Sibylle Scheuring

Prevalence of gyrA, gyrB, parC, and parE Mutations in Clinical Isolates of Streptococcus pneumoniae with Decreased Susceptibilities to Different Fluoroquinolones and Originating from Worldwide Surveillance Studies during the 1997-1998 Respiratory Season

ABSTRACT From 8,419 worldwide clinical isolates of Streptococcus pneumoniae obtained during 1997-1998, 69 isolates with reduced susceptibility or resistance to fluoroquinolones (FQs) were molecularly characterized. For the isolates in this prevalence study, onlyparC (Ser-79→Tyr) and gyrA (Ser-81→Phe or Tyr) mutations, especially in combination, were found to contribute significantly to resistance. These mutations influenced the FQ MICs to varying degrees, although the rank order of activity remains independent of mutation type, with ciprofloxacin the least active, followed by levofloxacin, gatifloxacin/grepafloxacin/moxifloxacin/sparfloxacin/trovafloxacin, and clinafloxacin/sitafloxacin. Efflux likely plays a crucial role in reduced susceptibility for new hydrophilic FQs.

Characterization of grlA,grlB, gyrA, and gyrB Mutations in 116 Unrelated Isolates of Staphylococcus aureus and Effects of Mutations on Ciprofloxacin MIC

ABSTRACT One hundred sixteen unrelated clinical isolates ofStaphylococcus aureus (70 ciprofloxacin resistant and 46 ciprofloxacin susceptible) from eight countries were studied for the presence of mutations in the grlA, grlB,gyrA, and gyrB gene loci. Two mutations withingrlA (located at codons 80 and 84) and two mutations withingyrA (located at codons 84 and 88) were clearly associated with ciprofloxacin resistance, although other mutations detected within the four genes studied may also contribute to decreased susceptibility.

In Vitro Development of Resistance to Six Quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus

ABSTRACT Streptococcus pneumoniae, Streptococcus pyogenes, andStaphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.

Comparative sequence and expression analyses of four mammalian VPS4 genes.

The VPS4 gene is a member of the AAA-family; it codes for an ATPase which is involved in lysosomal/endosomal membrane trafficking. VPS4 genes are present in virtually all eukaryotes. Exhaustive data mining of all available genomic databases from completely or partially sequenced organisms revealed the existence of up to three paralogues, VPS4a, -b, and -c. Whereas in the genome of lower eukaryotes like yeast only one VPS4 representative is present, we found that mammals harbour two paralogues, VPS4a and VPS4b. Most interestingly, the Fugu fish contains a third VPS4 paralogue (VPS4c). Sequence comparison of the three VPS4 paralogues indicates that the Fugu VPS4c displays sequence features intermediate between VPS4a and VPS4b. Using complete mammalian VPS4a and VPS4b cDNA clones as probes, genomic clones of both VPS4 paralogues in human and mouse were identified and sequenced. The chromosomal loci of all four VPS4 genes were determined by independent methods. A BLAST search of the human genome database with the human VPS4A sequence yielded a double match, most likely due to a faulty assembly of sequence contigs in the human draft sequence. Fluorescent in situ hybridization and radiation hybrid analyses demonstrated that human and mouse VPS4A/a and VPS4B/b are located on syntenic chromosomal regions. Northern blot and semi-quantitative reverse transcription analyses showed that mouse VPS4a and VPS4b are differentially expressed in different organs, suggesting that the two paralogues have developed different functional properties since their divergence. To investigate the subcellular distribution of the murine VPS4 paralogues, we transiently expressed various fluorescent VPS4 fusion proteins in mouse 3T3 cells. All tested VPS4 fusion proteins were found in the cytosol. Expression of dominant-negative mutant VPS4 fusion proteins led to their concentration in the perinuclear region. Co-expression of VPS4a-GFP and VPS4b-dsRed fusion proteins revealed a partial co-localization that was most prominent with mutant VPS4a and VPS4b proteins. A physical interaction between the mouse paralogues was also supported by two-hybrid analyses.

MEN I gene mutations in sporadic adrenal adenomas

Abstract. Loss of heterozygosity (LOH) on chromosome 11q13 occurs in about 20% of sporadic adrenal neoplasms. Adrenal lesions, mostly benign, occur in up to 40% of patients from MEN I kindreds. The MEN I gene, positioned on 11q13, has been considered a primary candidate gene in these lesions. We studied a group of 15 patients with sporadic adrenal adenoma, and 1 patient with multinodular hyperplasia. Of the 16 patients, 4 had incidentally discovered masses, 5 had Conns syndrome, 6 suffered from Cushings syndrome, and 9 had high sex hormone production. Studies with the markers D11S480, PYGM, D11S449, and D11S987 in 13 patients (12 of whom were from our group of 16) revealed 4 losses of heterozygosity on D11S480 on 11q13, but the deletion did not affect the MEN I gene in any case. We present complete direct DNA sequencing data of the menin gene in 14 sporadic adrenal adenomas and one with adrenal hyperplasia. We identified one heterozygous missense mutation, T552S, in a hormonally inactive adrenal adenoma. One base exchange was identified close to the intron-exon boundary in intron 9 of a nodular adrenal hyperplasia. mRNA expression studies found that MEN I was transcribed in all 13 samples analyzed. In summary, our study identified the second patient with sporadic benign adrenal tumor presenting a menin gene mutation. Our complete direct sequencing approach adds evidence that menin gene mutations may account only for a minority of benign adrenal tumors if at all. Another tumor-suppressor gene inactivated in sporadic adrenal neoplasms may be located on chromosome 11q13.

In Vitro Activities of Novel Des-Fluoro(6) Quinolone BMS-284756 against Mutants of Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus Selected with Different Quinolones

BMS-284756 (T-3811) is a novel quinolone that lacks a fluorine at the C-6 position. BMS-284756 has a broad spectrum of antibacterial activity (3, 7). The purpose of the present study was (i) to analyze the in vitro activity of the novel quinolone against mutants of Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus selected with marketed quinolones and (ii) to investigate whether this compound is a substrate for efflux pumps. In order to analyze these therapeutically important aspects, we repeatedly exposed six clinical strains of each species to ciprofloxacin, gatifloxacin, moxifloxacin, and BMS-284756, as described previously for several other quinolones (1). MICs were determined using microdilution methodology according to NCCLS guidelines (5). MIC determinations for BMS-284756 were conducted in the presence and absence of reserpine (20μg/ml; tests were repeated three times) for all of the original isolates (n = 18) as well as for all of the selected mutants (n = 72) (1, 2). In addition, isolates were analyzed before and after transfers for mutations in the quinolone-resistance determining regions of parC or grlA and gyrA (4, 6, 8). The MIC results from subculturing as well as the alterations in the quinolone resistance-determining regions are listed in Table ​Table11, TABLE 1. Resistance selection results for six Streptococcus pneumoniae, six Streptococcus pyogenes, and six Staphylococcus aureus isolatesa BMS-284756 (range, 0.004 to 0.06 μg/ml) exhibited the best in vitro activities against all 18 original isolates, followed by moxifloxacin (range, 0.03 to 0.25 μg/ml) and gatifloxacin (range, 0.06 to 0.5 μg/ml). Ciprofloxacin (range, 0.125 to 2 μg/ml) showed the lowest in vitro activities. Furthermore, BMS-284756 (range, 0.015 to 8 μg/ml) exhibited the best in vitro activities against all 72 selected mutants, followed by moxifloxacin (range, 0.06 to 64 μg/ml), gatifloxacin (range, 0.06 to 128 μg/ml), and ciprofloxacin (range, 0.25 to 256 μg/ml) (Table ​(Table11). None of the original 18 strains and none of the 72 selected mutants displayed lower MICs for BMS-284756 in the presence of reserpine. These results illustrate that the novel quinolone is not a good substrate for efflux pumps. Based on the results presented, i.e., two- to ninefold increases in MICs after passages in BMS-284756-containing medium, the new quinolone seems to have the same potential for resistance development as that of the C-8 methoxy quinolones gatifloxacin and moxifloxacin. Alterations in ParC (S79Y or S79F) and GyrA (S81F) contributed to the resistance seen in S. pneumoniae mutants selected by BMS-284756. In Streptococcus pyogenes mutants, alterations in ParC (S79A and S79F) and GyrA (S81Y or E85G) were found. In addition, classical alterations in GrlA (S80Y, S80F, or E84K) and GyrA (S84L) contributed to the resistance in S. aureus mutants selected by BMS-284756. These classical alterations are identical to those observed in isolates selected by other quinolones (4, 6, 8). In summary, the new quinolone BMS-84756 has good in vitro activity against wild-type isolates and selected mutants of Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. The novel quinolone is not a good substrate for efflux. BMS-84756 seems to have the same potential for resistance development as that of the C-8 methoxy quinolones gatifloxacin and moxifloxacin.

Structural alterations in the translational attenuator of constitutively expressed erm(A) genes in Staphylococcus aureus.

The expression of the predominant macrolide resistance genes in staphylococci, erm(A) and erm(C) (2, 7), is either inducible by 14- and 15-membered macrolides via translational attenuation or constitutive. Constitutively expressed erm(A) and erm(C) genes are of particular clinical relevance since they also confer resistance to 16-membered macrolides, lincosamides, streptogramin B antibiotics, and the new ketolide drugs. To date, very few data on the molecular basis of constitutive erm(A) gene expression in naturally occurring staphylococci are available (8). In this study, we examined 64 clinical Staphylococcus aureus isolates from humans, collected at 24 European university hospitals within the SENTRY program (5, 6). All 64 isolates carried constitutively expressed erm(A) genes and exhibited the respective resistance phenotype as confirmed by MIC determination (5, 6). A previously described PCR assay (8) served for the detection of structural alterations in the erm(A) regulatory region. All 64 PCR amplicons were sequenced and compared to the sequence of the inducibly expressed erm(A) gene of Tn554 (3). Five different types of structural alterations were seen: deletions of 83, 121, 123 bp and two closely related tandem duplications of 25 bp each (Fig. ​(Fig.1).1). The two tandem duplications of 25 bp (5′-TAAGGAGAAGGTTATAATGAACCAG-3′ and 5′-GGAGAAGGTTATAATGAACCAGAAA-3′) were detected in 1 and 51 isolates, respectively, and comprised the erm(A)-associated ribosome binding site (AGAAGG) as well as the inverted repeat 6 (IR6) sequence (GGTTATAATGAAC). The 83-bp deletion found in two of the isolates comprised the entire open reading frame (ORF) of the 19-amino-acid (aa) peptide including IR3. The 121-bp deletion detected in nine isolates and the 123-bp deletion observed in a single isolate are closely related. Both types of deletions comprise the ORF of the 19-aa peptide as well as the part immediately downstream of it which includes the IR4 and IR5 sequences. These deletions and tandem duplications favored the formation of mRNA secondary structures which allowed translation of the erm(A) transcripts in the absence of inducers. FIG. 1 Regulatory regions of the inducibly expressed erm(A) gene of Tn554 (2) and the constitutively expressed erm(A) genes analyzed in this study. SD1 to -3, Shine-Dalgarno sequences of the ORFs of the 15-aa peptide, the 19-aa peptide and the erm(A) gene, respectively; ... The data show that tandem duplications and deletions of different sizes account for constitutive erm(A) gene expression in naturally occurring S. aureus isolates. So far, deletions of 26 to 141 bp, a tandem duplication of 12 bp, and a point mutation, all of which cause constitutive erm(A) gene expression, have been derived under in vitro selection in the presence of noninducers (3, 4). Thus, these data on in vivo-occurring mutations in the erm(A) translational attenuator in human isolates complement both the in vitro-derived mutations (3, 4) and the mutation seen in a naturally occurring S. intermedius isolate of avian origin (8), thus confirming that similar mutations can arise in vivo and in vitro (1). The development of constitutive erm(C) and erm(A) mutants is a fast and irreversible process. Under in vitro conditions constitutive mutants can be obtained after overnight cultivation in the presence of noninducers (1, 3, 7, 9), and reversion to the inducible type has not been observed in any such mutants. Thus, noninducers, such as lincosamides or streptogramins, should not be recommended for the control of staphylococci which exhibit an inducible macrolide-lincosamide-streptogramin B resistance phenotype.

Sequence, organization, chromosomal localization, and alternative splicing of the human serine protease inhibitor gene hurpin (PI13) which is upregulated in psoriasis.

Hurpin (protease inhibitor 13; PI13) is the most recently identified member of the ovalbumin family of serine protease inhibitors (serpins). It is expressed in human epidermal keratinocytes and is downregulated by exposure to ultraviolet irradiation. A role for hurpin in the proliferation or differentiation of keratinocytes has been proposed because of its strong expression in proliferating cells and its deregulated expression in the lesional epidermis of psoriatic patients. Here, we report the cloning, chromosomal localization, and complete sequence of the human hurpin gene. By PCR-based screening of the GeneBridge 4 radiation hybrid panel, we mapped the gene to chromosome 18q21.3, close to a known cluster of ov-serpin genes. Using the full-length cDNA for hurpin, we identified two clones from an arrayed genomic P1 placental library that contain the entire hurpin gene. Sequencing revealed that the gene covers 12.253 kb and is comprised of eight exons and seven introns. The exon--intron boundaries are identical in position and phasing to those in other members of the 18q serpin gene cluster, and analysis of hurpin variants indicated that modified functional inhibitors, differing only in the CD interhelical loop, can be generated by differential splicing of exon 3. These data show that hurpin is a typical member of the 18q ovalbumin-serpins most closely related to the serpins squamous-cell carcinoma antigens 1 and 2.