Immunostimulatory Effect of Flagellin on MDR-Klebsiella-Infected Human Airway Epithelial Cells.

Pneumonia caused by multi-drug-resistant Klebsiella pneumoniae (MDR-Kpneu) poses a major public health threat, especially to immunocompromised or hospitalized patients. This study aimed to determine the immunostimulatory effect of the Toll-like receptor 5 ligand flagellin on primary human lung epithelial cells during infection with MDR-Kpneu. Human bronchial epithelial (HBE) cells, grown on an air-liquid interface, were inoculated with MDR-Kpneu on the apical side and treated during ongoing infection with antibiotics (meropenem) and/or flagellin on the basolateral and apical side, respectively; the antimicrobial and inflammatory effects of flagellin were determined in the presence or absence of meropenem. In the absence of meropenem, flagellin treatment of MDR-Kpneu-infected HBE cells increased the expression of antibacterial defense genes and the secretion of chemokines; moreover, supernatants of flagellin-exposed HBE cells activated blood neutrophils and monocytes. However, in the presence of meropenem, flagellin did not augment these responses compared to meropenem alone. Flagellin did not impact the outgrowth of MDR-Kpneu. Flagellin enhances antimicrobial gene expression and chemokine release by the MDR-Kpneu-infected primary human bronchial epithelium, which is associated with the release of mediators that activate neutrophils and monocytes. Topical flagellin therapy may have potential to boost immune responses in the lung during pneumonia.

Rapid and Accurate Detection of Aminoglycoside-Modifying Enzymes and 16S rRNA Methyltransferases by Targeted Liquid Chromatography-Tandem Mass Spectrometry.

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in curbing drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside-modifying enzymes and 16S rRNA methyltransferases in Escherichia coli and Klebsiella pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, AAC(6')-Ib-cr, ANT(2″)-I, APH(3')-VI, ArmA, RmtB, RmtC, and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole-genome sequencing results. Of the 2,460 isolate and resistance mechanism combinations tested, 2,416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin, and amikacin in only the E. coli and K. pneumoniae isolates (n = 191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

Therapeutic Efficacy of Novel Antimicrobial Peptide AA139-Nanomedicines in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Antimicrobial peptides (AMPs) have seen limited clinical use as antimicrobial agents, largely due to issues relating to toxicity, short biological half-life, and lack of efficacy against Gram-negative bacteria. However, the development of novel AMP-nanomedicines, i.e., AMPs entrapped in nanoparticles, has the potential to ameliorate these clinical problems. The authors investigated two novel nanomedicines based on AA139, an AMP currently in development for the treatment of multidrug-resistant Gram-negative infections. AA139 was entrapped in polymeric nanoparticles (PNPs) or lipid-core micelles (MCLs). The antimicrobial activity of AA139-PNP and AA139-MCL was determined in vitro The biodistribution and limiting doses of AA139-nanomedicines were determined in uninfected rats via endotracheal aerosolization. The early bacterial killing activity of the AA139-nanomedicines in infected lungs was assessed in a rat model of pneumonia-septicemia caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae In this model, the therapeutic efficacy was determined by once-daily (q24h) administration over 10 days. Both AA139-nanomedicines showed equivalent in vitro antimicrobial activities (similar to free AA139). In uninfected rats, they exhibited longer residence times in the lungs than free AA139 (∼20% longer for AA139-PNP and ∼80% longer for AA139-MCL), as well as reduced toxicity, enabling a higher limiting dose. In rats with pneumonia-septicemia, both AA139-nanomedicines showed significantly improved therapeutic efficacy in terms of an extended rat survival time, although survival of all rats was not achieved. These results demonstrate potential advantages that can be achieved using AMP-nanomedicines. AA139-PNP and AA139-MCL may be promising novel therapeutic agents for the treatment of patients suffering from multidrug-resistant Gram-negative pneumonia-septicemia.

Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. pneumon iae and P. aeruginosa.

SET-M33 is a multimeric antimicrobial peptide active against Gram-negative bacteria in vitro and in vivo. Insights into its killing mechanism could elucidate correlations with selectivity. SET-M33 showed concentration-dependent bactericidal activity against colistin-susceptible and resistant isolates of P. aeruginosa and K. pneumoniae. Scanning and transmission microscopy studies showed that SET-M33 generated cell blisters, blebs, membrane stacks and deep craters in K. pneumoniae and P. aeruginosa cells. NMR analysis and CD spectra in the presence of sodium dodecyl sulfate micelles showed a transition from an unstructured state to a stable α-helix, driving the peptide to arrange itself on the surface of micelles. SET-M33 kills Gram-negative bacteria after an initial interaction with bacterial LPS. The molecule becomes then embedded in the outer membrane surface, thereby impairing cell function. This activity of SET-M33, in contrast to other similar antimicrobial peptides such as colistin, does not generate resistant mutants after 24h of exposure, non-specific interactions or toxicity against eukaryotic cell membranes, suggesting that SET-M33 is a promising new option for the treatment of Gram-negative antibiotic-resistant infections.

Acquisition of Carbapenem Resistance by Plasmid-Encoded-AmpC-Expressing Escherichia coli.

Although AmpC ß-lactamases can barely degrade carbapenems, if at all, they can sequester them and prevent them from reaching their targets. Thus, carbapenem resistance in Escherichia coli and other Enterobacteriaceae can result from AmpC production and simultaneous reduction of antibiotic influx into the periplasm by mutations in the porin genes. Here we investigated the route and genetic mechanisms of acquisition of carbapenem resistance in a clinical E. coli isolate carrying blaCMY-2 on a plasmid by selecting for mutants that are resistant to increasing concentrations of meropenem. In the first step, the expression of OmpC, the only porin produced in the strain under laboratory conditions, was lost, leading to reduced susceptibility to meropenem. In the second step, the expression of the CMY-2 ß-lactamase was upregulated, leading to resistance to meropenem. The loss of OmpC was due to the insertion of an IS1 element into the ompC gene or to frameshift mutations and premature stop codons in this gene. The blaCMY-2 gene was found to be located on an IncIγ plasmid, and overproduction of the CMY-2 enzyme resulted from an increased plasmid copy number due to a nucleotide substitution in the inc gene. The clinical relevance of these genetic mechanisms became evident from the analysis of previously isolated carbapenem-resistant clinical isolates, which appeared to carry similar mutations.

Risk factors for resistance to ciprofloxacin in community-acquired urinary tract infections due to Escherichia coli in an elderly population.

BACKGROUND: Antimicrobial resistance to ciprofloxacin is rising worldwide, especially in bacteria causing urinary tract infections (UTIs). Prudent use of current antibiotic drugs is therefore necessary. OBJECTIVES: We analysed (modifiable) risk factors for ciprofloxacin-resistant Escherichia coli. METHODS: Urinary cultures of UTIs caused by E. coli were collected from participants in the Rotterdam Study, a prospective cohort study in an elderly population, and analysed for susceptibility to ciprofloxacin. Multivariate logistic regression was performed to investigate several possible risk factors for resistance. RESULTS: Ciprofloxacin resistance in 1080 E. coli isolates was 10.2%. Multivariate analysis showed that higher age (OR 1.03; 95% CI 1.00-1.05) and use of two (OR 5.89; 95% CI 3.45-10.03) and three or more (OR 3.38; 95% CI 1.92-5.97) prescriptions of fluoroquinolones were associated with ciprofloxacin resistance, while no association between fluoroquinolone use more than 1 year before culture and ciprofloxacin resistance could be demonstrated. Furthermore, a high intake of pork (OR 3.68; 95% CI 1.36-9.99) and chicken (OR 2.72; 95% CI 1.08-6.85) and concomitant prescription of calcium supplements (OR 2.51; 95% CI 1.20-5.22) and proton pump inhibitors (OR 2.04; 95% CI 1.18-3.51) were associated with ciprofloxacin resistance. CONCLUSIONS: Ciprofloxacin resistance in community-acquired UTI was associated with a high intake of pork and chicken and with concomitant prescription of calcium supplements and proton pump inhibitors. Modification of antibiotic use in animals as well as temporarily stopping the prescription of concomitant calcium and proton pump inhibitors need further evaluation as strategies to prevent ciprofloxacin resistance.

Environmental Spread of New Delhi Metallo-ß-Lactamase-1-Producing Multidrug-Resistant Bacteria in Dhaka, Bangladesh.

Resistance to carbapenem antibiotics through the production of New Delhi metallo-ß-lactamase-1 (NDM-1) constitutes an emerging challenge in the treatment of bacterial infections. To monitor the possible source of the spread of these organisms in Dhaka, Bangladesh, we conducted a comparative analysis of wastewater samples from hospital-adjacent areas (HAR) and from community areas (COM), as well as public tap water samples, for the occurrence and characteristics of NDM-1-producing bacteria. Of 72 HAR samples tested, 51 (71%) samples were positive for NDM-1-producing bacteria, as evidenced by phenotypic tests and the presence of the blaNDM-1 gene, compared to 5 of 41 (12.1%) samples from COM samples (P < 0.001). All tap water samples were negative for NDM-1-producing bacteria. Klebsiella pneumoniae (44%) was the predominant bacterial species among blaNDM-1-positive isolates, followed by Escherichia coli (29%), Acinetobacter spp. (15%), and Enterobacter spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes, including blaCTX-M-1 (80%), blaCTX-M-15 (63%), blaTEM (76%), blaSHV (33%), blaCMY-2 (16%), blaOXA-48-like (2%), blaOXA-1 (53%), and blaOXA-47-like (60%) genes. Around 40% of the isolates contained a qnr gene, while 50% had 16S rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30 to 50 MDa carrying blaNDM-1 were self-transmissible. Our results highlight a number of issues related to the characteristics and source of spread of multidrug-resistant bacteria as a potential public health threat. In view of the existing practice of discharging untreated liquid waste into the environment, hospitals in Dhaka city contribute to the potential dissemination of NDM-1-producing bacteria into the community.IMPORTANCE Infections caused by carbapenemase-producing Enterobacteriaceae are extremely difficult to manage due to their marked resistance to a wide range of antibiotics. NDM-1 is the most recently described carbapenemase, and the blaNDM-1 gene, which encodes NDM-1, is located on self-transmissible plasmids that also carry a considerable number of other antibiotic resistance genes. The present study shows a high prevalence of NDM-1-producing organisms in the wastewater samples from hospital-adjacent areas as a potential source for the spread of these organisms to community areas in Dhaka, Bangladesh. The study also examines the characteristics of the isolates and their potential to horizontally transmit the resistance determinants. The significance of our research is in identifying the mode of spread of multiple-antibiotic-resistant organisms, which will allow the development of containment measures, leading to broader impacts in reducing their spread to the community.

A Polymorphism in leuS Confers Reduced Susceptibility to GSK2251052 in a Clinical Isolate of Staphylococcus aureus.

GSK2251052 is a broad-spectrum antibacterial inhibitor of leucyl tRNA-synthetase (LeuRS) that has been evaluated in phase II clinical trials. Here, we report the identification of a clinical isolate of Staphylococcus aureus that exhibits reduced susceptibility to GSK2251052 without prior exposure to the compound and demonstrate that this phenotype is attributable to a single amino acid polymorphism (P329) within the editing domain of LeuRS.

Evaluation of different pretreatment protocols to detect accurately clinical carbapenemase-producing Enterobacteriaceae by MALDI-TOF.

OBJECTIVES: Carbapenemase-resistant bacteria are increasingly spreading worldwide causing public concern due to their ability to elude antimicrobial treatment. Early identification of these bacteria is therefore of high importance. Here, we describe the development of a simple and robust protocol for the detection of carbapenemase activity in clinical isolates of Enterobacteriaceae, suitable for routine and clinical applications. METHODS: The final protocol involves cellular lysis and enzyme extraction from a defined amount of bacterial cells followed by the addition of a benchmark drug (e.g. the carbapenem antibiotic imipenem or ertapenem). Carbapenem inactivation is mediated by enzymatic hydrolysis (cleavage) of the ß-lactam common structural motif, which can be detected using MALDI-TOF MS. RESULTS: A total of 260 strains were studied (208 carbapenemase producers and 52 non-carbapenemase producers) resulting in 100% sensitivity and 100% specificity for the KPC, NDM and OXA-48-like PCR-confirmed positive isolates using imipenem as benchmark. Differences between the benchmark (indicator) antibiotics imipenem and ertapenem, buffer constituents and sample preparation methods have been investigated. Carbapenemase activity was further characterized by performing specific inhibitor experiments. Intraday and interday reproducibility (coefficient of variation) of the observed hydrolysis results were 15% and 30%, respectively. A comparative study of our extraction method and a recently published method using whole bacterial cells is presented and differences are discussed. CONCLUSIONS: Using this method, an existing carbapenemase activity can be directly read from the mass spectrum as a ratio of hydrolysed product and substrate, setting an important step towards routine application in clinical laboratories.

Characterisation of clinical Staphylococcus aureus isolates harbouring mecA or Panton-Valentine leukocidin genes from four tertiary care hospitals in Indonesia.

OBJECTIVES: To determine the prevalence, antimicrobial susceptibility profiles and clonal distribution of either methicillin-resistant Staphylococcus aureus (MRSA) or Panton-Valentine leukocidin (PVL)-positive S. aureus obtained from clinical cultures in Indonesian hospitals. METHODS: S. aureus isolates from clinical cultures of patients in four tertiary care hospitals in Denpasar, Malang, Padang and Semarang were included. We assessed the antimicrobial susceptibility profiles using the Vitek2(®) system, determined the presence of the mecA gene and genes encoding PVL using PCR and analysed the clonal relatedness with Raman spectroscopy. SCCmec typing was performed for all MRSA isolates. Multilocus sequence typing (MLST) was performed for a subset of isolates. RESULTS: In total, 259 S. aureus strains were collected. Of these, 17/259 (6.6%) and 48/259 (18.5%) were MRSA and PVL-positive methicillin-susceptible S. aureus (MSSA), respectively. The prevalence of MRSA and PVL-positive MSSA ranged between 2.5-8.9% and 9.5-29.1%, respectively and depended on geographic origin. PVL-positive MRSA were not detected. Raman spectroscopy of the strains revealed multiple Raman types with two predominant clusters. We also showed possible transmission of a ST239-MRSA-SCCmec type III strain and a ST121 PVL-positive MSSA in one of the hospitals. CONCLUSIONS: We showed that MRSA and PVL-positive MSSA are of clinical importance in Indonesian hospitals. A national surveillance system should be set-up to further monitor this. To reduce the prevalence of MRSA in Indonesian hospitals, a bundle of intervention measures is highly recommended.

Salmonella subtypes with increased MICs for azithromycin in travelers returned to The Netherlands.

Antimicrobial susceptibility was analyzed for 354 typhoidal Salmonella isolates collected during 1999-2012 in the Netherlands. In 16.1% of all isolates and in 23.8% of all isolates that showed increased MICs for ciprofloxacin, the MIC for azithromycin was increased. This resistance may complicate empirical treatment of enteric fever.

The therapeutic effect of tigecycline, unlike that of Ceftazidime, is not influenced by whether the Klebsiella pneumoniae strain produces extended-spectrum ß-lactamases in experimental pneumonia in rats.

The efficacies of tigecycline and ceftazidime against fatal pneumonia in rats caused by an extended-spectrum ß-lactamase (ESBL)-positive Klebsiella pneumoniae strain or its wild-type (WT) progenitor were compared. Ceftazidime at 12.5 or 50 mg/kg of body weight twice daily (b.i.d.) was effective (50% or 100% rat survival) in pneumonia caused by the WT isolate but unsuccessful (100% rat mortality) in pneumonia caused by the ESBL-positive variant. In contrast, tigecycline at 6.25, 12.5, or 25 mg/kg b.i.d. showed dosage-dependent efficacy up to 100% rat survival irrespective of the ESBL character of the infecting organism.

Antibiotic trapping by plasmid-encoded CMY-2 ß-lactamase combined with reduced outer membrane permeability as a mechanism of carbapenem resistance in Escherichia coli.

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed bla(CTX-M-15) and bla(OXA-1). The CR E. coli strain additionally harbored bla(CMY-2) and demonstrated a >15-fold increase in ß-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated ß-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the ß-lactamase. This study, therefore, provides evidence that the mechanism of "trapping" by CMY-2 ß-lactamase plays a role in carbapenem resistance.

Colonization dynamics of antibiotic-resistant coagulase-negative Staphylococci in neonates.

Coagulase-negative staphylococci (CoNS) isolated in neonatal late-onset sepsis are often antibiotic resistant. We analyzed CoNS from skin and feces of neonates during hospitalization. Antibiotic resistance of skin isolates increased during hospitalization, especially in Staphylococcus haemolyticus. Staphylococcus warneri showed low antibiotic resistance. Our data suggest that different CoNS species may play distinct roles in colonization.

Characterization of ß-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry.

Plasmid-encoded ß-lactamases are a major reason for antibiotic resistance in gram negative bacteria. These enzymes hydrolyze the ß-lactam ring structure of certain ß-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of ß-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine ß-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the ß-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible ß-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of ß-lactamase activity in various bacterial strains by mass spectrometry.

Faecal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae among humans in Java, Indonesia, in 2001-2002.

OBJECTIVE: To characterise commensal Escherichia coli and other Enterobacteriaceae with reduced susceptibility to cefotaxime that were collected in a large survey carried out among 3995 patients and healthy persons in two urban regions on Java, Indonesia, in 2001-2002. METHODS: The putative extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae were analysed using double-disk synergy tests, isoelectric focusing, PCR assays, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). RESULTS: On the day of discharge after five or more days of hospitalisation, at least 95 of 999 (9.5%) patients carried ESBL-positive Enterobacteriaceae as dominant faecal flora. Six patients were simultaneously colonised with E. coli and Klebsiella pneumoniae isolates with ESBL activity. On admission, only 6 of 998 (0.6%) patients were colonised. Faecal carriage of ESBL-producing Enterobacteriaceae among healthy persons or persons visiting a public health centre was not detected. The 107 ESBL-positive strains included 68 E. coli, 35 K. pneumoniae, and four other Enterobacteriaceae. bla(CTX-M-15) was the most prevalent ESBL in both E. coli (47.1%) and K. pneumoniae (45.7%), but the E. coli O25b-ST131 clone was virtually absent. Other ESBL types found were: SHV-2, -2a, -5, -12, CTX-M-3, -9, -14, and TEM-19. PFGE revealed extensive genetic diversity among the isolates. CONCLUSIONS: In 2001-2002, faecal carriage of ESBL-producing Enterobacteriaceae as dominant flora in Indonesia was almost exclusively hospital-associated. The presence of various bla(ESBL) genes and the extensive genetic diversity among isolates argue against a single/dominant strain outbreak.

High Prevalence of ST502 Carrying an OXA-24 Carbapenemase gene in Carbapenem-Nonsusceptible Acinetobacter baumannii-calcoaceticus Isolates in Romania.

Background: Acinetobacter baumannii can cause difficult-to-treat infections because it can acquire extensive antimicrobial resistance mechanisms. We aim to describe the antimicrobial resistance pattern and the genetic basis of carbapenem-nonsusceptible A. baumannii isolates in a University Hospital in Romania, a country where multidrug-resistant A. baumannii is widespread. Methods: We collected 104 consecutive meropenem-nonsusceptible A. baumannii isolates from 104 patients (36% female, mean age [SD] of 63 [16] years) between May 2015 and August 2017 from a large tertiary center in Romania. Whole-genome sequencing of representative isolates from amplified fragment length polymorphism clusters was used to determine clonality and resistance patterns. Results: All isolates were resistant to piperacillin/tazobactam, ceftazidime, and ciprofloxacin; 88.5% to gentamicin; and 90.4% to trimethoprim/sulfamethoxazole. In contrast, 79.8% and 99.0% were susceptible to tobramycin and colistin, respectively. The only isolate resistant to colistin had an minimum inhibitory concentration (MIC) of ≥16 mg/L. The blaOXA-24 gene was detected in 79.1% and blaOXA-23 in 20.9% of the isolates. In one isolate, blaOXA-23 was copresent with blaOXA-24. ST502 (Oxford scheme) was the most prevalent sequence type and was exclusively associated with blaOXA-24. Conclusions: ST502 associated with blaOXA-24 was frequently observed in the region where carbapenem-nonsusceptible A. baumannii was found to be endemic. In these isolates, tobramycin and colistin might be the remaining therapeutic options. Due to differences in gentamicin and tobramycin resistance in these isolates, surveillance data should not group gentamicin, tobramycin, and amikacin together as aminoglycosides.

Using Targeted Liquid Chromatography-Tandem Mass Spectrometry to Rapidly Detect ß-Lactam, Aminoglycoside, and Fluoroquinolone Resistance Mechanisms in Blood Cultures Growing E. coli or K. pneumoniae.

New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the detection of ß-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing Escherichia coli or Klebsiella pneumoniae complex. Selected targets were the ß-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, ANT(2 ' ' )-I, and APH(3')-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6')-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the E. coli porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing E. coli or K. pneumoniae complex.

Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 ß-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem.

While Extended-Spectrum ß-Lactamases (ESBL) and AmpC ß-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding proteins, thereby inhibiting their activity. Further, it has been shown that Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC ß-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the Escherichia coli porins OmpC and OmpF, its chromosomal AmpC ß-lactamase, and the plasmid-mediated CMY-2 ß-lactamase. Bla CMY-2-like positive E. coli isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like ß-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant E. coli.

Clinical and molecular characteristics of extended-spectrum-beta-lactamase-producing Escherichia coli causing bacteremia in the Rotterdam Area, Netherlands.

We investigated the clinical and molecular characteristics of bacteremia caused by extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli over a 2-year period (2008 to 2009) in the Rotterdam region (including 1 teaching hospital and 2 community hospitals) of Netherlands. The majority of patients presented with community onset urinary and intra-abdominal infections, with an increase in prevalence during 2009. The majority of E. coli isolates produced CTX-M-15, and 4 sequence types (ST38, ST131, ST405, and ST648) predominated. There were significant differences in clinical and molecular characteristics between the 2 community hospitals.