Comparative efficacy of seven selective media for isolating Campylobacter jejuni.

Diarrheal stools from 263 patients were inoculated on seven selective media: Butzler selective medium, Blaser medium, Skirrow blood agar, Preston campylobacter selective medium, Preston campylobacter blood-free medium, Butzler Virion medium, and modified Preston medium (with amphotericin B [2 mg/liter]). A similar number of Campylobacter jejuni strains were isolated from all the media studied; nevertheless, the presence of competing fecal flora (FF) made the detection of suspect colonies difficult. Preston campylobacter blood-free medium with cefoperazone yielded the greatest number of C. jejuni isolations, and contaminating FF grew in only 9% of the plates showing C. jejuni growth; all the other media allowed the abundant growth of other FF, regardless of whether C. jejuni was isolated from them or not.     

Semisolid blood-free selective-motility medium for the isolation of campylobacters from stool specimens.

Isolation of Campylobacter jejuni and C. coli from stool specimens is done by growing campylobacter colonies on solid selective media with or without blood. However, recognition of these colonies can be difficult. Therefore, we decided to evaluate an isolation procedure based on the swarming of campylobacters through a semisolid medium. We developed a semisolid blood-free selective motility (SSM) medium which is composed of Mueller-Hinton broth with 0.4% agar and supplemented with cefoperazone (30 micrograms/ml) and trimethoprim (50 micrograms/ml). The SSM medium was compared with our previously described Butzler Medium Virion (Goossens et al., J. Clin. Microbiol. 24:840-843, 1986) and blood-free medium (Bolton and Coates, J. Appl. Bacteriol. 54:115-125, 1983) with cefoperazone (32 micrograms/ml) (Bolton et al., J. Clin. Pathol. 37:956-957, 1986). Of 1,890 routine stool specimens tested, 100 were found to be positive for campylobacters: 95 were recovered with the SSM medium, 94 with the Virion medium, and 90 with the blood-free medium. The SSM medium performed equally well whether it was incubated in the special incubator or the candle jar. Only 4.4 and 7.3% of the plates grew contaminating fecal flora when incubated in the special incubator and the candle jar, respectively. Clearly the SSM medium is easy, quick, cheap, sensitive, and more selective than any other medium which has been developed so far and does not require the addition of blood. We believe that this medium has a future in the routine microbiology laboratory in developed as well as in developing countries.     

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.     

Use of a selective medium and a membrane filter method for isolation ofCampylobacter species from Spanish paediatric patients

A study was conducted to assess the value of a combination of two culture methods for isolation ofCampylobacter spp. from Spanish children. Seven hundred twenty-nine diarrhoea] stool specimens from 599 patients were examined forCampylobacter spp. by culturing them on charcoal cefoperazone deoxycholate agar and on blood agar with a membrane filter. One hundred sixteenCampylobacter strains were isolated from a total of 108 specimens; 75 (64.6%) wereCampylobacter jejuni, 32 (27.5%) wereCampylobacter coli, 8 (6.8%) were non-typeable, and one (0.9%) wasCampylobacter upsaliensis. Campylobacters were isolated from 99 positive samples using charcoal cefoperazone deoxycholate agar alone. The filtration technique alone yielded only 86 positive samples. Seven specimens yielded differentCampylobacter spp. with different media. The only catalase-negative strain was recovered using the filter method. The combination of the selective medium with the filter method increased the isolation rate ofCampylobacter strains by 14.1%. Isolation rates of campylobacters using the filter method were similar to those reported in European studies, in which a similar frequency ofCampylobacter upsaliensis was observed. The addition of a filter method for routine laboratory isolation of campylobacters should be considered in selected age groups (in children <10 years of age) or in areas where catalase-negative or weakly-positiveCampylobacter strains may be of epidemiological significance.     

Comparison of two selective media and a membrane filter technique for isolation ofCampylobacter species from diarrhoeal stools

Diarrhoeal stool specimens from 415 patients were examined forCampylobacter spp. by culture on charcoal cefoperazone deoxycholate agar (CCDA), Skirrow medium and Columbia blood agar overlaid with a 0.65 µm pore size membrane filter. Forty-eightCampylobacter strains were isolated from 45 (10.8 %) specimens by all media; 44 wereCampylobacter jejuni (91.7 %), three wereCampylobacter coli (6.3 %) and one wasCampylobacter hyointestinalis (2.0 %). The percentages ofCampylobacter-positive specimens isolated on Skirrow medium, CCDA and the membrane filter were 62, 82 and 95 %, respectively. The recovery of moreCampylobacter spp. from the same stool sample was achieved by the membrane filter method only. The highest isolation rate (100 %) was observed when culture on CCDA and the membrane filter method were combined.     

The assay on a defined medium of the effects of β-2-thienylalanine on the growth of anaerobic bacterial isolates from phenylketonuric patients

Faecal samples were taken from three diet-managed phenylketonuric children to determine effects of-2-thienylalanine (-2-t) on indigenous bacteria. From sample swabs, 127 anaerobes were identified and tested for-2-t inhibition on a phenylalanine (Phe)-free medium, Anaerobe Inhibition Test (AIT) agar. Of the isolates, 77.9% grew sufficiently to assay reactions on at least 25% of AIT plates. Using Phe-containing Columbia agar, 86.5% of the strains could be assayed. None of 28Bacteroides cultures was inhibited by-2-t on AIT. Of the genera,Bifidobacterium, Eubacterium, Lactobacillus, Peptostreptococcus, andPropionibacterium, no isolates which would grow on AIT were inhibited. At least one isolate of each of the generaPeptococcus, Fusobacterium, andClostridium was inhibited. Of 127 total isolates, only nine were inhibited by-2-t on AIT, and inhibition was abolished on Columbia agar.Thirty-nine aerobes were isolated from the same patients. Strains of the genera tested reacted similarly to previously tested strains from non-PKU sources. Also, anaerobically isolatedEscherichia coli were inhibited, whileStreptococcus faecalis cultures were not, confirming results on aerobically-isolated non-PKU cultures of the same species.These studies, the first dealing with-2-t and anaerobic bacteria, suggest that little change in intestinal bacterial populations might be expected during in vivo-2-t treatment.     

Incidence and virulence ofAeromonas species in feces of children with diarrhea

Aeromonas spp. occurring in feces of children with diarrhea were studied. Forty-eight strains were isolated from 2,025 specimens during a one year period. Only 11 of 44 strains tested yielded virulence factors (cytotoxin, hemolysin and hemagglutinin). Six strains were identified as Aeromonas sobria and five as Aeromonas hydrophila.The other strains isolated were identified as Aeromonas caviae.The biochemical characteristics associated with virulence factors were a positive Voges-Proskauer reaction, production of gas from glucose, fermentation of mannose, and absence of -lactosidase. Beta-D-glucosidase and esculin hydrolysis were the main characteristics used to differentiate Aeromonas sobria from the other two species. The incidence of Aeromonas spp. with virulence factors in feces of children with diarrhea would seem to vary widely from one area to another.     

Beta-lactamase production in the upper respiratory tract flora

In order to determine the recovery rate of species of the genera Haemophilus and Moraxella (including subgenus Branhamella) from the upper respiratory tract and the incidence of -lactamase production within these genera, cultures were made of nose and throat swab specimens and adenoid tissue in 50 children undergoing adenoidectomy.Haemophilus influenzae was isolated from 92% of the children. All children harboured strains of Haemophilus spp. and in 46%, at least one strain produced the TEM-1 -lactamase.Branhamella catarrhalis and/or Moraxella nonliquefaciens were isolated from 82% of the children and strains producing the BRO-1 -lactamase from 34%. Overall, TEM-1 and/or BRO-1 producing strains were recovered from 60% of the investigated patients. The -lactamase production was found to be transferable by conjugation within the respective genera. It is suggested that the apathogenic species may be a source of transferable determinants mediating -lactamase production in the upper respiratory tract.     

Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli to identify potential sources of colonization in commercial turkey farms

Poultry are the main reservoir for thermophilic Campylobacter spp., which is the most common causative agent of human bacterial gastroenteritis. The epidemiology of Campylobacter in poultry, particularly in turkeys, is not completely understood. This study aimed at identifying potential sources and transmission routes of thermophilic Campylobacter spp. in commercial turkey farms. C. jejuni and C. coli isolates from breeders (n?=?29, 20 C. jejuni and 9 C. coli) and their progeny (n?=?51, 18 C. jejuni and 33 C. coli) reared in two different farms for three sequential production cycles were analysed by multilocus sequence typing (MLST). Strains (n?=?88, 42 C. jejuni and 46 C. coli) isolated from environmental (i.e. anteroom and in-house overshoes), water (i.e. drinkers and water line), and pest (i.e. flies, Alphitobius diaperinus, and mice) sources were also examined. MLST of C. jejuni and C. coli isolates resulted in 13 and 12 different sequence types (STs) belonging to six and one previously-described clonal complexes (CCs), respectively. Three novel STs were identified. Genetic similarities were detected between isolates from fattening turkeys and the considered environmental, water, and pest sources, and with the breeders to a lesser extent. Source attribution analysis estimated that environmental and water sources accounted for most (～75%) of fattening turkey isolates and were therefore identified as the most likely sources of flock colonization, followed by pests (～20%) and breeders (～5%). These sources may thus be targeted by control measures to mitigate the risk of Campylobacter colonization in commercial turkeys.

RESEARCH HIGHLIGHTS

• High occurrence of C. jejuni and C. coli in commercial turkey flocks.

• High genetic diversity of C. jejuni and C. coli in commercial turkey flocks.

• Horizontal transmission responsible for Campylobacter colonization of commercial turkey flocks.

• Environmental and water sources involved in Campylobacter colonization of commercial turkey flocks.

• Strategies for prevention and control of Campylobacter colonization of commercial turkey flocks are needed.

     

In vitro evaluation of the new Paulomycin antibiotic paldimycin

The comparative in vitro activity of paldimycin, a new antibiotic, was evaluated against 215 gram-positive bacteria. Activity of the compound was greater in nutrient agar of pH 6.8 than in Mueller-Hinton agar. All strains of staphylococci, streptococci, enterococci and Listeria monocytogenes were inhibited at concentrations 2 g/ml. Activity of the new drug was generally comparable to that of vancomycin.     

A new selective medium for the isolation ofCampylobacter jejuni from human faeces

A new selective medium, Butzler's medium Virion, for the isolation ofCampylobacter jejuni from human faeces is described. This medium contains the following antibiotics per liter: cefoperazone 15 mg, rifampicin 10 mg, colistin 10,000 IU, and amphotericin B 2 mg. At 42 °C there was no difference in the isolation rate on Butzler's medium Virion and Butzler's medium Oxoid, but the competing faecal flora was best suppressed by the new medium which allows easier reading of plates and better recognition of campylobacter colonies. Also, the isolation rate and growth of faecal flora were comparable when this new selective medium was incubated at both 37 °C and 42 °C in a candle jar or a special incubator. These results suggest that Butzler's medium Virion is a very reliable medium for the isolation of campylobacter. In comparison with other selective media it has important advantages for investigators who have little experience in isolating this enteropathogen, or for developing countries where special 42 °C incubators are often not available.     

Modified selective medium for isolation of Campylobacter spp. from feces: comparison with Preston medium, a blood-free medium, and a filtration system.

Our previously described (H. Goossens, M. De Boeck, and J. P. Butzler, Eur. J. Clin. Microbiol. 2:389-393, 1983) selective medium, consisting of cefoperazone (15 mg/liter), rifampin (10 mg/liter), colistin (10,000 IU/liter), and amphotericin B (2 mg/liter) (medium M1), for the isolation of Campylobacter jejuni and Campylobacter coli from stool specimens was modified as follows: cefoperazone (30 mg/liter), rifampin (10 mg/liter), and amphotericin B (2 mg/liter) (medium M2). A comparative study of the isolation of Campylobacter spp. from stool specimens was carried out with medium M1; medium M2; a selective blood-free medium consisting (per liter) of charcoal (4 g), ferrous sulfate (0.25 g), sodium pyruvate (0.25 g), casein hydrolysate (3 g), sodium deoxycholate (1 g), nutrient broth no. 2 (25 g), agar (12 g), and cefoperazone (32 mg) (medium M3); and Preston medium containing (per liter) trimethoprim (10 mg), rifampin (10 mg), polymyxin B (5,000 IU), and cycloheximide (100 mg) (medium M4). We also included a filtration system in which membrane filters were applied directly to the surface of the nonselective blood-free medium distributed in small petri dishes. A total of 5,276 stool specimens were tested: 2,788 stool specimens were tested on M1 and M3 in study 1; 2,488 stool specimens were inoculated on the four selective media in study 2, and the last 986 specimens of the 2,488 were tested in parallel with the filtration system. In study 2, 128 Campylobacter strains were isolated from 126 different patients; 85.0, 88.3, 82.5, and 66.6% of these strains were isolated on M1, M2, M3, and M4, respectively. No contaminating fecal flora was found on 65.4, 70.7, 62.4, and 40.3% of the M1, M2, M3, and M4 plates, respectively. Furthermore, C. coli was found to be more susceptible to antibiotics present in the selective media, particularly colistin and polymyxin B, than was C. jejuni. We therefore recommend M2 for the isolation of Campylobacter spp. Finally, the filtration method was found to be easy and cheap; although the sensitivity was low, this method allowed the isolation of new Campylobacter spp. which seem to be associated with diarrhea.     

Evaluation of novel antipseudomonal drugs using the serum bactericidal activity test

Serum bactericidal activity against Pseudomonas aeruginosa was determined in six volunteers 1 and 4 h after administration of 2 g ceftazidime, 4 g piperacillin, 500 mg imipenem, 80 mg tobramycin and four combinations of these agents. Ceftazidime produced the highest serum bactericidal titers, killing 100% and 86% of the 50 Pseudomonas aeruginosa strains tested after 1 and 4 h respectively at a serum dilution of 18. Imipenem had lower serum bactericidal titers than ceftazidime, killing 88 % of the isolates after 1 h at a serum dilution of 18. The combination showed only slightly higher titers. Killing curves were determined for nine strains of Pseudomonas aeruginosa using undiluted volunteer serum drawn 1 h after administration of the antibiotics. The combinations ceftazidime/tobramycin and piperacillin/ tobramycin exhibited higher killing activity than the single drugs. As the activity of the aminoglycosides could be underestimated on the basis of their low serum bactericidal titers, it is concluded that determination of these titers is inappropriate for evaluating the efficacy of the aminoglycosides.     

Evaluation of a new dipslide with a selective medium for the rapid detection of beta-glucuronidase-positiveEscherichia coli

A selective medium was incorporated into a new three-media dipslide (Uricult Trio, Orion Diagnostica) to allow rapid identification ofEscherichia coli. The medium is supplemented with a recently described chromogenic substrate, hydroxyquinoline--D-glucuronide, for -glucuronidase enzyme. The performance of the medium was compared to that of three other -glucuronidase detection methods in tests of 602 routine urine samples. Of 324Escherichia coli strains isolated, 92 % grew brown colonies on dipslide, thus being -glucuronidase positive. The proportion of -glucuronidase-positiveEscherichia coli detected by the three methods was 93 % for BGA II agar plates (Tammer-Tutka), 91 % for PGUA tablets (Rosco) and 84 % for Fluorocult Brolacin agar plates (Merck). No false-positive reactions were seen in the case of 209 significant isolates of species other thanEscherichia coli grown on the selective medium.     

The in vitro susceptibility of <Emphasis Type="Italic">Campylobacter</Emphasis> spp. to the antibacterial effect of manuka honey

We report the antimicrobial effect of manuka honey against Campylobacter spp. isolated by a diagnostic laboratory from specimens from a community in New Zealand. The isolates were differentiated according to species level using multiplex PCR. C. jejuni (20 strains) and C. coli (7 strains) were identified. The clinical isolates identified and type culture collection strains of these species were subjected to testing to determine the minimum inhibitory concentration (MIC) of manuka honey using a microdilution technique. The MIC of the manuka honey against all of the Campylobacter tested was found to be around 1% (v/v) honey. The low MIC values suggest that honey might still inhibit the growth of campylobacteria after dilution by fluid in the gut, but the actual concentration of honey that can be achieved in the intestine is unknown. Therefore, clinical investigation is required to establish the efficacy of honey against Campylobacter spp. in the gut environment.     

In vitro activity of LY146032 (Daptomycin), a new peptolide

The in vitro activity of LY146032, a new peptolide antibiotic, was compared with those of vancomycin, teicoplanin, imipenem, amoxicillin and erythromycin. LY146032 inhibited 90% ofStaphylococcus aureus andStaphylococcus epidermidis, including methicillin-resistant isolates at 1g/ml. Its activity was comparableto those of vancomycin and teicoplanin. MIC90s for the beta-hemolytic streptococci varied from 0.25 g/ml for group B streptococci to 4g/ml for some group C and F streptococci. MICs forStreptococcus faecalis were in the range of 0.5 to 8 g/ml,and the MIC90 4 g/ml,compared to 4 g/ml for vancomycin and 1g/ml for teicoplanin. For some viridans streptococci the MICs were 4g/ml, whereasStreptococcus pneumoniae were inhibited by 0. 5g/ml.Corynebacterium JK species were inhibited by 0. 5g/ml, similar to vancomycin, andListeria monocytogenes by 4g/ml.Neisseria species,Haemophilus species and enteric species were not inhibited. Most MBCs were within two-fold of the respective MICs. After 14 days passage in subinhibitory concentrations of LY146032,Staphylococcus aureus, Staphylococcus epidermidis andStreptococcus faecalis showed minimal increase in MICs. The activity of LY 146032 was increased by adding Ca²⁺ and was reduced in an anaerobic environment. Overall, LY146032 is an extremely interesting new agent that inhibits gram-positive species.     

In vitro activity of the new 4-quinolone compound RO 23-6240

The in vitro activity of the new 4-quinolone Ro 23-6240 was compared with that of pefloxacin, ciprofloxacin, norfloxacin, nalidixic acid and gentamicin against a total of 397 recent clinical isolates. An agar dilution procedure was used to determine MICs and two inocula (10⁴ and 10 ⁶ CFU)were used throughout. Ro 23-6240 inhibited most species of the Enterobacteriaceae, Haemophilus influenzae andStaphylococcus aureus (including methicillinresistant strains) at 1 mg/l.Pseudomonas aeruginosa was somewhat more resistant (MIC 90 4 mg/l) and the Bacteroides fragilis group were considerably more resistant (MIC 90 32 mg/l). Overall Ro 23-6240 was as active as pefloxacin but was two-to eight-fold less active than ciprofloxacin against most species tested.     

Use of a DNA hybridization method to verify results of screening for methicillin resistance in Staphylococci

Tests were performed by the disk diffusion method, agar dilution method and the E test to determine the susceptibility to methicillin and oxacillin of clinical isolates and control strains ofStaphylococcus aureus (n=106) and coagulase-negative species (n=131). Results were compared with those of a dot blot DNA hybridization test, in which themecA gene was detected using an oligonucleotide probe selected from themecA gene. Among theStaphylococcus aureus strains themecA gene was found in all but two strains inhibited by 8 mg/l of methicillin and all but two strains inhibited by 4 mg/l of oxacillin. A disk test using either 1 µg oxacillin or 10 µg methicillin and a tentative resistance breakpoint of 10 mm gave the best agreement with the hybridization test. For coagulase-negative staphylococci 34 of 35 strains inhibited by 8 mg/l methicillin hybridized with the probe as well as 58 of 82 strains inhibited by 1–4 mg/l; 93 of 97 strains inhibited by 0.5 mg/l oxacillin were also positive in the probe test. Using the 1 µg oxacillin disk and a resistance breakpoint of 10 mm good agreement was obtained between results of the disk diffusion and DNA hybridization tests. It is suggested that this genotypic method for detection of methicillin resistance is used as a reference method for routine methods.     

Interaction of the novel penem CGP 31 608 and Its enantiomer with type Id beta-lactamase and penicillin-binding proteins

The novel penem CGP 31 608 (5R, 6S, 8R) and its enantiomer CGP 32 879 (5S, 6R, 8S) were shown to be essentially stable against hydrolysis by type Id -lactamase isolated from Pseudomonas aeruginosa 18S/H. CGP 31 608 was a potent progressive inhibitor of this enzyme (I50=32 M), which was only weakly inhibited by CGP 32 879 (I50=460 M). CGP 31 608 had the highest affinity for penicillin-binding protein (PBP) 4 from Escherichia coli K-12 (I50= 1 g/ml), followed by PBPs 2 (10 g/ml) and 1A/1Bs (100 g/ml); CGP 32 879 did not inhibit binding of ¹⁴ C-benzylpenicillin to the PBPs. The steric configuration of the -lactam nucleus of penems appears to strongly influence their affinity for -Iactamases and target PBPs. The balanced spectrum of CGP 31 608 may be explained by its -lactamase stability and affinity for several vital PBPs.     

Specificity of Monoclonal Antibodies to Campylobacter jujuni Lipopolysaccharide Antigens

Monoclonal antibodies (Mabs) were produced to the lipopolysaccharide antigens of Campylobacter jejuni strain 1249 (Penner serotype 0:2/63). A polymyxin-cloth based enzyme immunoassay (pCEIA) was used for initial screening and for evaluating the specificity of these antibodies. Seven Mabs reacted with at least 11 and as many as 14 of 15 C, jejuni strains (representing 8 different Penner serotypes). These seven Mabs did not cross-react with any of 16 non-Campylobacter bacteria commonly encountered in food, with only two exceptions. Several combinations of these Mabs in pairs reacted with all 15 C jejuni strains. These results suggest that pCEIA employing two of these Mabs in combination is potentially useful for detection of Campylobacter jejuni in foods and other samples.