Patrick Descheemaeker

Identification and classification of Lactobacillus acidophilus, L. gasseri and L. johnsonii strains by SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization.

Thirty-two strains originally identified as Lactobacillus acidophilus and L. gasseri were screened for their taxonomic homogeneity by SDS-PAGE of whole-cell proteins. After numerical comparison of the resulting protein electrophoretic fingerprints, two well-delineated clusters were detected. The majority of the strains grouped in one electrophoretic cluster, which contained the type strain of L. acidophilus and corresponds to DNA group A1 of Johnson, J. L., Phelps, C. F., Cummins, C. S., London, J. & Gasser, F. (1980; International Journal of Systematic Bacteriology 30, 53-68). Another cluster corresponded to DNA group B. It contained two subclusters, which agreed perfectly with DNA subgroups B1 (L. gasseri) and B2 (L. johnsonii), respectively. The 23S rRNA genes were partially sequenced and 23S-rRNA-targeted oligonucleotide probes were designed for identification of DNA groups A1, B1 and B2. Probe Lbg reacted with all strains of electrophoretic cluster B1 (L. gasseri), probe Lbj hybridized with strains of cluster B2 (L. johnsonii) and probe Lba with strains of cluster A1 (authentic L. acidophilus). The probes were successfully used for the identification of strains belonging to the respective species. The phylogenetic relationship of a representative of L. johnsonii was determined by comparative sequence analysis of the 16S rRNA genes. It is very closely related to L. gasseri.

Evaluation of Arbitrarily Primed PCR Analysis and Pulsed-Field Gel Electrophoresis of Large Genomic DNA Fragments for Identification of Enterococci Important in Human Medicine

The increasing problems encountered with enterococcal nosocomial infections and the intrinsic and acquired resistance of the enterococci to different antimicrobial compounds highlight the need for a rapid identification technique. Enterococcus faecalis is readily identified by biochemical tests, but species differentiation within the Enterococcus faecium and Enterococcus gallinarum species groups is less well established. In the present study, 66 strains representing the most prevalent human enterococci were used to develop a PCR-based species-specific identification protocol. Whole-cell protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used as a reference method for species identification. In addition, the genomic SmaI macro-restriction fragment distribution of all of the strains was examined by pulsed-field gel electrophoresis (PFGE). Oligonucleotide D11344-primed PCR was as discriminative as whole-cell protein analysis and resulted in more easily interpreted band patterns. This PCR-based technique allowed identification of clinical isolates by visual examination of the DNA profiles obtained. The inability of both methods to discriminate between Enterococcus casseliflavus and Enterococcus flavescens brought into question the species status of E. flavescens. PFGE did not result in species-discriminative DNA bands or band patterns, but proved to be superior for interpretation of interstrain relationships.

Enterococcus villorum sp nov., an enteroadherent bacterium associated with diarrhoea in piglets.

The taxonomic positions of five enteroadherent bacterial pig isolates, showing phenotypic characteristics most similar to those of Enterococcus durans and Enterococcus hirae, were investigated in a polyphasic study that included 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determinations, whole-cell protein fingerprinting, D11344-primed PCR typing and an extensive examination of phenotypic properties. The results demonstrated that the organisms represent a new species in the Enterococcus faecium species group, for which the name Enterococcus villorum sp. nov. is proposed. The type strain is LMG 12287T (= CCM 4887T).

Differentiation and identification of Enterococcus durans, E. hirae and E. villorum

Aims: To compare different tests in the identification of Enterococcus durans, E. hirae and E. villorum strains. These bacteria belong to the E. faecium species group and are phylogenetically closely related, as evidenced by 16S rRNA sequence homologies of over 98·8%.

Molecular characterisation of group A streptococci from invasive and non-invasive disease episodes in Belgium during 1993-1994.

Five hundred clinical group A streptococcal (GAS) isolates were collected in Belgium during the period 1 Nov. 1993 to 31 Oct. 1994. Clinical and laboratory data were recorded and isolates were characterised. The presence of the genes encoding streptococcal pyrogenic exotoxin types A (speA), B (speB), C (speC), F (speF) and streptococcal superantigen (ssa) were determined by PCR to target specific sequences. These isolates were also emm-typed and analysed by pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments with the enzyme SmaI. In total, 136 unrelated GAS PFGE types were identified and genetic diversity was clearly demonstrated. Two GAS PFGE types predominated; a first PFGE type comprised 66 (13.2%) emm1 isolates characterised by speA-, speB+, speC-, speF+ and ssa-; the second PFGE type comprised 44 (8.8%) emm12 isolates characterised by speA-, speB+, speC+ (or speC-), speF+ and ssa-. Indistinguishable PFGE types were observed among both invasive and non-invasive isolates. Ten different PFGE types were found among 11 streptococcal toxic shock syndrome (STSS) isolates, and five of these lacked speA. Twenty-five (34.7%) of 72 invasive isolates gave negative results for speA, speC and ssa. This retrospective study confirmed the observation that the dissemination of one specific clone cannot be associated with invasive GAS disease and posed a question regarding the role of SPE A as a major virulence factor. Other streptococcal virulence factors in conjunction with host factors may determine the outcome of invasive GAS infection.

THE APPLICATION OF FATTY-ACID METHYL-ESTER ANALYSIS (FAME) FOR THE IDENTIFICATION OF HETEROTROPHIC BACTERIA PRESENT IN DECAYING LEDE-STONE OF THE ST-BAVO-CATHEDRAL IN GHENT.

Abstract The heterotrophic bacterial community present in decaying Lede-sandstone of the Cathedral of Ghent (Belgium) was studied. Two-hundred thirty-two heterotrophic bacterial strains were isolated of which 162 were studied by fatty acid methyl ester analysis (FAME). One-hundred forty isolates were Gram-positive; 101 strains belong to the genus Micrococcus ; nine strains were identified as M. kristinae . One large cluster of 83 strains was related to M. luteus / M. lylae . Other Gram-positive strains were related to Arthrobacter . Of the 22 Gram-negative strains, seven strains were related to Pseudomonas vesicularis . A number of Gram-negative and Gram-positive isolates formed separate clusters which could not be identified. The possible role of the bacterial isolates in stone degradation was examined by a zone-clearing test on a calcite-containing medium. Of the 152 strains examined, only nine showed clearing zones after 7 days of incubation at 25°C, 20 odd strains showed a dissolution of calcite after 14 days, whereas all other strains showed no clearing zones, even after prolonged incubation (up to 3 months). The zone-clearing test showed the same reactions when incubated at 24°C or at 19°C.

Comparison of the Lactococcus lactis differential medium (DCL) and SDS-PAGE of whole-cell proteins for the identification of lactococci to subspecies level

Summary In order to evaluate the practical usefulness of the DCL differential medium and highly standardised SDS-PAGE fingerprinting of whole-cell proteins for the identification of Lactococcus strains to subspecies level, a total of 99 Lactococcus lactis subsp. lactis and subsp. cremoris strains were analysed by both techniques. The SDS-PAGE protein patterns were compared to reference strains of lactic acid bacteria of the genera Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Tetragenococcus , and Vagococcus . Numerical analysis of the SDS-PAGE protein patterns clearly discriminated all species investigated. Seventy two Lactococcus lactis strains were assigned to their respective Lactococcus lactis subspecies. Twelve strains were originally incorrectly identified to subspecies level and two strains were identified as Lactococcus garvieae . Four Lactococcus lactis strains did not belong to the genus Lactococcus ; one strain remained unidentified. For eight Lactococcus lactis strains identification to subspecies level was not possible by SDS-PAGE. Identification of Lactococcus lactis subsp. lactis var. diacetilactis strains could not be achieved by SDS-PAGE. One strain received as Lactobacillus sp. and two Lactococcus sp. strains were also identified as Lactococcus lactis . Due to a very high electrophoretic similarity observed between Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris strains, subspecies discrimination required a high reproducibility level of the electrophoretic procedures used. The DCL differential medium failed for four Lactococcus lactis subsp. cremoris strains on solid medium but was correct when tested in broth. For three Lactococcus lactis subsp. lactis strains, including the type strain, the reaction was not conclusive. For 80 out of the 85 Lactococcus lactis strains identified to subspecies level by SDS-PAGE, the DCL differential medium was correct. One Lactococcus lactis strain, on repetition, displayed DCL test results contradictory to the SDS-PAGE identification. For four Lactococcus lactis strains no conclusion could be drawn from the DCL test results, while a good reaction was obtained (identified as Lactococcus lactis subsp. lactis ) for four non-lactococcal strains as well as for two Lactococcus garvieae strains. The test results for one unidentified isolate were not conclusive. Ten strains were identified as Lactococcus lactis subsp. lactis var. diacetilactis.

Diagnosis of hepatitis C virus genotype 2k/1b needs NS5B sequencing.

Hepatitis C virus (HCV) is probably the most common cause of liver cirrhosis and hepatocellular carcinoma worldwide. The correct identification of HCV genotype has important clinical implications as a marker of responsiveness to antiviral therapy and serves as a guideline for the duration of treatment. The VERSANT HCV Genotype 2.0 Assay failed to detect HCV genotype 2k/1b. HCV genotype 2k/1b detection requires NS5B sequencing.

Heads or Tails: Genotyping of Hepatitis C Virus Concerning the 2k/1b Circulating Recombinant Form

As different hepatitis C virus (HCV) genotypes respond differently to initiated therapy, correct HCV genotyping is essential. A potential risk for misclassification of the intergenotypic HCV circulating recombinant form (CRF) 2k/1b strains exists, depending on the genotyping method used. The aim was to investigate the differences in HCV genotyping methods with regard to CRF 2k/1b and to gain insight in the prevalence of the CRF 2k/1b. Genotyping results by Versant HCV Genotype Assay were compared with nonstructural protein 5B (NS5B) sequencing. In total, from November 2001 until March 2015, 3296 serum samples were analyzed by Versant HCV Genotype Assay. As misclassified CRF is harbored among HCV genotype 2, we further focused our search on 142 (4.3%) samples positive for HCV genotype 2. On 116 (81.7%) retrieved samples, the NS5B sequencing was performed. Twelve out of the 116 retrieved samples (10.3%) were classified as CRF 2k/1b by sequencing of the NS5B region. Ten of these 12 samples were originally misclassified as genotype 2a or 2c, while 2 of them were misclassified as genotype 2. Our results show that the current prevalence of CRF 2k/1b is underestimated. The importance of correct HCV genotyping is emphasized, considering the tailored choice of treatment regimen and overall prognosis.

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients

Abstract Background Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. Objectives First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. Study design We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. Results The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p <0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p >0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. Conclusions Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.