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Surveillance of antimicrobial agent resistance provides important information to guide microbiologists and infectious disease specialists understanding of the control and the spread of resistance mechanisms within the local environment. Continued monitoring of antimicrobial resistance patterns in the community and in local hospital environments is essential to guide effective empiric therapy. The Study for Monitoring Antimicrobial Resistance Trends (SMART) has monitored the in vitro susceptibility patterns of clinical Gram-negative bacilli to antimicrobial agents collected worldwide from intra-abdominal infections since 2002 and urinary tract infections since 2009. Resistance trends, with a particular focus on carbapenem resistance and the rate of extended-spectrum β-lactamases (ESBLs), were analyzed. Isolates from intra-abdominal infections (n = 92,086) and urinary-tract infections (n = 24,705) were collected and tested using Clinical and Laboratory Standards Institute methods. This review presents carbapenem susceptibility and ESBL rates over ten years of SMART study analysis, including key publications during this period. The SMART study has proved to be a valuable resource in determining pathogen prevalence and antibiotic susceptibility over the last ten years and continues to provide evidence for regulatory susceptibility breakpoints and clinical decision making.

To date there are no clear criteria to determine whether a microbe is susceptible to biocides or not. As a starting point for distinguishing between wild-type and resistant organisms, we set out to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) distributions for four common biocides; triclosan, benzalkonium chloride, chlorhexidine and sodium hypochlorite for 3319 clinical isolates, with a particular focus on Staphylococcus aureus (N = 1635) and Salmonella spp. (N = 901) but also including Escherichia coli (N = 368), Candida albicans (N = 200), Klebsiella pneumoniae (N = 60), Enterobacter spp. (N = 54), Enterococcus faecium (N = 53), and Enterococcus faecalis (N = 56). From these data epidemiological cut-off values (ECOFFs) are proposed. As would be expected, MBCs were higher than MICs for all biocides. In most cases both values followed a normal distribution. Bimodal distributions, indicating the existence of biocide resistant subpopulations were observed for Enterobacter chlorhexidine susceptibility (both MICs and MBCs) and the susceptibility to triclosan of Enterobacter (MBC), E. coli (MBC and MIC) and S. aureus (MBC and MIC). There is a concern on the potential selection of antibiotic resistance by biocides. Our results indicate however that resistance to biocides and, hence any potential association with antibiotic resistance, is uncommon in natural populations of clinically relevant microorganisms.

In this study, the distribution of macrolide resistance mechanisms was determined for isolates of Streptococcus pneumoniae and Streptococcus pyogenes obtained from the PROTEKT 1999-2000 study (a global, longitudinal study of the antibacterial susceptibility of bacterial pathogens associated with community-acquired lower respiratory tract infections). The global macrolide resistance mechanism distribution results for 1043 macrolide-resistant S. pneumoniae isolates collected from 25 countries were as follows: 35.3% mef(A), 56.2% erm(B), 6.8% both mef(A) and erm(B), 0.2% erm(A) subclass erm(TR) and 1.5% negative for mechanisms tested. Mechanisms of macrolide resistance were found to vary widely between countries and different geographical regions with mef(A) predominating in North America and erm(B) in Europe. Approximation of genotype from macrolide MIC without molecular determination of the mechanism of resistance resulted in an error of 10.2% (106 isolates). Overall, for 143 macrolide-resistant S. pyogenes isolates, 46.1% of the isolates tested were mef(A), 30.8% were erm(B), 23.1% were erm(A) subclass erm(TR) and no isolates were negative for all the genetic markers tested. Again, the distribution varied widely between countries and geographical regions. This study provides valuable baseline data for the continued monitoring of the evolution of macrolide resistance development in these important respiratory tract pathogens. The ketolide telithromycin retained excellent anti-pneumococcal activity irrespective of macrolide resistance mechanism, having a MIC(90) of 0.25, 0.5 and 0.5 mg/L against mef(A), erm(B) and mef(A)+erm(B) macrolide-resistant S. pneumoniae, respectively. It also exhibited potent activity against S. pyogenes that had become resistant to macrolides via either mef(A), (MIC(90 )0.5 mg/L) or erm(TR), (MIC(90) 0.03 mg/L).

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Sixteen (1.5%) of the 1,043 clinical macrolide-resistant Streptococcus pneumoniae isolates collected and analyzed in the 1999-2000 PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study have resistance mechanisms other than rRNA methylation or efflux. We have determined the macrolide resistance mechanisms in all 16 isolates by sequencing the L4 and L22 riboprotein genes, plus relevant segments of the four genes for 23S rRNA, and the expression of mutant rRNAs was analyzed by primer extension. Isolates from Canada (n ‫؍‬ 4), Japan (n ‫؍‬ 3), and Australia (n ‫؍‬ 1) were found to have an A2059G mutation in all four 23S rRNA alleles. The Japanese isolates additionally had a G95D mutation in riboprotein L22; all of these originated from the same collection center and were clonal. Three of the Canadian isolates were also clonal; the rest were not genetically related. Four German isolates had A2059G in one, two, and three 23S rRNA alleles and A2058G in two 23S rRNA alleles, respectively. An isolate from the United States had C2611G in three 23S rRNA alleles, one isolate from Poland had A2058G in three 23S rRNA alleles, one isolate from Turkey had A2058G in four 23S rRNA alleles, and one isolate from Canada had A2059G in two 23S rRNA alleles. Erythromycin and clindamycin resistance gradually increased with the number of A2059G alleles, whereas going from one to two mutant alleles caused sharp rises in the azithromycin, roxithromycin, and rokitamycin MICs. Comparisons of mutation dosage with rRNA expression indicates that not all alleles are equally expressed. Despite their high levels of macrolide resistance, all 16 isolates remained susceptible to the ketolide telithromycin (MICs, 0.015 to 0.25 g/ml).Macrolide, lincosamide, and streptogramin B (MLS B ) resistance in Streptococcus pneumoniae occurs either by modification of the drug-binding site or by active efflux of the drug. Target modification is usually the result of dimethylation of the adenine residue at position 2058 on the 23S rRNA by a methylase enzyme (30). In S. pneumoniae, Erm(B) [encoded by the erm(B) gene] is the enzyme mostly responsible (7, 30), although, more rarely, a methylase encoded by the erm(A) subclass erm(TR) gene is implicated (7,23).In vitro studies have demonstrated that target modification can also be achieved via mutations in domains II and V of 23S rRNA and in the genes encoding riboproteins L4 and L22 and can confer macrolide, lincosamide, streptogramin, and ketolide resistance (2, 24). Although previous reports are rare, such mutations have been found in MLS B -resistant clinical isolates (3,14,25). However, until now there have been no studies on the prevalence and epidemiology of these types of mutations in clinical isolates on a worldwide scale.PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) is a longitudinal, global, multicenter surveillance study of respiratory tract pathogens. We screened all macrolide-resistant S. pneumoniae isolat...

In order to examine the inhibitory activities of quinolones against topoisomerase IV, both subunits of this enzyme, ParC and ParE, were purified from Escherichia coli. The specific activity of topoisomerase IV decatenation was found to be more than five times greater than that of topoisomerase IV relaxation. Thus, the decatenation activity of topoisomerase IV seems the most relevant activity for use in studies of drug inhibition of this enzyme. Although topoisomerase IV was less sensitive to quinolones than DNA gyrase, the 50% inhibitory concentrations for decatenation were significantly lower than those for type I topoisomerases. Moreover, there was a positive correlation between the inhibitory activity against topoisomerase IV decatenation and that for DNA gyrase supercoiling. These results imply that topoisomerase IV could be a target for the quinolones in intact bacteria and that quinolones could inhibit not only supercoiling of DNA gyrase but also decatenation of topoisomerase IV when high concentrations of drug exist in bacterial cells.Four topoisomerases have been isolated from Escherichia coli so far: topoisomerase I (39), topoisomerase II (DNA gyrase) (9), topoisomerase III (34), and topoisomerase IV (16). Among these enzymes, topoisomerases I, II, and IV are implicated in the control of the intracellular supercoiling density of chromosomal or plasmid DNA, affecting the efficacy of DNA replication and transcription (4, 6-8, 20, 24, 29, 36, 37). In addition, some topoisomerases are required for the segregation of daughter chromosomes or plasmid DNA (1,21,35,40). Although all of these topoisomerases are capable of changing the topology of DNA by a cleaving and rejoining step, each enzyme appears to possess a favored reaction in vitro. Type I topoisomerases, topoisomerase I and topoisomerase III, show efficient relaxing and decatenating activity, respectively, in the absence of ATP (2,3,5,11,29,30,34 subunits of DNA gyrase), respectively (16). Similar sequences are located especially around the region of DNA gyrase known as the "quinolone resistance-determining region." The quinolone antibacterial agents, in particular the fluoroquinolones, strongly inhibit DNA gyrase, which results ultimately in bacterial death (32). However, the inhibitory activities of quinolones against type I topoisomerases, topoisomerase I (25) and topoisomerase III (2) in E. coli, are weak. The inhibitory activities of fluoroquinolones against topoisomerase IV have yet to be fully investigated. The similarity in amino acid sequence between DNA gyrase and topoisomerase IV, especially around sites in the GyrA protein of DNA gyrase at positions known to produce quinolone-resistant DNA gyrases (41), implies that quinolones may be capable of inhibiting the activity of topoisomerase IV as well as against DNA gyrase.In this study, in order to define the inhibitory activities of quinolones against topoisomerase IV, both subunits of topoisomerase IV, ParC and ParE, were purified, and optimum conditions for the decatenating activity of topoi...

The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.