Antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens by using an agar dilution procedure

An agar dilution procedure was used to evaluate the antimicrobial susceptibility of 2,126 anaerobic bacteria isolated from a broad spectrum of properly obtained clinical specimens. Significant numbers ofBacteroides species,Fusobacterium species, anaerobic Gram-positive cocci, and anaerobic nonsporeforming Gram-positive bacilli would not grow on Wilkins-Chalgren agar used in the procedure. The failure to obtain growth from theBacteroides species was of special concern since these organisms generally show a higher degree of resistance to antibiotics than the other anaerobic bacteria, which failed to grow or grew poorly on that medium. When compared to other investigations, the data indicated an emerging resistnace to clindamy a forB. thetaiotaomicron andB. distasonis. IndividualClostridium species showed a wide divergence in susceptibility.     

Unusual Fermentative, Gram-Negative Bacilli Isolated from Clinical Specimens: I. Characterization of Erwinia Strains of the “lathyri-herbicola Group”

Five strains of gram-negative, yellow chromogenic bacilli were recovered from clinical specimens which fit the characteristics of the "lathyri-herbicola group" within the genus Erwinia. The strains were facultatively anaerobic, fermentative, anaerogenic bacilli with peritrichous flagella which grew at 37 C, reduced nitrate to nitrite, and failed to produce oxidase, pectinase, arginine dihydrolase, and decarboxylases for lysine and ornithine. Aggregations of bacteria (symplasmata) were observed in the syneresis water of slant cultures, and analogous granular aggregates and biconvex, spindle-shaped bodies developed in colonies on plate cultures. Awareness of these characteristics should result in more frequent identification of Erwinia species from human sources.     

Comparison of Two Commercial Formulations of the MacConkey Agar Test for Mycobacteria

Recent evaluations of the MacConkey agar test for differentiation of rapidly growing mycobacteria have revealed that certain strains of Mycobacterium fortuitum and M. chelonei that were expected to grow on MacConkey agar failed to do so. Investigation of two formulations of MacConkey agar showed that these two species grew better on the medium when the crystal violet dye was omitted. Several possible reasons for this difficulty are discussed. It is recommended that clinical laboratories engaged in differential identification of mycobacteria utilize commercial MacConkey agar without crystal violet when testing rapidly growing species of this genus.     

Stimulation of the growth of cutaneous strains of Peptococcus saccharolyticus by iron, haematin and blood

The growth of nine strains of Peptococcus saccharolyticus was assessed quantitatively by culture Trypticase Soy/yeast extract/Tween 80 agar (TSY-TW) with and without supplementation with iron or haematin and on blood agar, in aerobic, reduced 02 (3% O2 with 8% CO2, 8% H2 and 81% N2) and anaerobic atmospheres. All strains grew better anaerobically and under reduced O2 conditions than aerobically on supplemented or unsupplemented TSY-TW.Supplementation of TSY-TW with iron or haematin resulted in an average 4.4-fold increase in bacterial count in a reduced O2 atmosphere and an average 4.2-fold increase under anaerobic conditions. Under aerobic conditions the increase in count ranged from O to greater than 5000-fold, as some strains failed to grow on unsupplemented TSY-TW but responded well to the supplements of iron or haematin. The highest bacterial counts were obtained on Columbia blood agar incubated anaerobically. However, P. saccharolyticus failed to grow aerobically on plain or heated Columbia blood agar with or without supplements. TSY-TW blood agar supported the growth of the one strain tested under all three atmospheric conditions. The type strain (ATCC 14953) differed from all others in its failure to grow aerobically or in a reduced O2 atmosphere on supplement or unsupplemented media. Colony size varied greatly on different media, in different atmospheres and from strain to strain, being greatest in a reduced O2 atmosphere on Columbia blood agar. There was no correlation between the viable bacterial count and colony size.     

Comparison of media for the isolation of Enterobacter sakazakii

Enterobacter sakazakii is associated with neonatal infections and is occasionally present at low levels (<1 CFU/g) in powdered infant formula milk (IFM). It has been previously reported that some E. sakazakii strains do not grow in standard media for Enterobacteriaceae and coliform bacteria; therefore, a reliable method is needed for recovery of the organism. Three E. sakazakii enrichment broths-Enterobacteriaceae enrichment broth (EE), E. sakazakii selective broth (ESSB), and modified lauryl sulfate broth (mLST)-were compared with a novel broth designed for maximum recovery of E. sakazakii, E. sakazakii enrichment broth (ESE). One hundred seventy-seven strains (100%) grew in ESE, whereas between 2 and 6% of strains did not grow in EE, mLST, or ESSB. E. sakazakii possesses alpha-glucosidase activity, and a number of selective, chromogenic agars for E. sakazakii isolation based on this enzyme have been developed. E. sakazakii isolation agar produced fewer false-positive colonies than did Druggan-Forsythe-Iversen agar. However, the latter supported the growth of more E. sakazakii strains. It was also determined that 2% of E. sakazakii strains did not produce yellow pigmentation on tryptone soya agar at 25 degrees C, a characteristic frequently cited in the identification of E. sakazakii. The recovery of desiccated E. sakazakii (0.2 to 2000 CFU/25 g) from powdered IFM in the presence of a competing flora was determined with various enrichment broths and differential selective media. Current media designed for the isolation and presumptive identification of E. sakazakii do not support the growth of all currently known E. sakazakii phenotypes; therefore, improvements in the proposed methods are desirable.     

A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group

A new selective medium, Bacteroides fragilis ammonium-sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram-positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.     

Anaerobic respiration of fumarate as a differential test betweenCampylobacter fetus andCampylobacter jejuni

The effect of formate and of various electron acceptors—fumarate, aspartate, or nitrate—on the growth of 36 catalase-producingCampylobacter strains was quantitatively investigated in a semisynthetic medium, under aerobic (5% oxygen, 10% carbon dioxide, 85% nitrogen) or anaerobic (10% carbon dioxide, 90% nitrogen) conditions. Under anaerobic conditions, 24 strains ofC. jejuni failed to grow, or grew poorly, in the presence of fumarate, whereas ten strains ofC. fetus subsp.fetus and two strains ofC. fetus subsp.venerealis grew abundantly, rather better than under aerobic conditions. The quantitative differences of growth yields were very significant between the two species with fumarate, but less pronounced with aspartate or nitrate. The activity of fumarate-reductase inC. fetus was substantiated by identification of relevant metabolites by gas liquid chromatography and by mass spectrometry. The anaerobic fumarate respiration in the genusCampylobacter has taxonomic implications.     

Selectivity of Mitis Salivarius agar and a new selective medium for oral streptococci in dogs

An evaluation on the applicability of Mitis Salivarius agar (MS) medium, commonly used for the detection of oral streptococci in human and animals, to dog specimens and the development of a new selective medium for isolating streptococci from the canine oral cavity are described. Oral samples from dogs were cultured on MS medium under anaerobic conditions. The predominant facultative anaerobic bacteria on MS plates were gram-negative rods. Selectivity of streptococci on MS medium was 21.2%. A new selective medium, designated MS-CAN-AE, was developed for the isolation of streptococci from the canine oral cavity. The average growth recovery of laboratory and clinically isolated strains of streptococci on MS-CAN-AE medium was 84.1% of that on MS medium. Gram-positive rods and gram-negative rods and cocci rarely grew on the MS-CAN-AE. The selectivity of MS-CAN-AE was 95.0% for clinical samples. MS-CAN-AE medium will be helpful for investigations of streptococci in the canine oral cavity.     

Anaerobic Spirochete from a Deep-Sea Hydrothermal Vent

An obligately anaerobic spirochete, designated strain GS-2, was selectively isolated from samples collected at a deep-sea (2,550 m) hydrothermal vent of the Galapagos Rift ocean floor spreading center. The morphological and physiological characteristics of strain GS-2 resembled those of Spirochaeta strains. However, strain GS-2 failed to grow consistently in any liquid medium tested. In addition, strain GS-2 grew more slowly and to lower yields than other Spirochaeta species. The occurrence of obligately anaerobic bacteria in hydrothermal vents indicates that the water in at least some of the vent areas is anoxic. The presence of strain GS-2 shows that these areas are favorable for anaerobic marine spirochetes.     

Role of pH, lactate, and anaerobiosis in controlling the growth of some fermentative Gram-negative bacteria on beef.

At 5 degrees C four strains of fermentative, gram-negative bacteria (Serratia liquefaciens, Yersinia enterocolitica, Enterobacter cloacae, and Aeromonas hydrophila) grew aerobically and anaerobically on adipose tissue removed from beef muscle of low pH (5.4 to 5.6). All four strains also grew aerobically and anaerobically on muscle tissue of high pH (6.0 to 6.3). However, none of the four grew anaerobically on beef muscle of low pH, and the aeromonad strain also failed to grow aerobically on such muscle. Growth of S. liquefaciens and E.cloacae on vacuum-packaged beef muscle was dependent on the pH of the tissue and the oxygen transmission rate of the packaging film. Although the four strains grew in broth buffered at pH 5.55, L-lactate, at the concentration found in muscle of low pH (ca. 100 mM), prevented anaerobic growth of all four isolates and prevented the aerobic growth of th aeromonad. At pH 6.1 in buffered broth, the concentration of L-lactate occurring in muscle of high pH did not prevent aerobic or anaerobic growth of any of the strains.     

Acidocella aromatica sp. nov.: an acidophilic heterotrophic alphaproteobacterium with unusual phenotypic traits

Three obligately heterotrophic bacterial isolates were identified as strains of a proposed novel species of extremely acidophilic, mesophilic Alphaproteobacteria, Acidocella aromatica. They utilized a restricted range of organic substrates, which included fructose (but none of the other monosaccharides tested), acetate and several aromatic compounds (benzoate, benzyl alcohol and phenol). No growth was obtained on complex organic substrates, such as yeast extract and tryptone. Tolerance of the proposed type strain of the species (PFBC) to acetic acid was much greater than that typically reported for acidophiles. The bacteria grew aerobically, and catalyzed the dissimilatory reductive dissolution of the ferric iron mineral schwertmannite under both micro-aerobic and anaerobic conditions. Strain PFBC did not grow anaerobically via ferric iron respiration, though it has been reported to grow in co-culture with acid-tolerant sulfidogenic bacteria under strictly anoxic conditions. Tolerance of strains of Acidocella aromatica to nickel were about two orders of magnitude greater than those of other Acidocella spp., though similar levels of tolerance to other metals tested was observed. The use of this novel acidophile in solid media designed to promote the isolation and growth of other (aerobic and anaerobic) acidophilic heterotrophs is discussed.     

Mannitol and Fructose Catabolic Pathways of Pseudomonas aeruginosa Carbohydrate-Negative Mutants and Pleiotropic Effects of Certain Enzyme Deficiencies

Mutant strains of Pseudomonas aeruginosa PAO were isolated on the basis of their inability to utilize mannitol as sole carbon source for growth. Four linkage groups (I through IV) among these mutant strains were resolved by two-factor crosses using the general transducing phage F116, and the strains appeared to contain point mutations as evidenced by ability to give rise to spontaneous revertants with wild phenotype on mannitol minimal agar. Group I strains were affected only in ability to grow on mannitol; all were deficient in inducible mannitol dehydrogenase activity, and all but one were deficient in inducible mannitol transport activity. Fructokinase was induced in group I strains and in wild-type bacteria during growth in the presence of mannitol but not fructose, indicating the presence of a pathway specific for endogenously generated fructose. Cells grown on fructose contained phosphoenolpyruvate:fructose-1-phosphotransferase activity, and mannitol-grown cells contained a lower level of this activity. Group II mutants were deficient in constitutive phosphoglucoisomerase, failed to grow on mannitol, grew very slowly on glycerol and fructose, but grew normally on glucose and gluconate. Group III strains were deficient in both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase activities that reside in a single enzyme species. 6-Phosphogluconate appeared to be the inductive effector for this enzyme, which was not required for aerobic growth on glucose or gluconate. A single mannitol-negative mutant in group IV also failed to grow on glycerol and glucose, but no biochemical lesion was identified.     

Anaerobic microbiology in 198 cases of pleural empyema: a Bulgarian study

The aim of the study was to evaluate the incidence of anaerobic bacteria in 198 patients with pleural empyema and the susceptibility of isolates to eight antibacterial agents. Isolates were identified by the Crystal anaerobes identification system, API System rapid ID 32 A and/or routine methods. Susceptibility was tested by Sceptor MIC system for anaerobic bacteria and limited agar dilution method. Anaerobic bacteria were found in 74.2% of the patients and included 247 strains within 21 genera. The predominant anaerobes were Gram-positive anaerobic cocci (52 isolates), Fusobacterium (51), microaerophilic streptococci (24), Prevotella (19) and Bacteroides species (11). Common species/groups were Fusobacterium nucleatum (in 27.2% of specimens yielding anaerobes), Micromonas micros (8.2%), Finegoldia magna (7.5%), Bacteroides fragilis group (6.8%), Peptostreptococcus anaerobius (6.1%) and F. necrophorum (5.4%). No resistance to chloramphenicol and ampicillin/sulbactam was detected. The susceptibility rates of Gram-negative anaerobic isolates to penicillin, cefoxitin, clindamycin, clarithromycin, metronidazole and tetracycline were 63.8%, 90.2%, 87.8%, 58.6%, 98.8% and 71%, and those of Gram-positive anaerobes were 79.2%, 100%, 84.3%, 68.4%, 41.9% and 75%, respectively. The wide diversity of isolated anaerobic genera and species and the susceptibility patterns of the isolates emphasize the role of the anaerobic microbiology in cases of pleural empyema.     

Masking of antibiotic-resistance upon recovery of endophytic bacteria

During studies on internal plant colonization by rhizosphere bacteria and endophytic bacteria over several years, we frequently observed lack of growth of rifampicin-resistant mutants (rif+) on tryptic soy agar amended with rifampicin (RTSA). Following seed treatment of cucumber with 6 species of rif+ rhizosphere bacteria in one experiment, all strains were recoverable on RTSA when external root colonization was monitored. Following trituration of surface-disinfested roots, only one strain grew directly on RTSA; however colonies isolated on tryptic soy agar (TSA) grew within 18 h after transfer to RTSA. We term this temporary loss of the antibiotic-resistant phenotype ‘antibiotic masking’. Antibiotic masking was also observed with isolation of 7 rif+ endophytic bacterial strains from inside stems of cotton and with isolation of mutants of bacterial endophytes resistant to polymyxin B sulfate from cotton plants. Rifampicin-masking was not accounted for in vitro by inhibitory compounds from cotton plant extracts, by bacterial growth on low nutrient agar, or by competition with other bacteria. Collectively, these results suggest that expression of antibiotic-resistance may be altered in planta, although causes for this antibiotic-masking remain to be elucidated, methods for quantifying internal plant colonization by rif+ bacteria should account for this possibility. ei]Section editor: R O D Dixon     

Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces

AIMS: To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces. METHODS AND RESULTS: Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g(-1) faeces) for 48-h incubation at 37 degrees C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTect SYBR green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R2 = 0.78, P < 0.01) and total anaerobic bacteria (R2 = 0.21, P < 0.05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0.83 x log(CFU) + 1.43 and log(DNA copy) = 1.62 x log(CFU) - 6.32 respectively. CONCLUSIONS: The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5-6 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.     

厌氧菌预还原琼脂平板培养方法

为简化厌氧菌分离培养方法,使其在普通实验条件下于固体培养基上形成单菌落,本研究增加庖肉培养基无氧溶液体积,用作无氧倍比稀释液,在琼脂柱下进行倍比稀释,将皿盖带有胶塞孔的厌氧琼脂平板进行预还原,注射接种倍比稀释菌液,通过厌氧指示剂监测无氧效果,初步试用于肠道厌氧菌分离培养。结果显示,该方法整个操作过程厌氧效果良好,无需专门厌氧设备即可以分离纯化培养肠道乳酸杆菌,甚至无芽胞专性厌氧菌,如双歧杆菌和韦荣球菌。     

Composition of toluene-degrading microbial communities from soil at different concentrations of toluene.

Toluene-degrading bacteria were isolated from hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was controlled by dilution with vacuum pump oil. Incubation in desiccators equilibrated with either 100, 10, or 1% (wt/wt) toluene in vacuum pump oil and testing for genomic cross-hybridization resulted in four genomically distinct strains (standards) capable of growth on toluene (strains Cstd1, Cstd2, Cstd5, and Cstd7). The optimal toluene concentrations for growth of these standards on plating media differed considerably. Cstd1 grew best in an atmosphere equilibrated with 0.1% (wt/wt) toluene, but Cstd5 failed to grow in this atmosphere. Conversely, Cstd5 grew well in the presence of 10% (wt/wt) toluene, which inhibited growth of Cstd1. 16S ribosomal DNA sequencing and cross-hybridization analysis indicated that both Cstd1 and Cstd5 are members of the genus Pseudomonas. An analysis of the microbial communities in soil samples that were incubated with 10% (wt/wt) toluene with reverse sample genome probing indicated that Pseudomonas strain Cstd5 was the dominant community member. However, incubation of soil samples with 0.1% (wt/wt) toluene resulted in a community that was dominated by Pseudomonas strain Q7, a toluene degrader that has been described previously (Y. Shen, L. G. Stehmeier, and G. Voordouw, Appl. Environ. Microbiol. 64:637-645, 1998). Q7 was not able to grow by itself in an atmosphere equilibrated with 0.1% (wt/wt) toluene but grew efficiently in coculture with Cstd1, suggesting that toluene or metabolic derivatives of toluene were transferred from Cstd1 to Q7.     

Usefulness of Chromogenic CromoCen® AGN agar medium for the identification of the genus Aeromonas: Assessment of faecal samples

Selective screening media for the detection and identification of Aeromonas strains are needed to guide primary isolation procedures in the clinical laboratory. This study compared the selective CromoCen? AGN chromogenic agar medium for the detection and identification of Aeromonas strains that were isolated from various samples against the conventional selective agar media that are commonly used for the isolation of this organism in food, environmental and clinical samples. The Miles and Misra and ecometric methods were used to evaluate the microbiological performance of CromoCen? AGN chromogenic agar medium, which was shown to be satisfactory. A total of 14 reference Aeromonas strains, 44 wild strains and 106 clinical stool specimens were examined using both non-chromogenic selective agars that are commonly used for Aeromonas isolation and CromoCen? AGN agar. The latter exhibited 94.73% sensitivity and 100% specificity for the various samples. On CromoCen? AGN agar medium, Aeromonas formed colonies with light green, greenish and salmon pigments with or without a surrounding wide transparent zone (halo) of 2-3mm in diameter around the entire border. This medium is recommended for the isolation and potential identification of the Aeromonas genus.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Characteristic of clinical strains of gram-negative obligate anaerobes

The aim of the study was to assess prevalence and antibiotic susceptibility profiles ofGram-negative strictly anaerobic bacteria isolated from clinical specimens taken from hospitalized patients in 2005-2006. Biochemical identification and antibiotic susceptibility were done in an automated system ATB Expression (bioMerieux sa). From 12262 specimens examined 867 strains of obligate anaerobes were isolated. Gram-negative strictly anaerobic bacteria were cultured in number of 138 strains (15,9%). All cultures were performed on Columbia agar and Schaedler agar media (bioMerieux sa) supplemented with 5% sheep blood and incubated at 37 degrees C for 48-120 h in 85% N2, 10% H2, 5% CO2. Most frequently isolated was Bacteroides spp. (41,3%). For this group beta-lactamase activity was evaluated by using nitrocefin disc test (Cefinase BBL, Becton Dickinson and Co., Cockeysville, MD, USA). Production of ESBLs was detected with the use of two disc diffusion methods: the double-disc synergy test (DDST) according to Jarlier et al. and the diagnostic disc (DD) test according to Appleton. ESBLs were produced by 5,3% strains of Bacteroides spp. For all Bacteroides spp. strains MIC values were determined by gradient diffusion method Etest (AB BIODISK, Sweden). ESBLs and MIC were performed on Wilkins-Chalgren solid medium supplemented with 5% sheep blood (Difco Lab., USA) and all plates were incubated at 35 degrees C for 48 hours in 85% N2, 10% H2, 5% CO2. Most Gram-negative obligate anaerobes isolated from clinical specimens are still susceptible to imipenem (100%), metronidazole (99,3%) and beta-lactam antibiotics with beta-lactamase inhibitors: piperacillin/tazobactam (99,3%), ticarcillin/clavulanate (99.3%), amoxicillin/clavulanate (97.8%).