Investigating and Treating a Corneal Ulcer Due to Extensively Drug-Resistant Pseudomonas aeruginosa.

Resistant Gram-negative bacteria are a growing concern in the United States, leading to significant morbidity and mortality. We identified a 72-year-old female patient who presented with unilateral vision loss. She was found to have a large corneal ulcer with hypopyon. Culture of corneal scrapings grew extensively drug-resistant Pseudomonas aeruginosa. Treatment involved a combination of systemic and topical antibiotics. Whole genome sequencing revealed the presence of blaVIM-80, blaGES-9, and other resistance determinants. This distinctive organism was linked to an over-the-counter artificial tears product.

Phage-antibiotic combinations against multidrug-resistant Pseudomonas aeruginosa in in vitro static and dynamic biofilm models.

Biofilm-producing Pseudomonas aeruginosa infections pose a severe threat to public health and are responsible for high morbidity and mortality. Phage-antibiotic combinations (PACs) are a promising strategy for combatting multidrug-resistant (MDR), extensively drug-resistant (XDR), and difficult-to-treat P. aeruginosa infections. Ten MDR/XDR P. aeruginosa strains and five P. aeruginosa-specific phages were genetically characterized and evaluated based upon their antibiotic susceptibilities and phage sensitivities. Two selected strains, AR351 (XDR) and I0003-1 (MDR), were treated singly and in combination with either a broad-spectrum or narrow-spectrum phage, phage EM-T3762627-2_AH (EM), or 14207, respectively, and bactericidal antibiotics of five classes in biofilm time-kill analyses. Synergy and/or bactericidal activity was demonstrated with all PACs against one or both drug-resistant P. aeruginosa strains (average reduction: -Δ3.32 log10 CFU/cm2). Slightly improved ciprofloxacin susceptibility was observed in both strains after exposure to phages (EM and 14207) in combination with ciprofloxacin and colistin. Based on phage cocktail optimization with four phages (EM, 14207, E20050-C (EC), and 109), we identified several effective phage-antibiotic cocktails for further analysis in a 4-day pharmacokinetic/pharmacodynamic in vitro biofilm model. Three-phage cocktail, EM + EC + 109, in combination with ciprofloxacin demonstrated the greatest biofilm reduction against AR351 (-Δ4.70 log10 CFU/cm2 from baseline). Of remarkable interest, the addition of phage 109 prevented phage resistance development to EM and EC in the biofilm model. PACs can demonstrate synergy and offer enhanced eradication of biofilm against drug-resistant P. aeruginosa while preventing the emergence of resistance.

Imipenem/Relebactam Resistance in Clinical Isolates of Extensively Drug Resistant Pseudomonas aeruginosa: Inhibitor-Resistant ß-Lactamases and Their Increasing Importance.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections are a major clinical challenge. Many isolates are carbapenem resistant, which severely limits treatment options; thus, novel therapeutic combinations, such as imipenem-relebactam (IMI/REL), ceftazidime-avibactam (CAZ/AVI), ceftolozane-tazobactam (TOL/TAZO), and meropenem-vaborbactam (MEM/VAB) were developed. Here, we studied two extensively drug-resistant (XDR) P. aeruginosa isolates, collected in the United States and Mexico, that demonstrated resistance to IMI/REL. Whole-genome sequencing (WGS) showed that both isolates contained acquired GES ß-lactamases, intrinsic PDC and OXA ß-lactamases, and disruptions in the genes encoding the OprD porin, thereby inhibiting uptake of carbapenems. In one isolate (ST17), the entire C terminus of OprD deviated from the expected amino acid sequence after amino acid G388. In the other (ST309), the entire oprD gene was interrupted by an ISPa1328 insertion element after amino acid D43, rendering this porin nonfunctional. The poor inhibition by REL of the GES ß-lactamases (GES-2, -19, and -20; apparent Ki of 19 ± 2 µM, 23 ± 2 µM, and 21 ± 2 µM, respectively) within the isolates also contributed to the observed IMI/REL-resistant phenotype. Modeling of REL binding to the active site of GES-20 suggested that the acylated REL is positioned in an unstable conformation as a result of a constrained Ω-loop.

Multidrug-Resistant Gram-Negative Bacteria in Burn Patients.

Patients with burn injuries are at high risk for infectious complications, and infections are the most common cause of death after the first 72 h of hospitalization. Hospital-acquired infections caused by multidrug resistant (MDR) Gram-negative bacteria (GNB) in this population are concerning. Here, we evaluated carriage with MDR GNB in patients in a large tertiary-care burn intensive care unit. Twenty-nine patients in the burn unit were screened for intestinal carriage. Samples were cultured on selective media. Median time from admission to the burn unit to first sample collection was 9 days (IQR 5 - 17 days). In 21 (72%) patients, MDR GNB were recovered; the most common bacterial species isolated was Pseudomonas aeruginosa, which was found in 11/29 (38%) of patients. Two of these patients later developed bloodstream infections with P. aeruginosa. Transmission of KPC-31-producing ST22 Citrobacter freundii was detected. Samples from two patients grew genetically similar C. freundii isolates that were resistant to ceftazidime-avibactam. On analysis of whole-genome sequencing, blaKPC-31 was part of a Tn4401b transposon that was present on two different plasmids in each C. freundii isolate. Plasmid curing experiments showed that removal of both copies of blaKPC-31 was required to restore susceptibility to ceftazidime-avibactam. In summary, MDR GNB colonization is common in burn patients and patient-to-patient transmission of highly resistant GNB occurs. These results emphasize the ongoing need for infection prevention and antimicrobial stewardship efforts in this highly vulnerable population.

Carbapenem Use Is Driving the Evolution of Imipenemase 1 Variants.

Metallo-ß-lactamases (MBLs) are a growing clinical threat because they inactivate nearly all ß-lactam-containing antibiotics, and there are no clinically available inhibitors. A significant number of variants have already emerged for each MBL subfamily. To understand the evolution of imipenemase (IMP) genes (blaIMP) and their clinical impact, 20 clinically derived IMP-1 like variants were obtained using site-directed mutagenesis and expressed in a uniform genetic background in Escherichia coli strain DH10B. Strains of IMP-1-like variants harboring S262G or V67F substitutions exhibited increased resistance toward carbapenems and decreased resistance toward ampicillin. Strains expressing IMP-78 (S262G/V67F) exhibited the largest changes in MIC values compared to IMP-1. In order to understand the molecular mechanisms of increased resistance, biochemical, biophysical, and molecular modeling studies were conducted to compare IMP-1, IMP-6 (S262G), IMP-10 (V67F), and IMP-78 (S262G/V67F). Finally, unlike most New Delhi metallo-ß-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase (VIM) variants, the IMP-1-like variants do not confer any additional survival advantage if zinc availability is limited. Therefore, the evolution of MBL subfamilies (i.e., IMP-6, -10, and -78) appears to be driven by different selective pressures.

Monitoring Ceftazidime-Avibactam and Aztreonam Concentrations in the Treatment of a Bloodstream Infection Caused by a Multidrug-Resistant Enterobacter sp. Carrying Both Klebsiella pneumoniae Carbapenemase-4 and New Delhi Metallo-ß-Lactamase-1.

In an infection with an Enterobacter sp. isolate producing Klebsiella pneumoniae Carbapenemase-4 and New Delhi Metallo-ß-Lactamase-1 in the United States, recognition of the molecular basis of carbapenem resistance allowed for successful treatment by combining ceftazidime-avibactam and aztreonam. Antimicrobial synergy testing and therapeutic drug monitoring assessed treatment adequacy.

NDM-5 and OXA-181 Beta-Lactamases, a Significant Threat Continues To Spread in the Americas.

Among Gram-negative bacteria, carbapenem-resistant infections pose a serious and life-threatening challenge. Here, the CRACKLE network reports a sentinel detection and characterization of a carbapenem-resistant Klebsiella pneumoniae ST147 isolate harboring blaNDM-5 and blaOXA-181 from a young man who underwent abdominal surgery in India. blaNDM-5 was located on an IncFII plasmid of ≈90 kb, whereas blaOXA-181 was chromosomally encoded. Resistome and genome analysis demonstrated multiple copies of the transposable element IS26 and a "hot-spot region" in the IncFII plasmid.

Nosocomial Outbreak of Extensively Drug-Resistant Acinetobacter baumannii Isolates Containing blaOXA-237 Carried on a Plasmid.

Carbapenem antibiotics are among the mainstays for treating infections caused by Acinetobacter baumannii, especially in the Northwest United States, where carbapenem-resistant A. baumannii remains relatively rare. However, between June 2012 and October 2014, an outbreak of carbapenem-resistant A. baumannii occurred in 16 patients from five health care facilities in the state of Oregon. All isolates were defined as extensively drug resistant. Multilocus sequence typing revealed that the isolates belonged to sequence type 2 (international clone 2 [IC2]) and were >95% similar as determined by repetitive-sequence-based PCR analysis. Multiplex PCR revealed the presence of a blaOXA carbapenemase gene, later identified as blaOXA-237 Whole-genome sequencing of all isolates revealed a well-supported separate branch within a global A. baumannii phylogeny. Pacific Biosciences (PacBio) SMRT sequencing was also performed on one isolate to gain insight into the genetic location of the carbapenem resistance gene. We discovered that blaOXA-237, flanked on either side by ISAba1 elements in opposite orientations, was carried on a 15,198-bp plasmid designated pORAB01-3 and was present in all 16 isolates. The plasmid also contained genes encoding a TonB-dependent receptor, septicolysin, a type IV secretory pathway (VirD4 component, TraG/TraD family) ATPase, an integrase, a RepB family plasmid DNA replication initiator protein, an alpha/beta hydrolase, and a BrnT/BrnA type II toxin-antitoxin system. This is the first reported outbreak in the northwestern United States associated with this carbapenemase. Particularly worrisome is that blaOXA-237 was carried on a plasmid and found in the most prominent worldwide clonal group IC2, potentially giving pORAB01-3 great capacity for future widespread dissemination.

Analysis of ß-Lactamase Resistance Determinants in Enterobacteriaceae from Chicago Children: a Multicenter Survey.

Multidrug-resistant (MDR) Enterobacteriaceae infections are increasing in U.S. children; however, there is a paucity of multicentered analyses of antibiotic resistance genes responsible for MDR phenotypes among pediatric Enterobacteriaceae isolates. In this study, 225 isolates phenotypically identified as extended-spectrum ß-lactamase (ESBL) or carbapenemase producers, recovered from children ages 0 to 18 years hospitalized between January 2011 and April 2015 at three Chicago area hospitals, were analyzed. We used DNA microarray platforms to detect ESBL, plasmid-mediated AmpC (pAmpC), and carbapenemase type ß-lactamase (bla) genes. Repetitive-sequence-based PCR and multilocus sequence typing (MLST) were performed to assess isolate similarity. Plasmid replicon typing was conducted to classify plasmids. The median patient age was 4.2 years, 56% were female, and 44% presented in the outpatient setting. The majority (60.9%) of isolates were Escherichia coli and from urinary sources (69.8%). Of 225 isolates exhibiting ESBL- or carbapenemase-producing phenotypes, 90.7% contained a bla gene. The most common genotype was the blaCTX-M-1 group (49.8%); 1.8% were carbapenem-resistant Enterobacteriaceae (three blaKPC and one blaIMP). Overall, pAmpC (blaACT/MIR and blaCMY) were present in 14.2%. The predominant E. coli phylogenetic group was the virulent B2 group (67.6%) associated with ST43/ST131 (Pasteur/Achtman MLST scheme) containing the blaCTX-M-1 group (84%), and plasmid replicon types FIA, FII, and FIB. K. pneumoniae harboring blaKPC were non-ST258 with replicon types I1 and A/C. Enterobacter spp. carrying blaACT/MIR contained plasmid replicon FIIA. We found that ß-lactam resistance in children is diverse and that certain resistance mechanisms differ from known circulating genotypes in adults in an endemic area. The potential impact of complex molecular types and the silent dissemination of MDR Enterobacteriaceae in a vulnerable population needs to be studied further.

Exploring the Role of Residue 228 in Substrate and Inhibitor Recognition by VIM Metallo-ß-lactamases.

ß-Lactamase inhibitors (BLIs) restore the efficacy of otherwise obsolete ß-lactams. However, commercially available BLIs are not effective against metallo-ß-lactamases (MBLs), which continue to be disseminated globally. One group of the most clinically important MBLs is the VIM family. The discovery of VIM-24, a natural variant of VIM-2, possessing an R228L substitution and a novel phenotype, compelled us to explore the role of this position and its effects on substrate specificity. We employed mutagenesis, biochemical and biophysical assays, and crystallography. VIM-24 (R228L) confers enhanced resistance to cephems and increases the rate of turnover compared to that of VIM-2 (kcat/KM increased by 6- and 10-fold for ceftazidime and cefepime, respectively). Likely the R → L substitution relieves steric clashes and accommodates the C3N-methyl pyrrolidine group of cephems. Four novel bisthiazolidine (BTZ) inhibitors were next synthesized and tested against these MBLs. These inhibitors inactivated VIM-2 and VIM-24 equally well (Ki* values of 40-640 nM) through a two-step process in which an initial enzyme (E)-inhibitor (I) complex (EI) undergoes a conformational transition to a more stable species, E*I. As both VIM-2 and VIM-24 were inhibited in a similar manner, the crystal structure of a VIM-2-BTZ complex was determined at 1.25 Å and revealed interactions of the inhibitor thiol with the VIM Zn center. Most importantly, BTZs also restored the activity of imipenem against Klebsiella pneumoniae and Pseudomonas aeruginosa in whole cell assays producing VIM-24 and VIM-2, respectively. Our results suggest a role for position 228 in defining the substrate specificity of VIM MBLs and show that BTZ inhibitors are not affected by the R228L substitution.

Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance.

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Col(r)) is increasingly reported from clinical settings. The genetic mechanisms that lead to Col(r) in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Col(r) clinical isolates. Col(r) was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-L-arabinose (Ara4N) modification of lipid A was found in all Col(r) strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Col(r) mechanisms may be dependent on the genetic background.

Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates.

This study compared the performance of the Carba NP assay, published by the Clinical and Laboratory Standards Institute, and the Rosco Rapid Carb Screen kit. Carba NP had superior sensitivity, but both assays required an increased inoculum to detect carbapenemase production in isolates with blaNDM, blaIMP, and blaOXA-48.

Population structure of KPC-producing Klebsiella pneumoniae isolates from midwestern U.S. hospitals.

Genome sequencing of carbapenem-resistant Klebsiella pneumoniae isolates from regional U.S. hospitals was used to characterize strain diversity and the bla(KPC) genetic context. A phylogeny based on core single-nucleotide variants (SNVs) supports a division of sequence type 258 (ST258) into two distinct groups. The primary differences between the groups are in the capsular polysaccharide locus (cps) and their plasmid contents. A strict association between clade and KPC variant was found. The bla(KPC) gene was found on variants of two plasmid backbones. This study indicates that highly similar K. pneumoniae subpopulations coexist within the same hospitals over time.

Extensively drug-resistant pseudomonas aeruginosa isolates containing blaVIM-2 and elements of Salmonella genomic island 2: a new genetic resistance determinant in Northeast Ohio.

Carbapenems are a mainstay of treatment for infections caused by Pseudomonas aeruginosa. Carbapenem resistance mediated by metallo-ß-lactamases (MBLs) remains uncommon in the United States, despite the worldwide emergence of this group of enzymes. Between March 2012 and May 2013, we detected MBL-producing P. aeruginosa in a university-affiliated health care system in northeast Ohio. We examined the clinical characteristics and outcomes of patients, defined the resistance determinants and structure of the genetic element harboring the blaMBL gene through genome sequencing, and typed MBL-producing P. aeruginosa isolates using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST). Seven patients were affected that were hospitalized at three community hospitals, a long-term-care facility, and a tertiary care center; one of the patients died as a result of infection. Isolates belonged to sequence type 233 (ST233) and were extensively drug resistant (XDR), including resistance to all fluoroquinolones, aminoglycosides, and ß-lactams; two isolates were nonsusceptible to colistin. The blaMBL gene was identified as blaVIM-2 contained within a class 1 integron (In559), similar to the cassette array previously detected in isolates from Norway, Russia, Taiwan, and Chicago, IL. Genomic sequencing and assembly revealed that In559 was part of a novel 35-kb region that also included a Tn501-like transposon and Salmonella genomic island 2 (SGI2)-homologous sequences. This analysis of XDR strains producing VIM-2 from northeast Ohio revealed a novel recombination event between Salmonella and P. aeruginosa, heralding a new antibiotic resistance threat in this region's health care system.

First report of KPC variants conferring ceftazidime-avibactam resistance in Colombia: introducing KPC-197.

Resistance to ceftazidime-avibactam (CZA) due to Klebsiella pneumoniae carbapenemase (KPC) variants is increasing worldwide. We characterized two CZA-resistant clinical Klebsiella pneumoniae strains by antimicrobial susceptibility test, conjugation assays, and WGS. Isolates belonged to ST258 and ST45, and produced a KPC-31 and a novel variant KPC-197, respectively. The novel KPC variant presents a deletion of two amino acids on the Ω-loop (del_168-169_EL) and an insertion of two amino acids in position 274 (Ins_274_DS). Continued surveillance of KPC variants conferring CZA resistance in Colombia is warranted. IMPORTANCE: Latin America and the Caribbean is an endemic region for carbapenemases. Increasingly high rates of Klebsiella pneumoniae carbapenemase (KPC) have established ceftazidime-avibactam (CZA) as an essential antimicrobial for the treatment of infections due to MDR Gram-negative pathogens. Although other countries in the region have reported the emergence of CZA-resistant KPC variants, this is the first description of such enzymes in Colombia. This finding warrants active surveillance, as dissemination of these variants could have devastating public health consequences.

The role of the plasmid-mediated fluoroquinolone resistance genes as resistance mechanisms in pediatric infections due to Enterobacterales.

Introduction: Fluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug-resistant (MDR) Enterobacterales (Ent) infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies. Methods: A case-control study of children (0-18 years) at three Chicago hospitals was performed. Cases had infections by FQ-susceptible, ß-lactamase-producing (bla) Ent harboring a non- or low-level expression of PMFQR genes (PMFQS Ent). Controls had FQR infections due to bla Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay®) and PMFQR genes by PCR. We performed rep-PCR, MLST, and E. coli phylogenetic grouping. Whole genome sequencing was additionally performed on PMFQS Ent positive isolates. Demographics, comorbidities, and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed. Results: Of 170 ß-lactamase-producing Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). Eighty-five (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent was 4.3 and 6.2 years, respectively (p = NS). Of 23 PMFQS Ent, 56% were Klebsiella spp., and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes detected in PMFQS Ent were bla SHV ESBL (44%) and oqxAB (57%), and the corresponding genes detected in PMFQR Ent were bla CTX-M-1-group ESBL (79%) and aac(6')-Ib-cr (83%). Whole genome sequencing of PMFQS Ent revealed the additional presence of mcr-9, a transferable polymyxin resistance gene, in 47% of isolates, along with multiple plasmids and mobile genetic elements propagating drug resistance. Multivariable regression analysis showed that children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6-22) and isolates containing multiple bla genes (OR 3.8, 95% CI 1.1-14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found. Conclusions: Paradoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, and additional silent, yet transferrable antibiotic resistance genes such as mcr-9, affecting therapeutic options and suggesting the need to address infection prevention strategies to control spread. Control of PMFQS Ent infections will require validating community and healthcare-based sources and risk factors associated with acquisition.

Characterization of a Novel Pathogen in Immunocompromised Patients: Elizabethkingia anophelis-Exploring the Scope of Resistance to Contemporary Antimicrobial Agents and ß-lactamase Inhibitors.

Background: Elizabethkingia anophelis is an emerging Gram-negative nonlactose fermenter in the health care setting, where it causes life-threatening infections in immunocompromised patients. We aimed to characterize the molecular mechanisms of antimicrobial resistance and evaluate the utility of contemporary antibiotics with the intent to offer targeted therapy against an uncommonly encountered pathogen. Methods: Whole-genome sequencing (WGS) was conducted to accurately identify isolate species and elucidate the determinants of ß-lactam resistance. Antimicrobial susceptibility testing was performed using broth microdilution and disk diffusion assays. To assess the functional contribution of the major metallo-ß-lactamase (MBL) encoding genes to the resistance profile, bla BlaB was cloned into pBCSK(-) phagemid vector and transformed into Escherichia coli DH10B. Results: WGS identified the organism as E. anophelis. MBL genes bla BlaB-1 and bla GOB-26 were identified, in addition to bla CME-2, which encodes for an extended-spectrum ß-lactamase (ESBL). Plasmids were not detected. The isolate was nonsusceptible to all commonly available ß-lactams, carbapenems, newer ß-lactam ß-lactamase inhibitor combinations, and to the combination of aztreonam (ATM) with ceftazidime-avibactam (CAZ-AVI). Susceptibility to the novel siderophore cephalosporin cefiderocol was determined. A BlaB-1 transformant E. coli DH10B isolate was obtained and demonstrated increased minimum inhibitory concentrations to cephalosporins, carbapenems, and CAZ-AVI, but not ATM. Conclusions: Using WGS, we accurately identified and characterized an extensively drug-resistant E. anophelis in an immunocompromised patient. Rapid evaluation of the genetic background can guide accurate susceptibility testing to better inform antimicrobial therapy selection.

Transcending the challenge of evolving resistance mechanisms in Pseudomonas aeruginosa through ß-lactam-enhancer-mechanism-based cefepime/zidebactam.

Multi-drug resistant (MDR) Pseudomonas aeruginosa harbor a complex array of ß-lactamases and non-enzymatic resistance mechanisms. In this study, the activity of a ß-lactam/ß-lactam-enhancer, cefepime/zidebactam, and novel ß-lactam/ß-lactamase inhibitor combinations was determined against an MDR phenotype-enriched, challenge panel of P. aeruginosa (n = 108). Isolates were multi-clonal as they belonged to at least 29 distinct sequence types (STs) and harbored metallo-ß-lactamases, serine ß-lactamases, penicillin binding protein (PBP) mutations, and other non-enzymatic resistance mechanisms. Ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/relebactam, and cefepime/taniborbactam demonstrated MIC90s of >128 mg/L, while cefepime/zidebactam MIC90 was 16 mg/L. In a neutropenic-murine lung infection model, a cefepime/zidebactam human epithelial-lining fluid-simulated regimen achieved or exceeded a translational end point of 1-log10 kill for the isolates with elevated cefepime/zidebactam MICs (16-32 mg/L), harboring VIM-2 or KPC-2 and alterations in PBP2 and PBP3. In the same model, to assess the impact of zidebactam on the pharmacodynamic (PD) requirement of cefepime, dose-fractionation studies were undertaken employing cefepime-susceptible P. aeruginosa isolates. Administered alone, cefepime required 47%-68% fT >MIC for stasis to ~1 log10 kill effect, while cefepime in the presence of zidebactam required just 8%-16% for >2 log10 kill effect, thus, providing the pharmacokinetic/PD basis for in vivo efficacy of cefepime/zidebactam against isolates with MICs up to 32 mg/L. Unlike ß-lactam/ß-lactamase inhibitors, ß-lactam enhancer mechanism-based cefepime/zidebactam shows a potential to transcend the challenge of ever-evolving resistance mechanisms by targeting multiple PBPs and overcoming diverse ß-lactamases including carbapenemases in P. aeruginosa.IMPORTANCECompared to other genera of Gram-negative pathogens, Pseudomonas is adept in acquiring complex non-enzymatic and enzymatic resistance mechanisms thus remaining a challenge to even novel antibiotics including recently developed ß-lactam and ß-lactamase inhibitor combinations. This study shows that the novel ß-lactam enhancer approach enables cefepime/zidebactam to overcome both non-enzymatic and enzymatic resistance mechanisms associated with a challenging panel of P. aeruginosa. This study highlights that the ß-lactam enhancer mechanism is a promising alternative to the conventional ß-lactam/ß-lactamase inhibitor approach in combating ever-evolving MDR P. aeruginosa.

"Silent" dissemination of Klebsiella pneumoniae isolates bearing K. pneumoniae carbapenemase in a long-term care facility for children and young adults in Northeast Ohio.

BACKGROUND: Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (bla(KPC)) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection. METHODS: Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed. RESULTS: The DNA microarray detected bla(KPC) in all 12 isolates, and bla(KPC-3) was identified by PCR amplification and sequencing of the amplicon. In addition, a bla(SHV-11) gene was detected in all isolates. Immunoblotting revealed "low-level" production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all bla(KPC-3)-positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with bla(KPC) carriage. Plasmids from 3 representative isolates readily transferred the bla(KPC-3) to Escherichia coli J-53 recipients. CONCLUSIONS: Our findings reveal the "silent" dissemination of bla(KPC-3) as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing bla(KPC-3) in an LTCF caring for neurologically impaired children and young adults.