Use of Dorset Egg Medium for Maintenance and Transport of Neisseria meningitidis and Haemophilus influenzae Type b

Studies of bacterial meningitis are hampered by the inability to maintain the viability of etiological agents during transport to reference laboratories. The long-term survival rate of 20 isolates of Neisseria meningitidis and Haemophilus influenzae type b (Hib) on Dorset egg medium, supplemented Columbia agar base medium, chocolate agar, and Amies medium was compared with that on 70% GC agar (chocolate) transport medium. N. meningitidis isolates were also inoculated onto 5% horse blood agar, and Hib was inoculated onto Haemophilus test medium. All of the N. meningitidis isolates remained viable on Dorset egg medium for 21 days; viability on the other media was poor after only 7 days. Recovery rates of Hib isolates were similar on Dorset egg and Haemophilus test media (100% after 21 days) and significantly better than on the other media. Dorset egg medium is inexpensive and easy to make and may be invaluable for studies of bacterial meningitis in developing countries.     

Simple, Inexpensive, Reliable Method for Differentiation of Candida dubliniensis from Candida albicans

Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans, including the ability to form germ tubes and chlamydospores. These similarities have caused significant problems in the identification of C. dubliniensis by the average clinical mycology laboratory. To facilitate the differentiation of these species, we investigated the growth of 120 isolates of C. dubliniensis and 98 C. albicans isolates at 42 and 45°C on Emmons’ modified Sabouraud glucose agar (SGA) and 10 isolates of each species in yeast-peptone-dextrose broth. None of the C. dubliniensis isolates grew on the agar or in the broth medium at 45°C, while 11 isolates were capable of growing on SGA at 42°C. In contrast, all of the C. albicans isolates but one grew at 45°C on or in either medium. These reproducible results clearly demonstrate that the incubation of isolates suspected to be C. dubliniensis or C. albicans at 45°C provides a simple, reliable, and inexpensive method for the differentiation of the two species.     

Isolation of Campylobacter fetus subsp. jejuni from Human Fecal Specimens by Incubation at 35 and 42°C

Campylobacter strains were isolated from human stool specimens that were cultured on modified Skirrow agar and incubated at 35 and 42°C. Incubation at 42°C resulted in more isolates, increased colony numbers, and earlier isolation than was found at 35°C.     

Quantitative survival of aerobic and anaerobic microorganisms in Port-A-Cul and Copan transport systems

Transport media should preserve the viability and stability of microorganisms in clinical specimens. In this study, the Port-A-Cul transport system and the Copan transport system without charcoal, both designed to preserve anaerobes, were evaluated. Dacron swabs were inoculated with two combinations of facultative and anaerobic organisms typically found in vaginal swab samples. Combination I contained Candida albicans, Escherichia coli, Enterococcus spp., group B streptococci, Lactobacillus crispatus, and Staphylococcus aureus. Combination II contained Lactobacillus iners, Peptoniphilus asaccharolyticus, Mycoplasma hominis, Prevotella bivia, Prevotella corporis, Porphyromonas asaccharolytica, Mobiluncus curtisii, Peptostreptococcus anaerobius, and Gardnerella vaginalis. Duplicate swabs were placed into the two transporters and held for 24, 48, 72, and 96 h at 4 and 24°C. Both transporters maintained the viability of organisms better at 4°C than at 24°C. Prevotella bivia and Prevotella corporis had a loss of viability in both transporters at both temperatures. However, at 24°C, there was a significantly greater loss of viability for Mycoplasma hominis, Prevotella bivia, Prevotella corporis, and Peptoniphilus asaccharolyticus when the organisms were stored in Copan transport medium than when they were stored in Port-A-Cul transport medium for 96 h (P < 0.002). Some organisms proliferated in the transport media, but when transporters were held at 24°C for 96 h, a significantly greater increase in the concentrations of group B streptococci and Candida albicans, Escherichia coli, and Enterococcus spp. organisms in Copan medium than in Port-A-Cul medium was observed (P < 0.002). At room temperature, the Port-A-Cul system is superior to the Copan system with respect to the preservation of fastidious microorganisms and the prevention of the proliferation of facultative organisms.     

A Novel Solid Medium for Culturing Mycobacterium tuberculosis Isolates from Clinical Specimens

The laboratory diagnosis of tuberculosis usually relies on culture-based isolation of the causative Mycobacterium tuberculosis bacteria. We developed and evaluated the performance of MOD9, a new blood-free derivative of the MOD4 solid medium on which we previously reported for the isolation and culture of mycobacteria. First, inoculation of Lowenstein-Jensen medium with 21 M. tuberculosis isolates at 10⁵, 10³, and 10 CFU yielded colonies in 5.7 ± 1.5 days, 7.6 ± 0.8 days, and 10.8 ± 1.7 days versus 1.5 ± 0.4 days, 3.5 ± 0.6 days, and 4.9 ± 1 days in MOD9 (P < 0.05, Student''s t test). Further, the time to detectable growth of M. tuberculosis was measured on MOD9 and Lowenstein-Jensen media after duplicate inoculation of 250 clinical specimens decontaminated with 0.7% chlorhexidine. The contamination rate was 1.6% (4/250) on MOD9 versus 4.4% (11/250) on Lowenstein-Jensen medium (P = 0.11, Fisher''s exact test). Chlorhexidine-MOD9 yielded 38/250 (15.2%) isolates versus 32/250 (12.8%) isolates for the chlorhexidine-LJ (P = 0.5195, Fisher''s exact test). All together, eight M. tuberculosis isolates were cultured solely from chlorhexidine-MOD9, and two M. tuberculosis isolates were cultured solely from chlorhexidine-LJ. The time to detection was 9.8 ± 3.9 (range, 5 to 18) days for chlorhexidine-MOD9 versus 17.4 ± 5.9 (range, 10 to 35) days for chlorhexidine-LJ (P < 0.05, Student''s t test). These data indicate the superiority of the MOD9 medium over the standard LJ medium following chlorhexidine decontamination for the recovery of M. tuberculosis.     

Evaluation of Silica Gel Packages for Transport of Neisseria meningitidis

Eight Neisseria meningitidis reference strains, representing six different serogroups, were plated on 57 blood agar plates each. The growth was harvested and stored in silica gel packages at different temperatures for up to 90 days. When held at 4°C, all strains were recovered after 90 days of storage. Strains held at room temperature or alternately at 4°C and room temperature survived for at least 10 and 17 days, respectively.     

Optochin Revisited: Defining the Optimal Type of Blood Agar for Presumptive Identification of Streptococcus pneumoniae

To determine the optimal media for optochin susceptibility testing of Streptococcus pneumoniae, we measured inhibition zones for 72 S. pneumoniae and 22 Streptococcus viridans isolates on three blood-containing media. Because 15.3, 0, and 22.2% of S. pneumoniae organisms were misidentified on Columbia agar, Trypticase soy agar (TSA), and Mueller-Hinton agar, respectively, each containing sheep blood, we recommend that TSA-sheep blood agar be used.     

New Transport Medium for Cultural Recovery of Helicobacter pylori

We developed a new transport medium (GESA—Helicobacter pylori transport medium [publication no. WO/2014/019696, patent pending no. PCT/EP2013/002292; Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy]) for recovery of Helicobacter pylori from gastric biopsy samples. GESA transport medium, in a semisolid state, provides the optimal conditions for maintaining the viability of the microorganism over time. The efficacy of the transport medium was assessed through in vitro and ex vivo experiments. We were able to recover different suspensions of H. pylori ATCC 43629 and H. pylori 13 A in GESA transport medium stored at 4°C for up to 10 days. In particular, with a starting inoculum of ∼10⁵ CFU, after 7 days of storage, 150 ± 25 CFU and 40 ± 7 CFU of the reference and clinical strains were detected, respectively. H. pylori colonies were isolated from gastric specimens taken from both the antrum and the fundus in 68 (90.66%) of 75 urea breath test (UBT)-positive patients. Moreover, GESA transport medium allowed the recovery and isolation of H. pylori colonies from additional biopsy samples from 13 of the 75 detected subjects at up to 10 days of biopsy sample storage at 4°C. Finally, GESA transport medium preserved its characteristics when stored at 4°C for 1 year from its preparation, thus allowing good recovery of H. pylori. GESA transport medium can be considered a standardized transport medium with high performance that optimizes the recovery rate of H. pylori grown by culture.     

Sensitivity of SARS‐CoV‐2 to different temperatures

This study was designed to investigate the sensitivity of SARS‐CoV‐2 to different temperatures, to provide basic data and a scientific basis for the control of COVID‐19 epidemic. The virus was dispersed in 1 mL basal DMEM medium at a final concentration of 10^3.2 TCID₅₀/mL and then incubated at 4, 22, 30, 35, 37, 38, 39 and 40°C for up to 5 days. The infectivity of residual virus was titrated using the Vero E6 cell line. The results showed that the virus remained viable for 5 days at 4°C, and for 1 day only at 22 and 30°C. We found that the infectivity of the virus was completely lost after less than 12 hours at 37, 38 and 39°C, while at 40°C, the inactivation time of the virus was rapidly reduced to 6 hours. We show that SARS‐CoV‐2 is sensitive to heat, is more stable at lower temperatures than higher temperature, remains viable for longer at lower temperatures, and loses viability rapidly at higher temperatures.     

Comparison of CHROMagar Salmonella Medium and Hektoen Enteric Agar for Isolation of Salmonellae from Stool Samples

CHROMagar Salmonella (CAS), a new chromogenic medium, was retrospectively compared to Hektoen enteric agar (HEA) with 501 Salmonella stock isolates and was then prospectively compared to HEA for the detection and presumptive identification of Salmonella spp. with 508 stool samples before and after enrichment. All stock cultures (100%), including cultures of H₂S-negative isolates, yielded typical mauve colonies on CAS, while 497 (99%) isolates produced typical lactose-negative, black-centered colonies on HEA. Following overnight incubation at 37°C, a total of 20 Salmonella strains were isolated from the 508 clinical samples. Sensitivities for primary plating and after enrichment were 95% (19 isolates) and 100% (20 isolates), respectively, for CAS and 80% (16 isolates) and 100% (20 isolates), respectively, for HEA. The specificity of CAS (88.9%) was significantly higher than that of HEA (78.5%; P < 0.0001). On the basis of its good sensitivity and specificity, CAS medium can be recommended for use for primary plating when human stool samples are screened for Salmonella spp.     

Colistin-sensitive Proteus Organisms: Including Indole-negative Proteus vulgaris. Non-swarming on First Isolation

Forty-eight isolates of Proteus vulgaris and 4 isolates of Proteus mirabilis were found by disc diffusion to be sensitive to colistin sulphomethate. The minimum inhibitory concentration (MIC) of colistin sulphomethate of the isolates ranged from 3·125-200 μg. per ml. The MIC was lower with a smaller inoculum, and for most strains the MIC in agar was lower than the MIC in broth. On first isolation only 48 per cent of the colistin-sensitive Pr. vulgaris strains exhibited swarming at 37°, and after passage through semi-solid agar 25 per cent of the strains still did not swarm. None of the Pr. vulgaris strains produced indole.     

Detection of Candida dubliniensis in Oropharyngeal Samples from Human Immunodeficiency Virus-Infected Patients in North America by Primary CHROMagar Candida Screening and Susceptibility Testing of Isolates

Candida dubliniensis has been associated with oropharyngeal candidiasis in patients infected with human immunodeficiency virus (HIV). C. dubliniensis isolates may have been improperly characterized as atypical Candida albicans due to the phenotypic similarity between the two species. Prospective screening of oral rinses from 63 HIV-infected patients detected atypical dark green isolates on CHROMagar Candida compared to typical C. albicans isolates, which are light green. Forty-eight atypical isolates and three control strains were characterized by germ tube formation, differential growth at 37, 42, and 45°C, identification by API 20C, fluorescence, chlamydoconidium production, and fingerprinting by Ca3 probe DNA hybridization patterns. All isolates were germ tube positive. Very poor or no growth occurred at 42°C with 22 of 51 isolates. All 22 poorly growing isolates at 42°C and one isolate with growth at 42°C showed weak hybridization of the Ca3 probe with genomic DNA, consistent with C. dubliniensis identification. No C. dubliniensis isolate but only 18 of 28 C. albicans isolates grew at 45°C. Other phenotypic or morphologic tests were less reliable in differentiating C. dubliniensis from C. albicans. Antifungal susceptibility testing showed fluconazole MICs ranging from ≤0.125 to 64 μg/ml. Two isolates were resistant to fluconazole (MIC, 64 μg/ml) and one strain was dose dependent susceptible (MIC, 16 μg/ml). MICs of other azoles, including voriconazole, itraconazole, and SCH 56592, for these isolates were lower. C. dubliniensis was identified in 11 of 63 (17%) serially evaluated patients. Variability in phenotypic characteristics dictates the use of molecular and biochemical techniques to identify C. dubliniensis. This study identifies C. dubliniensis in HIV-infected patients from San Antonio, Tex., and shows that C. dubliniensis is frequently detected in those patients by using a primary CHROMagar screen.     

Rifampin stability in 7H9 broth and Löwenstein-Jensen medium

The objectives of this study were to monitor the stability of rifampin (RIF) in Löwenstein-Jensen medium (L-J medium) and 7H9 broth, which are the media commonly used for drug susceptibility testing (DST) of Mycobacterium tuberculosis. Rifampin degradation in stock solution, 7H9 broth, and L-J medium and during the inspissation process for L-J medium preparation was serially monitored by high-performance liquid chromatography (HPLC). L-J medium-based DST was conducted to examine the effect of L-J medium storage on the DST outcome. The RIF stock solution was stable for at least 3 months when kept at either 4°C or −20°C; RIF in 7H9 broth and L-J medium was almost 50% decayed after 1 week of storage at 37°C, and rifampin could not be detected in 7H9 or L-J medium after 3 weeks or 6 weeks of storage at 37°C. Approximately half of the drug was decomposed after 4 months of storage at 4°C for both media, and after 6 months of storage at 4°C, RIF in L-J medium was undetectable, while 38% of RIF remained in 7H9 medium. Approximately 21, 24, 29, and 35% RIF degradations were detected when the L-J medium was coagulated at 75°C, 80°C, 85°C, and 90°C, respectively. The DST outcomes when using L-J medium stored for different periods of time were consistent with the RIF concentration monitoring data. Rifampin in stock solution is stable for at least 3 months at a reduced storage temperature. Media containing RIF should be prepared strictly according to validated standard operating procedures. RIF degradation is a possible reason for false resistance categorizations of M. tuberculosis isolates in the clinical laboratory.     

Lymphocytotoxins in infectious mononucleosis: a non-cytotoxic effect on their cytotoxicity in vitro

The lymphocytotoxic activity (LCA) of sera from patients with infectious mononucleosis (IM) was tested against lymphocytes under various experimental conditions. Firstly, lymphocytes from 11 healthy donors were preincubated with pools of normal human sera (NHS) or IM sera at 37°C and then tested for (a) reactivity with the same IM sera in a standard lymphocytotoxin assay at 15°C; (b) rosetting with various sheep erythrocyte (E) preparations (E, EA and EAC) and (c) stimulation by non-specific activators (phytohaemagglutinin, pokeweed mitogen and concanavalin A). These experiments showed that preincubation of normal cells with IM sera caused significant reduction in subsequent lymphocyte killing at 15°C (P<0·01) compared to unincubated cells or those preincubated with pooled NHS. There was no change in the binding of E, EA and EAC or mitogen stimulation following incubation. Culture of preincubated lymphocytes in lymphocytotoxin-free medium for 24 hr did not restore LCA at 15°C. Secondly, a pool of normal lymphocytes was incorporated into media containing either 2,4-dinitrophenol or sodium azide and tested for LCA against 11 acute IM sera and two NHS at both 15 and 37°C. No significant change in cell killing was observed at 15°C in the presence of these inhibitors, but there was a significant return of LCA at 37°C. Finally, normal lymphocytes and cells from two patients with IM were cultured at 37°C in lymphocytotoxin-free medium to determine the role of down-regulation of lymphocyte surface receptors in reducing autolymphocytotoxicity during the acute phase of the illness. There was no change in cell killing by IM sera after culture for 24 hr. These experiments show that lymphocytotoxic sera from patients with infectious mononucleosis interact with normal lymphocytes at 37°C without causing cell killing. This interaction caused a change in surface-binding characteristics that was not reversed by culture in ligand-free medium for 24 hr. Studies using metabolic inhibitors suggested that the failed lymphocytotoxicity at 37°C resulted, at least in part, from lymphocyte metabolism, although this did not inhibit the reaction between cytotoxic material and the lymphocyte surface.     

Phenotypic Differences of Cryptococcus Molecular Types and Their Implications for Virulence in a Drosophila Model of Infection

Compared to Cryptococcus neoformans, little is known about the virulence of the molecular types in Cryptococcus gattii. We compared in vitro virulence factor production and survival data using a Drosophila model of infection to further characterize the phenotypic features of different cryptococcal molecular types. Forty-nine different isolates were inoculated into wild-type flies and followed for survival. In vitro, isolates were assessed for growth at 30 and 37°C, melanin production, capsule size, resistance to H₂O₂, and antifungal susceptibility. A mediator model was used to assess molecular type and virulence characteristics as predictors of survival in the fly model. VGIII was the most virulent molecular type in flies (P < 0.001). At 30°C, VGIII isolates grew most rapidly; at 37°C, VNI isolates grew best. C. gattii capsules were larger than those of C. neoformans (P < 0.001). Mediator model analysis found a strong correlation of Drosophila survival with molecular type and with growth at 30°C. We found molecular-type-specific differences in C. gattii in growth at different temperatures, melanin production, capsule size, ability to resist hydrogen peroxide, and antifungal susceptibility, while growth at 30°C and the VGIII molecular type were strongly associated with virulence in a Drosophila model of infection.     

Evaluation of the Etest Method for Determining Fluconazole Susceptibilities of 402 Clinical Yeast Isolates by Using Three Different Agar Media

The performance of the Etest for fluconazole susceptibility testing of 402 yeast isolates was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and Mueller-Hinton agar (MHA) and were read after incubation for 48 h at 35°C. The yeast isolates included Candida albicans (n = 161), Candida glabrata (n = 41), Candida tropicalis (n = 35), Candida parapsilosis (n = 29), Candida krusei (n = 32), Candida lusitaniae (n = 31), Candida species (n = 19), Cryptococcus neoformans (n = 40), and miscellaneous yeast species (n = 14). The Etest results correlated well with reference MICs. Overall agreement was 94% with RPG, 97% with CAS, and 53% with MHA. When RPG was used, agreement ranged from 89% for Candida spp. to 100% for C. krusei. When CAS was utilized, agreement ranged from 93% for Cryptococcus neoformans to 100% for C. tropicalis, C. parapsilosis, C. lusitaniae, Candida spp., and miscellaneous yeast species. With MHA, agreement ranged from 17% for C. parapsilosis to 90% for C. krusei. Both RPG and CAS supported growth of all yeast species, whereas growth on MHA was comparatively weaker. Etest results were somewhat easier to read on CAS. The Etest method using either RPG or CAS, but not MHA, appears to be a viable alternative to the NCCLS reference method for determining fluconazole susceptibilities of yeasts.     

Viability of Trichomonas vaginalis in Copan universal transport medium and eSwab transport medium

We compared the use of universal transport medium and eSwab transport medium held at room temperature or 37°C to bedside inoculation and immediate incubation of culture media for the detection of Trichomonas vaginalis. There were no significant culturable differences in the sensitivity of either of the transport media to that of bedside inoculation.     

A rapid method for determining decarboxylase and dihydrolase activity

A total of 764 fresh clinical isolates were used to test a rapid method for determining lysine, arginine, and ornithine decarboxylase activity as well as arginine dihydrolase activity. The conventional Møller decarboxylase broth was tested in parallel with the rapid method on 234 Enterobacteriaceae and 140 non-fermentative Gram-negative rods. The 0·3% agar method was tested in parallel on 245 Enterobacteriaceae and 146 non-fermentors. All media were checked at half-hour or hourly intervals for up to eight hours, with the final reading taken after incubation for 24 hours at 37°C. The rapid method detected 17 positive decarboxylase or dihydrolase reactions that were not detected by the Møller broth and 16 more than the agar medium when testing Enterobacteriaceae. The corresponding figures for the nonfermentative Gram-negative rods were three and two respectively. Lysine and ornithine decarboxylase were generally detected by the rapid broth in two to four hours'' incubation while the arginine decarboxylase and dihydrolase were slower and required six to eight hours. This compares with overnight incubation as the general rule for the Møller broth and agar decarboxylases. The comparable accuracy of the rapid method with conventional techniques and the shorter incubation time required for detection of positive reactions make this procedure well suited to a routine clinical laboratory.     

New Assay for Measuring Cell Surface Hydrophobicities of Candida dubliniensis and Candida albicans

Hydrophobic interactions, based on cell surface hydrophobicity (CSH), are among the many and varied mechanisms of adherence deployed by the pathogenic yeast Candida albicans. Recently it was shown that, unlike C. albicans, C. dubliniensis is a species that exhibits an outer fibrillar layer consistent with constant CSH. Previously, C. dubliniensis grown at 25 or 37°C was shown to coaggregate with the oral anaerobic bacterium Fusobacterium nucleatum. C. albicans, however, demonstrated similar coaggregation only when hydrophobic or grown at 25°C. This observation implied that coaggregation of Candida cells with F. nucleatum is associated with a hydrophobic yeast cell surface. To test this hypothesis, 42 C. albicans and 40 C. dubliniensis clinical isolates, including a C. albicans hydrophobic variant, were grown at 25 and 37°C and tested with the established hydrophobicity microsphere assay, which determines CSH levels based on the number of microspheres attached to the yeast cells. The coaggregation assay was performed in parallel experiments. All C. dubliniensis isolates grown at either temperature, hydrophobic 25°C-grown C. albicans isolates, and the C. albicans hydrophobic variant, unlike the 37°C-hydrophilic C. albicans isolates, exhibited hydrophobic CSH levels with the microsphere assay and simultaneously showed maximum, 4+, coaggregation with F. nucleatum. The parallel results obtained for C. dubliniensis using both assays support the use of the CoAg assay both as a rapid assay to determine CSH and to differentiate between C. dubliniensis and C. albicans.     

Evaluation of tobacco agar for chlamydosporulation in Candida albicans and Candida dubliniensis

It had recently reported that tobacco agar (TA) could induce chlamydosporulation in Candida albicans and Candida dubliniensis. This new medium was compared with corn meal-Tween 80 agar. Twenty-nine of the 46 C. albicans isolates tested, produced chlamydospores on both media. Six C. albicans isolates produced chlamydospores only on TA, whereas three isolates produced chlamydospores only on corn meal agar. Eight isolates failed to chlamydosporulate on either media. All the reference strains of C. dubliniensis tested produced chlamydospores on both media.