Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

Background: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. Methods: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. Results: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. Conclusion: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis

Whole-genome sequencing to explore nosocomial transmission and virulence in neonatal methicillin-susceptible Staphylococcus aureus bacteremia

BACKGROUND: Neonatal Staphylococcus aureus (S. aureus) bacteremia is an important cause of morbidity and mortality. In this study, we examined whether methicillin-susceptible S. aureus (MSSA) transmission and genetic makeup contribute to the occurrence of neonatal S. aureus bacteremia. METHODS: A retrospective, single-centre study was performed. All patients were included who suffered from S. aureus bacteremia in the neonatal intensive care unit (NICU), Erasmus MC-Sophia, Rotterdam, the Netherlands, between January 2011 and November 2017. Whole-genome sequencing (WGS) was used to characterize the S. aureus isolates, as was also done in comparison to reference genomes. Transmission was considered likely in case of genetically indistinguishable S. aureus isolates. RESULTS: Excluding coagulase-negative staphylococci (CoNS), S. aureus was the most common cause of neonatal bacteremia. Twelve percent (n = 112) of all 926 positive blood cultures from neonates grew S. aureus. Based on core genome multilocus sequence typing (cgMLST), 12 clusters of genetically indistinguishable MSSA isolates were found, containing 33 isolates in total (2-4 isolates per cluster). In seven of these clusters, at least two of the identified MSSA isolates were collected within a time period of one month. Six virulence genes were present in 98-100% of all MSSA isolates. In comparison to S. aureus reference genomes, toxin genes encoding staphylococcal enterotoxin A (sea) and toxic shock syndrome toxin 1 (tsst-1) were present more often in the genomes of bacteremia isolates. CONCLUSION: Transmission of MSSA is a contributing factor to the occurrence of S. aureus bacteremia in neonates. Sea and tsst-1 might play a role in neonatal S. aureus bacteremia

A multi-center blinded study on the efficiency of phenotypic screening methods to detect glycopeptide intermediately susceptible Staphylococcus aureus (GISA) and heterogeneous GISA (h-GISA)

Contains fulltext : 52338.pdf (publisher's version ) (Open Access)BACKGROUNDS: To determine the true incidence of hGISA/GISA and its consequent clinical impact, methods must be defined that will reliably and reproducibly discriminate these resistant phenotypes from vancomycin susceptible S. aureus (VSSA). METHODS: This study assessed and compared the ability of eight Dutch laboratories under blinded conditions to discriminate VSSA from hGISA/GISA phenotypes and the intra- and inter-laboratory reproducibility of agar screening plates and the Etest method. A total of 25 blinded and unique strains (10 VSSA, 9 hGISA and 6 GISA) were categorized by the PAP-AUC method and PFGE typed to eliminate clonal duplication. All strains were deliberately added in quadruplets to evaluate intra-laboratory variability and reproducibility of the methods. Strains were tested using three agar screening methods, Brain Heart Infusion agar (BHI) + 6 microg/ml vancomycin, Mueller Hinton agar (MH) + 5 microg/ml vancomycin and MH + 5 microg/ml teicoplanin) and the Etest macromethod using a 2 McFarland inoculum. RESULTS AND DISCUSSION: The ability to detect the hGISA/GISA phenotypes varied significantly between methods and phenotypes. BHI vancomycin and MH vancomycin agar screens lacked the ability to detect hGISA. The MH teicoplanin agar screen was more sensitive but still inferior to Etest that had a sensitivity of 98.5% and 99.5%, for hGISA and GISA, respectively. Intra- and inter-laboratory reproducibility varied between methods with poorest performance seen with BHI vancomycin. CONCLUSION: This is the first multi-center blinded study to be undertaken evaluating various methods to detect GISA and hGISA. These data showed that the ability of clinical laboratories to detect GISA and hGISA varied considerably, and that screening plates with vancomycin have a poor performance in detecting hGISA

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

While Extended-Spectrum β-Lactamases (ESBL) and AmpC β-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 β-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 β-lactamase, and the plasmid-mediated CMY-2 β-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 β-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

Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. pneumoniae 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

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

Effect of Dosing and Dosing Frequency on the Efficacy of Ceftizoxime and the Emergence of Ceftizoxime Resistance during the Early Development of Murine Abscesses Caused by Bacteroides fragilis and Enterobacter cloacae Mixed Infection▿

The efficacy of β-lactams is thought to be dependent on the time that the unbound concentrations exceed the MIC (fT>MIC). However, the pharmacokinetic/pharmacodynamic index (PDI) that correlates best to the selection of resistance is not yet clear. The selection of ceftizoxime (CZX)-resistant Enterobacter cloacae mutant strains during the development of murine mixed-infection abscesses was studied to determine the PDI that is important for the emergence of resistance and the PDI value needed for the prevention of resistance. Studies were carried out 24 h after inoculation with Bacteroides fragilis ATCC 23745 and E. cloacae 22491. Six to 1,536 mg of CZX/kg of body weight/day given every 2 h (q2h), q4h, q6h, or q8h was started 30 min before inoculation and continued for 24 h. Resistant mutants were isolated to determine mutant frequencies (MF). The fT>MIC varied from 9 to 98% for E. cloacae, the peak concentration (unbound fraction) was 0.6 to 578 mg/liter, and the area under the concentration-time curve (unbound fraction) (fAUC) was 1.9 to 553 mg·h/liter. The fAUC-to-MIC ratio best explained the in vivo efficacy. CZX-resistant B. fragilis and E. cloacae mutants were isolated from untreated controls at an MF of 10−5 to 10−7. The MF of resistant B. fragilis did not increase during therapy. The selection of resistant E. cloacae strains at an MF of 10−1 to 10−2 was related to the fT>MIC and the ratio of fAUC to MIC following an inverse U shape. However, the ratio of fAUC to MIC was the stronger driver of resistance. The highest MFs were 0.7 to 0.9 at an fAUC-to-MIC ratio of approximately 250. We conclude that the ratio of fAUC to MIC is the PDI that correlated best to the in vivo efficacy of CZX and probably also to the emergence of resistant E. cloacae mutants. An fAUC-to-MIC ratio of 1,000 was needed to prevent the emergence of this resistance

Rapid Detection of Methicillin Resistance in Staphylococcus aureus Isolates by the MRSA-Screen Latex Agglutination Test

The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR (“gold standard”) for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant S. aureus (MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSA strains displayed positive results only after induction of the mecA gene by exposure to methicillin. Both mecA PCR and MRSA-Screen displayed negative results among the methicillin-susceptible S. aureus strains (n = 106), as well as for Micrococcus spp. (n = 10), members of the family Enterobacteriaceae (n = 10), Streptococcus pneumoniae (n = 10), and Enterococcus spp. (n = 10) (specificity = 100%). Producing the same PBP2a antigen, all 10 methicillin-resistant Staphylococcus epidermidis strains score positived in both the latex test and the mecA PCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain to the species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity and sensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in the microbiology laboratory

Effect of Treatment Duration on Pharmacokinetic/Pharmacodynamic Indices Correlating with Therapeutic Efficacy of Ceftazidime in Experimental Klebsiella pneumoniae Lung Infection

The pharmacokinetic/pharmacodynamic (PK/PD) indices that define the therapeutic effect of the beta-lactam ceftazidime in a rat model of Klebsiella pneumoniae lung infection were investigated in relation to treatment duration and treatment endpoint. Treatment was started 24 h after infection with dosing regimens of 3.1 up to 1,600 mg/kg of body weight/day and dosing every 6, 12, or 24 h. When animals were treated for a relatively short period of 48 h, the duration of time that unbound plasma ceftazidime levels exceeded the MIC of the antibiotic for the infecting strain was the index that best correlated with therapeutic efficacy in terms of significant bacterial killing in the infected lung (microbiological effect). The maximum effect was reached when plasma ceftazidime levels were above the MIC for 60 to 70% of the dosing interval. However, when the treatment duration was extended to a relatively long period of 18 days instead of 48 h and animal survival rate instead of microbiological efficacy was taken as the endpoint, the fAUC/MIC ratio (where AUC is the area under the concentration-time curve) was the PK/PD index that best correlated with therapeutic efficacy. The PK/PD indices that effect 50% survival of rats for the fAUC/MIC ratios were 18.0 (95% confidence interval [95% CI], 16.3 to 19.9), 20.2 (95% CI, 13.8 to 29.4), and 27.9 (95% CI, 21.3 to 36.5) for the schedules of administration of every 6, 12, and 24 h, respectively. The fAUC/MIC needed for 100% survival was >100. We conclude that the PK/PD index that best correlates with outcome is dependent on the duration of treatment and/or the parameter of outcome. The effect of long-term treatment should be studied more extensively in other models of infection