Carbapenem Susceptibility Research Articles

P-824. A Tertiary Care Hospital's Eight-Year Experience with Acinetobacter Bloodstream Infections in the Pediatric Population

Abstract Background Acinetobacter spp has emerged as an important nosocomial pathogen causing life-threatening infections in hospitalized patients. Since carbapenem-resistant Acinetobacter bloodstream infections have been increasingly reported in the pediatric population worldwide in recent years, we aimed to evaluate the clinical characteristics and identify possible risk factors in hospitalized patients with carbapenem-resistant bloodstream infections. Methods We retrospectively analyzed the results of blood-stream infections caused by Acinetobacter spp. in hospitalized children between 2014-2022. Demographic features and clinical and laboratory findings were evaluated. Results Bloodstream infections due to Acinetobacter spp. were detected in 70 hospitalized children. Carbapenem resistance was present in 38 (54.2%) of the patients. The mean age of patients with carbapenem-resistant (CR) Acinetobacter bloodstream infection (21 males and 17 females) was 5.7±0.95 years and 3.9±0.74 years in the carbapenem-susceptible (CS) group (13 males and 19 females) (p< 0.005). The most common pathogen in the CR group (97.4%) and CS group (56.3%) was Acinetobacter baumannii. While 15.3% of the CR patients were previously healthy, 3.1% of the CS group had no history of previous diseases. The most important predisposing risk factors for the development of carbapenem resistance were invasive mechanical ventilation (p< 0.005), previous antibiotic use (p< 0.005), and history of meropenem use (p< 0.005). Combination therapy was initiated in 64.6% of patients in the CR group and 53.1% in the CS group. The most frequently used antibiotic regimens in the CR group were meropenem-colistin (15.8%), colistin-amikacin (10.5%), and colistin-sulfamethoxazole and trimethoprim (SMZ-TMP) (7.9%). The mean treatment duration for the groups was 13.3±0.98 days for the CR group and 11.3±1 days for the CS group (p:0.523). The infection-related mortality rates for the CR and the CS groups were 36.8% and 21.9%, respectively, and there was no statistically significant difference between the groups (p:0.136). Conclusion Our single-center experience aims to emphasize the importance of rational antibiotic use and enhance the awareness of clinicians about the increasing carbapenem resistance. Disclosures All Authors: No reported disclosures

P-807. Evaluation of Risk Factors for Carbapenem-Discordant Enterobacterales Bacteremia at a Large Academic Cancer Institution

Abstract Background Carbapenem resistance in non-carbapenemase producing Enterobacterales is thought to be mediated by increased beta-lactamase production and outer membrane porin mutations. Cases of phenotypic discordance of carbapenem susceptibility (ertapenem-nonsusceptible, meropenem-susceptible) are documented. Given limited clinical data in immunocompromised patients, we aim to characterize risk factors that may lead to carbapenem-discordant Enterobacterales (CD-E) bloodstream infection (BSI) in patients with cancer. Methods This is a retrospective, single-center study comparing adults > 18 years of age from January 2016 through November 2023 with a history of a ceftriaxone-resistant Enterobacterales (CRO-R-E) BSI subsequently having either a CRO-R-E or CD-E BSI of the same index organism within one year. Fisher’s exact test was used to compare categorical variables. Wilcoxon rank-sum test was used for continuous variables. Univariate logistic regression models were used to evaluate risk factors associated with CD-E BSI development. Results We evaluated 829 patients with CRO-R-E BSI. 9.4% had a subsequent CRO-R-E BSI and 1.6% developed CD-E BSI with the same index organism. The most common organisms identified were Escherichia coli (n=70, 76.9%) and Klebsiella species (n=16, 17.6%). There was no difference in age and most patients had a hematologic malignancy (n=83, 91.2%). Subsequent CRO-R-E or CD-E BSI occurred most often within 90 days of the index BSI (n=61, 78% and n=11, 85%, respectively). Ceftazidime-avibactam (CZA) and latter generation cephalosporin exposure were each significantly associated with CD-E BSI (OR 10.29; p< 0.001 and OR 1.05; p=0.03, respectively). Severe neutropenia (absolute neutrophil count < 500 K/µL) trended towards significance for CD-E BSI development (OR 3.41, p=0.06). Of the 13 CD-E BSI, development of nonsusceptible carbapenem-concordant phenotype with the same organism in the following year occurred in 5 patients (38.5%). Conclusion These data suggest that risk factors associated with CD-E BSI development include exposure to CZA or latter generation cephalosporins. Severe neutropenia may also be an indicator, but further studies are warranted to validate this and other CD-E BSI risk factors. Disclosures All Authors: No reported disclosures

Correlation between carbapenem susceptibility in Pseudomonas aeruginosa and modified antibiotic heterogeneity index: a multicenter observational study using a surveillance platform

Abstract Objective: This study focused on exploring the relationship between antimicrobial use indicators, including the modified antibiotic heterogeneity index (mAHI), and the carbapenem susceptibility in Pseudomonas aeruginosa. Design: Survey-based observational study conducted across multiple facilities. Setting: Public community hospital institutions. Methods: This survey was conducted in 15 community hospitals in Japan. Indicators, such as the defined daily doses (DDDs), days of therapy (DOTs), antibiotic heterogeneity index (AHI), and mAHI, were analyzed for P. aeruginosa carbapenem susceptibility using Spearman’s rank correlation. The predictive accuracies of the AHI and mAHI for carbapenem susceptibility were compared using DeLong’s test for the 2 correlated receiver operating characteristic curves. Results: No significant correlations were observed between DDDs or DOTs and carbapenem susceptibility. However, a significant correlation was observed between carbapenem susceptibility and the mAHI (r = 0.261, P = .02), which also demonstrated a higher predictive accuracy for high susceptibility rates than that of the AHI (area under the curve: 0.75 vs 0.58, p < .01). The optimal mAHI cutoff value for predicting 90% susceptibility was 0.765, with a sensitivity of 67.7% and specificity of 76.5%. Conclusions: The mAHI may be a better predictor of carbapenem susceptibility than other commonly used indicators. This study underscores the utility of the mAHI as an effective indicator of antimicrobial usage patterns for managing carbapenem susceptibility in P. aeruginosa. Incorporating the mAHI into antimicrobial stewardship programs could enhance the effectiveness of antimicrobial interventions across diverse healthcare settings.

The Prevalence of Carbapenem and Colistin-Resistant Gram-Negative Bacteria Isolated from Hospital-Admitted Patients with Bacteremia

Carbapenem-resistance, a global health concern augmented with resistance against last-resort antibiotic colistin has become a great challenge. Bacteremia caused by Carbapenem-resistant (CR) Gram-negative bacteria (GNB) has been linked to prolonged hospitalization and high mortality. Colistin-resistant Acinetobacter baumannii (CoRAB) represent a serious threat to patients admitted to Intensive Care Unit (ICU) with mortality estimates up to 100%. This study aimed to estimate the current burden of CR and CoR GNB in admitted patients. In this prospective study, a total of 21,600 blood cultures were processed in automated BACTEC system. Conventional methods and Automated Profile Index (API) 20E and 20NE were used for GNB identification. Carbapenem and colistin Antimicrobial Susceptibility Testing (AST) was determined using disc-diffusion and broth microdilution (BMD) methods respectively. The pooled CR was 25.59%. CR was highest in Klebsiella spp. (44.2%) and Acinetobacter baumannii (34.66%). The overall CoR among 2903 GNB was 0.96% while among 743 CR-GNB was 3.76%. CoR rates are lower in Sindh compared to Punjab. CoR has reached up to 5.20% in Klebsiella spp. and up to 3.8% in A. baumannii. All Pseudomonas aeruginosa isolates were sensitive to colistin. Significant proportion of CoRKP and CoRAB in ICU alarms the situation and calls for to seek ways to minimize the emergence of CoR. Klebsiella spp. and A. baumannii remain the predominant CR and CoR GNB in Bloodstream infections (BSI). Presence of CoR-E. coli in pediatric wards highlight the poor hygienic practices and fecal transmission.

Phenotypic Profiling of Tigecycline-resistant Klebsiella pneumoniae Strains Induced In vitro.

Tigecycline is one of the last-resort treatment options for infections caused by carbapenem-resistant Klebsiella pneumoniae (KP). Unfortunately, tigecycline resistance is increasingly reported and causes an unprecedented public health crisis worldwide. Although studies on tigecycline resistance are expanding, the underlying mechanisms are not fully understood. The goal of this study is to investigate resistance-associated phenotypic changes in descendant tigecycline-resistant KP strains induced in vitro. Compared with the parental KP strains, descendant tigecycline-resistant strains grew slowly and reversed the susceptibility of carbapenems and aminoglycosides from resistance to sensitivity. The efflux pump inhibitor phenylalanyl-arginyl-β-naphthylamine (PAβN) could significantly decrease the MIC values of tigecycline in descendant strains, but the efflux pump inhibitor carbonyl cyanide-m-chlorophenylhydrazine (CCCP), verapamil, and reserpine could not. Although the descendant strains showed inconsistent (increased or decreased) biofilm formation and ethidium bromide uptake, they showed consistently decreased ethidium bromide efflux. As for the expression of efflux pumps and regulators determined by quantitative reverse transcript polymerase chain reaction (qRT-PCR), higher level of efflux pump acrAB-TolC and lower level of regulator ramA were observed in these descendant strains, while the efflux pump oqxAB and the other 6 regulators (acrR, rarA, marA, soxS, bpeT, and Rob) showed inconsistent (higher or lower) expression level. Thus, a global regulatory network driven by regulators (acrR, ramA, rarA, marA, soxS, bpeT, rob, etc.) alone or synergistically might play important roles in conferring tigecycline resistance in KP by regulation of efflux pumps (especially increasing acrAB-TolC) or other pathways.

Ceftolozane/tazobactam disrupts Pseudomonas aeruginosa biofilms under static and dynamic conditions.

Pseudomonas aeruginosa biofilms limit the efficacy of currently available antibacterial therapies and pose significant clinical challenges. Pseudomonal biofilms are complicated further when other markers of persistence such as mucoid and hypermutable phenotypes are present. There is currently a paucity of data regarding the activity of the newer β-lactam/β-lactamase inhibitor combination ceftolozane/tazobactam against P. aeruginosa biofilms. We evaluated the efficacy of ceftolozane/tazobactam against clinical P. aeruginosa isolates, the laboratory isolate PAO1 and its isogenic mutS-deficient hypermutator derivative (PAOMS) grown under static and dynamic biofilm conditions. The clinical isolate collection included strains with mucoid and hypermutable phenotypes. Ceftolozane/tazobactam exposure led to a bactericidal (≥3 log cfu/cm2) biofilm reduction in 15/18 (83%) clinical isolates grown under static conditions, irrespective of carbapenem susceptibility or mucoid phenotype, with greater activity compared with colistin (P < 0.05). Dynamically grown biofilms were less susceptible to ceftolozane/tazobactam with active biofilm reduction (≥1 log cfu/cm2) observed in 2/3 isolates. Hypermutability did not affect the antibiofilm efficacy of ceftolozane/tazobactam in either static or dynamic conditions when comparing PAO1 and PAOMS. Consistent with the activity of ceftolozane/tazobactam as a potent inhibitor of PBP3, dramatic impacts on P. aeruginosa morphology were observed. Our data demonstrate that ceftolozane/tazobactam has encouraging properties in the treatment of P. aeruginosa biofilm infections, and its activity is not diminished against mucoid or hypermutable variants at the timepoints examined.

Re-sensitization of imipenem-resistant Pseudomonas aeruginosa and restoration of cephalosporins susceptibility in Enterobacteriaceae by recombinant Esterase B.

Sphingobium sp. SM42 Esterase B (EstB) is an enzyme with a dual function in degrading dibutyl phthalate and catalyzing the cleavage of the C-S bond in C3-sidechains of the dihydrothiazine ring of cephalosporins, generating more active β-lactam derivatives. Global prokaryotic genome analysis revealed the existence of a gene identical to estB in Pseudomonas aeruginosa strain PS1 suggesting a horizontal gene transfer event involving estB. To investigate the effect of ectopic expression of EstB in the periplasm of P. aeruginosa and several Enterobacteriaceae on antibiotic susceptibility levels, plasmid, pEstB, carrying a recombinant EstB fused with the signal peptide from Escherichia coli outer membrane protein A (OmpA) for periplasmic localization was constructed. The expression of EstB in the periplasm of P. aeruginosa and the Enterobacteriaceae: E. coli, Klebsiella pneumoniae, and Salmonella enterica serovar Typhi, increased susceptibility to carbapenems and cephalosporins. EstB reversed the imipenem resistance of P. aeruginosa ΔmexS and restored the changes in susceptibility to cephalosporins conferred by the downregulation of the outer membrane proteins, OmpK35 and OmpK36, in K. pneumoniae ΔramR-ompK36 to wild-type level. The introduction of EstB to the periplasmic space of Gram-negative bacteria can increase carbapenem and cephalosporin susceptibility.

Selection and Evaluation of Suitable Quality Control Strains for Meropenem Antimicrobial Susceptibility Testing through Preliminary External Quality Assessment

BackgroundQuality control (QC) of carbapenem susceptibility testing for Gram-negative bacteria faces challenges due to limited measuring ranges and the lack of suitable QC strains. This study aimed to select and evaluate QC strains for meropenem antimicrobial susceptibility testing (AST) through a pilot external quality assessment (EQA). MethodsCandidate QC strains for meropenem AST were selected based on primary AST data and genomic information from the Japan Antimicrobial Resistant Bacterial Bank. Phenotype stability was verified through serial passaging and MIC comparison with original strains. The validated broth microdilution method was used to determine the target MIC value in a pilot EQA involving 47 clinical laboratories in Japan using ten different AST methods. ResultsTwo strains, Citrobacter freundii JBBDAJB-19-0032 (Strain-A) and Enterobacter hormaechei subsp. steigerwaltii JBEBAAB-19-0102 (Strain-B), both carrying blaIMP-1, were selected as candidate QC strains. The meropenem MICs for Strain-A and Strain-B were 4 mg/L and 2 mg/L, respectively. In the pilot EQA, 43 laboratories reported appropriate results for Strain-A and 40 laboratories reported appropriate results for Strain-B. The MIC range was 2-8 mg/L for Strain-A and 1-4 mg/L for Strain-B. However, 19 and 12 laboratories, respectively, reported out-of-range MICs using AST plates on the MicroScan WalkAway (Beckman Coulter). Inappropriate results were reported by four and seven laboratories, respectively, using common methods for Strain-A and Strain-B, respectively. ConclusionsThe candidate QC strains selected for this study are suitable for meropenem AST EQA, except when the measuring range of certain methods does not match their QC range.

Comparison of two commercial broth microdilution panels for multidrug-resistant Gram-negative bacteria: Thermo Scientific™ Sensititre DKMGN vs. Beckman Coulter MicroScan NMDRM1.

Antimicrobial susceptibility testing (AST) using broth microdilution (BMD) is usually the reference method to obtain accurate minimum inhibitory concentrations and optimally manage infections with resistant organisms. Several commercial dry BMD are available for AST in clinical laboratories. Two commercial BMD panels for testing of multidrug-resistant Gram-negative bacteria were compared: the Thermo Scientific™ Sensititre DKMGN and the Beckman Coulter NMDRM1, for 17 antimicrobial agents. A total of 207 isolates were tested: three ATCC strains and one NCTC strain, six quality control strains from the Belgian National Antimicrobial Committee, and 197 clinical isolates, including carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 breakpoints version 13.1 were used to assign susceptibility categories. Overall, the categorical agreement (CA) and essential agreement (EA) were both above 90%, but several useful antibiotics for the treatment of multi-resistant organisms showed CA and EA under 90%, that is, meropenem, imipenem, and colistin for Enterobacterales and meropenem and colistin for P. aeruginosa. For Enterobacterales, the NMDRM1 panel showed a significantly higher resistance rate for meropenem, imipenem, amikacin, and colistin. For carbapenems, the minimal inhibitory concentrations (MICs) were underestimated by the DKMGN panel, as already pointed out by a warning on the EUCAST website. To better assess carbapenem susceptibility in carbapenem-resistant organisms, the DKMGN panel now requires the use of a higher inoculum in the insert kit. However, for a given isolate whose susceptibility to carbapenems is not known, there is a risk of underestimating the MIC values. Our results show that colistin testing remains a challenge, highlighting the urgent need for the development of more accurate commercial methods. The use of a single commercial method cannot guarantee good precision in the determination of the MIC value for colistin.

The OprF porin as a potential target for the restoration of carbapenem susceptibility in Pseudomonas aeruginosa expressing acquired carbapenemases.

Carbapenem resistance in Pseudomonas aeruginosa is primarily due to the acquisition of carbapenemases and is often associated with a diminution of the membrane permeability. The outer membrane protein, OprD, is a well-known route, by which carbapenems, predominantly imipenem, can enter the cell, and its loss has been associated with reduced susceptibility to imipenem. In this study, we investigated the antibiotic susceptibility patterns of isogenic P. aeruginosa mutants containing various acquired β-lactamases, including carbapenemases, in a porin-depleted background. We identified that the deletion of oprF was associated with some recovery of susceptibility to carbapenems.

Phenotypic and Genetic Characteristics of Carbapenemase-Producing Enterobacterales Isolates at Okayama University Hospital.

Spread of carbapenemase-producing Enterobacterales (CPE) is an ongoing public health issue worldwide, including in Japan. In this study, we investigated the phenotypic and genetic characteristics of CPE isolates at Okayama University Hospital over the 5 years (2013-2018) prior to the outbreak of the 2019 coronavirus pandemic. Of 24 carbapenem-resistant Enterobacterales isolated during the study period, we identified 8 CPE isolates harboring blaIMP-1 (5 isolates) and blaIMP-6 genes (3 isolates). Bacterial species and carbapenem susceptibility patterns exhibited diversity. Minimum inhibitory concentrations (MICs) of meropenem were generally higher than those of imipenem and biapenem. Results of pulsed-field gel electrophoresis demonstrated that neither clonal nor plasmid-mediated outbreaks of blaIMP-harboring CPE isolates have developed at our hospital. One Klebsiella oxytoca isolate showed a high MIC (128 μg/mL) of meropenem, which could be explained by the high plasmid copy number. Subsequent analysis of this isolate may elucidate the intricacies of carbapenem resistance profiles among CPE isolates. Collectively, our findings underscore the necessity for ongoing genetic surveillance of CPE, complemented by tailored approaches for infection prevention and control.

Discovery of novel BfmR inhibitors restoring carbapenem susceptibility against carbapenem-resistant Acinetobacter baumannii by structure-based virtual screening and biological evaluation

ABSTRACT The emergence and spread of Acinetobacter baumannii pose a severe threat to public health, highlighting the urgent need for the next generation of therapeutics due to its increasing resistance to existing antibiotics. BfmR, a response regulator modulating virulence and antimicrobial resistance, shows a promising potential as a novel antimicrobial target. Developing BfmR inhibitors may propel a new therapeutic direction for intractable infection of resistant strains. In this study, we conducted a structure-based hierarchical virtual screening pipeline combining molecular docking, molecular dynamic simulation, and MM/GBSA calculation to sift the Specs chemical library and finally discover three novel potential BfmR inhibitors. The three hits can reduce the MIC of meropenem for the carbapenem-resistant Acinetobacter baumannii (CRAB) strain ZJ06. Similar to the BfmR knockout strain, Cmp-98 was demonstrated to downregulate the expression of K locus genes, indicating it as a BfmR inhibitor. Bacteria underwent harmful morphological changes after treatment with these inhibitors. Molecular dynamic simulations found that all the hits tend to dynamically bind to different positions of the phosphorylation site of BfmR. Wherein we identified a potential inhibitory-binding cleft, beside a possible activated binding cleft at the edge of the phosphorylation site. Restraining the ligand binding poses may help exert inhibitory effects. This study reports a group of new scaffold BfmR inhibitors, offering new insights for novel antibiotic therapeutics against CRAB.

Complete genome sequences of Escherichia coli KA0011 clinical isolate used as a quality control strain of carbapenem susceptibility testing in Japan.

Escherichia coli KA0011 had stable minimum inhibitory concentration values around the breakpoint range of meropenem and imipenem, making it suitable for use as a quality control strain for antimicrobial susceptibility testing. Here, we report the complete genomic sequence of KA0011.

The prevalence and mechanisms of heteroresistance to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae.

To investigate the prevalence and mechanisms of ceftazidime/avibactam heteroresistance in KPC-producing Klebsiella pneumoniae (KPC-KP) isolates, as well as the role of heteroresistance in the transition of ceftazidime/avibactam susceptibility to resistance. Clinical KPC-KP isolates were obtained from a tertiary hospital in China from 2016 to 2017 and 2019 to 2020. Antimicrobial susceptibility was determined by the broth microdilution method. Population analysis profiles were used to assess ceftazidime/avibactam heteroresistance. WGS and molecular cloning were conducted to reveal heteroresistance mechanisms and molecular characteristics. The findings indicated that the transition of ceftazidime/avibactam susceptibility to resistance during the treatment of KPC-KP infection is primarily attributed to the heteroresistance exhibited by KPC-KP isolates towards ceftazidime/avibactam. Among 355 ceftazidime/avibactam-susceptible KPC-KP isolates (indicating a resistance rate of 0%), 41 (11.55%) exhibited ceftazidime/avibactam heteroresistance, with the primary mechanism being the presence of KPC mutant subpopulations. These KPC variants, arising from point mutations, deletions and insertions, significantly increased ceftazidime/avibactam resistance while alongside enhanced carbapenem susceptibility. Notably, 11 new KPC variants were identified. Furthermore, four heteroresistant isolates were caused by mixed infection involving subpopulations carrying NDM-1 or NDM-5. Phylogenetic analysis indicated that the clonal spread of ST11-KL64 KPC-KP may be correlated with the prevalence of heteroresistance. Ceftazidime/avibactam heteroresistance, primarily driven by pre-existing KPC variants, underscores the importance of considering heteroresistance in ceftazidime/avibactam therapeutics. Awareness of these dynamics is crucial for the effective and sustainable clinical application of ceftazidime/avibactam.

Change in Klebsiella pneumoniae susceptibility profile after the arrival of ceftazidime-avibactam in an Argentinean intensive care unit: a new ecological landscape.

Ceftazidime-avibactam (CZA) is a good option for Gram-negative bacilli infections that produce carbapenemase Classes A (especially blaKPC) and D (blaOXA). However, it is unknown whether it would have an impact on metallo-β-lactamases (blaMBL) selection. The aim of the study was to compare carbapenem and CZA Klebsiella pneumoniae (KPN) susceptibility profiles for a period of two years following the introduction of CZA. The study was conducted in a 36-bed adult ICU of a tertiary hospital in Buenos Aires, Argentina. Antimicrobial consumption was expressed as days of treatment per 100 patients-day (DOT). A total of 123 KPN strains in the first year and 172 in the second year were analyzed. An alarming decrease in carbapenem susceptibility was detected in the second year (OR 0.5 [0.3-0.8] p<.001). In parallel, there was a decrease in CZA susceptibility (OR 0.5 [0.3-0.9] p<.05). These findings were linked to a rise in blaMBL-KPN (32.1% vs. 45.1%, OR 1.7 [1.1-2.9], p <.04) during the second year. This new KPN susceptibility profile promoted an increment in CZA (1.0 DOT vs. 6.6 DOT, OR 6.6 [4.9-9.1] p<.001) and aztreonam (0.3 DOT vs. 4.1 DOT, OR 16.3 [9.1-29.3] p<.001) consumption. Thus, there was a decrease in carbapenem prescription (17.8 DOT vs. 15.4 DOT, OR 0.8 [0.8-0.9] p<.001). There was an escalation of blaMBL-KPN rate two years after CZA introduction, leading to a decrease in CZA and carbapenem susceptibility and an increase in CZA and aztreonam prescriptions.

Complex evolutionary trajectories in vivo of two novel KPC variants conferring ceftazidime-avibactam resistance

More and more ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae have been reported with its widespread use, and the detection rate of KPC variants has increased dramatically. However, the evolutionary mechanism and fitness effects during KPC mutation remained unknown. Here, we report the complex in vivo evolutionary trajectories of two novel KPC variants, KPC-155 (L169P/GT242A) and KPC-185 (D179Y/GT242A), from K. pneumoniae in the same patient. The novel variants were shown to confer ceftazidime-avibactam resistance but restore carbapenem susceptibility based on the results of plasmid transformation assays, cloning experiments, and enzyme kinetic measurements. In vitro, competition experiments highlighted the adaptive advantage conferred by strains carrying these KPC variants, which could lead to the rapid spread of these ceftazidime-avibactam-resistant strains. The growth curve indicated that blaKPC-185 had better growth conditions at lower avibactam concentration compared to blaKPC-155, which was consistent with ceftazidime-avibactam use in vivo. In addition, replicative transposition of the IS26-flanked translocatable unit (IS26-ISKpn6-blaKPC-ISKpn27-IS26) also contributes to the blaKPC amplification and formation of two copies (blaKPC-2 and blaKPC-185), conferring both carbapenem and ceftazidime-avibactam resistance. However, strains with double copies showed reduced competitive advantage and configuration stability. The comparative plasmid analysis of IS26 group (IS26-blaKPC-IS26) and Tn1721 group (Tn1721-blaKPC-IS26) revealed that IS26-insertion could influence the distribution of resistance genes and ability of self-conjugation. The dynamic changes in blaKPC configuration highlight the need for consistent monitoring including antimicrobial susceptibility testing and determination of blaKPC subtypes – during clinical treatment, especially when ceftazidime-avibactam is administered.

Hospital mortality and length of stay associated with Enterobacterales positive blood cultures: a multicenter analysis.

Delayed time to antimicrobial susceptibility results can impact patients' outcomes. Our study evaluated the impact of susceptibility turnaround time (TAT) and inadequate empiric antibacterial therapy (IET) in patients with bloodstream infections (BSI) caused by Enterobacterales (ENT) species on in-hospital mortality and length of stay (LOS). This retrospective, multicenter investigation which included 29,570 blood ENT-positive admissions across 161 US healthcare facilities evaluated the association between antimicrobial susceptibility testing (AST) TAT, carbapenem susceptibility, and empiric therapy on post-BSI in-hospital mortality and LOS following an ENT BSI event in adult patients. After adjusting for outcomes covariates, post-BSI in-hospital mortality was significantly higher for patients in the IET vs adequate empiric therapy (AET) group [odds ratio (OR): 1.61 (95% CI: 1.32, 1.98); P < 0.0001], and when AST TAT was >63 h [OR:1.48 (95% CI: 1.16, 1.90); P = 0.0017]. Patients with carbapenem non-susceptible (carb-NS) ENT BSI had significantly higher LOS (16.6 days, 95% CI: 15.6, 17.8) compared to carbapenem susceptible (carb-S, 12.2 days, 95% CI: 11.8, 12.6), (P < 0.0001). Extended AST TAT was significantly associated with longer LOS for TAT of 57-65 h and >65 h (P = 0.005 and P< 0.0001, respectively) compared to TAT ≤42 h (reference). Inadequate empiric therapy (IET), carb-NS, and delayed AST TAT are significantly associated with adverse hospital outcomes in ENT BSI. Workflows that accelerate AST TAT for ENT BSIs and facilitate timely and adequate therapy may reduce post-BSI in-hospital mortality rate and LOS.IMPORTANCEFor patients diagnosed with bloodstream infections (BSI) caused by Enterobacterales (ENT), delayed time to antimicrobial susceptibility (AST) results can significantly impact in-hospital mortality and hospital length of stay. However, this relationship between time elapsed from blood culture collection to AST results has only been assessed, to date, in a limited number of publications. Our study focuses on this important gap using retrospective data from 29,570 blood ENT-positive admissions across 161 healthcare facilities in the US as we believe that a thorough understanding of the dynamic between AST turnaround time, adequacy of empiric therapy, post-BSI event mortality, and hospital length of stay will help guide effective clinical management and optimize outcomes of patients with ENT infections.

Complete Genome Sequence of a Klebsiella pneumoniae Strain Carrying Novel Variant blaKPC-203, Cross-Resistant to Ceftazidime/Avibactam and Cefiderocol, but Susceptible to Carbapenems, Isolated in Italy, 2023.

Klebsiella pneumoniae is a concerning pathogen, responsible for hospital-associated outbreaks. Multi drug resistant (MDR) strains are especially hard to treat. We conducted whole-genome sequencing on a MDR K. pneumoniae strain in order to identify genomic features potentially linked to its phenotype. DNA sequencing was performed on the Illumina iSeq 100 platform. Genome assembly was carried out with SPAdes. The genome was annotated with RASTtk. Typing was performed with MLST and Kaptive. Antibiotic resistance genes were detected with AMRFinderPlus and Abricate, and further verified with BLAST. The strain exhibited resistance to ceftazidime/avibactam and cefiderocol, but remained susceptible to carbapenems. The strain belonged to sequence type ST101, serotype O1:K17. The analysis of antibiotic resistance genes indicated that the strain carried a novel KPC variant, designated as KPC-203, featuring a EL deletion at amino acid position 166-167, within the Ω-loop, and a nine-amino-acid insertion (LAVYTRAPM) at position 259. Sequence alterations were found in porin genes ompK35 and ompK36. Unlike molecular testing, which was able to detect the KPC-203 variant, all phenotypic carbapenemase detection methods achieved negative results. KPC-203, a novel KPC variant, showed a sequence modification in a cephalosporin resistance-associated hotspot. Interestingly, such alterations typically correlate with the restoration of carbapenem susceptibility. We hypothesize that KPC-203 likely led to resistance to ceftazidime/avibactam and cefiderocol, while maintaining susceptibility to carbapenems.

Activity of propyl-propane-thiosulfinate and propyl-propane-thiosulfonate against carbapenem-resistant Gram-negative bacteria.

Organosulfur compounds derived from plants of the Allium genus, such as propyl-propane-thiosulfinate (PTS) and propyl-propane-thiosulfonate (PTSO), have been proposed as an alternative in antibiotic resistance. The aim of this study was to compare the activity of these substances with other antibiotics against clinical isolates of carbapenem-resistant (CAR-R) and carbapenem-susceptible (CAR-S) Gram-negative bacteria. A total of 126 clinical isolates of CAR-R and 155 CAR-S bacteria were selected, including Enterobacterales, A. baumannii and P. aeruginosa. The antibiotic susceptibility of all isolates was assessed using the microdilution and Kirby-Bauer methods for PTS, PTSO, amoxicillin/clavulanate, piperacillin/tazobactam, cefotaxime, ceftazidime, cefepime, imipenem, ciprofloxacin, and amikacin. Both PTS and PTSO demonstrated in vitro bactericidal activity against CAR-R Enterobacteriaceae and A. baumannii, with no significant difference in activity compared to their response against CAR-S isolates. However, both compounds were less active against P. aeruginosa than against any of the other bacteria, regardless of their resistance to carbapenems. In all cases, the minimum inhibitory concentration values of PTSO were significantly lower than those of PTS. These findings offer valuable information about the potential antibacterial use of these substances, particularly against infections that currently have limited therapeutic options.

Dihydrophenazine: a multifunctional new weapon that kills multidrug-resistant Acinetobacter baumannii and restores carbapenem and oxidative stress susceptibilities.

The current work aims to fully characterize a new antimicrobial agent against Acinetobacter baumannii, which continues to represent a growing threat to healthcare settings worldwide. With minimal treatment options due to the extensive spread of resistance to almost all the available antimicrobials, the hunt for new antimicrobial agents is a high priority. An Egyptian soil-derived bacterium strain NHM-077B proved to be a promising source for a new antimicrobial agent. Bio-guided fractionation of the culture supernatants of NHM-077B followed by chemical structure elucidation identified the active antimicrobial agent as 1-hydroxy phenazine. Chemical synthesis yielded more derivatives, including dihydrophenazine (DHP), which proved to be the most potent against A. baumannii, yet it exhibited a marginally safe cytotoxicity profile against human skin fibroblasts. Proteomics analysis of the cells treated with DHP revealed multiple proteins with altered expression that could be correlated to the observed phenotypes and potential mechanism of the antimicrobial action of DHP. DHP is a multipronged agent that affects membrane integrity, increases susceptibility to oxidative stress, interferes with amino acids/protein synthesis, and modulates virulence-related proteins. Interestingly, DHP in subinhibitory concentrations re-sensitizes the highly virulent carbapenem-resistant A. baumannii strain AB5075 to carbapenems providing great hope in regaining some of the benefits of this important class of antibiotics. This work underscores the potential of DHP as a promising new agent with multifunctional roles as both a classical and nonconventional antimicrobial agent that is urgently needed.