constant 2.5 ml volume of the support medium, and use of a sodium thymolphthalein monophosphate method (Worthington Diagnostics), vaginal acid phosphatase activity in non-coital women is less than 10 U/liter of broth, and in recently post-coital women is more than 50 U/liter (242 +/- 104 U/liter). In vivo degradation of vaginal activity follows a nearly logarithmic course until four days after intercourse, when it reaches nearly normal values.     

Minimal inhibitory concentrations of broad spectrum beta-lactam antibiotics for bacterial control strains

The minimal inhibitory concentrations of 21 broad spectrum beta-lactam antibiotics for five bacterial control strains, as determined by a microtube method in cation-supplemented Mueller-Hinton broth, are reported.     

Staphylococci, in vitro and in vivo

Strains of Staphylococcus aureus were grown in broth and by the membrane technique; both drug-free media and media containing cloxacillin were used. The staphylococci grown in broth containing cloxacillin showed one thick cross wall and were larger than those grown in drug-free broth: 1.6 micron in diameter as opposed to 0.9 micron. The staphylococci grown on membranes placed on agar containing cloxacillin were 2-3 microns in diameter and contained three or more cross walls. Mice were infected intraperitoneally with staphylococci. After treatment with cloxacillin, the peritoneal fluid and spleens contained staphylococci that were 2-3 microns in diameter with three or more cross walls. A staphylococcal endocarditis was induced in rabbits that were then treated with cloxacillin. The staphylococci in the vegetation of the treated rabbits were 2-3 microns in diameter and contained multiple cross walls. Large staphylococci with multiple cross walls were observed in specimens from patients with respiratory infections treated with beta-lactam antibiotics. It appears, therefore, that the ultrastructure of staphylococci in vivo is comparable to that of staphylococci grown on a solid support medium such as a membrane, and different from that of staphylococci grown in a liquid medium.     

Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool

Detection of diarrheagenic Escherichia coli (DEC) typically depends on identification of virulence genes from stool cultures, not on stool itself. We developed a multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA). The assay involved a multiplex PCR reaction followed by detection of amplicon(s) using Luminex beads. The assay was evaluated on over 100 colony and broth specimens. We then evaluated the assay using DNA extracted from stool, colony pools, and Gram-negative broths, using stool spiked with known quantities of DEC. Performance of the assay on stool DNA was most quantitative, while stool broth DNA offered the lowest limit of detection. The assay was prospectively evaluated on clinical specimens in Tanzania. Stool DNA yielded higher sensitivity than colony pools compared with broth DNA as the standard. We propose using this assay to screen for DEC directly in stool or stool broths.     

STUDIES ON OXIDATION AND REDUCTION BY PNEUMOCOCCUS : II. THE PRODUCTION OF PEROXIDE BY STERILE EXTRACTS OF PNEUMOCOCCUS.

In the present paper methods have been described for the preparation of sterile extracts of pneumococci. These extracts may be obtained by dissolving the bacteria in broth cultures by means of bile, or by extraction of the cellular substances by repeated freezing and thawing of broth or saline suspensions of unwashed cells. Under special precautions these extracts may be passed through Berkefeld filters without loss of potency. In this procedure, as in all other manipulations incident to their preparation, the extracts should be protected as far as possible from contact with air. All extracts were proved sterile by cultural and animal tests. Sterile extracts of unwashed pneumococcus cells promptly form peroxide on exposure to air. Peroxide formation is almost as active in extracts aerated at 2°C. as in those exposed to the air at room temperature. Detectable amounts of peroxide may be produced by these cell extracts within the reaction range of pH 5 to 9, the optimal zone lying at reactions less acid than pH 6. The peroxide-forming activity of the extracts is gradually diminished by prolonged exposure to 55°C., and is completely destroyed by heating at 65°C. for 5 minutes. Cell extracts of pneumococci which have been thoroughly washed prior to extraction in salt or phosphate solutions exhibit no peroxide-forming activity. These extracts of washed cells may be activated by the addition of the cell washings, yeast extract, or muscle infusion.     

Temocillin: In vitro activity against 734 selected clinical isolates,including β-lactamase-producing strains

The in vitro activity of temocillin against 734 clinical isolates was tested by broth microdilution. Good activity was demonstrated against Enterobacteriaceae and both β-lactamase-positive and -negative strains of $\text{Haemophilus influenzae}$ and $\text{Neisseria gonorrhoeae}$. There was little to no activity against gram-positive cocci and nonfermenting gram-negative bacilli. Bactericidal activity and effect of inoculum size on temocillin activity were comparable to that of ticarcillin. Temocillin was stable to commonly encountered β-lactamases and significantly inhibited Richmond-Sykes type 1 enzymes of $\text{Enterobacter cloacae}$.     

Activity in vitro of CGP 31608, a new penem antibacterial agent

The in-vitro activity of CGP 31608 (hereinafter termed CGP), a new penem, was tested by an agar dilution technique in comparison with imipenem, Sch 34343, cefotaxime, ceftazidime, aztreonam, ampicillin, gentamicin and ciprofloxacin. 480 clinical isolated were tested, some of which were selected because of their multiple resistance. CGP showed consistent activity against a wide range of species, having MIC90 values of 2-8 mg/l for almost all Enterobacteriaceae, Pseudomonas spp., Haemophilus spp., Corynebacterium spp. and Bacteroides spp. It was the most active agent tested against staphylococci having an MIC90 of 0.25 mg/l, showing no reduction in activity against methicillin-resistant strains. Lesser activity was observed against some streptococci, Proteus spp. and clostridia. Tests carried out in broth demonstrated that CGP activity was constant over a pH range of 6-8 and was unaffected by the presence of 50% serum or 50% urine. The rate of killing of CGP, gentamicin, cefotaxime and ciprofloxacin was investigated in broth against log and stationary-phase cultures of Staphylococcus aureus and Escherichia coli. The most rapid rate of kill was seen with ciprofloxacin, while CGP exhibited a more rapid bactericidal effect than cefotaxime against Staph. aureus. The stability of CGP was studied at two concentrations in serum, broth and phosphate buffer at 4 degrees C, room temperature and 37 degrees C. In serum the half-life was 112 h at 4 degrees C, 35 h at room temperature and 11.4 h at 37 degrees C. Protein binding tested at concentrations of 5-100 mg/l was 2-6.3%.     

Susceptibility testing with the sensititer breakpoint broth microdilution system

The antimicrobial susceptibility profiles of a total of 318 aerobic and facultatively anaerobic bacteria (255 gram-negative bacilli and 63 gram-positive cocci) were determined, using a new commercially available breakpoint broth microdilution procedure (Sensititer Breakpoint System (SBS), Gibco Diagnostics, Inc., Madison, WI) that categorizes test results in the form of susceptibility categories: susceptible, intermediate, and resistant. Results obtained with the SBS were compared with those achieved with a standardized disk diffusion procedure. Among a total of 4,414 organism-antimicrobic comparisons, concordance between the results of the SBS and the disk diffusion procedure was observed in 3,888 cases (88.1%). Four hundred twenty-three (9.6%) minor discrepancies, 45 (1.0%) major discrepancies, and 58 (1.3%) very major discrepancies were noted. Arbitration of major and very major discrepancies with a full-range minimum inhibitory concentration (MIC) procedure confirmed the results of the SBS in 53.4% of cases. A single organism-antimicrobial combination, the nonenterococcal streptococci tested against the aminoglycosides, yielded a significant number of very major errors which were arbitrated in favor of the disk diffusion result. These errors were probably due to poor growth of the test organism in the broth medium used for performing the SBS test (i.e., cation-supplemented Mueller-Hinton broth). With this exception, the SBS was found to be at least as accurate as the standardized disk diffusion procedure.     

泌尿生殖道解脲支原体感染的检测方法与诊断

目的 评价解脲支原体(Uu)液体培养法的诊断价值.方法 采集130例患者的泌尿生殖道分泌物、前列腺液,同时进行液体培养法和荧光定量聚合酶链反应(FQ-PCR)检测Uu,并对Uu阳性和判为污染的液体培养基进行细菌培养.然后,比较结果、分析原因.结果 130份标本中,液体培养法Uu阳性71份、判为污染的13份、阴性46份.FQ-PCR法Uu阳性66份,其中63份与液体培养法一致,另3份中,2份为液体培养法判为污染的标本、1份为液体培养法阴性标本.细菌培养结果,8份液体培养阳性而FQ-PCR法阴性的标本及13份判为污染的标本,均培养出细菌和/或真菌,菌株做脲酶和精氨酸分解试验,至少一项为阳性.结论 用液体培养法检测Uu时,阴性标本可能受分解尿素、精氨酸的细菌的影响,而误判为阳性,阳性标本可能受混浊生长的细菌影响,而误判为阴性,从而导致诊断上的错误.     

Multiple forms of renin substrate in human plasma

When adapting the immediate bromcresol green method for urinary albumin determination a correlation study with the Mancini single radial immunodiffuon (SRID) method was performed. This study showed large disparities between the two methods, SRID giving the higher results. The unsuitability of the currently used SRID methods is demonstrated and improvements to the method are suggested.

Known amounts of albumin were added to urine samples as well as to a “synthetic urine”, both giving falsely elevated results with the SRID method. On investigating the different components of the “synthetic urine”, it was found that the disparities were due to the influence of citrate and phosphate.

On addition of citric acid or phosphate to the dilution buffer and/or the gel buffer, the results of the SRID method agreed with those of other methods and with the expected values.

The findings presented in this paper can probably be extended to other immunological methods too since it seems to be the antigen-antibody reaction which is affected.     

Evaluation of a Commercial Microdilution System for Quantitative Susceptibility Testing of Aminoglycosides Against Multidrug-Resistant, Gram-Negative Bacilli

Susceptibility of clinical isolates of Pseudomonas aeruginosa (29 isolates), Klebsiella species (54 isolates), Escherichia coli (28 isolates), Serratia marcescens (28 isolates), and Enterobacter species (29 isolates) to gentamicin, tobramycin, and amikacin was determined by the following three methods: commercial broth microdilution trays, standard agar dilution, and disk diffusion susceptibility. A total of 504 tests were performed by each method, and overall susceptibility or resistance determined by the broth microdilution method agreed with that determined by the agar dilution method in 92.7% of the tests, whereas results from the disk diffusion method agreed with those from the agar dilution method in 91.9% of the tests. The broth microdilution and disk diffusion methods agreed with each other 88.7% of the time. The broth microdilution system results varied from the agar dilution method results by more than one dilution in 121 of 504 determinations (24%); however, this altered susceptibility determinations in only 7.3% of the assays. E. coli isolates were found to be quantitatively more resistant to the aminoglycosides with the broth microdilution method than with the agar dilution method. In contrast, the broth microdilution method demonstrated P. aeruginosa to be quantitatively more susceptible to the aminoglycosides than when the results were obtained by the agar dilution method. The Micro-Media Systems method is economical, reliable, rapid, and simple to perform and yields quantitative minimum inhibitory concentrations.     

The response to 1,25-dihydroxycholecalciferol and to dihydrotachysterol in adult-onset hypophosphataemic osteomalacia.

The biochemical changes observed in a patient with adult-onset hypophosphataemic osteomalacia after three weeks treatment with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) followed by dihydrotachysterol (DHT) are reported. The treatment with 1,25-(OH)2D3 resulted in restoration of intestinal phosphate absorption to normal with a small rise in plasma phosphate concentration; there was no significant change in tubular reabsorption of phosphate. The tubular reabsorption of bicarbonate, which was initially low, returned almost into the normal range with normalisation of plasma bicarbonate concentration. Aminoaciduria decreased. There were no changes in plasma or urinary calcium but immunoreactive parathyroid hormone (i-PTH) which was initially elevated fell but still remained above the normal range. These changes were maintained after replacing the 1,25-(OH)2D3 treatment with dihydrotachysterol (DHT).