Β-lactam Combination Research Articles

The combination of daptomycin with β-lactam antibiotics is more effective than daptomycin alone for vancomycin-resistant Enterococcus faecium bloodstream infection

BackgroundThe treatment options for vancomycin-resistant Enterococcus (VRE) are limited. A combination of daptomycin (DAP) and β-lactam (BL) has been suggested; however clinical studies supporting this are lacking. MethodsPatients with VR E. faecium bacteremia who received ≥ 8 mg/kg daptomycin for ≥ 72 h and initiated ≤ 5 days of culture collection between 2010 and 2021 were included. DAP+BL was defined as receiving BL for ≥ 24 h and within 24 h of DAP initiation. The primary endpoint was a composite clinical success (neither 14-day mortality, microbiological failure, nor change in the anti-VRE regimen). Outcomes were analyzed using multivariable logistic regression and augmented inverse probability weighting (AIPW). ResultsA total of 430 patients were enrolled (DAP, n = 45; DAP+BL, n = 385). Clinical success was achieved in 19 (42.2%) patients in the DAP group and 244 (63.4%) in the DAP+BL group [adjusted odds ratio, 3.19; 95% confidence interval (CI) 1.61–6.33; P = 0.001]. Marginal analysis showed that the efficacy of DAP+BL was particularly significant with DAP dose ≥ 9 mg/kg and DAP minimum inhibitory concentration (MIC) ≥ 2 mg/L. With the balance of AIPW, standardized mean clinical success rates for DAP and DAP+BL 37.3% and 63.5%, respectively. The difference between DAP+BL and DAP was of 26.2% in favor of DAP+BL (95% CI, 10.0–42.3%; P = 0.001). ConclusionsDAP+BL was associated with a significantly higher rate of compositive clinical success than DAP for treatment of VR E. faecium bacteremia. The study suggested BL in combination with high-dose DAP for VR E. faecium bacteremia treatment, especially when VRE showed a high DAP MIC.

Prevalence and Antimicrobial Susceptibility Pattern of Secondary Gram-negative Bacteria Isolated from Severe Acute Respiratory Syndrome Coronavirus Disease 2 Patients in A Tertiary Care Hospital

Prior to the Severe Acute Respiratory Syndrome Coronavirus Disease 2 (SARS-CoV-2) pandemic, the rise in antimicrobial resistance was a major source of concern in public health. However, due to the novelty of SARS-CoV-2 infection during the pandemic, antibiotics were administered prior to laboratory testing for secondary gram-negative bacteria (SGNB) in order to avoid or reduce the occurrence of SGNB infection. The purpose of this study was to investigate the etiology, prevalence, and antimicrobial susceptibility pattern of gram-negative bacteria (GNB) isolated from SARS-CoV-2 positive patients. Respiratory and blood samples were collected from confirmed SARS-CoV-2 positive patients. They were subsequently cultured and bacterial isolates identified according to standard microbiological protocols. Antimicrobial susceptibility testing (AST) was performed and interpreted according to Clinical & Laboratory Standards Institute (CLSI) 2021 guidelines. A total of sixty-four non-repetitive GNB were isolated from respiratory samples and twenty-two GNB from blood samples. K. pneumoniae was the major cause of SGNB, followed by Acinetobacter species. K. pneumoniae had over 60% resistance to β-Lactam combination agents, cephalosporin, and the carbapenem group of antibiotics. In the current study, we observed that K. pneumoniae was the major cause of SGNB and had high resistance to the antimicrobial agents. Hence, it is important that the epidemiology and susceptibility patterns of circulating organisms causing SGNB infection are always monitored to inform clinical treatment and decrease the occurrence of antibiotic-resistant bacteria.

Computational and data mining studies to understand the distribution and dynamics of Temoneria (TEM) β-lactamase and their interaction with β-lactam and β-lactamase inhibitors

β-lactams are large group of antibiotics widely used to suppress the bacterial growth by inhibiting cell wall synthesis. Bacterial resistance against β-lactam antibiotics is primarily mediated through the production of Temoneria (TEM) β-lactamase (BLs), with almost 474 variants identified in Lactamase Engineering Database (LacED). The present study aims to develop a model to track the evolution of TEM BLs and their interactions with β-lactam and BLs inhibitors through data mining and computational approaches. Further, the model will be used to predict the effective combinations of β-lactam and BLs inhibitors to treat the bacterial infection harbouring emerging variants of β-lactamase. The molecular docking study results demonstrated that most TEM mutants recorded the least binding energy to penicillin and cephalosporin (I/II/III/IV/V generations) class of antibiotics. On the contrary, the same mutants recorded higher binding energy to carbapenem and Monobactam class of antibiotics. Among the BLs inhibitors, tazobactam recorded the least binding energy against most of the TEM mutants, indicating that it can lower the catalytic activity of TEM BLs, thereby potentiating antibiotic action. Similarly, data mining work has assisted us in creating a database of TEM mutants that has comprehensive data on mutations, bacterial diversity, Km, MIC, and IRT types. It has been noted that earlier released antibiotics like amoxicillin and ampicillin had lower Km and higher MIC values, which indicates the prevalence of bacterial resistance. By analysing the differential binding energy (ΔBE) of the selected TEM mutants against β-lactam and BLs inhibitors, the most effective combination of β-lactam (carbapenem and monobactam class of antibiotics) and BLs inhibitors (tazobactam) was identified, to cure bacterial diseases/infections and to prevent similar antibiotic resistance outbreaks. Therefore, our study opens a new avenue in developing strategies to manage antibiotic resistance in bacteria.

In vitro effects of diazabicyclooctane β-lactamase inhibitors relebactam and nacubactam against three subspecies of Mycobacterium abscessus complex

BackgroundMycobacterium abscessus complex (MABC) pulmonary disease is notoriously difficult to treat due to intrinsic resistance to many common antibiotics. MABC is β-lactam-resistant as it produces class A β-lactamases, such as blaMab, which are inhibited by diazabicyclooctane (DBO) β-lactamase inhibitors. ObjectivesTo investigate the microbiological effects of the combination of β-lactam and DBO β-lactamase inhibitors (relebactam and nacubactam) against MABC and determine if the effects are associated with the MABC subspecies and colony morphotype. MethodsThe antimicrobial susceptibility of three type strains and 20 clinical isolates of MABC to the combination of seven β-lactams with relebactam or nacubactam was evaluated using broth microdilution checkerboard assays. For these strains, expression levels of blaMab were assessed using quantitative real-time polymerase chain reaction and genotypic diversity was evaluated using 18-locus variable number tandem repeat assay. ResultsRelebactam and nacubactam lowered the minimum inhibitory concentrations of β-lactams, particularly imipenem, meropenem, and tebipenem, against MABC. There was no difference in efficacy of combination treatment between three subspecies, but rough morphotypes tended to be less susceptible than smooth morphotypes. There were no differences in blaMab expression levels and genotypic diversity between the morphotypes. ConclusionsThe combination of β-lactam with relebactam or nacubactam improved the efficacy of β-lactams against all MABC subspecies, but higher concentrations of β-lactams were needed for rough morphotypes.

Proteomics profiling of ertapenem challenged major porin deficient carbapenem-resistant Klebsiella pneumoniae

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent threat to human health. Major outer membrane proteins (OMPs) porin mutation is one important resistance mechanism of CRKP, and may also affect the inhibition activity of β-lactam and β-lactamase inhibitor combinations. The ertapenem-resistant K. pneumoniae strain 2018B120 with major porin mutations was isolated from a clinical patient. Genomic and time-series proteomic analyses were conducted to retrieve the ertapenem-challenged response of 2018B120. The abundance changing of proteins from PTS systems, ABC transporters, the autoinducer 2 (AI-2) quorum sensing system, and antioxidant systems can be observed. Overexpression of alternative porins was also noticed to balance major porins' defection. These findings added a detailed regulation network in bacterial resistance mechanisms and gave new insights into bypass adaptation mechanisms the porin deficient bacteria adopted under carbapenem antibiotics pressure. SignificanceOuter membrane porins deficiency is an important mechanism of carbapenem resistance in K. pneumoniae. Comprehensive genomic and proteomic profiling of an ertapenem-resistant K. pneumoniae strain 2018B120 gives a detailed systematic regulation network in bacterial resistance mechanisms. Overexpression of alternative porins to balance major porins' defection was noticed, giving new insights into bypass adaptation mechanisms of porin deficient bacteria.

Strongly Bactericidal All-Oral β-Lactam Combinations for the Treatment of Mycobacterium abscessus Lung Disease.

ABSTRACTBioactive forms of oral β-lactams were screened in vitro against Mycobacterium abscessus with and without the bioactive form of the oral β-lactamase inhibitor avibactam ARX1796. Sulopenem was equally active without avibactam, while tebipenem, cefuroxime, and amoxicillin required avibactam for optimal activity. Systematic pairwise combination of the four β-lactams revealed strong bactericidal synergy for each of sulopenem, tebipenem, and cefuroxime combined with amoxicillin in the presence of avibactam. These all-oral β-lactam combinations warrant clinical evaluation.

Coexistence of bla NDM-1 and bla IMP-4 in One Novel Hybrid Plasmid Confers Transferable Carbapenem Resistance in an ST20-K28 Klebsiella pneumoniae.

ObjectivesWe identified a novel hybrid plasmid simultaneously carrying blaNDM-1 and blaIMP-4 in an ST20-K28 carbapenem-resistant Klebsiella pneumoniae (CRKP) strain AZS099 and reported its detailed genetic and phenotypic characterization.MethodsAntimicrobial susceptibility was characterized using broth microdilution method. Complete genome characteristics and plasmid detailed analysis were carried out by PacBio Sequel and Illumina sequencing and further bioinformatics analysis. Conjugation assay, S1-PFGE, Southern blot, plasmid stability, and fitness cost were conducted to the phenotypic characterization of this novel hybrid plasmid.ResultsAZS099 was isolated from a blood specimen obtained from a 3-month baby who presented with biliary tract infection. Susceptibility testing showed that AZS099 was resistant to almost all β-lactams examined, including cephalosporins, combinations of β-lactams and β-lactamase inhibitors, carbapenems, and aztreonam. PacBio and Illumina sequencing together with S1-PFGE and Southern blot showed that blaNDM-1 and blaIMP-4 were simultaneously located on a 296 kb IncFIB(K)/IncHI1B/IncX3 plasmid (pAZS099-NDM-IMP), which consists of four main parts that came from four different types of plasmids. The region harboring blaIMP-4 is located in a class 1 integron designated as In0, which is located in an IS6100-IS26 transposon-like structure with a total length of ~5 kb. The region harboring blaNDM-1 is located in the Tn125 transposon remnant. Conjugation and transformation assay confirmed that the plasmid pAZS099-NDM-IMP has the potential for horizontal transfer and displayed high stability (retention rate > 95%). Furthermore, growth curve assessment confirmed that the presence of pAZS099-NDM-IMP exhibits no growth pressure on bacteria.ConclusionOur research reported a hybrid plasmid coharboring blaNDM-1 and blaIMP-4 in an ST20-K28 CRKP strain. The emergence of novel hybrid plasmid could threaten the control of antimicrobial resistance and should be closely supervised.

P09 Activity of newer antibiotics against NDM- and OXA-48-producing organisms

BackgroundSeveral antibiotics active against carbapenemase-producing organisms have recently been licensed for clinical use. These organisms usually have multiple integrative and conjugative elements with different resistance genes. The positioning of these agents for empirical use in sepsis caused by different carbapenemase-producing organisms is important in optimizing the likelihood of appropriate treatment before susceptibility testing is complete. Here we present the activities of meropenem-vaborbactam, imipenem-relebactam, eravacycline and tigecycline against NDM- and OXA-48-producing clinical isolates in a London teaching hospital.MethodsSixty-two previously characterized carbapenemase-producing Gram-negative clinical isolates were studied. These comprised 31 NDM and 31 OXA-48 producers. OXA-48 producers comprised Klebsiella pneumoniae (17), Escherichia coli (10), Enterobacter aerogenes (3) and Serratia marcescens (1). NDM producers comprised K. pneumoniae (11), E. coli (10), Acinetobacter baumannii (3), Klebsiella oxytoca (1), Enterobacter asburiae (1), Enterobacter cloacae (1), Pseudomonas aeruginosa (1), Citrobacter koseri (1), Citrobacter freundii (1) and Leclercia adecarboxylata (1). MICs of meropenem/vaborbactam, imipenem/relebactam, eravacycline and tigecycline were determined using Etests. Interpretation was done using EUCAST breakpoints.Results OXA-48 producers: Eight of 17 K. pneumoniae, 8/10 E. coli and 2/3 E. aerogenes isolates were meropenem/vaborbactam susceptible. Of these, 2 K. pneumoniae, 5 E. coli and 1 E. aerogenes isolates were imipenem/relebactam susceptible. All imipenem/relebactam/susceptible isolates were also meropenem/vaborbactam susceptible. The S. marcescens isolate (1/1) was resistant to both antibiotics. The only isolates demonstrating susceptibility to the glycylcyclines were E. coli. Five of 10 were susceptible to both glycylcyclines. One of 10 was susceptible to one glycylcycline but resistant to the other. NDM producers: Five of 11 K. pneumoniae, 6/10 E. coli and 1/1 K. oxytoca, E. asburiae and C. koseri isolates were susceptible to meropenem/vaborbactam. Of these, two K. pneumoniae and the K. oxytoca (1/1) and E. asburiae (1/1) isolates were imipenem/relebactam susceptible. The L. adecarboxylata isolate (1/1) was meropenem/vaborbactam resistant but imipenem/relebactam susceptible. All P. aeruginosa, C. freundii, A. baumannii and E. cloacae isolates were resistant to both meropenem/vaborbactam and imipenem/relebactam. Seven of 10 E. coli isolates were tigecycline susceptible of which 6 were also eravacycline susceptible. One of 11 K. pneumoniae, 1/1 K. oxytoca and 1/1 L. adecarboxylata isolates were susceptible to both glycylcyclines. All other isolates were resistant to both glycylcyclines.ConclusionsIn both OXA-48 and NDM producers, the activity of the four antibiotics tested was better against E. coli than K. pneumoniae. Meropenem/vaborbactam provided better cover than imipenem/relebactam. Eravacycline and tigecycline showed similar activity to each other, but their activity was inferior compared with the β-lactam combinations, particularly against K. pneumoniae.

Combined PK/PD Index May Be a More Appropriate PK/PD Index for Cefoperazone/Sulbactam against Acinetobacter baumannii in Patients with Hospital-Acquired Pneumonia.

Cefoperazone/sulbactam (CPZ/SUL) is a β-lactam and β-lactamase inhibitor combination therapy for the treatment of respiratory tract infections. Using data from a prospective, multiple-center, open-label clinical trial in 54 patients with hospital-acquired pneumonia or ventilator-associated pneumonia caused by multidrug-resistant Acinetobacter baumannii (Ab), we showed that a combined PK/PD index %(T > MICcpz*T > MICsul) is a more appropriate PK/PD index against Ab, compared to the PK/PD index (%T > MIC) for a single drug. For a 2 h infusion, the PK/PD cutoff of CPZ/SUL (2 g/1 g, q8h) for clinical and microbiological efficacy was 4/2 and 1/0.5 mg/L, respectively. The corresponding cumulative fraction of response was 46.5% and 25.3%, respectively. Results based on the combined PK/PD index were quite similar to that based on the joint probability of target attainment. The two drugs have interaction from the viewpoint of PK/PD. When the dose of one drug was too high, the PK/PD cutoff was often determined by another drug in which the dose was maintained. In most cases, sulbactam exerted the main effect against infection by Ab in the complex CPZ/SUL, which was similar to the literature reports. When the MIC of CPZ was 8, 16, or 32 mg/L, a CPZ/SUL 2 g/1 g (q8h), 2 g/2 g (q8h), or 2 g/2 g (q6h) (infusion was all 3 h) was recommended, respectively. A clinical efficacy and safety study to confirm simulation results is warranted.

Efficacy and In Vitro Activity of Novel Antibiotics for Infections With Carbapenem-Resistant Gram-Negative Pathogens.

Infections by Gram-negative multi-drug resistant (MDR) bacterial species are difficult to treat using available antibiotics. Overuse of carbapenems has contributed to widespread resistance to these antibiotics; as a result, carbapenem-resistant Enterobacterales (CRE), A. baumannii (CRAB), and P. aeruginosa (CRPA) have become common causes of healthcare-associated infections. Carbapenems, tigecycline, and colistin are the last resource antibiotics currently used; however, multiple reports of resistance to these antimicrobial agents have been documented worldwide. Recently, new antibiotics have been evaluated against Gram-negatives, including plazomicin (a new aminoglycoside) to treat CRE infection, eravacycline (a novel tetracycline) with in vitro activity against CRAB, and cefiderocol (a synthetic conjugate) for the treatment of nosocomial pneumonia by carbapenem-non-susceptible Gram-negative isolates. Furthermore, combinations of known β-lactams with recently developed β-lactam inhibitors, such as ceftazidime-avibactam, ceftolozane-tazobactam, ceftazidime-tazobactam, and meropenem-vaborbactam, has been suggested for the treatment of infections by extended-spectrum β-lactamases, carbapenemases, and AmpC producer bacteria. Nonetheless, they are not active against all carbapenemases, and there are reports of resistance to these combinations in clinical isolates.This review summarizes and discusses the in vitro and clinical evidence of the recently approved antibiotics, β-lactam inhibitors, and those in advanced phases of development for treating MDR infections caused by Gram-negative multi-drug resistant (MDR) bacterial species.

Prevalence of High Level Gentamicin Resistance among the Clinical Isolates of Enterococci Species

Enterococci infections, have drawn attention of clinicians due to rapid increase in high level aminoglycoside resistance (HLAR). This resistance predicts failure of β-lactam antibiotic and aminoglycoside combination, which is the current treatment of choice for serious enterococci infections. This led us to investigate about the prevalence of HLAR enterococci in our hospital setting in Shimoga, Karnataka. All the clinical samples received at Microbiology laboratory, McGan hospital, Shivamogga between June 2016 and December 2016 was considered for the study. Enterococci were identified and further speciated by following conventional method. Antibiotic susceptibility pattern of all the isolates was determined according to CLSI guidelines. High level gentamicin resistance was detected by disc diffusion method using 120µg gentamicin disc. A total of 64 Enterococci species were isolated from the clinical specimens. On speciation, Enterococcus faecalis (E. faecalis) accounted for 72% (46) of the total isolates, 24%(16) of the isolates were Enterococcus faecium (E. faecium)and 4% (2) isolates were Enterococcus durans (E. durans). High level gentamicin resistance was noted in 42% (27) among the total isolates. E. faecium was found to be more resistant compared to E. faecalis. E. durans was a sensitive strain. Our study reconfirms the high prevalence of HLAR in the clinical setting. The study stresses on the need for regular screening of HLAR in all Enterococci infections and revise the battery of drugs for its treatment according to the report.

Characterization of carbapenem-resistant gram-negative bacteria collected in the Sentinel Surveillance Program, 2018–2019

Background: Carbapenem resistance in gram-negative organisms is an important public health problem. The CDC conducted Sentinel surveillance in 2018–2019 to characterize these organisms from 9 facilities in 9 different states. Methods: Carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa (CRPA), and Acinetobacter spp (CRA) obtained from clinical samples of patients in acute-care or long-term care facilities were submitted to the CDC. Identification was confirmed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and antimicrobial susceptibility testing (AST) was performed via broth microdilution for 27 antibiotics. All confirmed CRE and CRPA were tested for carbapenemase production (CP) using the modified carbapenem inactivation method (mCIM). The isolates that were mCIM-positive were assessed by real-time PCR for presence of blaKPC, blaNDM, blaVIM, and blaIMP. CP-CRE were also assessed for blaOXA-48-like. All confirmed CRA were tested for the same genes as CRPA and blaOXA-23–like, blaOXA-24/40-like, blaOXA-58–like, and blaOXA-235–like genes. Difficult-to-treat resistance (DTR) was defined as resistance to all β-lactams (excluding newer β-lactam combination agents) and quinolones tested. Results: The CDC confirmed 208 CRE, 161 CRPA, and 94 CRA. Table 1 summarizes AST results for a selection of drugs. We identified 112 (53.8%) mCIM-positive CRE and 6 (3.7%) mCIM-positive CRPA. The PCR results are summarized in Table 2. One mCIM-positive and PCR-negative isolate was positive in a metallo-β-lactamase screen. Conclusions: Resistance among CRE and CRPA to newer β-lactam combination agents was detected. Options for treating CRA are limited. Of 112 CP-CRE, 85.7% harbored blaKPC; CP-CRPA were rare (3.7%); and most CRA harbored blaOXA-23-like (55.3%) or blaOXA-24/40-like (30.9%). Whole-genome sequencing is planned to better understand gene variants, sequence types, and additional resistance markers present among the isolates.Funding: NoneDisclosures: None

Potential risks of treating bacterial infections with a combination of β-lactam and aminoglycoside antibiotics: A systematic quantification of antibiotic interactions in E. coli blood stream infection isolates.

SummaryBackgroundTreatment of Blood Stream Infections (BSIs) with a combination of a β-lactam and an aminoglycoside antibiotic is widely used in intensive care units (ICUs) around the world. However, no studies have systematically examined how these drugs interact and potentially influence the antimicrobial efficacy of the overall treatment.MethodsWe collected 500 E. coli isolates from the Uppsala University hospital that were isolated from blood of patients with suspicion of infection. Of those we tested the efficacy of combinations of 2 common β-lactam antibiotics (Ampicillin and Cefotaxime) combined with 2 common aminoglycosides (Gentamicin and Tobramycin) on 254 isolates. The efficacy of all 4 pairwise combinations in inhibiting bacterial growth was then examined on all susceptible strains. That was done by quantifying the Fractional Inhibitory index (FICi), a robust metric for antibiotic combinatorial behaviour, of all possible treatments on every strain. When non additive interactions were identified, results of the original screen were verified with time kill assays. Finally, combination behaviours were analysed for potential cross correlations.FindingsOut of the 4 antibiotic combinations screened none exhibited synergistic effects on any of the 254 strains. On the contrary all 4 exhibited important antagonistic effects on several isolates. Specifically, the combinations of AMP-GEN and CTX-GEN were antagonistic in 1.97% and 1.18% of strains respectively. Similarly, the combinations of AMP-TOB were antagonistic on 0.78% of all strains. PCA analysis revealed that an important factor on the responses to the combination treatments was the choice of a specific aminoglycoside over another. Subsequent cross correlation analysis revealed that the interaction profiles of combinations including the same aminoglycoside are significantly correlated (Spearman's cross correlation test p<0.001).InterpretationThe findings of this study elucidate potential risks of the common combination treatment for blood stream infections. They also demonstrate, previously unquantified metrics on how antibiotics in combination therapies are not interchangeable with others of the same class. Finally, they reiterate the need for case-by-case testing of antibiotic interactions in a clinical setting.FundingThis work was funded by grants to DIA from the Swedish Research Council, the Wallenberg foundation and the Swedish Strategic Research Foundation.

Aztreonam in combination with imipenem-relebactam against clinical and isogenic strains of serine and metallo-β-lactamase-producing enterobacterales

ObjectiveThe objective of this study was to evaluate the in vitro activity of aztreonam plus imipenem-relebactam against clinical and isogenic strains of Escherichia coli and Klebsiella pneumoniae co-harboring NDM and >1 serine β-lactamase. MethodsThirteen isolates were included: 4 clinical E. coli, 4 clinical K. pneumoniae, and 5 isogenic E. coli. Drugs were tested in time-kill analyses alone, in dual β-lactam combinations, and in triple drug combinations against all strains. ResultsAll isolates were resistant to imipenem and imipenem-relebactam, and 85% were aztreonam-resistant. Neither imipenem nor imipenem-relebactam was bactericidal alone while aztreonam was bactericidal against 54% of isolates. The combination of aztreonam+imipenem was bactericidal and synergistic against 7/13 and 10/13 isolates. The addition of relebactam to this combination resulted in synergy against all 11 aztreonam-resistant clinical isolates. ConclusionAztreonam plus imipenem-relebactam may be a viable treatment option for aztreonam-non-susceptible NDM and serine β-lactamase-producing E. coli and K. pneumoniae.

Analysis of diverse β-lactamases presenting high-level resistance in association with OmpK35 and OmpK36 porins in ESBL-producing Klebsiella pneumoniae

Emerging extensively drug-resistant (XDR) Klebsiella pneumoniae due to the production of β-lactamases and porin loss is a substantial worldwide concern. This study aimed to elucidate the role of outer membrane porin (OMP) loss, AmpC, and carbapenemases among extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains with XDR phenotype. This study analyzed 79 K. pneumoniae from several clinical sources and detected ESBLs in 29 strains co-harbored with other β-lactamases using standard microbiological practices and phenotypic procedures. Minimum inhibitory concentrations (MICs) were determined against several antibiotics using Microscan WalkAway plus. OMP analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ESBL, AmpC, and carbapenemase genes were detected using molecular methods. The microbiological analysis discovered 29 (36.7%) ESBL strains of K. pneumoniae, which showed the co-existence of 7 (24.1%) AmpC β-lactamases and 22 (75.9%) carbapenemases. Porin loss of OmpK35 was observed in 13 (44.8%) and OmpK36 in 8 (27.5%) K. pneumoniae strains. The strains were significantly associated with the intensive care unit (ICU) (p = 0.006) and urinary sources (p = 0.004). The most commonly detected gene variants in each β-lactamase class included 16 (55.2%) blaCTX-M−1, 7 (100%) blaCYM-2, 11 (50%) blaNDM-1, and integron-1 was detected in 21/29 (72.4%) strains. MICs of cephalosporin, fluoroquinolone, carbapenem, aminoglycoside, and β-lactam combinations demonstrated a high number of XDR strains. Tigecycline (2 µg/mL MIC50 and >32 µg/mL MIC90) and colistin (1 µg/mL MIC50 and 8 µg/mL MIC90) presented lower resistance. ESBL K. pneumoniae strains with OmpK35 and OmpK36 porin loss demonstrate conglomerate resistance mechanisms with AmpC and carbapenemases, leading to emerging XDR and pan drug resistance.

In-vitro activity of β-lactams/trimethoprim-sulfamethoxazole combinations against different strains of Burkholderia pseudomallei.

Trimethoprim-sulfamethoxazole is an active agent against Burkholderia pseudomallei and is being used in intensive and maintenance phases of melioidosis therapy. In this study, we evaluated the bactericidal activities of β-lactams (imipenem, ceftazidime and amoxicillin- clavulanate) alone and in combinations with trimethoprim-sulfamethoxazole against B. pseudomallei . Four clinical strains of B. pseudomallei were selected based on different genotypes that are frequently found in Malaysia. The minimum inhibitory concentrations of trimethoprim-sulfamethoxazole, ceftazidime, imipenem and amoxicillin-clavulanate were determined using microdilution broth method. The bactericidal activities and synergy effects of β-lactams and/or trimethoprim-sulfamethoxazole were evaluated by checkerboard and static time-kill analyses at 1×MIC concentration of each antibiotic. Using checkerboard method, the β-lactam/trimethoprim-sulfamethoxazole combinations exhibited ΣFIC of 0.75-4.00. In time-kill analysis, ceftazidime/trimethoprim-sulfamethoxazole combination demonstrated synergy against three strains (less 2.25-2.41 log 10 CFU/mL compared to the most active antibiotic monotherapy) whereas imipenem/trimethoprim-sulfamethoxazole combination regimen showed synergy against one strain (less 3.32 log 10 CFU/mL). No antagonist effect or major re-growth was observed in all combination regimens, whereas 11 out of 12 of β-lactam monotherapy regimens were associated with re-growth of bacteria. However, all β-lactam monotherapy regimens exhibited rapid and stronger killing activities against BUPS/07/14, in the initial 12 hours compared to β-lactam/ trimethoprim- sulfamethoxazole combination regimens. The combination of β-lactams with trimethoprim- sulfamethoxazole demonstrated better killing effect at 24 hours compared to monotherapy and no major bacterial regrowth was observed. Nevertheless, delay in killing activities of β-lactam/trimethoprim-sulfamethoxazole combination regimens against BUPS/07/14 need further examination because this phenomenon can lead to treatment failure in some patients.

Proteomic Correlates of Enhanced Daptomycin Activity following β-Lactam Preconditioning in Daptomycin-Resistant, Methicillin-Resistant Staphylococcus aureus.

Clinical treatment options for daptomycin (DAP)-resistant (DAP-R), methicillin-resistant Staphylococcus aureus (MRSA) infections are relatively limited. Current therapeutic strategies often take advantage of potential synergistic activity of DAP plus β-lactams; however, the mechanisms underlying their combinatorial efficacy are likely complex and remain incompletely understood. We recently showed that in vitro β-lactam passaging can resensitize DAP-R strains to a DAP-susceptible (DAP-S) phenotype. To further investigate the implications of selected β-lactam pretreatments on DAP plus β-lactam combination efficacy, we utilized DAP-R strain D712. We studied six such combinations, featuring β-lactams with a broad range of penicillin-binding protein-targeting profiles (PBP-1 to -4), using DAP-R strain D712. Of note, preconditioning with each β-lactam antibiotic (sequential exposures), followed by DAP exposure, yielded significantly enhanced in vitro activity compared to either DAP treatment alone or simultaneous exposures to both antibiotics. To explore the underpinnings of these outcomes, proteomic analyses were performed, with or without β-lactam preconditioning. Relative proteomic quantitation comparing β-lactam pretreatments (versus untreated controls) identified differential modulation of several well-known metabolic, cellular, and biosynthetic processes, i.e., the autolytic and riboflavin biosynthetic pathways. Moreover, these differential proteomic readouts with β-lactam preconditioning were not PBP target specific. Taken together, these studies suggest that the cellular response to β-lactam preconditioning in DAP-R MRSA leads to distinct and complex changes in the proteome that appear to resensitize such strains to DAP-mediated killing.

1237. Characterization and crystallization of OXA-935, a novel class D OXA-10-like beta-lactamase, found in Pseudomonas aeruginosa

BackgroundRecently, we described a collection of ST298 Pseudomonas aeruginosa (PA) isolates that caused a prolonged epidemic of XDR infections. Many of these contain derivatives of a new plasmid, pPABL048, that harbors an MDR integron, in1697. In1697 contains a series of antimicrobial resistance (AMR) genes, one of which is the class D β-lactamase blaOXA-10. Variants of blaOXA-10 have been described that confer both extended-spectrum β-lactamase (ESBL) and carbapenemase activity.MethodsOf all ST298 isolates, three were resistant to ceftazidime (CTZ). Genomic comparison of in1697 in CTZ-resistant and CTZ-sensitive strains revealed that all three strains harbored a blaOXA-10 allele with two single nucleotide variations resulting in amino acid changes at positions 153 (F153S) and 157 (G157D). Using the NCBI database, we identified this allele as unique and defined this β-lactamase as OXA-935. OXA-935 shares the G157D variation with OXA-14 which is known to confer resistance to ceftazidime. We sought to characterize the function of OXA-935 and to determine the crystal structures of OXA-14 and OXA-935. ResultsDeletion of blaOXA-935 phenotypically converted all three strains to CTZ-susceptible. Expression of blaOXA-14 and blaOXA-935 conferred CTZ-resistance to laboratory PA strains PA01 and PA14. Determination of the crystal structures of OXA-14 (PDB code 7L5R) and OXA-935 (PDB code 7L5V) revealed that the F153S variant resulted in increased flexibility in the enzyme’s Ω loop. Conformational changes in the Ω loop likely contributed to the lack of carbamylation at lysine-70 (K70) observed in OXA-935. Carbamylation of K70 is known to be critical for enzymatic activity of class D β-lactamases. ConclusionOXA-935 is very similar to OXA-14; however, comparison revealed that the F153S variant has unique structural features and is functionally distinct. Despite these differences, both enzymes confer high-level CTZ resistance. As we increasingly rely on β-lactam antimicrobial therapy (e.g. ceftazidime, cefepime) and combination (e.g. ceftazidime-avibactam) therapy to treat MDR PA infections, it is critical that we continue to explore the mechanistic basis of β-lactam AMR in an effort to preserve existing treatments and design novel ones. Disclosures All Authors: No reported disclosures

In-Vitro Selection of Ceftazidime/Avibactam Resistance in OXA-48-Like-Expressing Klebsiella pneumoniae: In-Vitro and In-Vivo Fitness, Genetic Basis and Activities of β-Lactam Plus Novel β-Lactamase Inhibitor or β-Lactam Enhancer Combinations.

Ceftazidime/avibactam uniquely demonstrates activity against both KPC and OXA-48-like carbapenemase-expressing Enterobacterales. Clinical resistance to ceftazidime/avibactam in KPC-producers was foreseen in in-vitro resistance studies. Herein, we assessed the resistance selection propensity of ceftazidime/avibactam in K. pneumoniae expressing OXA-48-like β-lactamases (n = 10), employing serial transfer approach. Ceftazidime/avibactam MICs (0.25–4 mg/L) increased to 16–256 mg/L after 15 daily-sequential transfers. The whole genome sequence analysis of terminal mutants showed modifications in proteins linked to efflux (AcrB/AcrD/EmrA/Mdt), outer membrane permeability (OmpK36) and/or stress response pathways (CpxA/EnvZ/RpoE). In-vitro growth properties of all the ceftazidime/avibactam-selected mutants were comparable to their respective parents and they retained the ability to cause pulmonary infection in neutropenic mice. Against these mutants, we explored the activities of various combinations of β-lactams (ceftazidime or cefepime) with structurally diverse β-lactamase inhibitors or a β-lactam enhancer, zidebactam. Zidebactam, in combination with either cefepime or ceftazidime, overcame ceftazidime/avibactam resistance (MIC range 0.5–8 mg/L), while cefepime/avibactam was the second best (MIC: 0.5–16 mg/L) in yielding lower MICs. The present work revealed the possibility of ceftazidime/avibactam resistance in OXA-48-like K. pneumoniae through mutations in proteins involved in efflux and/or porins without concomitant fitness cost mandating astute monitoring of ceftazidime/avibactam resistance among OXA-48 genotypes.

Sepsis-associated hospitalizations and antimicrobial use in Hong Kong

Our study was conducted to assess the sepsis-associated hospitalisations and antimicrobials prescribed for sepsis inpatients in Hong Kong. Demographic, diagnostic and antimicrobial prescription data were analysed for patients admitted to public hospitals with a diagnosis of septicaemia from 2000 to 2015. A total of 223 250 sepsis hospitalisations were recorded in Hong Kong from 2000 to 2015 during which the hospitalisation rate increased by 85.6%. The majority of the sepsis hospitalisations occurred in adults ≥65 years and children aged 0–4 years. Adults with a secondary diagnosis of sepsis were often admitted with a primary diagnosis of urological conditions or pneumonia; whereas diabetes mellitus was the most common secondary diagnosis among those with primary sepsis. Paediatric sepsis patients aged 0–4 years were often diagnosed with disorders relating to short gestation and low birthweight. Antimicrobial prescriptions increased by 51.1% and 34.4% for primary and secondary sepsis patients, respectively. β-Lactam and β-lactamase inhibitor combinations were the most used antibiotics whereas the usage of carbapenems increased more than 10 times over the study period. A substantial burden of hospitalisations was attributable to sepsis in Hong Kong, particularly in the extremes of age. Broad-spectrum and last-resort antibiotics had been increasingly dispensed for sepsis inpatients.