Molecular characterization and antimicrobial susceptibilities of extra-intestinal Clostridium difficile isolates

Amongst 25 extra-intestinal clinical isolates of Clostridium difficile, A(+)B(+) (72%) and A(-)B(+) (4%) toxigenic phenotypes, as well as the non-toxigenic phenotype (A(-)B(-)) (24%), were identified. The A(-)B(-) isolates did not express toxin, yet carried part of the tcdA and tcdB gene and are of a previously unreported toxinotype. Six A(+)B(+) isolates also carried binary toxin genes. Resistance to erythromycin (20%), clindamycin (48%), tetracycline (16%), moxifloxacin (16%) and imipenem (11%) occurred but with no apparent correlation to phenotype. None of the strains was resistant to vancomycin or metronidazole. Imipenem-resistance decreased by EDTA, but susceptibility to meropenem suggests the presence of an imipenem specific metalloenzyme.     

A survey of metronidazole and vancomycin resistance in strains of Clostridium difficile isolated in Warsaw, Poland

The drug of choice used to treat Clostridium difficile-associated diarroea (CDAD) are metronidazole and vancomycin. Information about emergence of antimicrobial resistance among C. difficile strains to metronidazole and intermediate resistance to vancomycin in some countries are alarming. This study was performed to determine the susceptibility to metronidazole and vancomycin of 193 C. difficile strains isolated in our diagnostic laboratory between year 1998 and 2003 from patients adults and children suffering from CDAD. Among these strains, 142 produced toxin A and B (TcdA(+)TcdB(+)), 43 only B (TcdA(-)TcdB(+)) and 8 were nontoxigenic. We have not observed any differences in susceptibility to metronidazole and vancomycin between all C. difficile strains under investigation (toxinogenic and non-toxinogenic). Resistance to metronidazole and vancomycin was not observed.     

艰难梭菌耐药性及耐药机制研究进展

艰难梭菌(Clostridium difficile)是医疗保健相关性腹泻最主要的病原菌。2002年起欧美地区艰难梭菌感染发病率和病死率均明显增高,耐药艰难梭菌的出现和传播更给临床治疗和预防带来了挑战。绝大多数临床分离菌对甲硝唑及万古霉素仍呈高度敏感,但已有异质性耐药或最低抑菌浓度上升的报道;对红霉素和莫西沙星等其他抗菌药物的耐药率在不同国家和地区则有较大差异。艰难梭菌对甲硝唑或万古霉素敏感性下降产生的耐药机制尚不明确,而对红霉素、氟喹诺酮类、四环素和利福霉素形成的耐药机制主要是因为作用靶点发生了改变。文章简述了近年来国际上艰难梭菌耐药性及耐药机制方面的研究进展。     

Assessment of susceptibility to metronidazole and vancomycin of Clostridium difficile strains isolated between 1998-2002

The drugs of choice used to treat C. diffcile associated diarrhoea (CDAD) are metronidazole and vancomycin. C. difficile strains isolated in most laboratories are susceptible to metronidazole and vancomycin. Communication about emergence of antimicrobial resistance among C. difficile strains in some countries to metronidazole and intermediate resistance to vancomycin are alarming. This study was performed to determine the susceptibility to metronidazole and vancomycin of 140 C. difficile strains isolated from patients with CDAD hospitalised in academic hospital between 1999-2002. Resistance to metronidazole and vancomycin was not observed.     

Occurrence of Clostridium difficile in feces of children with dysfunction of the digestive tract and other disorders

Thousand and six hundred ninety two fecal samples from children of few weeks old up to over ten years were tested for the presence of Clostridium difficile. Most of them were treated with antibiotics and showed diarrhea symptoms. Hundred and twenty three strains of C-difficile were submitted to serological typing and their sensitivity to 10 selected antibiotics and chemotherapeutic agents was determined. Among 109 strains of C. difficile tested for enterotoxin production by latex test 82 strains (75.2%) were positive. Almost half (48%) of the isolated strains belonged to serotype C. Most of the strains were resistant to cefoxitin (88%) and clindamycin (76%). Over 90% of strains were sensitive to vancomycin and azlocillin and 86% to chloramphenicol and metronidazole. In the majority of patients with positive C. difficile cultures diarrhea was present, however, it was difficult to find a direct link between these symptoms and antibiotic therapy.     

抗生素在体内外对厌氧菌和艰难梭菌细胞毒素生成的作用

氯林霉素、灭滴灵和甲砜霉素对大多数肠道厌氧菌的生长具抑制作用。氯林霉素还会破坏肠道菌群平衡,使原来受抑制的艰难梭菌得以定植,并在艰难梭菌浓度达10~8/g盲肠内含物时,检测到艰难梭菌细胞毒素。培养基中亚抑菌浓度的氯林霉素和灭滴灵会推迟艰难梭菌细胞霉素的生成。灭滴灵还可保护无菌小鼠及受氯林霉素处理的悉生小鼠免遭艰难梭菌细胞毒素的致死作用,从而证实了灭滴灵在伪膜性结肠炎临床治疗中的可用性。     

Use of the Etest method for antimicrobial susceptibility testing of obligate anaerobes]

The aim of this study was to evaluate Etest usefulness for antimicrobial susceptibility testing of obligate anaerobes and to compare the activity of five antibacterial drugs against clinical strains of anaerobes. One hundred strains of obligate anaerobes were tested: 2 reference strains (B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741) and 98 clinical strains isolated from patients of the Infant Jesus Clinical Hospital--Center for Trauma Treatment in Warsaw during the last three years (1997-1999). Strains of seven genera of obligate nonsporeforming anaerobes (Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Propionibacterium and Actinomyces) and strains of two sporeforming species (C. perfringens and C. difficile) were examined. The MIC values were determined by the gradient diffusion method Etest (AB BIODISK, Sweden). Wilkins-Chalgren solid medium supplemented with 5% of sheep blood was used. Test plates were incubated at 35 degrees C for 48 hours in glove-box (85% N2, 10% H2, 5% CO2). The MIC values for each strain and antimicrobial agent, and the MIC ranges for bacteria of the same species were established. Ten strains resistant to clindamycin, ten resistant to piperacillin, and ten resistant to imipenem were detected. Seven strains were resistant to metronidazole and two strains to piperacillin combined with tazobactam. Tazobactam restored the susceptibility of eight strains to piperacillin. Obtained results confirm that Etest method is useful for antimicrobial susceptibility testing of obligate anaerobes. Older (clindamycin and metronidazole) and newer (piperacillin, piperacillin/tazobactam and imipenem) antimicrobial agents revealed high and comparable activity against clinical strains of obligate anaerobes. The percentage of strains susceptible to tested antimicrobials was > or = 90. These antimicrobials may be still useful in the empiric treatment of infections caused by medically important anaerobes.     

Antimicrobial Susceptibility of Clostridium difficile from Different Sources

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic-associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. The strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline-resistant strains was highly toxigenic, whereas 42 (70%) of 60 sensitive strains were highly toxigenic.     

Antibiotic resistance among anaerobic Gram-negative bacilli: lessons from a French multicentric survey

Temporal changes of antibiotic susceptibilities among anaerobes in France are followed in our laboratory since 1992. For Bacteroides strains, resistance increased from 1992 to 1998 for amoxicillin-clavulanic acid, cefotetan and clindamycin. The present study evaluates the situation in 2000 for 434 Gram-negative anaerobic clinical isolates (obtained from 9 large university hospitals) by testing amoxicillin and ticarcillin alone or combined with clavulanic acid, cefoxitin, cefotetan, imipenem, clindamycin and metronidazole (using the NCCLS-approved method for MIC determination. The main genera tested included Bacteroides (359 strains of the fragilis group), Prevotella (40 strains), Fusobacterium (23 strains) and miscellaneous species (8 strains). Resistance rates within the B. fragilis group were: amoxicillin-clavulanic acid 5.6%, ticarcillin 33%, ticarcillin-clavulanic acid 2%, cefoxitin 13%, cefotetan 44%, clindamycin 33%, imipenem 1% and metronidazole <1%, respectively. Only one strain of B. fragilis was resistant to metronidazole (MIC=64 mg/L); due to the presence of the nimA gene on the chromosome. Resistance to imipenem or metronidazole was only found among the B. fragilis species. These two former drugs excepted, B. fragilis was less resistant to antibiotics than the other species. beta-lactamase production was detected for 357/359 strains of the fragilis group, 26/40 stains of Prevotella and 3/23 strains of Fusobacterium. Dynamic changes of antibacterial resistance are occurring within the B. fragilis group: decreased resistance to amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, imipenem while resistance for cefoxitin, cefotetan, clindamycin continues to increase. Regular antibiotic surveys are needed as a source of information to guide the empirical therapy of anaerobic infections.     

Identification of lactic acid bacteria isolated from human blood cultures

Fifteen lactic acid bacterial strains were isolated from blood cultures from 15 different patients in the Faculty Hospital in Brno, Czech Republic. All strains were identified using biochemical tests and repetitive PCR using the (GTG)5 primer. Doubtful identification results were confirmed by whole-cell protein analysis. The strains were assigned to the genera Lactobacillus (eight strains representing seven species), Leuconostoc (six strains representing four species) and Weissella (one strain). Antibiotic susceptibility testing was performed using the E-test and revealed high-level resistance to cotrimoxazol, metronidazole, vancomycin and teicoplanin, but nearly all strains were susceptible to erythromycin, clindamycin, ampicillin and penicillin.     

Susceptibility to antimicrobial drugs of enterotoxin producing strains of Bacteroides fragilis isolated from different countries

Twenty two Bacteroides fragilis strains isolated from clinical samples in different countries (England, France, the Netherlands, Poland and USA) were used in the experiments. In all strains the presence of enterotoxin (fragilysin) gene was found by PCR with primers 404/407. Drug susceptibility of B. fragilis strains was determined with Etest (MICs for penicillin G, ceftriaxone, amoxicillin/clavulanic acid, imipenem, clindamycin and metronidazole). MICs were estimated in accordance to the NCCLS recommendations (1997). All tested strains were susceptible to imipenem and metronidazole. Twenty one strains were susceptible and one was intermediate susceptible to amoxicillin/clavulanic acid. Fourteen strains were resistant to ceftriaxone and five were found highly resistant to clindamycin. All examined strains were resistant to penicillin G. Four tested strains were simultaneously resistant to penicillin G, ceftriaxone and clindamycin (three French human strains isolated from postoperative wound, peritoneal fluid and bone inflammation, and one strain isolated from a pig).     

医院感染葡萄球菌的耐药性及D-试验研究分析

目的了解医院感染葡萄球菌的耐药性及克林霉素诱导试验（D-试验）临床意义。方法从住院患者标本中分离到的539株葡萄球菌进行药敏试验和D-试验,所得结果进行统计分析。结果葡萄球菌中耐甲氧西林金黄色葡萄球菌（MRSA）和耐甲氧西林凝固酶阴性葡萄球菌（MPSE）的检出率高,分别为65．1％和83．6％,各种葡萄球菌对万古霉素敏感率为100％,对阿奠西林头孢菌素在内的各种β-内酰胺酶类抗生素敏感率低于35％,对红霉素耐克林霉素敏感的D-试验阳性率为57．O％。结论葡萄球菌耐甲氧西林检出率,呈多重耐药,在选用大环内酯类,克林霉素类抗生素时要注意D试验,合理用药,提高疗效。     

Antimicrobial susceptibility of some streptococci strains of anginosus group isolated from oral and maxillofacial infections

Streptococci strains of the anginosus group isolated from various oral and maxillofacial infections (OMF) were screened for their susceptibility to the following antimicrobial agents: benzylpenicillin, ampicillin, oxacillin, cephalothin, ceftazidime, cefotaxime, cefuroxime, erythromycin, clindamycin, tetracycline, chloramphenicol, vancomycin and trimethoprime-sulphamethoxazole. The isolates were susceptable to: clindamycin, chloramphenicol, vancomycin and all beta-lactam antibiotics, except ceftazidime to which 54.5% of the strains showed intermediate susceptibility. Intermediate susceptibility to tetracycline was found in 11.3% of the strains, whereas resistance to the same antibiotic was demonstrated in 61.4%. Resistance to erythromycin and trimethoprime-sulphamethoxazole was of 2.3% for both. In conclusion, penicillin is the drug of choice in infections caused by streptococci of the anginosus group.     

Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species from the Human Gastrointestinal Tract

One hundred and twenty-two strains of Bifidobacterium and Lactobacillus species have been tested against 12 antibiotics and two antibiotic mixtures by a commercial system (Sensititre Anaero3; Treck Diagnostic Systems). The upper limits of some minimum inhibitory concentrations (MICs) were completed on MRS agar plates by the NCCLS procedure. All strains were sensitive to chloramphenicol and imipenem and most of the strains were resistant to metronidazole. Bifidobacteria isolates were susceptible to cefoxitin, whereas about half of the lactobacilli were resistant. Approximately 30% of the Bifidobacterium isolates were resistant to tetracycline, as well as five Lactobacillus strains belonging to four different species. None of the tested Bifidobacterium isolates was resistant to vancomycin, whereas a species-dependent resistance was found among the lactobacilli. Single strains of Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus brevis were resistant to erythromycin and/or clindamycin. Most of the observed resistances seemed to be intrinsic, but some others could be compatible with transmissible determinants.     

Assessment of changes in the epidemiology of Clostridium difficile isolated from diarrheal patients in Hungary

150 Clostridium difficile strains isolated from diarrheal feces were collected from three parts of Hungary and the presence of genes responsible for toxin A and B, and binary toxin production were examined. MIC distribution against clindamycin, erythromycin, metronidazole, moxifloxacin and rifampin of 80 toxigenic strains selected from the above-mentioned strains and 20 large clostridial toxins (LCTs)-positive strains chosen from our earlier strain collection were determined. 80% of the examined 150 strains were positive for both tcdA and tcdB, and no toxin A-negative, toxin B-positive isolates were found during the study period. 5.3% of toxigenic strains proved to be positive for binary toxin too. Among binary toxin-positive strains, one strain showed the same pattern characteristic of PCR ribotype 027. Comparison of recent findings and our earlier results, the prevalence of toxin-producing and binary toxin-positive strains among C. difficile isolated from diarrheal specimens increased. No metronidazole resistant isolate was detected among strains isolated in 2002–2003 and 2006–2007. The rates of resistance to erythromycin, clindamycin, moxifloxacin and rifampin among strains isolated between 2006 and 2007 were 25%, 27.5%, 25% and 6.3%, respectively. Erythromycin resistance was frequently associated with clindamycin and moxifloxacin resistance, however this resistant phenotype was not found among strains isolated in 2002–2003.     

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.     

In vitro antibiotic susceptibility of Bacteroides fragilis strains isolated from excised appendix of patients with phlegmonous or gangrenous appendicitis

Out of 34 studied after-appendectomy tissues of adult and child patients 86 different strains of anaerobes were isolated. The antibiotic susceptibility of 30 isolated B. fragilis strains was tested using E tests. All studied strains were sensitive to imipenem, clindamycin and penicillin/tazobactam. Sensitivity to penicillin and cefoxitin was variable among these strains. One strain resistant to metronidazole (MIC--256 mg/L) and 3 strains with increased MIC to metronidazole were detected. Most of isolated strains were beta-lactamase producers.     

Antimicrobial susceptibility, β-lactamase and enterotoxin production in <Emphasis Type="Italic">Bacillus cereus</Emphasis> isolates from clinical and food samples

The antimicrobial susceptibility of 30 clinical and 30 food Bacillus cereus isolates was determined. All isolates were susceptible to streptomycin, ciprofloxacin and gentamicin, 90 % of them to clindamycin and vancomycin, and 67 % to erythromycin. All isolates were resistant to amoxicillin with clavulanic acid, ampicillin, cefotaxime, ciprofloxacin, cloxacillin, cefotaxime with clavulanic acid and penicillin. The MIC values (determined by E-tests) were 48–256 mg/L for ampicillin, 0.19–1.5 mg/L for gentamicin, 0.125–1.0 mg/L for clindamycin, 0.047–4.0 mg/L for erythromycin and 1.5–16 mg/L for vancomycin. The MICs 4.6–18.75 g/L were observed for penicillin using the microdilution method. The presence of metallo-β-lactamases was detected by E-test for 100 % of strains. Nonhemolytic diarrheal enterotoxin (NHE) was produced by 98.3 % of strains, while 31.7 % of them produced hemolytic diarrheal enterotoxin (HBL). Clinical isolates produced 10 % more HBL than food isolates. The psychrotrophic strains isolated from food samples produced NHE at 6.5 °C in 73 % of cases.     

New PCR ribotypes of Clostridium difficile detected in children in Brazil

A total of 35 Brazilian isolates of Clostridium difficile from faecal stools and four isolates from hospital environments were analyzed by PCR ribotyping. A whole cell protein profile (as an alternative for serogrouping), in vitro toxin production and susceptibility to vancomycin, metronidazole and clindamycin were also investigated. All strains were typeable by both phenotypic and genotypic methods, and a total of 13 different PCR ribotypes were identified, of which seven (132, 133, 134, 135, 136, 142 and 143) were considered new types and accounted for 78.5% of all samples evaluated (including hospital environments). A non-toxigenic C. difficile PCR ribotype 133 was detected in all children groups examined (inpatients, outpatients and healthy children), whilst toxigenic PCR ribotypes 015, 131, 134 and 135 were associated mostly with symptomatic children. Serogroups G and D were disseminated both in patients from the community and from the pediatric hospital, with group G prevalent among outpatient children. All strains were susceptible to vancomycin and metronidazole but high levels of resistance to clindamycin were found, especially among serogroups G and D. Co-existence of different ribotypes and serogroups in the same individual was observed. The new seven ribotypes found in this investigation may represent strains characteristic of this region of Brazil.     

Clostridium difficile in emergency room

Clostridium difficile strains are known as etiological agents of pseudomembranous colitis (PMC), antibiotic-associated diarrhea (AAC) and colitis (AAC) and hospital-acquired infections. The aim of this study was to determine the frequency of C. difficile infection among patients in the emergency room and to compare isolated strains by phenotypic and genotypic characteristics. During a period of 11 months, 56 stool samples taken from diarrheic patients hospitalized in the emergency room of the Medical Center UC Davis and 14 environmental samples were cultured for isolation of C. difficile strains. Eighteen C. difficile strains were isolated from stool samples cultured on selective TCCCA plates and 5 strains from environmental samples using Rodac plates. Eleven toxigenic (TcdA+/TcdB+), 6 non-toxigenic (TcdA-/TcdB-) and unique toxin A-negative/toxin B-positive (TcdA-/TcdB+) C. difficile strains were detected among patients' isolates and 3 toxigenic and 2 non-toxigenic strains-among environmental samples. The majority of C. difficile-positive patients were treated previously by antibiotics. Four strains isolated from patients' fecal samples and one strain isolated from the environment demonstrated high-level resistance to erythromycin and clindamycin (MIC >256mug/mL). The results obtained by AP-PCR and PCR-ribotyping revealed genetic heterogeneity among the strains isolated from patients' fecal samples. However, similarity was observed among environmental strains and strains isolated from patients' fecal samples. Considering the importance of emergency room patients as a potential source of C. difficile strains, it appears to be important examine these patients for C. difficile before transfer to the other hospital units.