Recovery of Bilophila wadsworthiafrom Clinical Specimens in Italy

This report is the first survey in Italy to evaluate the incidence of recovery of Bilophila wadsworthia in clinical situations. The survey was carried out at the departments of Microbiology in two Northern Italian hospitals over a one-year period. Tests for B. wadsworthia were carried out on a range of specimens from different body sites, when etiology by anaerobes was suspected. Out of a total of 350 samples examined, 67% were positive in bacteriological tests. Mixed anaerobic infections were detected in 53 specimens, corresponding to 23% of all cases. Strains of B. wadsworthia were isolated from 12 samples, equivalent to 5% and 22% of total and mixed/anaerobic infections, respectively. Bilophila wadsworthia was always isolated in mixed infections, mainly from the large intestine (67% of cases). The infectious process of B. wadsworthia was often complicated by abscess formation, regardless of body site. Interestingly, a strain was isolated from one case of bacteremia. The microorganisms most frequently isolated with B. wadsworthia were Escherichia coli for facultative species (38%), and Bacteroides fragilis, from anaerobic isolates (25%). Production of beta-lactamases by B. wadsworthia isolates was found in ten strains (83%), which appeared to be penicillin G resistant at concentration equal to or greater than the break-point (4 microg/mL). Epidemiological and clinical data from this and previous studies point to the involvement of B. wadsworthia in mixed infections. To assess the specific contribution of the species to the disease, studies of pathogenetic factors are to be considered in parallel. Nonetheless, production of beta-lactamases by most B. wadsworthia isolates could easily interfere with the therapeutical approach to infections involving the new species. The addition of a selective medium to culture specimens from the abdominal cavity should be considered in order to detect the presence of B. wadsworthia.     

Evaluation of culture results of specimens from patients with suspected anaerobic infection.

In order to establish the etiological agents of anaerobic infections, 154 clinical specimens collected from patients with suspected anaerobic infection were cultured under both aerobic and anaerobic conditions. Bacteria were isolated from 111 (72%) of these specimens. Only aerobes were recovered from 48 (43%), only anaerobes from 31 (28%) and both aerobes and anaerobes from 32 (29%) of the 111 specimens. A total 83 anaerobic bacteria including Bacteroides fragilis group (28%), Porphyromonas (19%), Peptostreptococci (10%), Prevotella (17%), Clostridia (12%), Fusobacteria (10%), Veillonella (2%) and Eubacteria (2%) were identified. Anaerobes were most commonly isolated from peritoneal fluid followed by joint fluid, abscess and endometrial materials, blood, soft-tissue biopsy and draining material.     

Comparison of Methods for Isolation of Anaerobic Bacteria from Clinical Specimens

Five different anaerobic culture methods and several different media were compared for their ability to recover anaerobes from clinical specimens. Specimens were obtained from patients with documented infections, avoiding contamination with normal flora, and immediately placed in an anaerobic transporter. Each specimen was cultured by all methods and on all the various media. The comparative data indicate that anaerobic jars (GasPak and evacuation-replacement types) are just as effective in the recovery of clinically significant anaerobes as the more complex roll-tube and chamber methods employing prereduced media. Liquid media were disappointing as a "back-up" system but chopped-meat glucose was superior to two thioglycolate formulations. Growth of all anaerobes was poorer on selective media, but these media were very helpful in the workup of specimens containing mixed growth of anaerobic and facultative organisms. A variety of different anaerobes was isolated, but no very fastidious or extremely oxygen-sensitive organisms were recovered. This suggests that such organisms may not play a significant role in causing clinical infections.     

Retrospective Analysis of Anaerobic Bacterial Findings at a University Hospital in Rosario,Argentina from 1995 to 1997

Even though considerable progress has been made during recent years in anaerobic bacteriology, processing of anaerobes is, for small laboratories, a challenge. In order to evaluate all steps of anaerobic processing, we retrospectively analysed indications for anaerobic culture, the proportion of specimens yielding anaerobes on group and genus level and the distribution according to site of infection. We recovered anaerobes in 41 of 143 specimens. Twenty-five specimens with a positive finding for anaerobes were processed on clinicians' request and 16 were tested on the microbiologist's initiative. Anaerobes were mostly recovered from soft tissue infections, blood culture and intra-abdominal specimens. The most frequently isolated genera were Clostridium and Porphyromonas. Our results suggest the necessity of discussion between microbiologists and clinicians in order to analyse procedures, provide guidelines and consider the advantage and limitations of anaerobic bacteriology.     

Anaerobic bacteria colonizing the lower airways in lung cancer patients

Anaerobes comprise most of the endogenous oropharyngeal microflora, and can cause infections of airways in lung cancer patients who are at high risk for respiratory tract infections. The aim of this study was to determine the frequency and species diversity of anaerobes in specimens from the lower airways of lung cancer patients. Sensitivity of the isolates to conventional antimicrobial agents used in anaerobe therapy was assessed. Respiratory secretions obtained by bronchoscopy from 30 lung cancer patients were cultured onto Wilkins-Chalgren agar in anaerobic conditions at 37°C for 72-96 hours. The isolates were identified using microtest Api 20A. The minimal inhibitory concentrations for penicillin G, amoxicillin/clavulanate, piperacillin/tazobactam, cefoxitin, imipenem, clindamycin, and metronidazole were determined by E-test. A total of 47 isolates of anaerobic bacteria were detected in 22 (73.3%) specimens. More than one species of anaerobe was found in 16 (53.3%) samples. The most frequently isolated were Actinomyces spp. and Peptostreptococcus spp., followed by Eubacterium lentum, Veillonella parvula, Prevotella spp., Bacteroides spp., Lactobacillus jensenii. Among antibiotics used in the study amoxicillin/clavulanate and imipenem were the most active in vitro (0% and 2% resistant strains, respectively). The highest resistance rate was found for penicillin G and metronidazole (36% and 38% resistant strains, respectively). The results obtained confirm the need to conduct analyses of anaerobic microflora colonizing the lower respiratory tract in patients with lung cancer to monitor potential etiologic factors of airways infections, as well as to propose efficient, empirical therapy.     

Anaerobic Bacteriology in 75 Cases of Thoracic Empyema in Sofia,Bulgaria

The aim of this study was to evaluate the prevalence of anaerobes in patients with thoracic empyema over a period of 30 months and to assess the susceptibility of the isolates to penicillin, clindamycin and metronidazole. Seventy-nine pleural fluid specimens were obtained from 75 adult patients with empyema. Anaerobic isolates were identified by Crystal anaerobes identification system and routine methods. Susceptibility testing was conducted using broth microdilution method and limited agar dilution test. Anaerobic bacteria were found in 50 (66.7%) of the patients and included 96 isolates representing 16 genera. The predominant Gram-positive anaerobes were Peptostreptococcus species (19 isolates) and Streptococcus intermedius (10), and the commonest Gram-negative species were Fusobacterium nuleatum (13),Fusobacterium necrophorum (6) and Prevotella inermedia (3). From two to four anaerobes per specimen were present in 57.4% of the specimens yielding anaerobic bacteria. The susceptibility of the Gram-negative anaerobic isolates to penicillin and that of the Gram-positive anaerobes to clindamyin and metronidazole were unpredictable. The variable resistance patterns among anaerobes and the predominance of mixed anaerobic infections highlight the role of the anaerobic dignostics in case of serious pleuropulmonary diseases.     

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%).     

Identification and susceptibility to antimicrobial agents of strictly anaerobic bacteria isolated from hospitalized patients

The aim of this study was to identify anaerobic strains isolated in 2001 from clinical specimens obtained from patients of Warsaw hospital and to evaluate a susceptibility of these strains to antimicrobial agents. In 2001 two hundred and twenty five clinical strains of obligate anaerobes were cultured, which were identified in the automatic ATB system (bioMérieux, France) using biochemical tests API 20 A. Drug-susceptibility of strains was determined also in ATB system with the use of ATB ANA strips. C. difficile strains were isolated on selective CCCA medium. Toxins A/B of C. difficile directly in stool specimens were detected by means of ELISA test (TechLab, USA). Fifty four strains of Gram-negative anaerobes (B. fragilis strains dominated) and 171 strains of Gram-positive anaerobes (the greatest number of strains belonged to genus Peptostreptococcus) were cultured from clinical specimens. In the cases of antibiotic-associated diarrhea 28 C. difficile strains were isolated and C. difficile toxins A/B were detected in 39 stool samples. The most active in vitro antimicrobials against Gram-negative anaerobes were metronidazole, imipenem, ticarcillin combined with clavulanic acid and piperacillin with tazobactam. Gram-positive, clinical strains of anaerobes were the most susceptible in vitro to beta-lactam antibiotics combined with beta-lactamase inhibitors (amoxicillin/clavulanate, piperacillin/tazobactam, ticarcillin/clavulanate) and imipenem.     

Bacteremia After Genitourinary Tract Manipulation: Bacteriological Aspects and Evaluation of Various Blood Culture Systems

A total of 300 patients undergoing various types of urological procedures was studied for incidence of bacteremia. An osmotically stabilized anaerobic broth with sodium polyanethol sulfonate (Liquoid) yielded more positive blood cultures than any other culture system and was also the best system by far for recovery of anaerobes. The membrane filter showed faster growth and, therefore, facilitated faster identification of the infecting organism. There was a 31% incidence of bacteremia in the patients having transurethral resection of the prostate, 17% in the cystoscopy group, 24% in the urethral dilation group, and 8% in the urethral catheterization group. The organisms found most frequently isolated in blood cultures were enterococci and Klebsiella pneumoniae. Notable were a relatively large number of anaerobes and two protoplasts. The major source of the bacteremia was previous urinary tract infection, but evidence is presented which indicates that the prostate gland and the normal urethral flora are other significant sources.     

Comparison of recovery of anaerobic bacteria using the Anoxomat, anaerobic chamber, and GasPak jar systems

The Anoxomat system provides an automated evacuation-replacement technique to create an anaerobic or microaerophilic environment in a jar. We evaluated the Anoxomat system for the growth of obligate anaerobes and for the recovery of anaerobic organisms from clinical specimens, and compared its performance to that of an anaerobic chamber and the GasPak System. Of the 54 stock strains tested, the Anoxomat, the chamber, and the GasPak recovered 95%, 95% and 93% at 24 h, respectively. On 29 occasions (51%), the colonies on the Anoxomat plates were slightly larger than those in the chamber and on 17 (30%) occasions larger than the colonies on the GasPak jar plates. At 48 h, the Anoxomat, the chamber, and the GasPak recovered 93.5%, 94.4% and 88.9%, respectively; of 108 anaerobes isolated from 31 clinical specimens. Methylene blue indicators became decolorized (average of 10 tests) within 2 h inside the Anoxomat jars, 2 h 10 min inside the anaerobic chamber, and 2 h 30 min inside the GasPak jars.     

Beta-lactamases with a wide substrate spectrum in gram negative strictly anaerobic rods

This study was performed to determine the susceptibility of the clinical strains of Gram-negative strictly anaerobic rods to newer beta-lactam antibiotics. Also, the trial was undertaken to detect strains producing extended-spectrum beta-lactamases (ESBLs) and inducible beta-lactamases (IBLs) among Bacteroides spp. and Prevotella spp. rods isolated from hospitalized patients. One hundred strains of Gram-negative, obligatory anaerobic rods were applied in the study. The strains were identified in automatic ATB system using API 20 A strips. beta-lactamase-positive strains were determined with disc nitrocefin test. ESBL-producing strains were detected with double disc test according to Jarlier et al. (1988). Clavulanate was applied as the inhibitor of these beta-lactamases (AMO/CLAV disc). ESBL-positive strains were confirmed with the use of E test (TZ/TZL strip). Inducible beta-lactamases were determined by double disc method according to Sanders and Sanders (1979). Cefoxitin was the inducer of these beta-lactamases (FOX disc). Among 93 Bacteroides spp. strains and 7 Prevotella spp. strains, 91 strains (91%) produced beta-lactamases. Two ESBL-producing strains (2%) were detected. Strains producing inducible beta-lactamases (IBL) were not found. A high activity of the examined beta-lactam antibiotics against strains of Gram-negative anaerobes was found. The majority of strains were susceptible to piperacillin (95%), piperacillin combined with tazobactam (99%), ticarcillin combined with clavulanic acid (99%), meropenem (97%) and imipenem (99%). The obtained results indicate the necessity of ESBL determination among strains of the genus Bacteroides, isolated from clinical specimens. Newer beta-lactam antibiotics, especially penicillins in combination with beta-lactamase inhibitors and carbapenems, are useful in empiric therapy of infections caused by Bacteroides spp. and Prevotella spp. anaerobic rods.     

Prevalence of aerobic and anaerobic uterine bacteria during peripartum period in normal and dystocia-affected buffaloes

Parturition complications predispose establishment of uterine infections, which in turn affect subsequent fertility. The aim of present study was to characterize and compare the type of bacterial flora prevalent within the uterine lumen of dystocia-affected buffaloes and compare them with the normally calving buffaloes. The study was conducted on 40 buffaloes; of which 10 calved normally (Group I) and 30 were treated for dystocia (Group II). Bacteriological examination was performed using uterine swabs, which were collected before delivery, immediately after delivery and day's 24-60 postpartum. A total of 30 uterine swabs from Group I and 79 swabs from Group II were collected, of which 19 (63.3%) and 71 (89.9%) yielded significant bacterial growth, respectively. A total of 205 isolates belonging to 10 different genera of bacteria were identified, 8 facultative anaerobes and 2 obligate anaerobes. In Group II, 91.6% of the bacteria positive swabs (n = 71) yielded mixed cultures, whereas the remainder being pure cultures. In contrast, 89.5% of the bacteria positive swabs of Group I (n = 19) yielded pure cultures. Mixed infections comprised mostly Arcanobacter (Actinomyces) pyogenes together with obligate anaerobes, Fusobacterium spp. and Bacteroides spp. In Group II, the frequency of incidental and coliform group bacteria was highest at the time of parturition, i.e., before and immediately after delivery, and decreased to nil during the 24-60-day postpartum period. However, in Group I, the incidental and coliform group of bacteria present at the time of parturition apparently persisted beyond the period when uterine involution is complete. The frequency of obligate anaerobes and A. pyogenes at the time of parturition was nil in the Group I while they predominated in dystocia-affected buffaloes (Group II). During the postpartum period of 24-60 days, the frequency of both obligate anaerobes and A. pyogenes increased significantly in Group II, whereas in Group I, only occasional isolates were obtained. To conclude, at the time of calving the prevalence of obligate anaerobes and A. pyogenes occurring in combination was highest in dystocia-affected buffaloes, and further increased in the postpartum period suggesting that these infections act synergistically.     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

Anaerobes isolated from clinical material over three years

Data on anaerobic bacteria isolated from clinical specimens at the bacteriology department within the 3-year period (1992-1994) were analysed. Anaerobic cultivation was carried out in all aspirates and swabs were transferred in transport media in syringes or blood cultures. Established growth occurred in all samples cultivated in thioglycollate broth after 4 days of incubation. Cultivation methods included enrichment media, GasPak jar, and API (BioMerieux) for final identification. A sulfite-reduction test using the Wilson-Blair medium and the Ellner-Smith sporulation medium was also used for the isolation of Clostridium perfringens. Anaerobes were diagnosed in 899 samples. Wound swabs (266 samples) and aspirates (106 samples) were the most common clinical material used. In total, 964 anaerobes were isolated: Peptostreptococcus species (299 strains), Eubacterium species (188 strains), Propionibacterium species (153 strains), Bacteroides fragilis(149 strains), Bacteroides species (95 strains) and Clostridium perfringens(80 strains).     

National hospital survey of anaerobic culture and susceptibility methods: III

To assess the current status of anaerobic bacteriology in the United States, we surveyed, by means of a questionnaire, 150 hospitals selected at random with bed capacities of 200-1000 and we received responses from 98 (65%). Ninety-eight percent processed anaerobic culture specimens with 21% sending them to reference laboratories. Almost all these hospitals processed blood and wound cultures for anaerobes and all used selective media for identification, including BBE (52%), LKV (77%), and PEA (53%) agars. All hospital laboratories attempted identification of blood culture isolates including 80% that attempted speciation. Wound cultures for anaerobic bacteria and sterile site cultures were also processed for anaerobes by almost all labs. Identification of B. fragilis group species to species level was performed only in 56% of labs always and 37% sometimes. Preformed enzyme kits were used by 66% of labs and 30% used special potency disks for identification. Susceptibility testing was performed in-house by 21% of hospital labs and sent out to reference labs an additional 20%. Susceptibility testing was attempted for all blood culture isolates by both hospital (21% of total labs) and reference laboratories, but only performed by 17% for sterile body site and 14% of the time for wound isolates. Etest was used most often followed by broth microdilution. No labs used the agar dilution or disk elution methods. The antimicrobials most often tested in hospital labs, predicated on the commercial panel used, were penicillin/ampicillin and clindamycin (15/18; 83%; 15% of total labs), metronidazole (16/18; 89%; 16% of total labs) and cefotetan and ampicillin/sulbactam (12/18; 67%; 12% of total labs), piperacillin/tazobactam (7/18; 39%; 7% of total labs), cefoxitin (9/18; 50%), imipenem (8/18; 44%), and chloramphenicol (6/18; 33%). Our current survey suggests that while many labs are processing anaerobic cultures, especially blood cultures, the identification of isolates and the performance of antimicrobial susceptibility testing of isolates are in disarray and in dire need of improvement.     

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.     

Imipenem in the treatment of patients with severe surgical infection]

The clinical efficacy and safety of intravenously administered imipenem/cilastatin in the treatment of 45 patients with severe bacterial septicemia due to intra-abdominal abscesses, respiratory and urinary tract as well as skin, soft tissue and bone infections was studied in the prospective and open trial. The in vitro antimicrobial activity of imipenem has been assessed on the basis of 909 bacterial strains isolated from patients treated and non-treated with imipenem/cilastatin. Among them were 526 Gram-negative, 370 Gram-positive aerobic bacteria and 13 Gram-negative anaerobic bacteria (Bacteroides sp.). Pathogen susceptibility to imipenem was determined with a disc-diffusion technique using Merck, Sharp Dohme sensitive discs containing 10 mcg of imipenem. Highly sensitive to imipenem were 96.8% of Gram-negative 82.7% of Gram-positive aerobic bacteria and 100% of Bacteroides sp. All patients, in whom evident foci of infection e.g. intra-abdominal abscesses were discovered, were operated on. The dosage of imipenem/cilastatin ranged from 1.5 to 2.0 g/24 h. Clinical cure and bacteriological elimination was achieved in 39 (86.7%) of patients while 6 (13.3%) showed marked clinical improvement. Before and during therapy, aerobic and anaerobic cultures were taken from accessible sites. All specimens were worked up using conventional bacteriological techniques. Before during and after therapy, samples for hematology, biochemistry and urinanalysis were obtained. Adverse clinical effects were noted in 2 (4.4%) patients. One had nausea and vomiting which were probably related to rapid infusion and disappeared after increasing the administration time, and one had transient diarrhea. In conclusion, imipenem/cilastatin was a well tolerated and effective drug in the treatment of life-threatening surgical infections.     

38例慢性化脓性中耳炎厌氧菌分析

对广东省湛江农垦第二医院３８例慢性化脓性中耳炎患者脓性标本,进行厌氧菌和需氧菌培养,结果３２例培养阳性,总检出率８４．２％。其中单纯厌氧菌和需氧菌检出率,分别占阳性标本的２１８％和４０．６％。两者混合阳性占３７．５％。厌氧菌中以革兰氏阳性球菌多见４８．０％（１２／２５）,需氧菌以绿脓杆菌多见４２．８％（１２／２８）。并对感染机理,细菌种类和药敏试验进行了讨论。     

An Evaluation of the Gaspak System in the Culture of Anaerobic Bacteria

S ummary : Comparable results were obtained with a commercially available disposable system for the production of H₂ and CO₂ in an anaerobic jar (the Gaspak system) and with a standard anaerobic jar procedure. Both systems were used with the Baird & Tatlock anaerobic jar and a room temperature catalyst. The test organisms included strict anaerobes such as Clostridium oedematiens type D and Cl. tetani. Comparative tests were also made with Bacteroides species from laboratory stock cultures and with freshly isolated strains of Bacteroides. On occasion, higher recoveries were obtained with the standard system when relatively demanding models were set up but it has limitations, having been developed initially for research purposes. The Gaspak system probably gives more reliable and more reproducible results in a laboratory in which experience with strict anaerobes is limited or sporadic.     

Participation of gram-negative anaerobes in infections in hospitalized patients

The aim of the study was to assess the prevalence and antibiotic susceptibility profiles of strictly anaerobic Gram-negative bacteria isolated from clinical samples taken from hospitalized patients from 01.01.2007 to 31.12.2008. The specimens were cultured using media, incubated at 37 degrees C under anaerobic conditions. Biochemical identification and antibiotic susceptibility were done in an automated system ATB Expression (bioMerieux S.A, France). For selected strains of Bacteroides sp. sensitivity was determined using E-test (AB BIODISK, Sweden). Overall 1274 strains of obligate anaerobes were isolated. Gram-negative bacteria were cultured in number of 333 strains. Most frequently isolated was Bacteroides sp. (46,9%) and Prevotella sp. (29,7%). Isolated bacteria are still susceptible to imipenem (100%), metronidazole (100%) and beta-lactam antibiotics with beta-lactamase inhibitors: amoxicillin/clavulanate (97,8%) piperacillin/tazobactam (99,1%), ticarcillin/clavulanate (99,1%).