Biomedical subjects
Publications and source records attributed to M F Palepou.
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Fifteen of 22 clinical isolates of Enterococcus faecalis with high-level aminoglycoside resistance isolated in Greece, which were tested for mating ability, co-transferred pheromone response genes together with aminoglycoside resistance determinants to a sensitive recipient strain. Nine of them belonged to the same pulsotype, while the remaining six isolates were genetically unrelated. The prgB gene, which encodes aggregation substance, was detected in all the clinical isolates and transconjugants by both PCR and DNA hybridization but prgA, which encodes the surface exclusion protein, was only detected in two isolates, whereas it is present in most pheromone response plasmids from other sources.
Enterococcus faecium 10/96A from Brazil was resistant to vancomycin (MIC, 256 microg/ml) but gave no amplification products with primers specific for known van genotypes. A 2,368-bp fragment of a van cluster contained one open reading frame encoding a peptide with 83% amino acid identity to VanH(D), and a second encoding a D-alanine-D-lactate ligase with 83 to 85% identity to VanD. The divergent glycopeptide resistance phenotype was designated VanD4.
A Tn1546-related element with IS1216V at position 8839 underwent a structural change after storage of the host strain of Enterococcus faecium at 4 degrees C. The element acquired IS1542 at position 3932, nucleotides 8732 to 8831 were deleted, and the first 3417 nucleotides were lost and replaced by an inverted copy of the IS1216V-vanY-vanZ-inverted-repeat block from the 3' end. Insertion sequence movement is likely to play a key role in the evolution of VanA resistance elements.
Genes encoding carbapenemases in 15 reference strains of Chryseobacterium (Flavobacterium) meningosepticum from the United Kingdom National Collection of Type Cultures and in one recent clinical isolate were investigated. All the strains hydrolyzed imipenem, but their levels of resistance to carbapenems varied, with imipenem and meropenem MICs ranging from 2 to >32 microg/ml. The blaB gene, which encodes a molecular-class B carbapenemase, was detected in only six reference strains and in clinical isolate 97/P/5448. The gene from 97/P/5448 had 98% nucleotide identity with the published sequence of blaB (from strain NCTC 10585) and was designated blaB2. A distinct carbapenemase gene, designated blaB3, was cloned from the type strain of C. meningosepticum, NCTC 10016. blaB3 had an open reading frame of 750 bp with 82% nucleotide identity to blaB and blaB2 and encoded a beta-lactamase of 249 amino acids, including the putative signal peptide. This beta-lactamase showed 87.6 and 86.7% amino acid homology with BlaB and BlaB2, respectively. blaB3 was detected in one other reference strain besides NCTC 10016, but the genetic basis of the carbapenemase activity detected in the other seven reference strains was not defined. Thus, neither blaB nor blaB3 was ubiquitous in the strains of C. meningosepticum studied, indicating that the reference strains may represent more than one bacterial species, each with its own intrinsic metallo-beta-lactamase. Further taxonomic studies of C. meningosepticum are necessary to resolve this topic. Chryseobacterium spp. are environmental organisms and occasional opportunist pathogens. They apparently represent a reservoir of diverse metallo-beta-lactamases, which potentially spread to gram-negative bacteria of greater clinical significance.
Resistance to imipenem and meropenem was observed in 211 (16.5%) isolates of Pseudomonas aeruginosa recovered in a Greek university hospital during 1996 to 1998. In six isolates selected from throughout this period, high-level resistance to both carbapenems (MICs >/= 128 microg/ml) was associated with production of the class B beta-lactamase VIM-1. bla(VIM)-bearing isolates belonged to serotype O:12 and were indistinguishable by pulsed-field gel electrophoresis.
IS1542 is an insertion sequence-like element which was originally identified in the orf2-vanR intergenic region of VanA resistance elements of glycopeptide-resistant Enterococcus faecium from hospital patients and from non-human sources in the UK. The nucleotide sequence of IS1542 was determined. It showed 81% homology with IS256 and contained an open reading frame sufficient to encode a 390 amino acid peptide predicted to have 87.2% identity with the transposase of IS256. By PCR, IS1542 was detected in the genomes of 26 of 28 (93%) VanA enterococci isolated from poultry in the UK and Ireland, but in only two of 65 (3%) glycopeptide-sensitive or VanC enterococci from hospital patients in the UK and Brazil. IS1542 may be a useful marker for evolutionary and epidemiological studies of VanA glycopeptide resistance in enterococci.
Faeces received in a diagnostic laboratory were screened for glycopeptide-resistant enterococci (GRE) on modified Lewisham medium, with and without enrichment in Enterococcosel broth. Colonization by GRE was detected in 102/838 patients (12.2%). In 74 (73%) of colonized patients GRE were detected by both methods and in 28 (27%) they were detected only after enrichment. The carriage rate in hospitalized patients was 32% (93/289) compared with 2.3% (11/425) in the community (GP patients and food-handlers). Carriage of GRE increased with age. Clostridium difficile isolation was associated with GRE colonization, odds ratio 6.76 (P<0.001). Fifty-nine percent (60/102) of the GRE had the VanA phenotype and 41% (42/102) had the VanB phenotype. In the community VanA predominated (91%), whereas 64% (57/89) of the isolates from hospitalised patients were of the VanB phenotype.
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The susceptibilities to mupirocin of 102 selected clinical isolates of Staphylococcus aureus and of control strain S. aureus NCTC 6571 were determined by disc diffusion (using discs containing 5, 15, 25, 30, 50 and 200 microg of mupirocin) and Etest and the results were compared with MICs determined using an agar incorporation method. On the basis of agar incorporation MICs, 42 isolates were sensitive to mupirocin (MIC < or = 4 mg/L), 39 showed low-level resistance (MICs = 8-128 mg/L) and 22 were highly resistant (MICs > or = 256 mg/L) and contained the mupA resistance gene. Using Stokes' criteria, none of the discs used gave major errors (sensitive isolates classified as highly resistant) or very major errors (highly resistant isolates classified as sensitive) in assigning a category of susceptibility, but minor errors (a difference of one category) were noted with all strengths. The best correlation with agar incorporation MIC was obtained with 25 microg mupirocin discs, which classified correctly 98 (95%) isolates, while worse correlations were noted with 5 microg and 200 microg discs which are the only types currently available commercially, for which there were 47 and 30 minor errors, respectively. The MICs found by Etest were the same as, or lower than, those determined by agar incorporation. Etests classified correctly all 42 mupirocin-sensitive isolates, 19 (49%) low-level resistant isolates and 16 (73%) highly resistant isolates. Two isolates that contained the mupA gene and showed agar incorporation MICs of 256 mg/L and 512 mg/L were not classified as highly resistant by any of the diffusion methods used. Agar incorporation MIC determination, possibly supported by detection of the mupA gene, offers the most effective means of identifying high-level mupirocin resistance in S. aureus, although the Etest also proved to be reproducible. However, we conclude that 25 microg discs warrant further evaluation for possible use in clinical laboratories, as they appear to be more reliable than the discs currently available.
Differences were examined among 24 distinct elements mediating VanA-type glycopeptide resistance in enterococci isolated from hospital patients and non-human sources in the UK. The methods used included long-PCR restriction fragment length polymorphism (L-PCR RFLP) analysis and DNA hybridization. All elements had conserved vanRSHAX genes, but variation occurred upstream of vanR and downstream of vanX. Twenty-one VanA elements had significant alterations upstream of vanR in the transposition genes orf1 and orf2: either parts of these genes were absent or they were disrupted by IS1216V or IS3-like insertion sequences. Among VanA elements with alterations downstream of vanX, seven lacked vanY, one lacked both vanY and vanZ, and ten had copies of insertion sequence IS1216V between vanX and vanY. All VanA elements of group D (from geographically and temporally diverse enterococci) were characterized by the presence of an IS1216V/IS3-like/orf1 complex and a point mutation in vanX, both of which were absent from the other 23 groups of VanA elements. This finding is consistent with the dissemination of a stable resistance element. We conclude that L-PCR RFLP analysis, combined with DNA hybridization, merits further development for studying the evolution and epidemiology of VanA resistance elements in enterococci.
Elements mediating VanA glycopeptide resistance in 106 diverse enterococci from humans and nonhuman sources were compared with the prototype VanA transposon, Tn1546, in Enterococcus faecium BM4147. The isolates included 64 from individual patients at 15 hospitals in the United Kingdom (isolated between 1987 and 1996) and 42 from nonhuman sources in the United Kingdom (27 from raw meat, 7 from animal feces, and 8 from sewage). VanA elements were assigned to 24 groups (designated groups A to X) with primers that amplified 10 overlapping fragments of Tn1546. Ten groups of elements were found only in human enterococci, eight groups of elements were unique to nonhuman strains, and six groups of elements were common in enterococci from all sources. Elements indistinguishable from Tn1546 (group A) were observed more frequently in enterococci from nonhuman sources (34 versus 9%) but were identified in enterococci that caused outbreaks in hospital patients between 1987 and 1995. The most common group found in human enterococci (group H; 33%) was rarely observed in enterococci from other sources (5%). Group H elements differed from Tn1546 in three regions and included a novel insertion sequence, designated IS1542, between orf2 and vanR. The VanA elements of 14 other groups had a similar insertion at this position and/or distinct insertions at other positions. We conclude that VanA elements in enterococci are heterogeneous, although all show regions of homology with Tn1546. Furthermore, the elements most common among the human and nonhuman enterococci studied were different. This approach may be useful for monitoring the evolution of VanA resistance and may also be applicable in local "snapshot" epidemiological studies. However, as transposition events involving insertion sequences accounted for the differences observed between several groups, the stability of the elements must be assessed before their true epidemiological significance can be determined.
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An unusual case of discitis and vertebral osteomyelitis due to Haemophilus aphrophilus is described. Infections due to this organism have usually responded to treatment with beta-lactam antibiotics. However, our isolate was resistant to third-generation cephalosporins which has not been reported previously in the world literature. The patient made a good clinical response to ciprofloxacin treatment.
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