OmpK36 Expression Research Articles

Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae, modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. An analysis of large K. pneumoniae genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine-to-thymine transition at position 25 (25c > t) in ompK36. We show that the 25c > t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates K. pneumoniae in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c > t transition tips the balance toward treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c > t transition mediates an intramolecular messenger RNA (mRNA) interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

High Osmotic Stress Increases OmpK36 Expression through the Regulation of KbvR to Decrease the Antimicrobial Resistance of Klebsiella pneumoniae.

ABSTRACTKlebsiella pneumoniae is a pathogen known for its high frequency of antimicrobial resistance. Responses to various environmental stresses during its life can influence the resistance to antibiotics. Here, we demonstrate the role and mechanism of KbvR regulator in the response to environmental osmotic stress and in the effect of osmotic stress on antimicrobial resistance. The kbvR mutant strain exhibited increasing tolerance to high osmotic stress and certain antibiotics, including β-lactams. The expression levels of KbvR and outer membrane porin OmpK36 were upregulated in response to high osmotic stress in the wild type (WT), and the deletion of kbvR decreased the expression level of ompK36. The membrane permeability of the kbvR mutant strain was decreased, which was partly restored through the upregulated expression of OmpK36. The DNA affinity purification sequencing (DAP-seq) and microscale thermophoresis (MST) assay disclosed the binding of KbvR to the promoter of the ompK36 gene, indicating that KbvR directly and positively regulated the expression of OmpK36. The high osmotic stress increased the susceptibility to β-lactams and the expression of ompK36 in the WT strain. However, the increased ompK36 expression and the susceptibility to β-lactams in the kbvR mutant strain under high osmotic stress were lower than those of WT. In conclusion, our study has identified that high osmotic stress in the environment influenced the resistance of K. pneumoniae to antibiotics and that the regulation of KbvR with OmpR on the expression of OmpK36 was involved in countering high osmotic stress to change the antimicrobial resistance.IMPORTANCE Klebsiella pneumoniae is considered a global threat because of the rising prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. The sensing and adaption abilities of bacteria to the environmental osmotic stress can change the expression of their outer membrane porins, membrane permeability, and resistance to antibiotics. This study reports that KbvR is a newly found regulator that can be upregulated under high osmotic stress and directly regulate the expression of OmpK36 to change the resistance of K. pneumoniae to β-lactam antibiotics. The results demonstrate how adaptation to high osmotic stress changes the sensitivity of K. pneumoniae to antibiotics. The mechanism can be used to sensitize bacteria to antibiotics and highlight new potential strategies for exploiting shared constraints in governing adaptation to diverse environmental challenges.

Within-host evolution of a Klebsiella pneumoniae clone: selected mutations associated with the alteration of outer membrane protein expression conferred multidrug resistance.

A patient repeatedly developed bacteraemia despite the continuous use of antibiotics. We obtained two Klebsiella pneumoniae isolates from the patient's blood on Days 72 and 105 after hospitalization. Each of the two isolates belonged to ST45, but while the first isolate was susceptible to most antibiotics, the second one was resistant to multiple drugs including carbapenems. To identify the genetic differences between the two isolates and uncover alterations formed by the within-host bacterial evolution leading to the antimicrobial resistance. Whole-genome comparison of the two isolates was carried out to identify their genetic differences. We then profiled their outer membrane proteins related to membrane permeability to drugs. To characterize a ramR gene mutation found in the MDR isolate, its WT and mutant genes were cloned and expressed in the MDR isolate. The two isolates showed only three genomic differences, located in mdoH, ramR and upstream of ompK36. In the MDR isolate, a single nucleotide substitution in the ompK36 upstream region attenuated OmpK36 expression. A single amino acid residue insertion in RamR in the MDR isolate impaired its function, leading to the down-regulation of OmpK35 and the subsequent up-regulation of the AcrAB-TolC transporter, which may contribute to the MDR. We identified very limited genomic changes in the second K. pneumoniae clone during within-host evolution, but two of the three identified mutations conferred the MDR phenotype on the clone by modulating drug permeability.

4-Chloromercuribenzoic acid enhances carbapenem sensitivity among pathogenic Gram negative bacteria by altering blaVIM, adeB and ompC expression

BackgroundRapid global dissemination of carbapenem resistant Gram negative bacteria (CRGNB) is supposed to be clinically most alarming. Since, p-chloromercuribenzoic acid (pCMB) is a well known metallo-beta-lactamase inhibitor; evaluation of its bactericidal and carbapenem resistance reversing potential would be important. MethodsIn this study, bactericidal and meropenem resistance reversing potential of pCMB was investigated against CRGNB by MIC determination, checkerboard assay, time-kill assay and cellular viability assay. Effect of pCMB on cellular morphology was visualized by Scanning Electron Microscopy. Further, quantitative Real Time-PCR was performed to evaluate effects of pCMB on clinically relevant metallo-beta-lactamases, major efflux pumps and outer membrane proteins expression. ResultspCMB exhibited at least four fold reduced MIC value (2–256μg/ml) than that of meropenem against CRGNB. Moreover, pCMB exhibited synergism with meropenem against 86.06% of CRGNB. MIC of pCMB (16−32μg/ml) could kill upto 99.96% bacteria within 6−8h of dosing. pCMB exerted bactericidal activity by severely disrupting cell wall integrity. Reversal of carbapenemase property of CRGNB by pCMB might have developed through alteration of blaVIM, acrB, mexB and ompk36 expression. ConclusionsHence, the current study identified pCMB as a potential bactericidal agent which enhanced meropenem sensitivity by altering blaVIM, acrB, mexB and ompk36 expression.

1523. Antimicrobial Activity of Ceftazidime–Avibactam and Comparator Agents Tested Against Gram-Negative Organisms Isolated from Complicated Urinary Tract Infections: Results from the International Network for Optimal Resistance Monitoring (INFORM) Program

BackgroundUrinary tract infections (UTIs) are among the most frequent healthcare-associated infections and represent a major source of Gram-negative (GN) bacteremia. We evaluated the antimicrobial susceptibility (S) of GN bacteria isolated from patients with complicated UTIs (cUTIs) in United States medical centers.MethodsUnique patient isolates were consecutively collected from patients with cUTIs in 83 medical centers in 2015–2017, and the GN organisms (n = 9,403) were S tested against CAZ-AVI and comparators by reference broth microdilution Methods. Ceftolozane-tazobactam (C-T) was tested in 2017 only. Enterobacteriaceae (ENT) with an extended-spectrum β-lactamase (ESBL) phenotype was evaluated by whole genome sequencing for the presence of genes encoding β-lactamases.ResultsThe most common organisms were E. coli (EC; 53.2%), K. pneumoniae (KPN; 12.5%), E. faecalis (6.0%), P. mirabilis (PM; 5.3%), and P. aeruginosa (PSA; 4.9%). An ESBL phenotype was observed among 13.2, 13.4 and 7.0% of EC, KPN, and PM, respectively. CAZ-AVI inhibited >99.9% of all ENT, including all EC, PM and E. cloacae (ECL) isolates, at the S breakpoint of ≤8 µg/mL (table). CAZ-AVI was also highly active against KPN, including ESBL-phenotype (MIC50/90, 0.25/1 µg/mL; 99.5%S) and meropenem (MEM)-non-S isolates (MIC50/90, 1/2 µg/mL; 98.0%S). In contrast, only 72.9 and 73.1% of ESBL-phenotype KPN isolates were S to C-T and MEM, respectively. Only one ENT isolate was CAZ-AVI-resistant, a KPN with a CAZ-AVI MIC of 16 μg/mL that produced a KPC-2 and an SHV-12 and exhibited decreased expression of OmpK36. Among ECL (27.2% CAZ-non-S), S to CAZ-AVI, C-T, and MEM were 100.0, 80.0, and 98.8%, respectively. CAZ-AVI was also highly active against PSA (MIC50/90, 2/4 µg/mL; 99.1%S), including isolates expressing a multidrug-resistant (MDR) phenotype (MIC50/90, 4/8 µg/mL; 93.8%S). Further 86.2% (25/29) of PSA isolates non-S to MEM, CAZ, and piperacillin–tazobactam were CAZ-AVI-S.ConclusionCAZ-AVI demonstrated potent activity against a large collection of contemporary (2015–2017) GN bacteria isolated from patients with cUTIs in US hospitals, including MDR isolates, and provided greater coverage than the agents currently available in the United States for treatment of cUTIs. Disclosures H. S. Sader, Allergan: Research Contractor, Research support. M. Castanheira, Allergan: Research Contractor, Research support. R. E. Mendes, Allergan: Research Contractor, Research support. R. K. Flamm, Allergan: Research Contractor, Research support.

Insertion Sequence-Dependent <i>OmpK</i>36 Mutation Associated Ertapenem Resistance in Clinical <i>Klebsiella pneumoniae</i>

Extended-spectrum β-lactamases (ESBLs) and/or AmpC enzymes combined with deficiency of porins OmpK35 and OmpK36 are important for the development of carbapenem-resistant Klebsiella pneumoniae. We characterized the clinical K. pneumoniae human isolates and investigated the effect of meropenem induction on the ompK35 and ompK36 mutation to develop carbapenem resistance from six carbapenem-susceptible ESBL-producing K. pneumoniae strains. 163 clinical K. pneumoniae isolates were grouped mostly into the ESBL + AmpC (44.2%) and ESBL (42.9%) phenotypes. The resistance rate differed between cephalosporins (52.1% for cefepime - 97.5% for cefotaxime) and carbapenems (16% for meropenem - 28.2% for imipenem) (P blaTEM, blaSHV, blaCTX-M-3-like, and blaCTX-M-14-like of AmpA β-lactamase genes and blaDHA and blaCMY of AmpC β-lactamase genes. Compared to all 163 clinical isolates, the 56 carbapenem-resistant isolates carried less frequently of blaTEM, blaCTXM-14-like, and blaCTXM-3-like and more frequently of blaDHA-1 and blaCMY-2. The carbapenem-resistant isolates differed in prevalence against imipenem, ertapenem, and meropenem and lacked OmpK35 more frequently than OmpK36, but abnormal PCR amplicons were detected fewer in the Omp K35-deficient group than in the OmpK36-deficient group (32.5% vs. 68.4%, respectively). The carbapenem-resistant isolate mostly carried blaDHA (91.1%) and three isolates carried blaKPC-2. Following induction with meropenem insertion sequences in ompK36, not ompK36, were identified as IS5 for KP08, IS1 for KP15, and IS903 for KP16 isolates. OmpK36 deficiency increased resistance to ertapenem, but not imipenem and meropenem. Clinical isolates belonged mainly to ESBL + AmpC group and ESBL group with difference in resistance to cephalosporins and carbapenems, the bla genes. Carbapenem resistant isolates lacked OmpK35 expression, than the OmpK36 expression, Meropenem induction developed the carbapenem resistant isolates with insertion of different insertion sequences in ompK36, not ompK35.

Low Frequency of Ceftazidime-Avibactam Resistance among Enterobacteriaceae Isolates Carrying blaKPC Collected in U.S. Hospitals from 2012 to 2015.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae isolates have been increasingly reported worldwide, and therapeutic options to treat infections caused by these organisms are limited. We evaluated the activity of ceftazidime-avibactam and comparators against 456 Enterobacteriaceae isolates carrying blaKPC collected from 79 U.S. hospitals during 2012 to 2015. Overall, ceftazidime-avibactam (MIC50/90, 0.5/2 μg/ml; 99.3% susceptible) and tigecycline (MIC50/90, 0.5/1 μg/ml; 98.9% susceptible at ≤2 μg/ml) were the most active agents. Only 80.5% and 59.0% of isolates were susceptible to colistin and amikacin, respectively. All three isolates (0.7%) displaying resistance to ceftazidime-avibactam (K. pneumoniae; MICs, ≥16 μg/ml) were evaluated using whole-genome sequencing analysis and relative quantification of expression levels of porins and efflux pump. Two isolates carried metallo-β-lactamase genes, blaNDM-1 or blaVIM-4, among other β-lactam resistance mechanisms, and one displayed a premature stop codon in ompK35 and decreased expression of ompK36 Ceftazidime-avibactam was active against 100.0 and 99.3% of isolates carrying blaKPC-3 (n = 221) and blaKPC-2 (n = 145), respectively. Isolates carrying blaKPC were more commonly recovered from pneumonia (n = 155), urinary tract (n = 93), and skin/soft tissue (n = 74) infections. Ceftazidime-avibactam (97.8 to 100.0% susceptible) was consistently active against isolates from all infection sites. K. pneumoniae (83.3% of the collection) susceptibility rates were 99.2% for ceftazidime-avibactam, 98.9% for tigecycline, and 80.1% for colistin. Ceftazidime-avibactam susceptibility did not vary substantially when comparing isolates from intensive care unit (ICU) patients to those from non-ICU patients. Ceftazidime-avibactam was active against this large collection of isolates carrying blaKPC and represents a valuable addition to the armamentarium currently available for the treatment of infections caused by KPC-producing Enterobacteriaceae.

Outbreak of KPC-3-producing ST15 and ST348 Klebsiella pneumoniae in a Portuguese hospital.

To date, only a few sporadic cases of infections due to Klebsiella pneumoniae carbapenemase (KPC) producers have been reported in Portugal. Here, we report for the first time an outbreak of K. pneumoniae KPC-3 producers in a tertiary-care hospital during 2013. Twenty-seven ertapenem-resistant K. pneumoniae were identified in patients at a tertiary-care hospital during 2013 isolated predominantly from urine (48·1%) and blood (25·9%) cultures. All isolates were highly resistant to β-lactam antibiotics and most showed intermediate resistance to imipenem. The more frequent β-lactamases were TEM- (77·7%), CTX-M- (70·3%) and KPC-type (66·6%). KPC-3 was identified by sequencing. The bla KPC-3 gene was associated with an IncF plasmid, and efficiently transferred to E. coli J53. Pulsed-field gel electrophoresis typing revealed three clusters of isolates which were further characterized by multi-locus sequence typing as ST11, ST15 and ST348. Ertapenem-resistant ST15 was already in circulation in the hospital, related to expression of OmpK36 modified porin, but the other two sequence types had not been previously found in the hospital. We conclude that the IncF plasmid mediated transfer of KPC-3 in the outbreak and that implementation of carbapenemase gene screening in isolates from patients on admission to hospital is advisable in order to control dissemination of these antimicrobial resistance elements.

Study of <i>OmpK</i>35 and <i>OmpK</i>36 Expression in Carbapenem Resistant ESBL Producing Clinical Isolates of <i>Klebsiella pneumoniae</i>

Background: Carbapenem resistant extended spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae (K. pneumoniae) is increasing worldwide. Carbapenem resistance (CR) has been attributed not only to production of carbapenemases but also to permeability barriers due to outer membrane proteins (OmpK35 and OmpK36) disruption. Objective: Phenotypic detection of CR among ESBL producing K. pneumoniae isolates, followed by the evaluation of the role of ompK35 and ompK36 gene expression among carbapenem resistant K. pneumoniae (CR-KP) isolates. Methods: 100 ESBL producing K. pneumoniae isolates were included in this study. Minimum inhibitory concentration (MIC) of imipenem was performed for all isolates by broth microdilution method. For CR-KP isolates, phenotypic detection of K. pneumoniae carbapenemase (KPC), metallo-β-lactamase (MBL) and AmpC enzymes was performed followed by Realtime qRT-PCR to detect and quantify ompK35 and ompK36 gene expression. Results: 42% of our isolates were carbapenem resistant, and all of them were KPC producers either singly or in combination with MBL and/or AmpC production. Reduced expression of both ompK35 and ompK36 was detected in (52.38%) of CR-KP isolates, while reduced expression of ompK36 or ompK35 alone was found in (2.38%) and (33.33%) respectively. Twenty of 42 CR-KP isolates (47.62%), showing reduced ompK35 and ompK36 expression, exhibited high level resistance (HLR) (>32 μg/ml) to imipenem. There was a significant correlation between reduced expression of ompK36 and increase MIC values (p ompK35 and/or ompK36 resulted in significant increase in imipenem MIC (p K. pneumoniae showing carbapenemase and/or AmpC production together with loss of OmpK35 and/or OmpK36.

An Increase in the Clinical Isolation of Acquired AmpC β-Lactamase-Producing Klebsiella pneumoniae in Korea from 2007 to 2010

We investigated the occurrence and genetic basis of AmpC β-lactamase (AmpC)-mediated antibiotic resistance, by examining Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a university hospital, from 2007 to 2010. The ampC genes were detected by multiplex AmpC PCR, and AmpC-positive strains were subjected to DNA sequencing. Extended-spectrum β-lactamase (ESBL) production was assessed using the ESBL disk test based on the utilization of boronic acid. Carbapenem-resistant isolates were further investigated by the modified Hodge test, a carbapenemase inhibition test and SDS-PAGE experiments. AmpC expression was detected in 1.6% of E. coli (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, 7.2% of K. pneumoniae (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, and 2.5% of P. mirabilis (8 CMY-2 and 1 CMY-1) isolates. Of the 198 acquired AmpC producers, 58 isolates (29.3%) also produced an ESBL enzyme. Among the acquired AmpC-producing K. pneumoniae isolates, the minimum inhibitory concentration (MIC) MIC50/MIC90 values for cefoxitin, cefotaxime, cefepime, imipenem, and meropenem were >32/>32, 16/>32, 1/16, 0.25/0.5, and <0.125/0.125 µg/mL, respectively. The MIC values for carbapenem were ≥2 µg/mL for 2 K. pneumoniae isolates, both of which carried the blaDHA-1 gene with a loss of OmpK36 expression, but were negative for carbapenemase production. The acquisition of AmpC-mediated resistance in K. pneumoniae isolates increased, as did the proportion of AmpC and ESBL co-producers among the hospital isolates. The accurate identification of isolates producing AmpCs and ESBLs may aid in infection control and will assist physicians in selecting an appropriate antibiotic regimen.

Prevalence of OmpK35 and OmpK36 porin expression in beta-lactamase and non-betalactamase- producing Klebsiella pneumoniae.

BackgroundThe aims of this study were to confirm the presence of OmpK35 and OmpK36 in extended-spectrum beta-lactamase-producing and nonextended-spectrum beta-lactamase-producing Klebsiella pneumoniae and to determine the relationship between porin expression and resistance to third-generation cephalosporins.MethodsFifty-two K. pneumoniae isolates were obtained and analyzed for extended-spectrum beta-lactamase and for OmpK35 and OmpK36.ResultsTwenty-two (42.3%) isolates of K. pneumoniae were extended-spectrum beta-lactamase producers. The OmpK35 profile in K. pneumoniae producing extended-spectrum beta-lactamase showed the presence of porin protein in ceftazidime-sensitive K. pneumoniae (six isolates), and the OmpK36 profile in K. pneumoniae producing extended-spectrum beta-lactamase revealed isolates sensitive to cefotaxime (n = 8) and ceftriaxone (n = 6). All nonextended-spectrum beta-lactamase-producing K. pneumoniae showed the presence of OmpK35 and OmpK36 porin proteins.ConclusionThe presence of OmpK35 is mostly related to ceftazidime susceptibility and less to cefotaxime and ceftriaxone susceptibility, while OmpK36 expression is seen more often in cefotaxime-sensitive isolates. OmpK35 and OmpK36 indicate nonextended-spectrum beta-lactamase producing strains, and their presence is important when selecting an antimicrobial agent.

OmpK26, a Novel Porin Associated with Carbapenem Resistance in Klebsiella pneumoniae

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems are being isolated with increasing frequency. Loss of the expression of the major nonspecific porins OmpK35/36 is a frequent feature in these isolates. In this study, we looked for porins that could compensate for the loss of the major porins in carbapenem-resistant organisms. Comparison of the outer membrane proteins from two K. pneumoniae clinical isogenic isolates that are susceptible (KpCS-1) and resistant (KpCR-1) to carbapenems revealed the absence of OmpK35/36 and the presence of a new 26-kDa protein in the resistant isolate. An identical result was obtained when another pair of isogenic isolates that are homoresistant (Kpn-3) and heteroresistant (Kpn-17) to carbapenems were compared. Mass spectrometry and DNA sequencing analysis demonstrated that this new protein, designated OmpK26, is a small monomeric oligogalacturonate-specific porin that belongs to the KdgM family of porins. Insertion-duplication mutagenesis of the OmpK26 coding gene, yjhA, in the carbapenem-resistant, porin-deficient isolate KpCR-1 caused the expression of OmpK36 and the reversion to the carbapenem-susceptible phenotype, suggesting that OmpK26 is indispensable for KpCR-1 to lose OmpK36 and become resistant to these antibiotics. Moreover, loss of the major porin and expression of OmpK26 reduced in vitro fitness and attenuated virulence in a murine model of acute systemic infection. Altogether, these results indicate that expression of the oligogalacturonate-specific porin OmpK26 compensates for the absence of OmpK35/36 and allows carbapenem resistance in K. pneumoniae but cannot restore the fitness of the microorganism.

First Outbreak of a Plasmid-Mediated Carbapenem-Hydrolyzing OXA-48 β-Lactamase in Klebsiella pneumoniae in Spain

Twenty Klebsiella pneumoniae isolates producing OXA-48 were collected from April 2009 to September 2010. Strains were clonally related and coproduced a CTX-M-15 β-lactamase. A conjugative plasmid of circa 70 kb carrying bla(OXA-48) was identified. Eleven isolates showed low-level resistance to carbapenems, whereas nine showed high-level resistance. Decreased expression of OmpK36 was related to high-level resistance to carbapenems. The isolates belonged to sequence type 101 (ST101). This is the first outbreak caused by an OXA-48-producing K. pneumoniae strain in Spain.

Prevalence and Characteristics of Ertapenem-ResistantKlebsiella pneumoniaeIsolates in a Taiwanese University Hospital

This study was conducted to investigate the prevalence and characteristics of ertapenem-resistant (ETP-R) Klebsiella pneumoniae isolates at a Taiwanese hospital. The disk-diffusion tests revealed that the rate of ertapenem resistance among all isolates collected in 2008 was 13.5%, and the resistance rate among bloodstream isolates increased from 0% to 13.6% between 2001 and 2008. Eighty-two nonduplicate ETP-R isolates collected in 2008 were examined. Seventy-four (90.2%) isolates of them had extended-spectrum β-lactamases (CTX-M- and SHV-type), AmpC enzymes (DHA-1 and CMY-2), and IMP-8 metallo-β-lactamase alone or in combination, and an extremely high prevalence of fluoroquinolone resistance (95.1%) and plasmid-mediated quinolone resistance determinants (90.2%) were also observed. Eighteen ETP-R but imipenem-susceptible isolates were selected and compared with 18 imipenem-nonsusceptible isolates collected before 2008. Sequence analyses revealed genetic disruptions of OmpK36 in 11 imipenem-nonsusceptible and 6 imipenem-susceptible isolates, respectively, and OmpK35 disruptions in 10 isolates for both groups. For the isolates with intact ompK36, sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests decreased expression of OmpK36 in 5 of 7 imipenem-nonsusceptible isolates and 3 of 12 imipenem-susceptible isolates. In conclusion, the increasing prevalence of ertapenem resistance that was predominantly attributed to noncarbapenemase-mediated resistance mechanisms in K. pneumoniae is becoming a serious treat to patients in Taiwan.

An Ertapenem-Resistant Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae Clone Carries a Novel OmpK36 Porin Variant

Carbapenem-resistant Klebsiella pneumoniae caused an outbreak in a hospital in Rome, Italy. The clinical isolates were tested by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, multilocus sequence typing, plasmid typing, and β-lactamase identification. The OmpK35 and OmpK36 porins were analyzed by SDS-PAGE, and their genes were amplified and sequenced. Complementation experiments were performed using a recombinant unrelated ompK36 gene. An ertapenem-resistant and imipenem- and meropenem-susceptible clone was identified and assigned to the sequence type 37 lineage by MLST; it carried SHV-12 and CTX-M-15 ESBLs, did not produce the OmpK35 due to a nonsense mutation, and expressed a novel OmpK36 variant (OmpK36V). This variant showed two additional amino acids located within the L3 internal loop, one of the highly conserved domains of the protein. Two isolates of the same clone also exhibited resistance to imipenem and meropenem, due to the loss of OmpK36 expression by a nonsense mutation occurring in the ompK36V variant gene. These were the first carbapenem-resistant K. pneumoniae isolates identified within the hospital. Screening for the ompK36V gene of unrelated K. pneumoniae isolates derived from patients from 2006 to 2009 demonstrated the high frequency of this gene variant as well as its association with ertapenem resistance, reduced susceptibility to meropenem, and susceptibility to imipenem.

In vivo selection of imipenem-resistant Klebsiella pneumoniae producing extended-spectrum β-lactamase CTX-M-15 and plasmid-encoded DHA-1 cephalosporinase

Four Klebsiella pneumoniae isolates (KP1–4) were recovered sequentially from an infected patient. Whilst KP1–3 were resistant to all β-lactams except carbapenems, KP4 recovered after 24 days of imipenem-containing treatment showed additional resistance to carbapenems. No carbapenem hydrolysis could be identified in KP4. Molecular characterisation revealed that KP1–4 were indistinguishable by pulsed-field gel electrophoresis, contained a 95-kb self-transferable plasmid harbouring bla CTXM-15 and bla TEM-1 genes and a 65-kb plasmid that was not transferred by conjugation into Escherichia coli, and harboured the plasmid-mediated bla DHA-1 AmpC β-lactamase gene. In addition, KP4 failed to express OmpK36 owing to a point mutation leading to a premature stop of the protein. This study demonstrates development of carbapenem resistance related to loss of OmpK36 expression in a K. pneumoniae isolate harbouring extended-spectrum β-lactamase and plasmid-mediated cephalosporinase genes following prolonged imipenem exposure.

Contribution of OmpK36 to carbapenem susceptibility in KPC-producing Klebsiella pneumoniae

Isolates of Klebsiella pneumoniae harbouring the carbapenemase KPC may have carbapenem MICs that remain in the susceptible range, and may therefore go unrecognized. To understand the mechanisms contributing to the variability in carbapenem MICs, 20 clinical isolates, all belonging to either of two clonal groups of KPC-possessing K. pneumoniae endemic to New York City, were examined. Expression of genes encoding KPC, the porins OmpK35 and OmpK36, and the efflux pump AcrAB was examined by real-time RT-PCR. Outer-membrane profiles of selected KPC-producing isolates were examined by SDS-PAGE, and proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry. The identification of SHV and TEM beta-lactamases and the genomic sequences of ompK35 and ompK36 were determined by PCR and DNA sequencing, respectively. For one clonal group, carbapenem MICs increased with decreasing expression of ompK36. A second clonal group also had carbapenem MICs that correlated with ompK36 expression. However, all of the isolates in this latter group continued to produce OmpK36, suggesting that porin configuration may affect entry of carbapenems. For isolates that had the greatest expression of ompK36, carbapenem MICs tended to be lower when determined by the broth microdilution technique, and scattered colonies were seen around the Etest zones of inhibition. All of the KPC-producing isolates were highly resistant to ertapenem, regardless of ompK36 expression. In conclusion, isolates of KPC-possessing K. pneumoniae that express ompK36 tend to have lower MICs to carbapenems and therefore may be more difficult to detect by clinical laboratories. Regardless of ompK36 expression, all of the KPC producers were consistently resistant to ertapenem.

Collateral damage of flomoxef therapy: in vivo development of porin deficiency and acquisition of blaDHA-1 leading to ertapenem resistance in a clinical isolate of Klebsiella pneumoniae producing CTX-M-3 and SHV-5 β-lactamases

The study aimed to characterize the genetic basis of flomoxef and collateral ertapenem resistance in a clinical isolate of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) after flomoxef exposure. Four ESBL-KP isolates (Lkp11-14) were recovered sequentially from four episodes of bacteraemia in an elderly patient. Laboratory investigations included genotyping by PFGE, resistance gene analysis by PCR and sequencing, and outer membrane protein analysis by SDS-PAGE. Plasmid analysis by DNA-DNA hybridization, electroporation and conjugation was also performed. Lkp14 was recovered after 21 days of flomoxef therapy. It demonstrated an indistinguishable PFGE pattern when compared with those produced by Lkp11-13. However, resistance to both flomoxef and ertapenem emerged in Lkp14. Molecular characterization revealed that, in addition to the pre-existing ESBL production (CTX-M-3 and SHV-5) and OmpK35 deficiency found in Lkp11-13, Lkp14 had acquired an extra plasmid-mediated AmpC beta-lactamase gene (blaDHA-1) and failed to express OmpK36, because of insertional inactivation by an insertion sequence IS5. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Conjugational transfer of the plasmid-mediated blaDHA-1 gene into Lkp11 resulted in a significant increase in the MICs of cephamycins and beta-lactamase inhibitors, but not in those of carbapenems. Lkp14 was apparently derived from the previously flomoxef-susceptible isolates, Lkp11-13. After flomoxef exposure, the in vivo acquisition of the plasmid-mediated blaDHA-1 gene has led to flomoxef resistance in Lkp14, and the concomitant depletion of OmpK36 expression has resulted in a collateral effect of ertapenem resistance and diminished susceptibilities to imipenem and meropenem.

Characterization of a Large Outbreak by CTX-M-1-Producing Klebsiella pneumoniae and Mechanisms Leading to In Vivo Carbapenem Resistance Development

All extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolates from patients admitted to and adult intensive care unit were prospectively documented from 2002 to 2005, when a large outbreak (51 patients affected) of multiresistant ESBL-producing Klebsiella pneumoniae infection was detected. The involvement of a single K. pneumoniae clone was demonstrated by pulsed-field gel electrophoresis. In addition to the ESBL-mediated resistance, the epidemic strain uniformly showed cross-resistance to ciprofloxacin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, and tetracycline, whereas resistance to the beta-lactam-beta-lactamase inhibitor combinations was variable. The ESBL involved was CTX-M-1, as demonstrated by isoelectric focusing, PCR amplification, and sequencing. CTX-M-1 as well as the aminoglycoside resistance determinants were encoded in a 50-kb plasmid that could be transferred to Escherichia coli only by transformation. In two of the infected patients, carbapenem resistance development (MICs of 8 to 12, 16, and >32 microg/ml for imipenem, meropenem, and ertapenem, respectively) was documented, both in clinical samples and in intestinal colonization studies. The analysis of the outer membrane proteins of the carbapenem-susceptible and -resistant isolates revealed that the former expressed only one of the two major porins, OmpK36, whereas the latter did not express either of them. In one of the cases, the lack of expression of OmpK36 was demonstrated to be mediated by the interruption of the coding sequence by the insertion sequence IS26. This is the first report of a large outbreak of CTX-M-1-producing Enterobacteriaceae and, curiously, the first documented description in the literature of CTX-M-1 in K. pneumoniae, despite the fact that this enzyme has been found in multiple species. Furthermore, we document and characterize for the first time carbapenem resistance development in CTX-M-1-producing Enterobacteriaceae.

Outer membrane protein alterations and blaTEM-1 variants: their role in beta-lactam resistance in Klebsiella pneumoniae.

The aim of the study was to characterize the genetic basis of resistance to selected beta-lactam antibiotics in two clinical isolates of Klebsiella pneumoniae. K. pneumoniae strains were isolated from two hospitalized patients. One of the strains was resistant to amoxicillin, co-amoxiclav, cefuroxime, piperacillin and cefoxitin but susceptible to all the other cephalosporins tested. The second strain displayed a similar phenotype except that it was resistant to piperacillin/tazobactam and susceptible to cefoxitin. PCR assays and DNA sequencing showed that the cefoxitin-susceptible strain contained a novel blaTEM-1 variant downstream of the strong Pa/Pb promoter. SDS-PAGE analysis of the outer membrane proteins (OMPs) did not identify OmpK35 and suggested reduced expression of OmpK36 in this strain. Following passage in non-selective media, expression of OmpK36 was restored with a concomitant increase in cefuroxime susceptibility. A similar experimental approach identified blaTEM-1C in the cefoxitin-resistant K. pneumoniae strain. This strain was deficient in OmpK35 and OmpK36; absence of the latter protein was due to the presence of IS1 in the ompK36 regulatory region. Resistance to selected beta-lactams in two clinical isolates of K. pneumoniae was due to interplay between the expression of OMPs and TEM-1.