Possible impact of revisions in disc diffusion breakpoints for aminoglycosides and piperacillin/tazobactam in the 33rd edition of CLSI M100 document on clinical reporting and use in Indian settings with low susceptibility.

PURPOSE: The study explores the impact of significant interpretative breakpoint changes for aminoglycosides and piperacillin-tazobactam in Enterobacterales and Pseudomonas aeruginosa, considering PK/PD, clinical data, and susceptibility on clinical reporting and use. PROCEDURE: Between January 2021 and June 2023, a total of 189,583 samples were processed for bacterial pathogens and antimicrobial susceptibility testing was performed using disc diffusion method/VITEK® 2 Compact system/broth microdilution. WHONET software was utilised to capture and analyse the changes in the interpretation of disc diffusion method, following updates to CLSI M100 documents in comparison to previous editions. Antimicrobial consumption data was collected and interpreted as DDD/100 bed days using AMC tool software. Here, we present data for 13,615 members of Order Enterobacterales and 1793 Pseudomonas aeruginosa isolates. FINDING: Enterobacterales exhibited a significant susceptibility drop of 14.7% for gentamicin and 21.7% for amikacin. Pseudomonas aeruginosa showed an increase in isolates with intermediate tobramycin susceptibility, from 0.6% to 29.7%, with relatively minor changes in piperacillin-tazobactam interpretation. CONCLUSION: The changes indicate a shift toward increased 'resistance' and 'intermediate susceptibility' for these antibiotics, emphasizing the need for cautious use and leveraging PK/PD knowledge for improved antibiotic utilization, patient outcomes, and antimicrobial stewardship.

Clinical outcomes in patients with piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae bloodstream infection.

BACKGROUND: A small proportion of Escherichia coli and Klebsiella pneumoniae demonstrate in vitro non-susceptibility to piperacillin/tazobactam but retain susceptibility to ceftriaxone. Uncertainty remains regarding how best to treat these isolates. OBJECTIVES: We sought to compare clinical outcomes between patients with piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae bloodstream infection receiving definitive therapy with ceftriaxone versus an alternative effective antibiotic. METHODS: We retrospectively identified patients with a positive blood culture for piperacillin/tazobactam-non-susceptible but ceftriaxone-susceptible E. coli or K. pneumoniae between 1 January 2013 and 31 December 2022. Patients were divided into one of two definitive treatment groups: ceftriaxone or alternative effective antibiotic. Our primary outcome was a composite of 90 day all-cause mortality, hospital readmission, or recurrence of infection. We used Cox proportional hazards models to compare time with the composite outcome between groups. RESULTS: Sixty-two patients were included in our analysis. Overall, median age was 63 years (IQR 49.5-71.0), the most common source of infection was intra-abdominal (25/62; 40.3%) and the median total duration of therapy was 12.0 days (IQR 9.0-16.8). A total of 9/22 (40.9%) patients in the ceftriaxone treatment group and 18/40 (45.0%) patients in the alternative effective antibiotic group met the composite endpoint. In an adjusted time-to-event analysis, there was no difference in the composite endpoint between groups (HR 0.67, 95% CI 0.30-1.50). The adjusted Bayesian posterior probability that the HR was less than or equal to 1 (i.e. ceftriaxone is as good or better than alternative therapy) was 85%. CONCLUSIONS: These findings suggest that ceftriaxone can be used to effectively treat bloodstream infections with E. coli or K. pneumoniae that are non-susceptible to piperacillin/tazobactam but susceptible to ceftriaxone.

Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan.

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ2 test). Only 4.1% of CRPA isolates carried the carbapenemase genes: blaGES (2) and blaIMP (13). One ST235 isolate carried the novel blaIMP variant blaIMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

Lessons learned from an external quality assurance program in applying CLSI interpretive criteria for reporting piperacillin/tazobactam susceptibility.

We evaluated the performance of automated susceptibility testing for piperacillin/tazobactam (PTZ) MICs against the reference microbroth dilution method. The Minimum Inhibitory Concentration of PTZ against a clinical isolate of Klebsiella pneumoniae was determined by reference broth micro-dilution method in 10 replicates which yielded a modal MIC of 16 mg/L (susceptible dose-dependent). Out of 434 laboratories who obtained MIC of 16 mg/L correctly, only 301 interpreted the result as susceptible dose dependent as per 2022 revised CLSI criteria. Educating the clinical laboratories in validating AST methods as per latest CLSI guidelines is of utmost important.

In vitro development of resistance against antipseudomonal agents: comparison of novel ß-lactam/ß-lactamase inhibitor combinations and other ß-lactam agents.

We subjected seven P. aeruginosa isolates to a 10-day serial passaging against five antipseudomonal agents to evaluate resistance levels post-exposure and putative resistance mechanisms in terminal mutants were analyzed by whole-genome sequencing analysis. Meropenem (mean, 38-fold increase), cefepime (14.4-fold), and piperacillin-tazobactam (52.9-fold) terminal mutants displayed high minimum inhibitory concentration (MIC) values compared to those obtained after exposure to ceftolozane-tazobactam (11.4-fold) and ceftazidime-avibactam (5.7-fold). Fewer isolates developed elevated MIC values for other ß-lactams and agents belonging to other classes when exposed to meropenem in comparison to other agents. Alterations in nalC and nalD, involved in the upregulation of the efflux pump system MexAB-OprM, were common and observed more frequently in isolates exposed to ceftazidime-avibactam and meropenem. These alterations, along with ones in mexR and amrR, provided resistance to most ß-lactams and levofloxacin but not imipenem. The second most common gene altered was mpl, which is involved in the recycling of the cell wall peptidoglycan. These alterations were mainly noted in isolates exposed to ceftolozane-tazobactam and piperacillin-tazobactam but also in one cefepime-exposed isolate. Alterations in other genes known to be involved in ß-lactam resistance (ftsI, oprD, phoP, pepA, and cplA) and multiple genes involved in lipopolysaccharide biosynthesis were also present. The data generated here suggest that there is a difference in the mechanisms selected for high-level resistance between newer ß-lactam/ß-lactamase inhibitor combinations and older agents. Nevertheless, the isolates exposed to all agents displayed elevated MIC values for other ß-lactams (except imipenem) and quinolones tested mainly due to alterations in the MexAB-OprM regulators that extrude these agents.

Win Ratio Analyses of Piperacillin-Tazobactam Versus Meropenem for Ceftriaxone-Nonsusceptible Escherichia coli or Klebsiella pneumoniae Bloodstream Infections: Post Hoc Insights From the MERINO Trial.

BACKGROUND: Clinical trials of treatments for serious infections commonly use the primary endpoint of all-cause mortality. However, many trial participants survive their infection and this endpoint may not truly reflect important benefits and risks of therapy. The win ratio uses a hierarchical composite endpoint that can incorporate and prioritize outcome measures by relative clinical importance. METHODS: The win ratio methodology was applied post hoc to outcomes observed in the MERINO trial, which compared piperacillin-tazobactam with meropenem. We quantified the win ratio with a primary hierarchical composite endpoint, including all-cause mortality, microbiological relapse, and secondary infection. A win ratio of 1 would correspond to no difference between the 2 antibiotics, while a ratio <1 favors meropenem. Further analyses were performed to calculate the win odds and to introduce a continuous outcome variable in order to reduce ties. RESULTS: With the hierarchy of all-cause mortality, microbiological relapse, and secondary infection, the win ratio estimate was 0.40 (95% confidence interval [CI], .22-.71]; P = .002), favoring meropenem over piperacillin-tazobactam. However, 73.4% of the pairs were tied due to the small proportion of events. The win odds, a modification of the win ratio accounting for ties, was 0.79 (95% CI, .68-.92). The addition of length of stay to the primary composite greatly minimized the number of ties (4.6%) with a win ratio estimate of 0.77 (95% CI, .60-.99; P = .04). CONCLUSIONS: The application of the win ratio methodology to the MERINO trial data illustrates its utility and feasibility for use in antimicrobial trials.

Introducing the new face of CLSI M100 in 2023: An explanatory review.

BACKGROUND: The CLSI annual update of its M100 document is eagerly awaited every year. This year's update, the M100-Ed33, was published in February, and will significantly affect clinical practices. OBJECTIVE: To highlight and explain the rationale of the changes and their clinical impact. CONTENT: The major changes this year are mostly focused on PK/PD data, selective and cascade reporting of the antibiotics and therapy related comments. The CLSI has moved away from its classical grouping of antibiotics (A, B, U, O) to a tier-based approach (Tier 1, 2, 3, 4) which will aid in cascade reporting during an antibiotic susceptibility testing (AST). Rather than non-fastidious, fastidious and anaerobe grouping, the tables have been made organism specific. The aminoglycosides breakpoints have been changed for both Enterobacterales and Pseudomonas aeruginosa while for P. aeruginosa, the breakpoints of piperacillin - tazobactam (TZP) are also updated. These updates are mostly based on attainment of drug plasma level for bacterial stasis rather than bactericidal effect of the antibiotics. It is noteworthy, that these breakpoint changes are made, keeping in view that the aminoglycosides for all organisms should be used in combination therapy. For P. aeruginosa, gentamicin has been removed, while amikacin has been restricted for urinary isolates only.

Identification of antibiotic consumption targets for the management of Clostridioides difficile infection in hospitals- a threshold logistic modelling approach.

BACKGROUND: This study aims to demonstrate the utility of a threshold logistic approach to identifying thresholds for specific antibiotic use associated with Clostridioides difficile infection (CDI) in an English teaching hospital. METHODS: A combined approach of nonlinear modeling and logistic regression, named threshold logistic, was used to identify thresholds and risk scores in hospital-level antibiotic use associated with hospital-onset, healthcare-associated (HOHA) CDI cases. RESULTS: Using a threshold logistic regression approach, an incidence greater than 0.2645 cases/1000 occupied bed-days (OBD; 85th percentile) was determined as the cutoff rate to define a critical (high) incidence rate of HOHA CDI. Fluoroquinolones and piperacillin-tazobactam were found to have thresholds at 84.8 and 54 defined daily doses (DDD)/1000 OBD, respectively. Analysis of data allowed calculating risk scores for HOHA CDI incidence rates exceeding the 85th percentile, i.e. entering critical incidence level. The threshold-logistic model also facilitated performing 'what-if scenarios' on future values of fluoroquinolones and piperacillin-tazobactam use to understand how HOHA CDI incidence rates may be affected. CONCLUSION: Using threshold logistic analysis, critical incidence levels and antibiotic use targets to control HOHA CDI were determined. Threshold logistic models can be used to inform and enhance the effective design and implementation of antimicrobial stewardship programs.

Strategies for Updating Piperacillin-Tazobactam Breakpoints.

In this issue of the Journal of Clinical Microbiology, C. Manuel, R. Maynard, A. Abbott, K. Adams, et al. (J Clin Microbiol 61:e01617-22, 2023, https://doi.org/10.1128/JCM.01617-22) describe a multisite study evaluation of piperacillin-tazobactam (TZP) MIC testing on three U.S. Food and Drug Administration (FDA)-cleared antimicrobial susceptibility testing (AST) devices compared to the reference broth microdilution method for organisms belonging to Enterobacterales. Although overall performance of each of the three devices was comparable when applying either FDA or Clinical and Laboratory Standards Institute (CLSI) TZP breakpoints, failure to update to the current CLSI breakpoints may result in falsely categorizing as many as 20% of the TZP-resistant isolates as susceptible. The impact of not updating clinical breakpoints and strategies for implementation of updated breakpoints are discussed.

A Placebo-Controlled Trial to Evaluate Two Locally Delivered Antibiotic Gels (Piperacillin Plus Tazobactam vs. Doxycycline) in Stage III-IV Periodontitis Patients.

Background and objectives: this study aims to evaluate the clinical and microbiological effects of a single subgingival administration of a locally delivered antibiotic gel containing piperacillin plus tazobactam and compare it with a slow-release doxycycline (14%) gel and a placebo gel, following subgingival instrumentation (SI) in patients with severe periodontitis. Materials and methods: sixty-four patients diagnosed with stage III-IV periodontitis were enrolled, were randomly assigned into three groups, and were treated additionally with a single subgingival administration of piperacillin plus tazobactam gel (group A); doxycycline gel (group B); and placebo gel (group C). The primary outcome variable was the change in mean probing pocket depth (PPD) 6 months after the intervention. Secondary outcome variables were changes in mean full-mouth bleeding score (FMBS); full-mouth plaque score (FMPS); overall bleeding index (BOP); pocket closure; and clinical attachment level (CAL), along with changes in the numbers of five keystone bacteria: Aggregatibacter actinomycetemcomitans (A.a.), Porphyromonas gingivalis (P.g.), Prevotella intermedia (P.i.), Tannerella forsythia (T.f.), and Treponema denticola (T.d.). Intergroup and intragroup differences were evaluated at 3 and 6 months. Results: at baseline, the three groups were comparable. An improvement in clinical parameters such as PPD, CAL, and BOP between groups was observed at 3 and 6 months, but without statistical significance (p > 0.05). At 6 months, the intragroup analysis showed a significant reduction in clinical parameters. Even though the piperacillin plus tazobactam group showed slightly higher PPD reduction, this was not statistically significant when compared to both control groups. Conclusions: The groups had similar results, and subgingival instrumentation can be executed without adjunctive antimicrobials, reducing the costs for the patient and the working time/load of the professional.

Evaluation of Piperacillin-Tazobactam ETEST for the Detection of OXA-1 Resistance Mechanism among Escherichia coli and Klebsiella pneumoniae.

Globally, piperacillin-tazobactam resistance among Escherichia coli and Klebsiella pneumoniae is driven by OXA-1 beta-lactamases. Expression of blaOXA-1 yields piperacillin-tazobactam MICs of 8 to 16 µg/mL, which straddle the susceptible/susceptible-dose dependent breakpoint set by the Clinical and Laboratory Standards Institute in 2022. Variability of the reference broth microdilution method (BMD) was evaluated by manufacturing BMD panels using 2 brands of piperacillin, 2 brands of tazobactam and 2 brands of cation-adjusted Mueller-Hinton broth. In addition, ETEST, which harbors an intermediate dilution of 12 µg/mL was evaluated for the ability to differentiate isolates with and without blaOXA-1. A collection of 200 E. coli and K. pneumoniae, of which 82 harbored a blaOXA-1 gene, were tested. BMD variability was on average 1.3-fold, within the accepted 2-fold variability of MICs. However, categorical agreement (CA) between BMD reads was 74.0% for all isolates and 63.4% for those with a blaOXA-1 gene and 81.3% for those without blaOXA-1 detected (P = 0.004, Pearson's Chi Square). ETEST overall CA with the BMD mode was 68.0% and essential agreement (EA) was 80.5%. For isolates with blaOXA-1, CA was 50.0% and EA was 69.5%, versus 80.5% and 88.1%, respectively, for isolates without blaOXA-1 (P < 0.0001 for both comparisons). All ETEST errors were major errors (false resistance) compared to BMD mode. However, the negative predictive value of the ETEST for the presence of blaOXA-1 was 94.1%, compared to only 74.2% negative predictive value for BMD. Clinicians and microbiologists should be aware of the challenges associated with testing piperacillin-tazobactam in regions where blaOXA-1 is prevalent.

Activity of Ceftolozane-Tazobactam Against Gram-Negative Isolates from Australia and New Zealand as part of the PACTS Surveillance 2016-2018.

OBJECTIVES: Ceftolozane-tazobactam (C-T) is an anti-pseudomonal cephalosporin combined with a well-described ß-lactamase inhibitor. Ceftolozane-tazobactam has enhanced activity against Pseudomonas aeruginosa, and activity against Enterobacterales isolates that produce extended-spectrum ß-lactamases (ESBLs) or AmpC cephalosporinases. In this study, we analysed the susceptibility of Gram-negative isolates to C-T and comparators collected in Australia and New Zealand from 2016 to 2018 as part of the Program to Assess Ceftolozane-Tazobactam Susceptibility (PACTS) surveillance. METHODS: A total of 1693 nonduplicate Enterobacterales and 435 P. aeruginosa isolates were collected prospectively from hospitalized patients in six medical centres in Australia and two in New Zealand. Susceptibilities (S) to C-T and comparators were determined using broth microdilution. EUCAST breakpoints were used. Isolates with multi-drug resistant (MDR), extensively drug resistant (XDR), extended-spectrum ß-lactamase non-carbapenem resistant (ESBL, non-CRE) phenotype, and CRE were analysed. RESULTS: For P. aeruginosa, 97.5% were S to C-T while 89.9% were S to meropenem. According to EUCAST criteria, 86.4% were susceptible-increased exposure to piperacillin-tazobactam. MDR and XDR P. aeruginosa isolates had 76.7% and 65.4% S to C-T, respectively; 34.9% and 19.2% S to meropenem, respectively; and 23.3% and 15.4% were susceptible-increased exposure to piperacillin-tazobactam, respectively. Meropenem (99.8% S), amikacin (99.1% S), and C-T (96.5% S) were the most active against Enterobacterales. Susceptibilities to C-T were 94.3% for ESBL, non-CRE phenotype, and 78.4% for MDR isolates. Only three CRE and five XDR isolates were identified. CONCLUSIONS: These in vitro data indicate that C-T is a potent antimicrobial with activity against MDR and XDR P. aeruginosa, as well as ESBL, non-CRE phenotype isolates and MDR Enterobacterales.

Assessing Rates of Co-Resistance and Patient Outcomes in Multidrug-Resistant Pseudomonas aeruginosa.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections are associated with poor patient outcomes due to complex co-resistance patterns. We described common co-resistance patterns, clinical characteristics, and associated outcomes in patients admitted with an MDR P. aeruginosa. This national, multicenter, retrospective cohort study within the Veterans Affairs included adults hospitalized with a MDR P. aeruginosa infection (January 2015-December 2020) per Centers for Disease Control definition. Clinical outcomes were compared among those with differing MDR P. aeruginosa co-resistance: resistant to carbapenems and extended-spectrum cephalosporins and piperacillin-tazobactam (CARB/ESC/PT) versus without CARB/ESC/PT resistance; resistant to carbapenems and extended-spectrum cephalosporins and fluoroquinolone (CARB/ESC/FQ) versus without CARB/ESC/FQ resistance. We included 3,763 hospitalized patients. Co-resistance to CARB/ESC/PT was observed in 42.7%, and to CARB/ESC/FQ in 40.7%. The lowest co-resistance rates were observed with ceftolozane-tazobactam (6.2%, n = 6/97; 12.5%, n = 10/80, respectively) and ceftazidime-avibactam (5.2%, n = 5/97; 12.5%, n = 10/80, respectively). Overall, 14.2% of patients died during hospitalization, 59.7% had an extended length of stay, and 14.9% had reinfection with hospitalization. Outcomes were similar between patients with MDR P. aeruginosa strains with and without co-resistance to CARB/ESC/PT and CARB/ESC/FQ. Among a national cohort of patients hospitalized with MDR P. aeruginosa infections, co-resistance to three classes of standard of care antibiotics, such as carbapenem, extended-spectrum cephalosporins, and piperacillin-tazobactam or fluoroquinolones, exceeded 40% in our study population, posing great concerns for selecting appropriate empirical therapy. Clinical outcomes were poor for all patients, regardless of different co-resistance patterns. New treatment options are needed for hospitalized patients with suspected or confirmed MDR P. aeruginosa infections. IMPORTANCE We studied antibiotic co-resistance patterns in a national group of hospitalized patients with infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa, a type of bacteria that resists treatment to at least three classes of antibiotics. Co-resistance to antibiotic classes most typically used for treatment was common, which makes selecting appropriate antibiotics to successfully treat the infections difficult. Outcomes, including death, were poor for all patients in our study, regardless of the different patterns of co-resistance to common antibiotic classes. New antibiotics are needed to help treat hospitalized patients with MDR P. aeruginosa infections.

Comparison of the Treatment Outcome of Piperacillin-Tazobactam versus Carbapenems for Patients with Bacteremia Caused by Extended-Spectrum ß-Lactamase-Producing Escherichia coli in Areas with Low Frequency of Coproduction of OXA-1: a Preliminary Analysis.

Although piperacillin-tazobactam (TZP) was shown to be less effective than carbapenems in treating bacteremia due to extended-spectrum ß-lactamase-producing (ESBL)-producing organisms in a randomized controlled trial, the fact that many of the causative organisms co-produced inhibitor-resistant OXA-1 along with ESBLs may have influenced the results. In this study, we compared the therapeutic effectiveness of TZP and carbapenem in treating ESBL-producing Escherichia coli bacteremia in areas with low frequency of OXA-1 co-production. Forty patients, 14 in the TZP treatment group and 26 in the carbapenem treatment group, were included in the analysis. There were no significant differences in patient background between the two groups. Urinary tract infection or cholangitis was the source of bacteremia in 26 patients (65%), and the Pitt bacteremia score was zero or one in 35 patients (87.5%). Only four (11.4%) of the 35 causative isolates available for microbiological analysis harbored blaOXA-1, and only three (8.6%) were non-susceptible to TZP. Seventeen (48.6%) isolates carried blaCTX-M-27, none of which carried other ß-lactamase genes. No significant difference in the frequency of treatment failure on day 14 of bacteremia was documented between the TZP and carbapenem treatment groups in both the crude analysis and the inverse probability of treatment weighting-adjusted analysis. This study demonstrates that TZP may be a treatment option for non-severe cases of ESBL-producing E. coli bacteremia in areas with low frequency of OXA-1 co-production. IMPORTANCE Although carbapenems are considered the drug of choice for severe infections caused by extended-spectrum ß-lactamase-producing (ESBL)-producing organisms, other therapeutic options are being explored to avoid increasing the selective pressure for carbapenem-resistant organisms. In this study, it was suggested that piperacillin-tazobactam may be as effective as carbapenems for the treatment of mild bacteremia caused by ESBL-producing Escherichia coli in areas where OXA-1 co-production by ESBL-producing E. coli is rare. The genetic background of each regional epidemic clone differs even among multidrug-resistant bacteria classified under the same name (e.g., ESBL-producing organisms), resulting in possible differences in the efficacy of therapeutic agents. Exploration of treatment options for multidrug-resistant organisms according to local epidemiology is worthwhile from the perspective of antimicrobial stewardship.

Evolutionary Trajectories toward High-Level ß-Lactam/ß-Lactamase Inhibitor Resistance in the Presence of Multiple ß-Lactamases.

ß-Lactam antibiotics are the first choice for the treatment of most bacterial infections. However, the increased prevalence of ß-lactamases, in particular extended-spectrum ß-lactamases, in pathogenic bacteria has severely limited the possibility of using ß-lactam treatments. Combining ß-lactam antibiotics with ß-lactamase inhibitors can restore treatment efficacy by negating the effect of the ß-lactamase and has become increasingly important against infections caused by ß-lactamase-producing strains. Not surprisingly, bacteria with resistance to even these combinations have been found in patients. Studies on the development of bacterial resistance to ß-lactam/ß-lactamase inhibitor combinations have focused mainly on the effects of single, chromosomal or plasmid-borne, ß-lactamases. However, clinical isolates often carry more than one ß-lactamase in addition to multiple other resistance genes. Here, we investigate how the evolutionary trajectories of the development of resistance to three commonly used ß-lactam/ß-lactamase inhibitor combinations, ampicillin-sulbactam, piperacillin-tazobactam, and ceftazidime-avibactam, were affected by the presence of three common ß-lactamases, TEM-1, CTX-M-15, and OXA-1. First-step resistance was due mainly to extensive gene amplifications of one or several of the ß-lactamase genes where the amplification pattern directly depended on the respective drug combination. Amplifications also served as a stepping-stone for high-level resistance in combination with additional mutations that reduced drug influx or mutations in the ß-lactamase gene blaCTX-M-15. This illustrates that the evolutionary trajectories of resistance to ß-lactam/ß-lactamase inhibitor combinations are strongly influenced by the frequent and transient nature of gene amplifications and how the presence of multiple ß-lactamases shapes the evolution to higher-level resistance.

Antimicrobial resistance, virulence factors, and genotypes of Pseudomonas aeruginosa clinical isolates from Gorgan, northern Iran.

Pseudomonas aeruginosa is an important nosocomial pathogen with a capacity of resistance to multiple antibiotics and production of various extracellular and cell-associated virulence factors that clearly contribute to its pathogenicity. The objective of this study was to investigate the antibiotic susceptibility, virulence factors, and clonal relationship among clinical isolates of P. aeruginosa. Different clinical specimens from hospitalized patients were investigated for P. aeruginosa. Susceptibility of the isolates was evaluated by disc diffusion and broth microdilution methods, as described by the Clinical and Laboratory Standards Institute (CLSI) guideline. A total of 97 P. aeruginosa isolates were recovered from clinical specimens. The percentage of isolates resistant to antimicrobials was imipenem 25.77%, meropenem 15.46%, gentamicin 16.49%, tobramycin 15.46%, amikacin 16.49%, ciprofloxacin 20.61%, levofloxacin 24.74, ceftazidime 20.61%, piperacillin 15.46%, piperacillin/tazobactam 12.37%, colistin 9.27%, and polymyxin B 11.34%. Of isolates, 87.62% possessed ß-hemolytic activity, 78.35% lecithinase, 59.8% elastase, 37.11% DNase, and 28.86% twitching motility. The frequency of virulence genes in isolates was lasB 82.47%, plcH 82.47%, exoA 58.76%, exoS 56.7%, and pilA 10.3%. ERIC-PCR typing clustered P. aeruginosa isolates to 19 common types (CT1-CT19) containing isolates from different hospitals and 43 single types (ST1-ST43). Colistin and polymyxin B were the most effective agents against the majority of P. aeruginosa isolates, emphasizing the effort to maintain their antibacterial activity as last-line therapy. The frequency of some virulence factors and genes was noticeably high, which is alarming. In addition, more effective strategies and surveillance are necessary to confine and prevent the inter-hospital and/or intra-hospital dissemination of P. aeruginosa between therapeutic centers.