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ABSTRACTWe evaluated the in vitroactivity of delafloxacin against a panel of 117 Neisseria gonorrhoeaestrains, including 110 clinical isolates collected from 2012 to 2015 and seven reference strains, compared with the activities of seven antimicrobials currently or previously recommended for treatment of gonorrhea. We examined the potential for delafloxacin to select for resistant mutants in ciprofloxacin-susceptible and ciprofloxacin-resistant N. gonorrhoeae. We characterized mutations in the gyrA, gyrB, parC, and parEgenes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) by PCR and sequencing and by whole-genome sequencing. The MIC50, MIC90, and MIC ranges of delafloxacin were 0.06 μg/ml, 0.125 μg/ml, and ≤0.001 to 0.25 μg/ml, respectively. The frequency of spontaneous mutation ranged from 10−7to <10−9. The multistep delafloxacin resistance selection of 30 daily passages resulted in stable resistant mutants. There was no obvious cross-resistance to nonfluoroquinolone comparator antimicrobials. A mutant with reduced susceptibility to ciprofloxacin (MIC, 0.25 μg/ml) obtained from the ciprofloxacin-susceptible parental strain had a novel Ser91Tyr alteration in the gyrAgene. We also identified new mutations in the gyrAand/or parCand parEgenes and the multidrug-resistant efflux pumps (MtrC-MtrD-MtrE and NorM) of two mutant strains with elevated delafloxacin MICs of 1 μg/ml. Although delafloxacin exhibited potent in vitroactivity against N. gonorrhoeaeisolates and reference strains with diverse antimicrobial resistance profiles and demonstrated a low tendency to select for spontaneous mutants, it is important to establish the correlation between these excellent in vitrodata and treatment outcomes through appropriate randomized controlled clinical trials.

ABSTRACTActinobacillus pleuropneumoniaeis the etiological agent of porcine pleuropneumonia. Tetracycline is used for therapy of this disease, and A. pleuropneumoniaecarrying the tet(B) gene, coding for an efflux protein that reduces the intercellular tetracycline level, has been described previously. Of the 46 tetracycline-resistant (Tcr) Spanish A. pleuropneumoniaeisolates used in this study, 32 (70%) carried the tet(B) gene, and 30 of these genes were associated with plasmids. Eight (17%) isolates carried the tet(O) gene, two (4%) isolates carried either the tet(H) or the tet(L) gene, and all these genes were associated with plasmids. This is the first description of these tetgenes in A. pleuropneumoniae. The last two Tcrisolates carried none of the tetgenes examined. Except for tet(O)-containing plasmids, the other 34 Tcrplasmids were transformable into an Escherichia colirecipient. Two plasmids were completely sequenced. Plasmid p11745, carrying the tet(B) gene, was 5,486 bp and included a repgene, encoding a replication-related protein, and two open reading frames (ORFs) with homology to mobilization genes of Neisseria gonorrhoeaeplasmid pSJ7.4. Plasmid p9555, carrying the tet(L) gene, was 5,672 bp and, based on its G+C content, consisted of two regions, one of putative gram-positive origin containing the tet(L) gene and the other comprising four ORFsorganized in an operon-like structure with homology to mobilization genes in other plasmids of gram-negative bacteria.

ABSTRACTSeventy-six Neisseria gonorrhoeaeisolates, isolated between 1940 and 1987, and seven Neisseria meningitidisisolates, isolated between 1963 and 1987, were screened for the presence of acquired mef(A), erm(B), erm(C), and erm(F) genes by using DNA-DNA hybridization, PCR analysis, and sequencing. The mef(A), erm(B), and erm(F) genes were all identified in a 1955 N. gonorrhoeaeisolate, while the erm(C) gene was identified in a 1963 N. gonorrhoeaeisolate. Similarly, both the mef(A) and erm(F) genes were identified in a 1963 N. meningitidisisolate. All four acquired genes were found in later isolates of both species. The mef(A) gene from a 1975 N. gonorrhoeaeisolate was sequenced and had 100% DNA and amino acid identity with the mef(A) gene from a 1990s Streptococcus pneumoniaeisolate. Selected early isolates were able to transfer their acquired genes to an Enterococcus faecalisrecipient, suggesting that these genes are associated with conjugative transposons. These isolates are the oldest of any species to carry the mef(A) gene and among the oldest to carry these ermgenes.

ABSTRACTSixty-three recent Italian clinical isolates of Streptococcus pyogenesresistant to both erythromycin (MICs ≥ 1 μg/ml) and tetracycline (MICs ≥ 8 μg/ml) were genotyped for macrolide and tetracycline resistance genes. We found 19 isolates carrying the mef(A) and the tet(O) genes; 25 isolates carrying the erm(A) and tet(O) genes; and 2 isolates carrying the erm(A), tet(M), and tet(O) genes. The resistance of all erm(A)-containing isolates was inducible, but the isolates could be divided into two groups on the basis of erythromycin MICs of either >128 or 1 to 4 μg/ml. The remaining 17 isolates included 15 isolates carrying the erm(B) gene and 2 isolates carrying both the erm(B) and the mef(A) genes, with all 17 carrying the tet(M) gene. Of these, 12 carried Tn916-Tn1545-like conjugative transposons. Conjugal transfer experiments demonstrated that the tet(O) gene moved with and without the erm(A) gene and with the mef(A) gene. These studies, together with the results of pulsed-field gel electrophoresis experiments and hybridization assays with DNA probes specific for the tet(O), erm(A), and mef(A) genes, suggested a linkage of tet(O) with either erm(A) or mef(A) in erythromycin- and tetracycline-resistant S. pyogenesisolates. By amplification and sequencing experiments, we detected the tet(O) gene ca. 5.5 kb upstream from the mef(A) gene. This is the first report demonstrating the presence of the tet(O) gene in S. pyogenesand showing that it may be linked with another gene and can be moved by conjugation from one chromosome to another.

ABSTRACTWe screened 615 gram-positive isolates from 150 healthy children for the presence of the erm(A), erm(B), erm(C), erm(F), and mef(A) genes. The mef(A) genes were found in 20 (9%) of the macrolide-resistant isolates, including Enterococcusspp., Staphylococcusspp., and Streptococcusspp. Sixteen of the 19 gram-positive isolates tested carried the other seven open reading frames (ORFs) described in Tn1207.1, a genetic element carrying mef(A) recently described in Streptococcus pneumoniae. The three Staphylococcusspp. did not carry orf1to orf3. A gram-negative Acinetobacter juniiisolate also carried the other seven ORFs described in Tn1207.1.A Staphylococcus aureusisolate, a Streptococcus intermediusisolate, a Streptococcussp. isolate, and an Enterococcussp. isolate had their mef(A) genes completely sequenced and showed 100% identity at the DNA and amino acid levels with the mef(A) gene from S. pneumoniae.

ABSTRACTSulfonamide resistance in Streptococcus pneumoniaeis due to changes in the chromosomal folP(sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniaeisolate had a 6-bp repetition, the sul-dmutation, leading to a repetition of the amino acids Ile66and Glu67in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folPgene, identical to the sul-dmutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in Kifor sulfathiazole but changed the Kmfor p-aminobenzoic acid only 2.5-fold and did not significantly change theKmfor 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser61had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.

ABSTRACTThe mefgene, originally described for gram-positive organisms and coding for an efflux pump, has been identified in clinical isolates of Acinetobacter juniiandNeisseria gonorrhoeae. These strains could transfer themefgene at frequencies ranging from 10−6to 10−9into one or more of the following recipients: gram-negative Moraxella catarrhalis, Neisseria perflava/siccaand Neisseria mucosaand gram-positiveEnterococcus faecalis. Three Streptococcus pneumoniaestrains could transfer the mefgene intoEikenella corrodens, Haemophilus influenzae,Kingella denitrificans, M. catarrhalis,Neisseria meningitidis, N. perflava/sicca, andN. mucosaat similar frequencies. The mefgene can thus be transferred to and expressed in a variety of gram-negative recipients.

ABSTRACTTwo Neisseria gonorrhoeaeisolates from Seattle and two isolates from Uruguay were resistant to erythromycin (MIC, 4 to 16 μg/ml) and had reduced susceptibility to azithromycin (MIC, 1 to 4 μg/ml) due to the presence of the self-mobile rRNA methylase gene(s)ermFor ermBand ermF. The two Seattle isolates and one isolate from Uruguay were multiresistant, carrying either the 25.2-MDa tetM-containing plasmid (Seattle) or a β-lactamase plasmid (Uruguay). Sixteen commensal Neisseriaisolates (10 Neisseria perflava-N. sicca, 2 N. flava, and 4N. mucosa) for which erythromycin MICs were 4 to 16 μg/ml were shown to carry one or more known rRNA methylase genes, includingermB, ermC, and/or ermF. Many of these isolates also were multiresistant and carried thetetMgene. This is the first time that a complete transposon or a complete conjugative transposon carrying an antibiotic resistance gene has been described for the genus Neisseria.

The staphylococcal msr(A) gene, coding for a macrolide efflux protein, was identified in three new gram-positive genera and one gram-negative genus. These msr(A) genes shared 99 to 100% identity with each other and the staphylococcal gene. This study demonstrates that the msr(A) gene has a wider host range than previously reported.

ABSTRACTThe staphylococcal msr(A) gene, coding for a macrolide efflux protein, was identified in three new gram-positive genera and one gram-negative genus. These msr(A) genes shared 99 to 100% identity with each other and the staphylococcal gene. This study demonstrates that the msr(A) gene has a wider host range than previously reported.

ABSTRACTThe susceptibilities of 143 Porphyromonas gingivalis,Prevotella intermedia, and Prevotella nigrescensisolates to 18 antimicrobial agents were tested. AllP. gingivalisisolates were susceptible. In contrast, somePrevotellaspp. (17%) were resistant to β-lactams, erythromycin, clindamycin, or tetracycline and carried resistance genes, ermFor tetQ, or β-lactamases.