OmpK36 Porin Research Articles

Extensively Drug-Resistant Klebsiellapneumoniae Counteracts Fitness and Virulence Costs That Accompanied Ceftazidime-Avibactam Resistance Acquisition.

ABSTRACTThe ability of extensively drug-resistant (XDR) Klebsiella pneumoniae to rapidly acquire resistance to novel antibiotics is a global concern. Moreover, Klebsiella clonal lineages that successfully combine resistance and hypervirulence have increasingly occurred during the last years. However, the underlying mechanisms of counteracting fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we investigated whether and how an XDR sequence type (ST)307 K. pneumoniae strain developed resistance against the novel drug combination ceftazidime-avibactam (CAZ-AVI) using experimental evolution. In addition, we performed in vitro and in vivo assays, molecular modeling, and bioinformatics to identify resistance-conferring processes and explore the resulting decrease in fitness and virulence. The subsequent amelioration of the initial costs was also addressed. We demonstrate that distinct mutations of the major nonselective porin OmpK36 caused CAZ-AVI resistance that persists even upon following a second experimental evolution without antibiotic selection pressure and that the Klebsiella strain compensates the resulting fitness and virulence costs. Furthermore, the genomic and transcriptomic analyses suggest the envelope stress response regulator rpoE and associated RpoE-regulated genes as drivers of this compensation. This study verifies the crucial role of OmpK36 in CAZ-AVI resistance and shows the rapid adaptation of a bacterial pathogen to compensate fitness- and virulence-associated resistance costs, which possibly contributes to the emergence of successful clonal lineages.IMPORTANCE Extensively drug-resistant Klebsiella pneumoniae causing major outbreaks and severe infections has become a significant challenge for health care systems worldwide. Rapid resistance development against last-resort therapeutics like ceftazidime-avibactam is a significant driver for the accelerated emergence of such pathogens. Therefore, it is crucial to understand what exactly mediates rapid resistance acquisition and how bacterial pathogens counteract accompanying fitness and virulence costs. By combining bioinformatics with in vitro and in vivo phenotypic approaches, this study revealed the critical role of mutations in a particular porin channel in ceftazidime-avibactam resistance development and a major metabolic regulator for ameliorating fitness and virulence costs. These results highlight underlying mechanisms and contribute to the understanding of factors important for the emergence of successful bacterial pathogens.

Increased blaKPC Copy Number and OmpK35 and OmpK36 Porins Disruption Mediated Resistance to Imipenem/Relebactam and Meropenem/Vaborbactam in a KPC-Producing Klebsiella pneumoniae Clinical Isolate.

Herein, we report the in vivo evolution of imipenem/relebactam-resistance in KPC-producing K. pneumoniae (KPC-Kp) isolated from a critically-ill patient treated with multiple combination therapies based on ceftazidime-avibactam or meropenem-vaborbactam. Imipenem/relebactam-resistance was associated to meropenem-vaborbactam cross-resistance and was due to truncated OmpK35 and OmpK36 porins and increased copy of blaKPC copy number. Genome analysis demonstrated that imipenem/relebactam-resistant KPC-Kp harbored a second copy of blaKPC-carrying Tn4401 in a ColRNAI plasmid as a consequence of a transposition event.

Effect of sigma E on carbapenem resistance in OXA-48-producing Klebsiella pneumoniae.

Resistance levels of Gram-negative bacteria producing OXA-48 carbapenemase can vary greatly and some of them can even be categorized as susceptible to imipenem and meropenem according to EUCAST breakpoints. This study aimed to reveal resistance mechanisms leading to varying levels of resistance to carbapenems in Klebsiella pneumoniae with blaOXA-48 submitted to the German National Reference Centre for MDR Gram-negative bacteria. Meropenem-susceptible clinical blaOXA-48-bearing K. pneumoniae isolates were put under gradually increasing selective pressure of meropenem. Clinical isolates and spontaneous meropenem-resistant mutants were whole-genome sequenced with Illumina and Oxford Nanopore Technology. Identified mutations apart from porin mutations were genetically constructed in the original clinical isolates using CRISPR/Cas. Clinical isolates and mutants were analysed for MICs, growth rates and expression of porins on mRNA and protein levels. Mutations associated with meropenem resistance were predominantly found in ompK36, but in some cases ompK36 was unaffected. In two mutants, ISs within the rpoE (sigma factor E; σE) operon were detected, directly in or upstream of rseA. These IS1R elements were then inserted into the same position of the susceptible clinical isolates using CRISPR/Cas. CRISPR-rseA-rseB-rseC mutants showed higher resistance levels to carbapenems and cephalosporins, reduced growth rates and reduced expression of major porins OmpK36 and OmpK35 in quantitative RT-PCR and SDS-PAGE. Enhanced synthesis of σE leads to increased resistance to cephalosporins and carbapenems in clinical K. pneumoniae isolates. This effect could be based upon remodelling of expression patterns of outer membrane proteins. The up-regulated σE stress response also leads to a significant reduction in growth rates.

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.

Impact of Carbapenem Heteroresistance Among Multidrug-Resistant ESBL/AmpC-Producing Klebsiella pneumoniae Clinical Isolates on Antibiotic Treatment in Experimentally Infected Mice.

PurposeAntibiotic resistance is a growing health crisis that is further complicated by treatment failures caused by bacteria that exhibit heterogeneous susceptibility to antibiotics. The aim of this study was to describe imipenem (IPM)-heteroresistant strains among multidrug-resistant (MDR) ESBL/AmpC-producing Klebsiella pneumoniae clinical isolates, investigate their molecular phenotypic characteristics, and elucidate the outcome of antibiotic treatment in mice infected with the heteroresistant isolates.Materials and MethodsAntimicrobial susceptibility of K. pneumoniae isolates was determined by the disk diffusion and E-test methods. Heteroresistance to IPM was confirmed by population analysis profile (PAP) assays. PCR and sequencing were employed to detect MDR determinants. Molecular differences between the susceptible and resistant subpopulations were evaluated by sequencing and quantitative real-time reverse transcription PCR (qRT-PCR) analysis. The effect of the carbapenem-heteroresistant strains on antibiotic treatment was assessed using a mouse model of peritonitis with heteroresistant K. pneumoniae and subsequent treatment with IPM.ResultsIn total, 37 MDR ESBL/AmpC-producing clinical isolates of K. pneumoniae were identified between September 2018 and December 2019. These strains were notably resistant to conventional antimicrobials other than carbapenems. Among the isolates, three strains exhibited heteroresistance to IPM and carried several ESBL and/or AmpC genes. Mice infected with a lethal dose of any of the three heteroresistant isolates were unable to survive in the presence of IPM treatment, as the percentage of the IPM-resistant subpopulation of each strain was increased in the peritoneum of these mice at 24 h after infection. The resistant subpopulation of the strains presented pulsed-field gel electrophoresis (PFGE) profiles that were identical to those of the susceptible subpopulation, but ompK36 porin showed a reduction in gene expression (0.09- to 0.50-fold) in the resistant subpopulation.ConclusionCarbapenem-heteroresistant strains were present among the MDR K. pneumoniae isolates producing ESBL/AmpC β-lactamases, and these heteroresistant strains failed IPM therapy in experimentally infected mice.

Molecular Characterization of Carbapenem-resistant, Colistin-resistant Klebsiella pneumoniae Isolates from a Tertiary Hospital in Jeonbuk, Korea

Klebsiella pneumoniae resistant to both carbapenem and colistin has become a major clinical challenge. Although such resistant isolates are rare, they have been sporadically reported from many areas of the world. To investigate the prevalence and resistant mechanisms to carbapenem-resistant, colistin-resistant K. pneumoniae among Korean patients, we selected these resistant K. pneumoniae from clinical isolates collected over a period of five years at a tertiary hospital in Jeonbuk, Korea. The minimum inhibitory concentration of a variety of antibiotics against the resistant isolates was determined by the macrodilution method or the E-test. PCR analysis was used to determine sequence types (STs) and identify the genes involved in resistance to carbapenem and colistin. In 338 K. pneumoniae clinical isolates, two exhibited both carbapenem and colistin resistance. The ST of these belonged to ST11 and ST258, the most prevalent STs with K. pneumoniae carbapenemases, but the isolates did not carry any prevalent carbapenem-hydrolyzing β-lactamase genes. The carbapenem resistance was mediated by loss of porins OmpK35 and/or OmpK36 associated with DHA-1 AmpC β-lactamase. The colistin resistance was caused by amino acid changes in PmrB, PmrC, PmrE, PmrK, and MgrB, including novel amino acid changes in PmrE and PmrK. We first reported novel carbapenem-resistant and colistin-resistant K. pneumoniae ST11 and ST258 with mutations within ompK35, ompK36, mgrB, and pmr genes in Korea. The results suggest that non- carbapenemase-producing carbapenem-resistant, colistin-resistant K. pneumoniae might be prevalent in Korea, and that better measures are necessary to control the spread of the resistant pathogen.

Activity of β-lactam plus β-lactam-enhancer combination cefepime/zidebactam against Klebsiella pneumoniae harbouring defective OmpK35/36 porins and carbapenemases

Cefepime/zidebactam is a β-lactam/β-lactam-enhancer based novel antibiotic which is in clinical development for treating infections caused by multidrug-resistant Gram-negative bacteria. Here, in vitro activity of cefepime/zidebactam was determined against multicentre Klebsiella pneumoniae clinical isolates co-expressing serine and/or metallo-carbapenemases and defective OmpK35 and OmpK36 porins. The MICs were determined using the reference broth microdilution method. Outer membrane protein expression was assessed using SDS-PAGE and mutations in the genes encoding OmpK35 and OmpK36 were identified by DNA sequencing. Among 34 isolates studied, carbapenemase genes, blaKPC and blaOXA-48-like, were present in 18 and 11 isolates, respectively; 5 isolates harboured both blaOXA-48-like and blaNDM. Point mutations, insertions, and duplications in OmpK35 and OmpK36, which are known to impact the activity of carbapenems, were detected. Against these isolates, cefepime/zidebactam (1:1) showed a consistent activity (MICs ≤4 mg/L). In conclusion, cefepime/zidebactam overcomes enzymatic, and non–enzymatic carbapenem-impacting resistance mechanisms concurrently expressed in K. pneumoniae.

An outbreak of ertapenem-resistant, carbapenemase-negative and porin-deficient ESBL-producing Klebsiella pneumoniae complex.

Carbapenem-resistant Klebsiella pneumoniae is an emerging healthcare-associated pathogen with dynamic molecular epidemiology. This study presents a retrospective analysis of the distribution and antibiotic resistance patterns of ertapenem-resistant ESBL-producing K. pneumoniae strains recovered during an outbreak from 2012 to 2014 in a Croatian University hospital. We aimed to estimate genetic relatedness of clinical isolates and underlying mechanisms that conferred the ertapenem-resistant phenotype. Expression analysis of genes involved in the antibiotic resistance showed reduced expression of major non-selective porin channel OmpK35. Reduced expression of OmpK36 porin channel in isolates resistant to at least one more carbapenem, apart from the ertapenem, was found to a lesser degree. Pulsed-field gel electrophoresis analysis of genomic DNA revealed that almost all isolates belonged to the same genetic clone. Caution regarding ertapenem-resistant, carbapenemase-negative porin-deficient mutants of K. pneumoniae is required as they are widespread, and under selective pressure this could result in a local clonal outbreak.

From Turkey: First Report of KPC-3- and CTX-M-27-Producing Multidrug-Resistant Klebsiella pneumoniae ST147 Clone Carrying OmpK36 and Ompk37 Porin Mutations.

Aim: The aim of this study was to identify antimicrobial resistance genes, virulence factor genes, and porin loss or mutations exhibited by the multidrug-resistant Klebsiella pneumoniae strain. Materials and Methods: Whole-genome sequencing was done via the Illumina NovaSeq 6000 platform. Strain identification and antibiotic susceptibility testing of strains were performed by the Vitek 2 automated system. Multilocus sequence typing analysis was carried out using seven conserved housekeeping genes. Results: The strain was resistant to penicillins, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, fosfomycin, and trimethoprim/sulfamethoxazole. The isolate was found to carry KPC-3, CTX-M-27, SHV-11, SHV-67, and TEM-1 β-lactamases. The clonal subtype of the isolate was ST147, and it possessed wzi64 and wzc38 alleles. Fifteen different point mutations (N49S, L59V, R146H, V178P, G189T, F198Y, V202L, F207Y, A217S, T222L, D223G, H235N, A280V, N304E, and S346N) were detected in the OmpK36 porin. A frame shift was observed in OmpK35 and two different point mutations (I70M and I128M) were found in the OmpK37 porin, in addition to seven mutations observed on the AcrR. Conclusions: This study demonstrated for the first time that the ST147 clone produced CTX-M-27 as well as KPC-3. In addition, new mutations were detected in the outer membrane proteins. These mutations together with the production of extended-spectrum β-lactamase and carbapenemase were found to contribute to the resistance of the ST147 clone to carbapenem and other antibiotics.

Mutations in porin LamB contribute to ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae

Ceftazidime-avibactam (CAZ-AVI) shows promising activity against carbapenem-resistant Klebsiella pneumoniae (CRKP), however, CAZ-AVI resistance have emerged recently. Mutations in KPCs, porins OmpK35 and/or OmpK36, and PBPs are known to contribute to the resistance to CAZ-AVI in CRKP. To identify novel CAZ-AVI resistance mechanism, we generated 10 CAZ-AVI-resistant strains from 14 CAZ-AVI susceptible KPC-producing K. pneumoniae (KPC-Kp) strains through in vitro multipassage resistance selection using low concentrations of CAZ-AVI. Comparative genomic analysis for the original and derived mutants identified CAZ-AVI resistance-associated mutations in KPCs, PBP3 (encoded by ftsI), and LamB, an outer membrane maltoporin. CAZ-AVI susceptible KPC-Kp strains became resistant when complemented with mutated bla KPC genes. Complementation experiments also showed that a plasmid borne copy of wild-type lamB or ftsI gene reduced the MIC value of CAZ-AVI in the induced resistant strains. In addition, bla KPC expression level increased in four of the six CAZ-AVI-resistant strains without KPC mutations, indicating a probable association between increased bla KPC expression and increased resistance in these strains. In conclusion, we here identified a novel mechanism of CAZ-AVI resistance associated with mutations in porin LamB in KPC-Kp.

Molecular genetic characteristics of the carbapenem resistant <i>Klebsiella pneumoniae KP254</i> strain as a representative of the highly virulent strain evolutionary branch

Here we provide molecular and genetic characteristics of the Klebsiella pneumoniae KP254 clinical strain belonging to clonal group 23 based on the genome-wide sequencing data. It is known that representatives of such clonal group exert highly virulent properties and cause community-acquired infections. Phenotypically, K. pneumoniae KP254 strain is characterized by multidrug resistance, including carbapenems. The determinants of antibiotic resistance (blaSHV-1, oqxAB, fosA) and pathogenicity encoding fimbriae 1, 3 types and the siderophore yersineobactin synthesis were found in the chromosome structure. However, there was uncovered the lack of conjugative element ICEKp1, the pathogenicity island KPHPI208, and the allantoin regulon genes which are often found in highly virulent strains. Analyzing nucleotide sequences in silico allowed to reveal the replicons of incompatibility group plasmids for FII, FIAHI1/FIIK, Col440I, ColpVC, FIBK, FIIpCRY. Combining contigs relative to reference sequences by using the BLASTN service allowed to identify two putative antibiotic resistance plasmids IncFII and IncFIIpCRY as well as one virulence plasmid IncFIBK. The determinants of the aerobactin siderophore, the RmpA2 mucoid phenotype regulator as well as heavy metal resistance genes constitute the virulence plasmid structure. The virulence plasmid nucleotide sequence coverage comprised 93% relative to the virulence plasmid pK2044 with 99.38% identity level; the genomic regions responsible for the salmochelin and RmpA protein synthesis were deleted. The set of antibiotic resistance determinants identified in the mobilome structure includes the genes for beta-lactamase LAP-2 (IncFIIpCRY plasmid) — a TEM-1 analogue, as well as extended-spectrum beta-lactamase CTX-M-55 (IncFII plasmid), both of which are rarely recorded in the Russian Federation. Additionally, widespread genes blaOXA-1, aac(3’)-IIa, ΔcatB4, aac (6’)-Ib-cr, tet(A), qnrSI, sul2, catA2 were also found in the plasmid DNA. The carbapenemase genes are absent in the resistome structure, whereas the examined strain exerts carbapenem resistance. The analysis of the ompK35 and ompK36 porin gene translated sequences revealed mutational changes which resulted in emerged stop codon within the ompK35 gene, whereas OmpK36 amino acid sequence contains a large number of substitutions, insertions, and deletions. The changes identified serve as one of the factors determining the carbapenem resistance. A synergistic effect may be accounted for by activity of the efflux pumps found in the structure of the K. pneumoniae KP254 genome, particularly AcrAB-TolC and KpnEF. Thus, the strain examined by us preserves the most significant signs specific to the highly virulent evolutionary branch Klebsiella strains, and at the same time, acquires the multidrug resistance genetic determinants.

Mutation of kvrA Causes OmpK35 and OmpK36 Porin Downregulation and Reduced Meropenem-Vaborbactam Susceptibility in KPC-Producing Klebsiella pneumoniae.

Meropenem-vaborbactam resistance in Klebsiella pneumoniae isolates is associated with loss-of-function mutations in the OmpK35 and OmpK36 porins. We identify two previously unknown loss-of-function mutations that confer cefuroxime resistance in K. pneumoniae isolates. The proteins lost were NlpD and KvrA; the latter is a transcriptional repressor that controls capsule production. We demonstrate that KvrA loss reduces OmpK35 and OmpK36 porin production, which confers reduced susceptibility to meropenem-vaborbactam in a KPC-3-producing K. pneumoniae isolate.

Characterization of the Genetic Background of KPC-2-Producing Klebsiella pneumoniae with Insertion Elements Disrupting the ompK36 Porin Gene.

Carbapenemase-producing combined porin loss is one of the primary mechanisms for carbapenem resistance. Although mutations in ompK35 and ompK36 genes have often been identified in carbapenem-resistant Klebsiella pneumoniae, reports on the porin protein gene disruption by insertion sequence (IS) elements are varied. The ompK36 porin protein gene disruption by IS elements and OmpK36 production loss in six blaKPC-2-carrying K. pneumoniae isolates were detected in this study. IS903, ISEc68, and IS1 insertions were noted in the 3, 2, and 1 isolates, respectively. The six K. pneumoniae isolates showed five different pulsed-field gel electrophoresis patterns and belonged to four multilocus sequence typing types, ST4, ST11, ST15, and ST39. This study increases our understanding of the genetic background of KPC-2 carbapenemases in porin-defective clinical isolates and the contribution of OmpK36 production loss to carbapenem resistance.

Early Experience With Meropenem-Vaborbactam for Treatment of Carbapenem-resistant Enterobacteriaceae Infections.

Twenty patients with carbapenem-resistant Enterobacteriaceae infections were treated with meropenem-vaborbactam. Thirty-day clinical success and survival rates were 65% (13/20) and 90% (18/20), respectively. Thirty-five percent of patients had microbiologic failures within 90 days. One patient developed a recurrent infection due to meropenem-vaborbactam-nonsusceptible, ompK36 porin mutant Klebsiella pneumoniae.

Bloodstream infection caused by KPC-producing Klebsiella pneumoniae resistant to ceftazidime/avibactam: epidemiology and genomic characterization

ObjectivesThe aim of this study was to evaluate the incidence of ceftazidime/avibactam resistance among Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) strains isolated from patients with bloodstream infection. MethodsWe collected 120 carbapenemase producing Enterobacteriaceae (CPE) strains from unique patients hospitalized in two Italian hospitals between January 2018 to February 2019. Strains were phenotypically characterized for the type of carbapenemase production and susceptibility to ceftazidime/avibactam. Ceftazidime/avibactam-resistant strains were characterized by whole-genome sequencing. ResultsDuring the study period, we characterized 105 (87.5%) KPC producers among a total of 120 CPE strains. Ceftazidime/avibactam resistance was found in three KPC-Kp strains isolated from patients with no history of previous ceftazidime/avibactam-based treatment. Of note, two out of three ceftazidime–avibactam-resistant KPC-Kp were also resistant to meropenem/vaborbactam. Genomic characterization showed that a ceftazidime/avibactam-resistant KPC-Kp harboured a mixed population with D179Y mutated KPC-2, while the other two ceftazidime–avibactam-resistant KPC-Kp possessed non-functional ompK35-ompK37 and mutated ompK36 porins associated with higher copy number of blaKPC gene. ConclusionsOur results showed that incidence of ceftazidime/avibactam resistance emerged in KCP-Kp strains independently from previous antimicrobial exposure. Resistance to ceftazidime/avibactam was associated with mutations within the blaKPC gene or porin deficiency associated with higher blaKPC copy number and is also related to the meropenem/vaborbactam resistance.

234. Reversal of Carbapenem and Amikacin Susceptibilities in Isogenic Klebsiella pneumoniae From a Patient with Persistent Bacteriuria

BackgroundGenomic tools permit a detailed analysis of antibiotic resistance determinants in bacteria, or resistome. Here we discuss variations in antibiotic resistance in K. pneumoniae (Kp) not explained by changes in the resistomeMethodsWe compared Kp strains with divergent carbapenem and aminoglycoside susceptibilities. After identification of bacteria, antibiotic susceptibility testing was performed according to CLSI guidelines. Draft genome sequences were generated using Illumina MiSeq (Nextera paired-end library) and assembled using CLC Genomics Workbench (CLC bio, Cambridge, MA). Resistome, plasmid types and MLST were investigated using the CGE platform (http://cge.cbs.dtu.dk), while capsular type and virulence genes were investigated using the Pasteur BIGsDB database (https://bigsdb.pasteur.fr).ResultsWhile receiving amoxicillin-clavulanate, a 44-year old man with diabetes mellitus and paraplegia with neurogenic bladder grewKp resistant to carbapenems and amikacin from urine. He was treated with fosfomycin and amikacin, followed by imipenem and plazomicin, prior to lithotripsy. Three months later, while off antibiotics, urine cultures grew Kp susceptible to carbapenems and amikacin (figure). Genetic comparison between resistant (November 20, 2018) and susceptible (January 30, 2019) strains revealed they were isogenic, only differing by 559 SNPs (table). Both were ST14, presented capsular type 16, and shared cehalosporinase (blaSHV-28, blaCTX-M-15, blaTEM-1B, blaOXA-1) and aminoglycoside modifying enzyme (AME) (aph(3’’)-Ib, aph(6)-Id, aac(6’)-Ib-cr) genes. Although both had mutations in the outer membrane porin OmpK36, these differed (stop AA125 and frameshift AA183, respectively)ConclusionCarbapenem resistance in the initial Kp is likely explained by overexpression of cephalosporinases in combination with changes in membrane permeability, while amikacin resistance is likely due to AMEs. Since no significant gene variation was observed in the susceptible Kp, reversal of resistance was likely due to decreased expression of cephalosporinases and AMEs after antibiotics were stopped. Incorporation of antibiotic history and host factors can explain clinically important changes in antibiotic resistance Disclosures All authors: No reported disclosures.

OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo

Carbapenem-resistance in Klebsiella pneumoniae (KP) sequence type ST258 is mediated by carbapenemases (e.g. KPC-2) and loss or modification of the major non-selective porins OmpK35 and OmpK36. However, the mechanism underpinning OmpK36-mediated resistance and consequences of these changes on pathogenicity remain unknown. By solving the crystal structure of a clinical ST258 OmpK36 variant we provide direct structural evidence of pore constriction, mediated by a di-amino acid (Gly115-Asp116) insertion into loop 3, restricting diffusion of both nutrients (e.g. lactose) and Carbapenems. In the presence of KPC-2 this results in a 16-fold increase in MIC to Meropenem. Additionally, the Gly-Asp insertion impairs bacterial growth in lactose-containing medium and confers a significant in vivo fitness cost in a murine model of ventilator-associated pneumonia. Our data suggests that the continuous selective pressure imposed by widespread Carbapenem utilisation in hospital settings drives the expansion of KP expressing Gly-Asp insertion mutants, despite an associated fitness cost.

Genomic characterization of a KPC-23-producing Klebsiella pneumoniae ST258 clinical isolate resistant to ceftazidime-avibactam

ObjectivesWe characterized the first ceftazidime-avibactam-resistant KPC-producing-Klebsiella pneumoniae clinical isolate detected in Greece, before the introduction of ceftazidime-avibactam in clinical practice. MethodsK. pneumoniae KP-90 was isolated from a hospitalized patient in Thessaloniki during a nationwide surveillance study conducted between 2014 and 2016. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing (Ion Torrent TM platform) of the isolate was carried out to identify the acquired resistance genes and mutations that were associated with ceftazidime-avibactam resistance. ResultsThe K. pneumoniae isolate belonged to multilocus sequence type ST258 and harboured blaKPC-23 as the only carbapenemase gene. The isolate had a minimum inhibitory concentration (MIC) of 16 mg/L to ceftazidime-avibactam and was highly resistant to imipenem, meropenem (MICs, 512 mg/L) and ceftazidime (MIC, >1024 mg/L). blaKPC-23 was detected on a Tn4401a transposon, located on a pKPQIL-type plasmid. A non-functional outer membrane protein OmpK35 and an OmpK36 variant that had been previously associated with K. pneumoniae isolates of ST258 were detected. Transformation studies with Escherichia coli TOP10 showed that KPC-23 offered similar carbapenem MICs as KPC-2 and KPC-3. However, KPC-23 conferred a four-fold higher ceftazidime MIC (>1024 mg/L), which in the presence of avibactam was reduced (>7-fold) to 8 mg/L, which is just within the limit of the susceptibility breakpoint. ConclusionsCeftazidime-avibactam resistance in a KPC-23- producing K. pneumoniae clinical isolate was due to increased ceftazidime hydrolysis and was likely enhanced by OmpK35 porin deficiency.

1378. Evaluation of the In vitro Activity of Meropenem-Vaborbactam Against Carbapenem-Resistant Enterobacteriaceae, Including Isolates Resistant to Ceftazidime–Avibactam

BackgroundMeropenem-vaborbactam (M-V) is a novel antibiotic for treatment of carbapenem-resistant Enterobacteriaceae (CRE) infections. Our objective was to determine the in vitro activity of meropenem-vaborbactam against genetically-diverse CRE isolates, including those that have developed resistance to Ceftazidime–Avibactam (C-A).MethodsMinimum inhibitory concentrations (MICs) were determined for meropenem (MER), M-V, and C-A by reference broth microdilution (BMD) methods in triplicate. Vaborbactam and avibactam were tested at fixed concentrations of 8 and 4 µg/mL, respectively. Quality control strains were used and within expected ranges. Polymerase chain reaction (PCR) with DNA sequencing was used to detect resistance determinants, including Klebsiella pneumoniae carbapenemase (KPC) subtypes and porin mutations.ResultsA total of 117 CRE isolates were tested, including K. pneumoniae (Kp; n = 83), E. cloacae (n = 17), E. coli (n = 10), and E. aerogenes (n = 7). Seventy-nine percent harbored blaKPC. KPC subtypes included KPC-2 (n = 32), KPC-3 (n = 41), KPC-3 variants (n = 16), and KPC [not typed] (n = 4, all E. coli). Among 74 K. pneumoniae, 95% had a premature stop codon in ompk35 and ompK36 genotypes included wild type (n = 48), IS5 insertion (n = 13), 135–136 DG duplication (n = 9), and other mutations (n = 4). The median (range) MICs for MER, C-A, and M-V were 8 (0.06 to ≥128), 1 (0.25 to ≥512), and 0.03 (0.015––16), respectively. Corresponding rates of susceptibility were 23, 84, and 98%, respectively. Fifty-three percent and 95% of C-A-resistant isolates were susceptible to MER and M-V, respectively. Among Kp, C-A MICs did not vary by KPC subtype or porin genotype. On the other hand, median M-V MICs were higher among KPC-2 than KPC-3 Kp (0.12 vs. 0.03; P = 0.002), and among Kp with ompK36 porin mutations compared with wild type (0.25 vs. 0.03; P < 0.001). Among Kp with KPC-3 variants (n = 16), the median M-V MIC was 0.03 (0.015––2); 100% were M-V susceptible. Median M-V MICs did not vary by CRE species. Only two isolates were M-V resistant, both were E. cloacae that did not harbor blaKPC.ConclusionM-V demonstrates high rates of in vitro susceptibility against diverse CRE isolates, including those that are resistant to C-A. As this agent is introduced into the clinic, it will be important to identify K. pneumoniae isolates harboring KPC-2 with ompK36 porin mutations that demonstrate higher MICs.Disclosures M. H. Nguyen, Merck: Grant Investigator, Research grant. Astellas: Grant Investigator, Research grant.

Meropenem heteroresistance in clinical isolates of OXA-48–producing Klebsiella pneumoniae

OXA-48–producing Klebsiellapneumoniae isolates often show growth of colonies within inhibition zones in carbapenem diffusion assays. The nature of these colonies was investigated in a series of clinical isolates of OXA-48–producing K. pneumoniae obtained in the context of a hospital outbreak, and they were found to be persistent colonies that reproduced again the same phenotype when they were collected and tested in diffusion assays again. The frequency of mutations conferring resistance to meropenem (8 μg/mL) was determined for the same isolates. The average mutation frequency was 5.47·10−6 (range: 2.59·10−8–5.87·10−5), and the analysis of several resistant mutants showed that all of them had mutations in the ompK36 porin gene. Heteroresistance was investigated using population analysis profiling. The profiles were compatible with mutation frequency assays, and all the colonies analyzed were resistant mutants. In OXA-48–producing K. pneumoniae, the growth of persisters seems to be specific of diffusion assays.