Bacterial resistance studies using in vitro dynamic models are highly dependent on the starting inoculum that might or might not contain spontaneously resistant mutants (RMs). To delineate concentration-resistance relationships with linezolid-exposed Staphylococcus aureus, a mixed inoculum containing both susceptible cells and RMs was used. An RM selected after the 9th passage of the parent strain (MIC, 2 g/ml) on antibiotic-containing media (RM9; MIC, 8 g/ml) was chosen for the pharmacodynamic studies, because the mutant prevention concentration (MPC) of linezolid against the parent strain in the presence of RM9 at 10 2 (but not at 10 4 ) CFU/ml did not differ from the MPC value determined in the absence of the RMs. Five-day treatments with twice-daily linezolid doses were simulated at concentrations either between the MIC and MPC or above the MPC. A specific mechanism of oxazolidinone action blocks functional initiation complexes in bacterial translation systems. Given the lack of analogues among available antimicrobials, a low probability of preexisting, naturally occurring resistance mechanisms has been hypothesized (1, 2). For example, resistance of Staphylococcus aureus rarely occurred through spontaneous mutations at frequencies of only 10 Ϫ9 to 10 Ϫ11 (3-5). However, the first report on the detection of a clinical isolate of S. aureus with a linezolid MIC of Ͼ32 g/ml was published as early as 2001 (6). Analysis of 23S rRNA encoding DNA sequences showed that linezolid-resistant S. aureus had a G-to-T mutation at position 2576 (Escherichia coli numbering). The same point mutations also were observed in S. aureus isolated in other clinical studies (7-14). Later, amino acid substitutions in ribosomal protein L3 (50S large-subunit ribosomal protein) were associated with oxazolidinone resistance (linezolid MIC, 8 g/ml) in a clinical S. aureus strain (15). Thus, clinical data indicate that resistance to linezolid mediated by mutations in ribosomal genes may emerge more readily than was initially predicted by routine in vitro studies performed early in the development of oxazolidinones, because they were not specifically designed to simulate antibiotic exposures that would allow the enrichment of resistant mutants.The resistance studies with linezolid cited above were not supported by concomitant pharmacokinetic studies to relate antibiotic concentrations to the selection of resistant mutants. To be sure, some in vitro model studies of the enrichment of resistant S. aureus simulated linezolid pharmacokinetics (16, 17).However, these attempts were unsuccessful because resistant mutants were not enriched, at least in simulations of oscillating antibiotic concentrations that mimic the usual linezolid dosing in humans. Linezolid-resistant staphylococci also were not enriched in other in vitro studies that were not designed to establish concentration-resistance relationships (18)(19)(20), probably because of the lack of spontaneous mutants in the starting inocula.To more clearly delineate concentration-resistance rel...

The bell-shaped pattern of AUC24/MIC and AUC24/MPC relationships with S. aureus resistance to linezolid is consistent with the MSW hypothesis. 'Antimutant' AUC24/MIC ratios were predicted based on the AUC24/MIC relationship with AUBCM.

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Macrolide (MLR) and fluoroquinolone (FQR) resistance in Mycoplasma genitalium (MG) has recently become a major problem worldwide. The available data on the prevalence of MLR and FQR in MG in Russia are limited. In this study, we aimed to evaluate the prevalence and pattern of mutations in 213 MG-positive urogenital swabs from patients in Moscow between March 2021 and March 2022. MLR- and FQR-associated mutations were searched in 23S rRNA as well as in the parC and gyrA genes using Sanger sequencing. The prevalence of MLR was 55/213 (26%), with A2059G and A2058G substitutions being the two most common variants (36/55, 65%, and 19/55, 35%, respectively). FQR detection showed 17% (37/213); two major variants were D84N (20/37, 54%) and S80I (12/37, 32.4%) and three minor variants were S80N (3/37, 8.1%), D84G (1/37, 2.7%), and D84Y (1/37, 2.7%). Fifteen of the fifty-five MLR cases (27%) simultaneously harbored FQR. This study revealed the high frequency of MLR and FQR. We conclude that the improvement of patient examination algorithms and therapeutic approaches should be combined with the routine monitoring of antibiotic resistance based on the sensitivity profiles presented. Such a complex approach will be essential for restraining the development of treatment resistance in MG.

There is growing evidence of applicability of the hypothesis of the mutant selection window (MSW), i.e., the range between the MIC and the mutant prevention concentration (MPC), within which the enrichment of resistant mutants is most probable. However, it is not clear if MPC-based pharmacokinetic variables are preferable to the respective MIC-based variables as interstrain predictors of resistance. To examine the predictive power of the ratios of the area under the curve (AUC 24 ) to the MPC and to the MIC, the selection of ciprofloxacin-resistant mutants of three Klebsiella pneumoniae strains with different MPC/MIC ratios was studied. Each organism was exposed to twice-daily ciprofloxacin for 3 days at AUC 24 /MIC ratios that provide peak antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC. Resistant K. pneumoniae mutants were intensively enriched at an AUC 24 /MIC ratio of 60 to 360 h (AUC 24 /MPC ratio from 2.5 to 15 h) but not at the lower or higher AUC 24 /MIC and AUC 24 /MPC ratios, in accordance with the MSW hypothesis. AUC 24 /MPC and AUC 24 /MIC relationships with areas under the time courses of ciprofloxacin-resistant K. pneumoniae (AUBC M ) were bell shaped. These relationships predict highly variable "antimutant" AUC 24 /MPC ratios (20 to 290 h) compared to AUC 24 /MIC ratios (1,310 to 2,610 h). These findings suggest that the potential of the AUC 24 /MPC ratio as an interstrain predictor of K. pneumoniae resistance is lower than that of the AUC 24 /MIC ratio.A n increasing number of reports on the isolation of resistant pathogens (1, 2) combined with a weak antibiotic pipeline suggests that optimization of antibiotic therapy should be aimed at the suppression of resistance (3, 4). In this regard, dynamic models that mimic antimicrobial pharmacokinetics in vitro have been proven to be a useful tool in predicting the amplification of resistant mutants at clinically achievable antibiotic concentrations (5). Using these models, bell-shaped relationships have been established between the emergence of resistance to fluoroquinolones and the ratios of 24-hour area under the concentration-time curve (AUC 24 ) to the MIC (6-17). Such relationships have been reported with moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin, the investigational fluoroquinolone ABÒ492, pazufloxacin, tosufloxacin, and garenoxacin against Staphylococcus aureus (6-8, 14-16); moxifloxacin against Streptococcus pneumoniae (12, 13); garenoxacin against Klebsiella pneumoniae (15); ciprofloxacin and moxifloxacin against Pseudomonas aeruginosa (11, 12); and ciprofloxacin, marbofloxacin, and enrofloxacin against Escherichia coli (9,10,17). In some of these studies (6-15, 17, 18), changes in susceptibility of antibiotic-exposed bacteria and/or their enrichment with resistant mutants was observed at fluoroquinolone concentrations above the MIC but below the mutant prevention concentration (MPC), i.e., inside the mutant selection window (MSW) but not outside the MSW, in accordance with the MSW hypo...

Objective. To assess the in vitro activity of ceftazidime-avibactam against clinical Enterobacterales and Pseudomonas aeruginosa isolates in various regions of Russia based on results of local susceptibility testing by disk diffusion method. Materials and Methods. Overall, 160 laboratories located in 61 Russian cities participated in this surveillance during 2018-2020. All consecutive clinical isolates of Enterobacterales and Pseudomonas aeruginosa in each participating laboratory were included in the study. Ceftazidime-avibactam susceptibility testing was done by disc-diffusion method in accordance with current EUCAST recommendations. Susceptibility data for carbapenems and III-IV generation cephalosporins, as well as results of carbapenemases detection, were also reported, if available. All the data were recorded in electronic case report form developed on the OpenClinica online platform (www.openclinica.com). Data analysis and reporting were done using AMRcloud online platform (https://amrcloud.net/). Results. In total, we received information on antimicrobial susceptibility of 22,121 isolates, including 17,456 (78.9%) Enterobacterales and 4,665 (21.1%) P. aeruginosa. Less than 9% of Enterobacterales isolates were resistant to ceftazidime-avibactam. At the same time rates of resistance to ceftazidime, cefotaxime, cefepime, ertapenem, imipenem, and meropenem were 54.1%, 58.9%, 59.4%, 41.4%, 23.9%, and 21.3%. Among Enterobacterales the highest level of resistance to ceftazidime-avibactam was detected in K. pneumoniae (16.5%), lowest – in E. coli (2.1%). Some increase of resistance to ceftazidimeavibactam was noted during the study – from 7.8% in 2018-2019 to 9.6% in 2020 (p = 0.0001). Rate of resistance to ceftazidime-avibactam in P. aeruginosa was 33.1%. At the same time rates of resistance to ceftazidime, cefepime, imipenem, and meropenem were 51.1%, 54.5%, 50%, and 47.3%. During the study there was statistically significant decrease in resistance to ceftazidime-avibactam in P. aeruginosa (p = 0.0001). Resistance rates for all beta-lactams for both Enterobacterales and P. aeruginosa were higher in nosocomial isolates than in community-acquired isolates. Conclusions. Ceftazidime-avibactam demonstrated significantly higher in vitro activity against Enterobacterales and P. aeruginosa Russian clinical isolates comparing with commonly used carbapenems and extended spectrum cephalosporins. Access for all study data available at the AMRcloud online platform (https://amrcloud.net/ru/project/cazavi-1-2/).

Mycoplasma genitalium is one of the obligate pathogens that cause sexually transmitted diseases. To detect this pathogen in routine practice, only molecular genetic methods are used that are also used to identify the resistance of MGE to antibiotics. The first-line drugs for the treatment of diseases caused by MGE, are tetracycline and macrolides. In recent years, many countries have increasingly recorded cases of unsuccessful therapy macrolides. Mutations that confer antibiotic resistance to macrolides for Mycoplasm genitalium are concentrated in nucleotide positions 2058 and 2059 in region V of the 23S rRNA gene. Unknown status of macrolide resistance M. genitalium can lead to the development of a persistent infection. We describe the first reported cases of clinical josamycin treatment failure from patient with ure - thritis. The reason for antibiotic resistance was a mutation in the 23S rRNA of MGE as a nucleotide substitution in position A2058G.

Objective. In this study, we examined the prevalence of macrolide-resistant M. genitalium in two Russian cities, Smolensk and Tula, between 2013 and 2017. Materials and Methods. DNA’s samples were isolated from urethral and cervicovaginal swabs using primary screening and tested for macrolide resistance-associated mutations by real-time PCR. This technology makes it possible to identify any nucleotide substitutions in the 23S rRNA M. genitalium gene at positions 2058, 2059, 2611 M. genitalium by melting curve analysis after the amplification. Results. According to the study in two cities (Smolensk and Tula) macrolide resistance-associated mutations were found in 3, 65% of isolates (21⁄574). The A2058G transition 23S rRNA MGE was the most common mutation that is associated with macrolide resistance: 5⁄12 (41.6%) – Smolensk, 8⁄9 (88.8%) – Tula. Rare substitutions have been reported at position A2058T 23S rRNA MGE and at position C2611T23S rRNA MGE. All received data is published at the AntiMicrobial Resistance Map (AMRmap) (http://AMRmap.com). Conclusions. According to our study, the frequency of macrolide-resistance mutations in M. genitalium was not more than 4% in two cities of Central Russia during 2013–2017. Despite the relatively low rates of resistance of M. genitalium to macrolides in Smolensk and Tula, our data emphasize the need for epidemiological surveillance of resistance in this pathogen.