Comparison of atmospheres of incubation for primary isolation of Campylobacter fetus subsp. jejuni from animal specimens: 5% oxygen versus candle jar.

An atmosphere with reduced oxygen tension is required for the primary isolation of Campylobacter fetus subsp. jejuni. Therefore, we compared use of the conventional atmosphere of 5% oxygen and 8% carbon dioxide with use of a candle jar (17% oxygen and 3% carbon dioxide) for primary isolation of C. fetus subsp. jejuni from 263 positive canine, cattle, and turkey fecal or cecal specimens. At an incubation temperature of 42 degrees C, the atmosphere with 5% oxygen resulted in more Campylobacter colonies per plate (P less than 0.005) and consistently larger Campylobacter colonies (P less than 0.005) than did the candle jar, whereas the growth of interfering flora was similar. Overall, 96% of the 263 specimens were positive for C. fetus subsp. jejuni with 5% oxygen, and 90% were positive with the candle jar (P less than 0.02). More striking differences in isolation rates were seen when both the temperature and the atmosphere were varied: 5% oxygen at 42 degrees C enabled recovery of 93% of the isolates from 70 positive specimens, versus 46% recovery with the candle jar at 37 degrees C. Results with 5% oxygen at 37 degrees C were intermediate. The addition of FBP supplement (0.25% each of ferrous sulfate, sodium metabisulfite, and sodium pyruvate) to Campy-BAP selective medium made no improvement over unsupplemented medium at 42 degrees C (whether in 5% oxygen or in the candle jar), but there was significant improvement over unsupplemented medium when both media were incubated at 37 degrees in the candle jar.     

Comparison of CampyPak II with standard 5% oxygen and candle jars for growth of Campylobacter jejuni from human feces

To determine optimal temperature and atmospheric conditions for isolating Campylobacter jejuni from fecal specimens of humans, we studied six laboratory isolates and 19 fecal specimens that were known to contain C. jejuni. We compared incubations in 5% oxygen, the CampyPak II (BBL Microbiology Systems, Cockeysville, Md.) with 6 plates per jar (CP-6) and 12 plates per jar (CP-12), and candle jars at 37 and 42 degrees C. At both temperatures, the colony sizes for the laboratory strains were larger in the 5% O2 and the CP-6 than under the other two conditions. For the primary isolations, CP-12 failed to detect one and two campylobacters at 42 and 37 degrees C, respectively, whereas the candle jar failed to detect one at 42 degrees C and four at 37 degrees C. Colony size was again larger in the 5% O2 and the CP-6. For all four atmospheric conditions tested, colonies were significantly larger at 42 degrees C than at 37 degrees C. These studies showed that incubation at 42 degrees C in either 5% O2 or the CampyPak II with six plates per jar was optimal for primary isolation of C. jejuni from fecal specimens of humans. The candle jars incubated at 42 degrees C appeared to be satisfactory for primary isolation of C. jejuni from human feces, but incubation at 37 degrees C was not acceptable.     

Growth of non-Campylobacter, oxidase-positive bacteria on selective Campylobacter agar.

A total of 67 oxidase-positive, gram-negative bacteria were tested for growth on selective Campylobacter agar (Blaser formulation, BBL Microbiology Systems, Cockeysville, Md.) at 42 degrees C under microaerophilic conditions. Although the growth of most of these bacteria was prevented, all strains of Achromobacter xylosoxidans, Pseudomonas aeruginosa, Pseudomonas putrefaciens, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes grew as well as Campylobacter fetus subsp. jejuni.     

Development of a standardized susceptibility test for campylobacter with quality-control ranges for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem

A standardized agar dilution susceptibility testing method was developed for Campylobacter that consisted of testing on Mueller-Hinton medium supplemented with 5% defibrinated sheep blood in an atmosphere of 10% CO2, 5% O2, and 85% N2. Campylobacter jejuni ATCC 33560 was identified as a quality-control (QC) strain. Minimal inhibitory concentration (MIC) QC ranges were determined for two incubation time/temperature combinations: 36 degrees C for 48 hr and 42 degrees C for 24 hr. Quality-control ranges were determined for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem. For all antimicrobial agents tested at both temperatures, 95-100% of the QC MIC results fell within recommended QC ranges. Twenty-one Campylobacter clinical isolates, encompassing five species of Campylobacter (C. jejuni, C. coli, C. jejuni, subsp. doylei, C. fetus, and C. lari) were tested in conjunction with the C. jejuni QC strain. While C. jejuni and C. coli could be reliably tested under both test conditions, growth of C. jejuni subsp. doylei, C. fetus, and C. lari isolates was inconsistent when incubated at 42 degrees C. Therefore, it is recommended that these species only be tested at 36 degrees C.     

Comparative efficacy of liquid enrichment medium for isolation of Campylobacter jejuni.

Isolation of Campylobacter jejuni from 1,249 human fecal specimens by direct inoculation on selective Columbia agar and liquid enrichment medium was compared. The use of liquid enrichment medium provided a 30% higher isolation rate of C. jejuni. The overall isolation rate achieved by using direct plating and enrichment together was 8.2%.     

Comparison of broth enrichment and direct plating for the isolation of Campylobacter jejuni from dogs.

Two techniques for the isolation of Campylobacter jejuni from feces, direct plating and thioglycolate broth enrichment, were compared. A total of 272 rectal swab cultures were performed on 156 laboratory dogs. Campylobacter blood agar plates and Campylobacter thioglycolate broth were inoculated immediately upon sampling of the dogs. After incubation at 4 degrees C for 12 to 16 h, material from the Campylobacter thioglycolate medium was inoculated onto Campylobacter blood agar plates. A total of 157 samples were positive for C. jejuni; 154 were positive by the direct method and 112 were positive by the enrichment technique. Forty-five samples which were negative by the enrichment were positive by the direct method, and three samples which were negative by the direct method were positive for C. jejuni by the enrichment method. The use of the enrichment step resulted in an increase in the isolation rate from 56.6 to 57.7%.     

Laboratory and clinical evaluation of isolation media for Campylobacter jejuni.

Six selective isolation media were evaluated for their ability to support the growth of Campylobacter jejuni. Colony counts of 70 isolated strains of C. jejuni and recovery studies on these strains in simulated positive feces samples demonstrated that Bolton and Hutchinson' charcoal, cefoperazone, deoxycholate agar and Karmali's charcoal-based selective medium produced the highest recovery rates with the greatest suppression of other fecal flora. C. jejuni colonies were more easily recognized on charcoal-based selective medium. A clinical evaluation performed on 2,780 human, animal, and avian feces specimens confirmed the results of the laboratory investigation. From human samples, 4 more strains of C. jejuni were isolated on charcoal-based selective medium than were isolated on Skirrow medium, and 19 more strains of C. jejuni or C. coli were isolated on charcoal-based selective medium from animal specimens. Suppression of normal fecal flora was also greater on charcoal-based selective medium.     

Effects of sample holding time, temperature, and atmosphere on the isolation of Campylobacter jejuni from dogs.

Stool specimens were collected from 39 dogs, inoculated onto Campylobacter blood agar plates, and divided into four subsamples. Subsamples were held at 4 and 25 degrees C in room air and in a microaerobic environment and were reinoculated at 1, 2, 3, 4, 6, and 8 h. C. jejuni survived at least 3 h when it was held at 4 degrees C, but less than 2 h when it was held at 25 degrees C. The holding atmosphere was not associated with a difference in isolation rates.     

Evaluation of transport and storage techniques for isolation of Campylobacter fetus subsp. jejuni from turkey cecal specimens

Immediate culturing of fecal specimens is not always possible, and appropriate methods for transport and storage of Campylobacter fetus subsp. jejuni specimens have not been fully evaluated. Using nine techniques, we studied the survival of C. fetus subsp. jejuni in cecal specimens from infected turkeys. The organisms survived in specimens held without transport medium for 3 to 15 days (median, 9 days) at 4 degrees C, and 2 to 9 days (median, 4 days) at 25 degrees C. Only 20% of specimens frozen for 24 h at either -20 or -70 degrees C yielded C. fetus subsp. jejuni. Specimens dried on filter paper strips were negative for C. fetus subsp. jejuni within 1.5 h. Cary-Blair medium with decreased agar was the best of the six transport media tested, it enabled recovery of the organism from 100% (3 days) and 71% (7 days) of cecal samples held at 4 degrees C and 94% (3 days) and 85% (7 days) of cecal specimens held at 25 degrees C. In contrast, more than half of all cecal specimens held at 4 or 25 degrees C in Culturettes or buffered glycerol saline were negative by 3 days, and all were negative at 7 days. Results with the other three media studied (Campy-thio, thioglycolate medium, and alkaline peptone water) were intermediate. Overnight incubation of specimens in alkaline peptone water at 37 or 42 degrees C did not enhance recovery of C. fetus subsp. jejuni. Therefore, refrigeration without a transport medium is satisfactory for up to 3 days for recovery of C. fetus subsp. jejuni from specimens, however, we recommend the use of Cary-Blair medium with decreased agar for specimens that must be transported or stored for longer than 3 days and for rectal swabs, to prevent drying.     

Reassessment of selective agars and filtration techniques for isolation ofCampylobacter species from faeces

Four different studies were conducted in order to re-evaluate conventional methods and assess the efficacy of new selective agars and a filtration method for the isolation of campylobacters. Skirrow's medium, Preston agar, modified CCD agar and Fennell's medium, incubated microaerobically at 37 °C for 48 h, gave similarCampylobacter isolation rates from 225 faecal samples, but the latter two media were more selective. Evaluation of modified CCD agar demonstrated that campylobacters could be isolated from that medium more successfully after incubation at 37 °C (173/177 positive samples) than at 42 °C (152/177 positive samples). In a larger study 1286 faecal specimens were cultured using modified CCD agar, Fennell's medium and a 0.45 µm membrane filtration technique, all incubated at 37 °C. Campylobacters were isolated from 89 % (178), 86 % (171) and 60 % (130) of 199 positive samples respectively. Modified CCD agar was most successful in isolation of the majority of campylobacters, but Fennell's medium was essential for recovery of Campylobacter cinaedi andCampylobacter fennelliae, whereas the 0.45 µm membrane technique was the only method to isolate all of the catalase-negative campylobacter strains. Further evaluation of the 0.45 µm and 0.65 µm pore size membranes showed that more strains ofCampylobacter jejuni andCampylobacter coli were isolated using the larger pore size membranes.     

Direct isolation of atypical thermophilic Campylobacter species from human feces on selective agar medium.

Campylobacter upsaliensis is the name which has been proposed for a new group of thermophilic campylobacter strains which differ from C. jejuni and C. coli in having a negative or weak catalase reaction. Primary isolation of these strains from human feces has been achieved only by use of filtration techniques. We report here direct isolation of strains corresponding to C. upsaliensis from stools of six children. The strains were isolated on a newly described campylobacter-selective medium. The strains were oxidase positive, hippurate negative, nitrate positive, negative for H2S in triple sugar iron, and susceptible to cephalothin (30-micrograms disk) and nalidixic acid (30-micrograms disk), and they grew at 37 and 43 degrees C, but not at 25 degrees C. The selective medium used was a blood-free, charcoal-based medium consisting of Columbia agar base, activated charcoal, cefoperazone (32 micrograms/ml), vancomycin (20 micrograms/ml), and cycloheximide (100 micrograms/ml). The medium supported the growth of the weakly reacting or catalase-negative strains, with colony counts equivalent to those obtained on antibiotic-free horse blood agar. These strains could not be isolated directly from stool on Skirrow medium, and colony counts confirmed that this medium could not support a low inoculum of these organisms. The clinical significance of these strains is unknown. We conclude that C. upsaliensis can be isolated directly from stool by using a selective medium, without the need for filtration.     

CASA chromogenic medium for enteric Campylobacter species

We prospectively assessed stool samples from 370 patients for Campylobacter species by comparing three selective agar media incubated at two temperatures: 42°C and 37°C. Twenty patients (5.4%) were found positive. The chromogenic medium CASA (AES Chemunex, France) proved highly efficient for C. jejuni and C. coli recovery, while lessening the workload in the lab.     

Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp. in human fecal samples as estimated by a reevaluation of isolation methods for Campylobacters

The aims of this study were to investigate the prevalence of campylobacteria including Campylobacter jejuni subsp. jejuni (C. jejuni) and Campylobacter coli in human clinical samples and in samples from healthy individuals and to reevaluate the efficacies of conventional selective methods for isolation of Campylobacter spp. Two charcoal-based selective media, modified charcoal cefoperazone deoxycholate agar (mCCDA) and cefoperazone-amphotericin-teicoplanin (CAT) agar, were compared with Skirrow's blood-based medium and with a filter method (filter) applied to a yeast-enriched blood agar. A total of 1,376 specimens were tested on all four media, and the percentages of thermophilic Campylobacter-positive specimens isolated on Skirrow's medium, filters, CAT agar, and mCCDA were 82, 83, 85, and 95%, respectively. When additional samples were processed with the three selective media, mCCDA recovered significantly more thermophilic Campylobacter spp. than Skirrow's medium (P = 0.0034). No significant difference between Skirrow's medium and CAT agar was observed in this study. Another six taxa were identified, namely, Campylobacter concisus, Campylobacter curvus-like bacteria, Arcobacter butzleri, Arcobacter cryaerophilus, Helicobacter cinaedi, and Sutterella wadsworthensis. Most of these strains were isolated after 5 to 6 days of incubation by use of the filter technique. This paper provides evidence for the existence of S. wadsworthensis in human feces from clinical cases of gastrointestinal disorders and in feces from a healthy individual. Furthermore, C. concisus was isolated from a large number of diarrheal cases, particularly those at the extremes of age, but was additionally isolated from the feces of healthy people. Further investigations to establish the role of C. concisus and S. wadsworthensis in enteric disease is needed. We conclude that a range of campylobacteria may cause infections in Denmark.     

Retention of Campylobacter (Campylobacterales: Campylobacteraceae) in the house fly (Diptera: Muscidae)

The house fly (Musca domestica L.) may transmit Campylobacter to broiler flocks. We assessed the retention time of house flies for Campylobacter jejuni at five temperatures and three doses. Flies were inoculated individually at their proboscis with 1.6 x 10(7) CFU (colony forming units) of C. jejuni and incubated at 15, 20, 25, 30, and 35 degrees C. Furthermore, a dose experiment was conducted at 25 degrees C where individual flies were inoculated in three series: 6.5 x 10(6), 6.0 x 10(4), and 8.2 x 10(2) C.jejuni CFU. Whole flies were tested for C. jejuni carriage at 0, 6, 12, 18, and 24 h by initial preenrichment in Bolton broth, which afterwards was streaked on modified mCCDA agar plates and incubated under micro-aerobic conditions. The results showed that the time C. jejuni remained in flies declined over time with ascending temperatures and when reducing the inoculation dose. All flies stayed Campylobacter positive 24 h postinoculation at 15 degrees C whereas only one-third of the flies were positive at 20 degrees C and few to none at 25, 30, and 35 degrees C. When combinations of temperature and retention time were expressed as accumulated day-degrees, data could be adequately fitted using a generalized linear mixed model that included a linear effect of day-degrees and the difference between the lowest and the two highest doses. Based on model predictions of selected combinations of temperature and dose, the time for 50% and 1% of flies containing Campylobacter was calculated. It is suggested that house flies are mainly short distance carriers of C. jejuni.     

Combined effect of heat, essential oils and salt on fungicidal activity against Trichophyton mentagrophytes in a foot bath.

This work was originally undertaken to determine the effective conditions of essential oils against Trichophyton mentagrophytes in vitro for the treatment of tinea pedis in a foot bath. Agar blocks implanted with T. mentagrophytes were immersed in 0.1% aqueous agar containing two-fold dilutions of essential oils with or without sodium chloride at 27 degrees C, 37 degrees C and 42 degrees C for 10 and 20 min. The number of surviving mycelia on the agar blocks was determined from the standard curves of the colony diameter and original inocula of the conidia. At the same time, the thermal effect on the cellular morphology was examined using SEM. Most fungal mycelia (99.7%) were killed after treatment at 42 degrees C for 20 min without essential oil. The fungicidal activity of essential oils was markedly enhanced by treating at 42 degrees C for 20 min as compared with that at 27 degrees C, showing 1/4 - 1/32-fold reduction of minimum fungicidal concentration (MFC to kill 99.99%). The order of the fungicidal activity of 11 essential oils was oregano, thyme thymol, cinnamon bark > lemongrass > clove, palmarose, peppermint, lavender > geranium Bourbon, tea tree > thyme geraniol oils. MFCs were further reduced to 1/2 - 1/8 by the addition of 10% sodium chloride. The salt effect was explained, at least partly, by an increase in mycelial adsorption of antifungal constituents in the presence of sodium chloride. Considerable hyphal damage was done at 27 degrees C by the essential oils, but no further alteration in morphology of the hyphae treated at 42 degrees C with or without oil was observed by SEM. The inhibitory effect of heat and oils was also observed against mycelia of T. rubrum and conidia of T. mentagrophytes. Thermotherapy combined with essential oils and salt would be promising to treat tinea pedis in a foot bath.     

The use of a combined enrichment–filtration technique for the isolation of Campylobacter spp. from clinical samples

A combined non-selective enrichment-filtration technique was investigated for the isolation of Campylobacter spp. from clinical samples. In total, 479 samples were tested by direct culture, enrichment subculture and enrichment-filtration. The enrichment-filtration technique was used with both selective and non-selective media. Direct culture and enrichment subculture yielded 13 and seven isolates, respectively, while enrichment-filtration yielded 18 and 14 isolates on selective and non-selective agar, respectively. Thus, the combination of enrichment-filtration with selective agar produced a 38.5% increase in the number of isolates (p <0.05). All isolates were identified as Campylobacter jejuni.     

Growth of Aeromonas spp. on Butzler Campylobacter selective agar and evaluation of the agar for the primary isolation of Aeromonas spp. from clinical specimens

The fortuitous finding that Aeromonas spp. grew well on Butzler Campylobacter selective medium (BCSA) in a microaerobic atmosphere at 42 degrees C prompted us to evaluate the performance of BCSA for selective isolation of aeromonads in comparison with ampicillin (30 micrograms/ml) sheep blood agar (ASBA30). Overall recovery rates of aeromonads from 563 stool samples from patients with acute diarrhea were higher on ASBA30 (70.4%) than on BCSA (56.3%); however, 21 (29.5%) grew only on BCSA. The three human-associated Aeromonas spp. could be recovered on BCSA and ASBA30. We recommend the use of BCSA to laboratories reluctant to include a specific selective medium for aeromonads.     

Effects of trimethoprim-sulfamethoxazole and incubation atmosphere on isolation of group A streptococci.

The effects of selective media and incubation atmosphere on the isolation of group A beta-hemolytic streptococci were evaluated. A higher percentage of group A streptococci was isolated on sheep blood agar incubated in air than in CO2 or anaerobic atmospheric conditions. Fewer non-group A beta-hemolytic streptococci were isolated on sheep blood agar incubated in air than in CO2 or anaerobically. Group A streptococcal isolation was not significantly affected by different incubation atmospheres on sheep blood agar containing trimethoprim-sulfamethoxazole, but detection time was longer than on sheep blood agar alone. No significant difference was found between isolation of group A streptococci on sheep blood agar incubated in air and that on sheep blood agar containing trimethoprim-sulfamethoxazole and incubated in 5 to 10% CO2; however, more group A streptococci were isolated on sheep blood agar in air within 24 h. Sheep blood agar incubated at 35 degrees C in air is, therefore, recommended for the isolation of group A streptococci from throat swabs.     

Comparison of a blood-free medium and a filtration technique for the isolation of Campylobacter spp. from diarrhoeal stools of hospitalised patients in central Australia.

Single specimens of diarrhoeal stool from 676 patients, mostly aboriginals aged less than 5 years, admitted to Alice Springs Hospital, central Australia, for diarrhoea between Sept. 1988 and Feb. 1989, were examined for Campylobacter spp. by culture on a blood-free medium with selective supplement (BFM; Oxoid) and blood agar overlaid with a membrane filter (FM). Campylobacter spp. were isolated on either BFM or FM or both from 225 patients. Campylobacter spp. were isolated on BFM alone from 75 patients and on FM alone from 213 patients (p less than 0.001; chi 2 test). Most campylobacters isolated on BFM were C. jejuni. All C. jejuni subsp. doylei, all "C. upsaliensis" except one, all C. laridis, C. fetus subsp. fetus and several uncharacterised Campylobacter isolates were isolated on FM only. C. jejuni was isolated on BFM but not FM from several patients, and vice versa. Serotyping of C. jejuni and C. coli isolated from both media showed the serotypes recovered from the two media to be different in some patients. In some patients concurrent infection with several species or serotypes (up to five) of Campylobacter, or both, was shown for the first time by the use of FM. We conclude that the use in combination of a selective medium and a non-selective medium with a filtration technique are better than either medium alone for the isolation of Campylobacter spp.     

Trimethoprim activity in media selective for Campylobacter jejuni.

The activity of trimethoprim (TMP) in two selective media used for isolation of Campylobacter jejuni was evaluated. The two selective media, Campy-BAP and Skirrow medium, contain TMP in addition to other antimicrobial agents. The minimal inhibitory concentrations of TMP in blood agar base (basal agar for Skirrow medium) or brucella agar (basal agar for Campy-BAP) for three sensitive control organisms were compared with those in Mueller-Hinton agar, which contains low levels of thymidine. TMP was inactive in both blood agar base and brucella agar, even when lysed horse blood or thymidine phosphorylase was added. TMP had activity when used in combination with the other antimicrobial agents normally included in Skirrow medium and Campy-BAP, probably indicating synergism between TMP and one or more of the other antimicrobial agents. Sheep blood could be substituted for lysed horse blood in Skirrow medium without compromising the activity of TMP.