Bacteroides fragilis: A whole MALDI-based workflow from identification to confirmation of carbapenemase production for routine laboratories.

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level. The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well. The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections.

Fungaemia caused by Candida pulcherrima.

Although neonatal bloodstream infections may be caused by a variety of fungi, invasive fungaemia due to Candida pulcherrima in a premature neonate has not been previously reported. We describe such a case in which antifungal susceptibility test data led to successful therapy. A colonized catheter used for parenteral nutrition is presumed to have been the main source of this persistent infection.

Examination of cfiA-mediated carbapenem resistance in Bacteroides fragilis strains from a European antibiotic susceptibility survey.

Of 1284 Bacteroides strains collected in Europe in 2000 for antibiotic susceptibility surveillance, 65 isolates displayed imipenem minimum inhibitory concentrations (MICs) > or =1 mg/L and were chosen for a thorough analysis of their resistance mechanism. Twenty-five of the isolates were positive for the cfiA carbapenem resistance gene. The resistance rates were 0.8% and 1.3% for imipenem and meropenem, respectively. In six of the strains, insertion sequence (IS) elements (IS613, IS614B, IS1186 and IS1187) activated the cfiA gene. However, other strains displayed at least elevated carbapenem MICs or were carbapenem resistant and produced measurable carbapenemase activities but did not harbour IS elements in the region upstream of the cfiA gene. The major determinant of carbapenem resistance in Bacteroides fragilis is production of CfiA metallo-beta-lactamase via activation of the cfiA gene by IS elements (higher level resistance) or by activation of its putative own promoter.

Characterisation of a 5.5-kb cryptic plasmid present in different isolates of Bacteroides spp. originating from Hungary.

The plasmid profiles of 97 Bacteroides isolates collected during screening for different pathogenic markers of this genus were investigated. In all, 48% of 69 isolates from infections that belonged to six species harboured low mol.wt plasmids (2.8-11.0 kb). Similar plasmids were also found in 39% of 28 isolates, belonging to eight species, from faeces of healthy persons. The two most frequently obtained types were the 5.5- and the 4.2-kb plasmids, which were present in 70% and 52% of all plasmid-bearing isolates, respectively. Restriction endonuclease analysis revealed that the 5.5-kb plasmids found in the different Bacteroides spp. exhibited the same restriction map, with the exception that pBVP61 lacked the PstI recognition site. The two plasmid types (4.2 and 5.5 kb) seem to be most widely distributed among Bacteroides isolates independent of the site of isolation and with some differences depending on geographic regions.

Prevalence of the carbapenemase gene (cfiA) among clinical and normal flora isolates of Bacteroides species in Hungary.

The carbapenemase gene (cfiA) was detected in 4 (5.7%) of 70 clinical isolates of Bacteroides fragilis from different parts of Hungary. Among 24 other Bacteroides species isolated from infectious processes or from normal faecal flora, none was cfiA-positive. The MIC of imipenem and meropenem for all cfiA-positive B. fragilis isolates was < or =0.25 mg/L, but 17% of the B. fragilis and 46% of the non-fragilis Bacteroides isolates exhibited reduced susceptibility to imipenem (MICs 0.5-2 mg/L). Only one of these isolates produced increased levels of beta-lactamase. No difference was observed in the outer-membrane proteins of B. fragilis isolates that harboured the cfiA gene and those with reduced susceptibility to imipenem.

Occurrence of metronidazole and imipenem resistance among Bacteroides fragilis group clinical isolates in Hungary.

During the period between 1987 and 1997, various surveillances of the antibiotic resistance of B. fragilis group isolates revealed that practically all the isolates tested were susceptible to imipenem, metronidazole and chloramphenicol; very few isolates (2.5%) exhibited resistance to amoxicillin/clavulanic acid. However, similarly as in some southern European countries, the percentages of the isolates that were resistant to ampicillin, tetracycline and clindamycin were high throughout this period, and the resistance to cefoxitin increased from 6% to 16%. In 2000, isolates with intermediate or high resistance to imipenem and isolates with increased MICs to metronidazole were emerging among the clinical isolates of B. fragilis. The presence of the cfiA gene was demonstrated by PCR in 7 of 242 isolates (2.9%); 2 of them with high MICs to carbapenems harboured the IS942 element immediately upstream of the resistance genes. In the 2 B. fragilis isolates with increased MICs to metronidazole, the nim gene could be detected by PCR. The IS1186 element was found in these isolates upregulating the metronidazole resistance gene.

Multidrug-resistant Bacteroides fragilis group on the rise in Europe?

We report a case of multidrug-resistance (MDR) in a strain of Bacteroides fragilis from a blood culture and abdominal fluid in a Danish patient. The patient had not been travelling for several years and had not received antibiotics prior to the present case. We also summarize the cases that have been reported to date of MDR B. fragilis group in Europe. As far as we know, a case like this with MDR B. fragilis has not been described in Scandinavia before.

Incidence, antibiotic resistance and clonal relations of MRSA strains isolated from a Romanian university hospital.

Our aim was to estimate the frequency and characteristics ofmethicillin-resistant Staphylococcus aureus (MRSA) strains occurring in a Romanian teaching hospital. We retrospectively studied isolates from infected or colonized patients treated at the intensive care and surgical units during January 2004-December 2005. The antibiotic susceptibility of MRSA strains and the presence of mecA gene were determined. Consecutively occurring strains isolated through a three-month period were typed using pulsed field gel electrophoresis. A total of 423 S. aureus strains were identified, methicillin-resistance was detected in 211 (49.9%) strains. Most of them were multiresistant. One of the MRSA genotypes identified by PFGE was commonly recovered from patients treated in the intensive care unit. According to our results, MRSA strains were frequently isolated pathogens in our hospital and there is an urgent need to enhance infection control efforts.

The place of molecular genetic methods in the diagnostics of human pathogenic anaerobic bacteria. A minireview.

Anaerobic infections are common and can cause diseases associated with severe morbidity, but are easily overlooked in clinical settings. Both the relatively small number of infections due to exogenous anaerobes and the much larger number of infections involving anaerobic species that are originally members of the normal flora, may lead to a life-threatening situation unless appropriate treatment is instituted. Special laboratory procedures are needed for the isolation, identification and susceptibility testing of this diverse group of bacteria. Since many anaerobes grow more slowly than the facultative or aerobic bacteria, and particularly since clinical specimens yielding anaerobic bacteria commonly contain several organisms and often very complex mixtures of aerobic and anaerobic bacteria, considerable time may elapse before the laboratory is able to provide a final report. Species definition based on phenotypic features is often time-consuming and is not always easy to carry out. Molecular genetic methods may help in the everyday clinical microbiological practice in laboratories dealing with the diagnostics of anaerobic infections. Methods have been introduced for species diagnostics, such as 16S rRNA PCR-RFLP profile determination, which can help to distinguish species of Bacteroides, Prevotella, Actinomyces, etc. that are otherwise difficult to differentiate. The use of DNA-DNA hybridization and the sequencing of special regions of the 16S rRNA have revealed fundamental taxonomic changes among anaerobic bacteria. Some anaerobic bacteria are extremely slow growing or not cultivatable at all. To detect them in special infections involving flora changes due to oral malignancy or periodontitis, for instance, a PCR-based hybridization technique is used. Molecular methods have demonstrated the spread of specific resistance genes among the most important anaerobic bacteria, the members of the Bacteroides genus. Their detection and investigation of the IS elements involved in their expression may facilitate following of the spread of antibiotic resistance among anaerobic bacteria involved in infections and in the normal flora members. Molecular methods (a search for toxin genes and ribotyping) may promote a better understanding of the pathogenic features of some anaerobic infections, such as the nosocomial diarrhoea caused by C. difficile and its spread in the hospital environment and the community. The investigation of toxin production at a molecular level helps in the detection of new toxin types. This mini-review surveys some of the results obtained by our group and others using molecular genetic methods in anaerobic diagnostics.

Overexpression and purification of enzymatically active recombinant integrase protein of Rous sarcoma virus.

The carboxy-terminal domain of polymerase gene of Rous sarcoma virus was cloned into an expression vector under the control of lac regulatory elements, resulting in the plasmid pMF1413. Upon isopropyl-beta-D-thiogalactopyranoside induction, viral integration (IN) protein was expressed in large quantity in Escherichia coli. The expressed recombinant protein was prepurified by successive washing of the bacterial pellet with 0.1 M NaCl and detergents. Further purification was performed in high yield by standard chromatography methods. The purified enzyme revealed selective DNA cleaving activity on supercoiled plasmid with the LTR-LTR junction fragment. The reaction was metal ion dependent, with a preference for Mn2+ over Mg2+, and showed substrate specificity at 1 mM MnCl2.