What Contributes to the MIC? Beyond ß-Lactamase Gene Detection in Klebsiella pneumoniae.

BACKGROUND: K. pneumoniae is capable of resistance to ß-lactam antibiotics through expression of ß-lactamases (both chromosomal and plasmid-encoded) and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. The purpose of this study was to determine the extent to which ß-lactamases and outer membrane porins affected ß-lactam resistance. METHODS: MICs to ß-lactams and inhibitor combinations were determined by agar dilution or E-test. Outer membrane porin production was evaluated by western blot of outer membrane fractions. ß-lactamase carriage was determined by whole genome sequencing and expression evaluated by RT-qPCR. RESULTS: Plasmid-encoded ß--lactamases were important for cefotaxime and ceftazidime resistance. Elevated expression of chromosomal SHV was important for ceftolozane/tazobactam resistance. Loss of outer membrane porins was predictive of meropenem resistance. ESßLs and pAmpCs in addition to porin loss were sufficient to confer resistance to the third generation cephalosporins, pipercillin/tazobactam, ceftolozane/tazobactam, and meropenem. pAmpCs (CMY-2 and DHA) alone conferred resistance to pipercillin/tazobactam. DISCUSSION: Detection of a resistance gene by whole genome sequencing was not sufficient to predict resistance to all antibiotics tested. some ß-lactam resistance was dependent on the expression of both plasmid-encoded and chromosomal ß-lactamases and loss of porins.

Draft Genome Sequences of the Clinical Isolates Kp 23 and KPM 20.

Klebsiella pneumoniae strains are capable of becoming resistant through multiple mechanisms. Here, we announce draft sequences for Kp 23, a clinical isolate with no plasmid-encoded ß-lactamases, and KPM 20, a clinical isolate with no plasmid-encoded ß-lactamases and no detectable OmpK35, OmpK36, or PhoE in the outer membrane.

OmpC regulation differs between ST131 and non-ST131 Escherichia coli clinical isolates and involves differential expression of the small RNA MicC.

BACKGROUND: Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq. Modifications in the translatability of porin proteins could impact the fitness and therefore the success of E. coli ST131 isolates in the presence of antibiotic. OBJECTIVES: To identify differences in the translatability of OmpC and OmpF porins for different STs of E. coli by comparing steady-state RNA levels, mRNA half-life, regulatory sRNA expression and protein production. METHODS: RNA expression was evaluated using real-time RT-PCR and OmpC mRNA half-life by northern blotting. OmpC, OmpF and Hfq protein levels were evaluated by immunoblotting. RESULTS: Differences between ST131 and non-ST131 isolates included: (i) the level of OmpC RNA and protein produced with mRNA expression higher for ST131 but OmpC protein levels lower compared with non-ST131 isolates; (ii) OmpC mRNA half-life (21-30 min for ST131 isolates compared with <2-23 min for non-ST131 isolates); and (iii) levels of the sRNA MicC (2- to 120-fold for ST131 isolates compared with -4- to 70-fold for non-ST131 isolates). CONCLUSIONS: Mechanisms involved in the translatability of porin proteins differed among different STs of E. coli. These differences could provide a selective advantage to ST131 E. coli when confronted with an antibiotic-rich environment.

A Standard Numbering Scheme for Class C ß-Lactamases.

Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) ß-lactamases, which complicates communication in the field. Here, we propose a scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser64, Lys67, Tyr150, and Lys315), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.

lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa.

PURPOSE: In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34. METHODS: PA34 was exposed to subinhibitory concentrations of doripenem or meropenem during growth to mid-log phase. Antibiotic susceptibility of surviving colonies was determined by agar dilution. Two carbapenem-resistant colonies hypersusceptible to non-carbapenem antibiotics were selected for further analysis. Antibiotic resistance gene expression was evaluated by RT-rtPCR and OprD production by SDS-PAGE. PA34 and isogenic mutants were evaluated with whole genome sequencing. Sequence variants were confirmed by Sanger sequencing, and cognate genes in eight carbapenem-resistant clinical isolates hypersusceptible to non-carbapenem antibiotics were sequenced. Lipopolysaccharide preparations of PA34 and hypersusceptible mutants were evaluated with ProQ-Emerald stain. RESULTS: Isogenic mutants showed 4- to 8-fold MIC increase for imipenem, meropenem, and doripenem. However, they were hypersusceptible (≥4-fold MIC decrease) to aminoglycosides, fluoroquinolones, and non-carbapenem ß-lactams. Expression of ampC or mex-opr efflux pumps was unchanged, but OprD production was decreased. Mutations causing Q86H AlgU and G77C LptG amino acid substitutions and nonsense mutations within OprD were observed in both mutants. Lipopolysaccharide modifications were observed between isogenic mutants and PA34. Non-synonymous mutations in LptF or LptG were observed in 6/8 hypersusceptible clinical isolates resistant to carbapenem antibiotics. CONCLUSION: Evaluation of hypersusceptible mutants identified the association between lptG and a hypersusceptible phenotype. Modifications in lipopolysaccharide profiles suggests LptG modification interferes with lipopolysaccharide transport and contributes to hypersusceptibility.

Draft Genome Assemblies of Clinical Isolates of Klebsiella pneumoniae V9011662 and Enterobacter hormaechei Entb306.

Enterobacter hormaechei and Klebsiella pneumoniae are pathogenic Enterobacteriaceae that have been associated with the spread of antibiotic resistance. Here, we report draft genome assemblies of an Enterobacter hormaechei clinical isolate and a multidrug-resistant clinical isolate of Klebsiella pneumoniae.

Draft Genome Sequence of the Mucoid Pseudomonas aeruginosa Clinical Isolate PA34.

Pseudomonas aeruginosa is a serious threat to patients suffering from cystic fibrosis. These organisms are exposed to a unique set of selective pressures within the lung. Here, we report the draft genome sequence of a mucoid P. aeruginosa clinical isolate obtained from a cystic fibrosis patient colonized with P. aeruginosa.

Impact of CLSI and EUCAST Cefepime breakpoint changes on the susceptibility reporting for Enterobacteriaceae.

OBJECTIVE: We analyzed the effects of different cefepime MIC breakpoints on Enterobacteriaceae cefepime susceptibility and the presence of AmpC and extended-spectrum ß-lactamase (ESBL) genes within the cefepime MIC interpretative categories. METHODS: Using Enterobacteriaceae susceptibility data from 2013 comparisons of MIC breakpoints were performed using Pearson's chi-squared test. Molecular testing on a subset of isolates was done. RESULTS: Among 3784 non-duplicate clinical isolates, cefepime susceptibility decreased from 97.6% to 96.1% to 93.7% for CLSI 2013, CLSI 2014, and EUCAST 2011, respectively. In ceftriaxone non-susceptible isolates, cefepime susceptibility decreased from 79% to 66% (P<0.0001) using CLSI 2013 and 2014, respectively, which was greater and statistically significant for Escherichia coli and Klebsiella spp. but not for Enterobacter spp. (P=0.06). Isolates with MIC ≤1µg/mL more often harbored AmpC (77%) than ESBL (18%) genes. CONCLUSIONS: Lower cefepime MIC breakpoints decrease cefepime susceptibility for isolates harboring ESBLs, while sparing the majority of those with AmpCs.

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and ß-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.

IMP-27, a Unique Metallo-ß-Lactamase Identified in Geographically Distinct Isolates of Proteus mirabilis.

A novel metallo-ß-lactamase gene, blaIMP-27, was identified in unrelated Proteus mirabilis isolates from two geographically distinct locations in the United States. Both isolates harbor blaIMP-27 as part of the first gene cassette in a class 2 integron. Antimicrobial susceptibility testing indicated susceptibility to aztreonam, piperacillin-tazobactam, and ceftazidime but resistance to ertapenem. However, hydrolysis assays indicated that ceftazidime was a substrate for IMP-27.

Structural and Mutagenic Analysis of Metallo-ß-Lactamase IMP-18.

IMP-type metallo-ß-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of ß-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned from Pseudomonas aeruginosa, at 2.0-Å resolution. The overall structure of IMP-18 resembles that of IMP-1, with an αß/ßα "folded sandwich" configuration, but the loop that covers the active site has a distinct conformation. The relationship between IMP-18's loop conformation and its kinetic properties was investigated by replacing the amino acid residues that can affect the loop conformation (Lys44, Thr50, and Ile69) in IMP-18 with those occupying the corresponding positions in the well-described enzyme IMP-1. The replacement of Thr50 with Pro considerably modified IMP-18's kinetic properties, specifically those pertaining to meropenem, with the kcat/Km value increased by an order of magnitude. The results indicate that this is a key residue that defines the kinetic properties of IMP-type ß-lactamases.

Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs of Escherichia coli.

OBJECTIVES: High levels of ß-lactamase production can impact treatment with a ß-lactam/ß-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam. METHODS: Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT-PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives. RESULTS: mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2-48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5-15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2-3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm. CONCLUSIONS: CTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels.

The OpdQ porin of Pseudomonas aeruginosa is regulated by environmental signals associated with cystic fibrosis including nitrate-induced regulation involving the NarXL two-component system.

Pseudomonas aeruginosa is a versatile opportunistic pathogen that causes chronic infections in immunocompromised hosts. Multiple porins modulate outer membrane permeability under various environmental conditions. The lung environment of cystic fibrosis (CF) patients is unique with changes occurring in nutrient availability, osmolarity, and oxygen content. Although P. aeruginosa gene expression is modified under these conditions, little is known about how they influence porin regulation. In this study, we evaluated the regulation of the outer membrane porin OpdQ, a member of the OprD family of porins, with regard to oxygen, nitrate, and/or NaCl levels. We demonstrated using promoter::fusion clones of P. aeruginosa PAO1 and clinical strains collected from CF patients that OpdQ was transcriptionally repressed under low oxygen but increased in the presence of nitrate. The nitrate-induced regulation of OpdQ was found to be dependent on the transcription factor NarL via the NarXL two-component system. In addition, NaCl-induced osmotic stress increased OpdQ production among most of the clinical strains evaluated. In conclusion, these data identify for the first time that specific environmental cues associated with the CF microenvironment influence porin regulation, and that the nitrate-induced regulation of OpdQ is associated with nitrate metabolism via the NarXL two-component system of P. aeruginosa.

Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay.

Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; blaCTX-M, 98.9%; blaKPC, 100%; blaNDM, 96.2%; blaOXA, 94.3%; blaVIM, 100%; and blaIMP, 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths. The sample-to-result processing and automated reading of the detection microarray results enables results within 2 h of culture positivity.

Whole genome mapping of the first reported case of KPC-2-positive Klebsiella pneumoniae ST258 in Nebraska.

Three ertapenem-resistant Klebsiella pneumoniae carrying bla(KPC-2) were isolated from a single patient in Nebraska over a span of 5 months. A comparative analysis of the genetic relatedness of these isolates was investigated using pulsed-field gel electrophoresis, multilocus sequence typing, and whole genome mapping.

Emergence of carbapenem resistance due to the novel insertion sequence ISPa8 in Pseudomonas aeruginosa.

Chronic lung infections due to the persistence of Pseudomonas aeruginosa in cystic fibrosis patients are typically associated with the emergence of antibiotic resistance. The purpose of this study was to investigate the mechanisms responsible for the emergence of carbapenem resistance when a clinical isolate of P. aeruginosa collected from a patient with cystic fibrosis was challenged with meropenem. Nine carbapenem-resistant mutants were selected with subinhibitory concentrations of meropenem from a clinical isolate of P. aeruginosa and characterized for carbapenem resistance. Increased carbapenem MICs were associated with the identification of the novel insertion sequence ISPa8 within oprD or its promoter region in all the mutants. The position of ISPa8 was different for each of the mutants evaluated. In addition, Southern blot analyses identified multiple copies of ISPa8 within the genomes of the mutants and their parent isolate. These data demonstrate that transposition of IS elements within the Pseudomonas genome can influence antibiotic susceptibility. Understanding the selective pressures associated with the emergence of antibiotic resistance is critical for the judicious use of antimicrobial chemotherapy and the successful treatment of bacterial infections.

Multiplex high-resolution melting analysis as a diagnostic tool for detection of plasmid-mediated AmpC ß-lactamase genes.

High-resolution melting (HRM) analysis can be a diagnostic tool to evaluate the presence of resistance genes with the added bonus of discriminating sequence modifications. A real-time, multiplex PCR assay using HRM was designed for the detection of plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 96% and 100%, respectively.

Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.

Class B ß-lactamases are known as metallo-ß-lactamases (MBLs) and they hydrolyze most ß-lactams, including carbapenems. IMP-18, an MBL cloned from Pseudomonas aeruginosa, was overexpressed, purified and crystallized by vapour diffusion for X-ray crystallographic analysis. Preliminary X-ray analysis showed that the crystal diffracted to 2.4 Å resolution and belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 120.77, c = 96.54 Å, α = ß = γ = 90°, suggesting the presence of two molecules in the asymmetric unit.

Rapid PCR amplification protocols decrease the turn-around time for detection of antibiotic resistance genes in Gram-negative pathogens.

A previously designed end-point multiplex PCR assay and singleplex assays used to detect ß-lactamase genes were evaluated using rapid PCR amplification methodology. Amplification times were 16-18 minutes with an overall detection time of 1.5 hours. Rapid PCR amplifications could decrease the time required to identify resistance mechanisms in Gram-negative organisms.