•Carbapenem-non-susceptible P. aeruginosa (CNPA) were found to be endemic in a large referral hospital in Indonesia.•Acquisition of CNPA was associated with significantly longer ICU length of ...stay.•Carriage of CNPA may affect ICU survival.•Carbapenem-non-susceptibility among P. aeruginosa is largely due to IMP, VIM and GES-5 carbapenemases.•Most CNPA isolates belonged to the international clones ST235, ST357, ST446 and ST823.
The aim of this study was to describe the epidemiology and clinical impact of carbapenem-non-susceptible Pseudomonas aeruginosa (CNPA) in intensive care units (ICUs) of the national referral hospital of Indonesia. Adult patients admitted to ICUs were prospectively included. Pseudomonas aeruginosa were from clinical cultures and systematic screening. Environmental niches and healthcare workers (HCWs) were also screened. Susceptibility was determined phenotypically and the presence of carbapenemase genes was determined by PCR. Multiple loci variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) were used for genotyping. Of the patients included in the study, 17/412 (4.1%) carried CNPA on admission and 34/395 (8.6%) became positive during their ICU stay. The acquisition rate was 18/1000 patient-days at risk. Of 16 environmental isolates, 12 (75.0%) were CNPA. HCWs screened negative. Acquisition of CNPA was associated with longer ICU stay (adjusted hazard ratio = 1.89, 99% confidence interval 1.12–3.13). Mortality was >40% among patients with CNPA versus <30% among those without CNPA (P = 0.019). Moreover, 83/119 (69.7%) CNPA carried either blaVIM (n = 36), blaIMP (n = 23) or blaGES-5 (n = 24). Four sequence types (STs) dominated (ST235, ST823, ST446 and ST357). Five major MLVA clusters were distinguished, two belonging to ST235 and the other three to ST823, ST446 and ST357. CNPA are introduced into these ICUs and some strains expand clonally among patients and the environment, creating endemic CNPA. VIM-, IMP- and GES-5 genes are prevalent. CNPA acquisition was associated with prolonged ICU stay and may affect ICU survival.
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.
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.
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.
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.
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
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
porins OmpC and OmpF, its chromosomal AmpC β-lactamase, and the plasmid-mediated CMY-2 β-lactamase.
positive
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
.
A prospective observational study was performed to assess the epidemiology and clinical impact of carbapenem-non-susceptible Klebsiella pneumoniae (CNKP) in intensive care units (ICUs) of the ...national referral hospital in Jakarta, Indonesia.
Adult patients consecutively hospitalized for > 48 h in two ICUs of the national referral hospital were included from April until October 2013 and from April until August 2014. K. pneumoniae from clinical cultures and standardized screening of rectum and throat on admission, discharge and weekly if hospitalized > 7 days were collected. Environmental niches and healthcare workers (HCWs) were also screened. Susceptibility was determined phenotypically and the presence of carbapenemase genes by PCR. Raman spectroscopy as well as multiple-locus variable number tandem repeat analysis (MLVA) were used for typing.
Twenty-two out of 412 (5.3%) patients carried CNKP on admission and 37/390 (9.5%) acquired CNKP during ICU stay. The acquisition rate was 24.7/1000 patient-days at risk. One out of 31 (3.2%) environmental isolates was a CNKP. None of the HCWs carried CNKP. Acquisition of CNKP was associated with longer ICU stay (adjusted Hazard Ratio: 2.32 CI
: 1.35-3.68). ICU survival was lower among patients with CNKP compared to patients with carbapenem-susceptible K. pneumoniae (aHR 2.57, p = 0.005). Ninety-six of the 100 (96%) CNKP isolates carried a carbapenemase gene, predominantly bla
. Raman typing revealed three major clusters among 48 Raman types identified, whereas MLVA distinguished six major clusters among a total of 30 different genotypes.
NDM-producing CNKP are introduced into these ICUs and some strains expand clonally among patients and the environment, resulting in endemic CNKP. CNKP acquisition was associated with prolonged ICU stay and may affect ICU survival.
The study was registered at Netherlands Trial Register http://www.trialregister.nl. Candidate number: 23527, NTR number: NTR5541, NL number: NL5425 (https://www.trialregister.nl/trial/5424), Retrospectively registered: NTR: 22 December 2015.
Carbapenem-nonsusceptible
complex have emerged worldwide, but the epidemiology in Indonesian hospitals has not been studied.
A prospective observational study was performed on the intensive care ...units (ICUs) of the national referral hospital in Jakarta-Indonesia, in 2013 and 2014. All consecutive adult patients admitted and hospitalized for >48 h in ICUs were included. Basic and clinical data at admission were recorded. Carbapenem-nonsusceptible
complex from clinical cultures and standardized screening were included. Environmental niches and healthcare workers (HCWs) were also screened. PCR was used to detect carbapenemase genes, and Raman spectroscopy as well as multilocus sequence typing (MLST) for typing.
Of 412 included patients, 69 (16.7%) carried carbapenem-nonsusceptible
complex on admission, and 89 (25.9%) became positive during ICU stay. The acquisition rate was 43 per 1000 patient-days at risk. Six isolates were cultured from environment and one from a HCW. Acquisition of carbapenem-nonsusceptible
complex was associated with longer ICU stay (median interquartile range IQR: 11 days 5-18, adjusted hazard ratio aHR: 2.56 99% confidence interval (CI):1.76-3.70), but not with mortality (adjusted odds ratio: 1.59 99%CI: 0.74-3.40 at the chosen level of significance). The
-like gene was detected in 292/318 (91.8%) isolates, including isolates from the environment and HCW. Typing revealed five major clusters. Sequence types (ST)195, ST208, ST218, ST642 as well as new STs were found. The dominant clone consisted of isolates from patients and environment throughout the study period.
Carbapenem-nonsusceptible
complex are endemic in this setting. Prevention requires source control and limiting transmission of strains.
The study was retrospectively registered at www.trialregister.nl (No:5541). Candidate number: 23,527, NTR number: NTR5541, Date registered NTR: 22nd December 2015.
Infections with multidrug-resistant (MDR) microorganisms are an increasing threat to hospitalized patients. Although rapid typing of MDR microorganisms is required to apply targeted prevention ...measures, technical barriers often prevent this. We aimed to assess whether extended-spectrum beta-lactamase (ESBL)-producing Klebsiella species are transmitted between patients and whether routine, rapid typing is needed.
For 43 months, the clonality of all ESBL-producing Klebsiella isolates from patients admitted to Erasmus MC University Medical Center in Rotterdam, the Netherlands was assessed with Raman spectroscopy. A cluster was defined as n ≥ 2 patients who had identical isolates. Primary patients were the first patients in each cluster. Secondary patients were those identified with an isolate clonally related to the isolate of the primary patient.
Isolates from 132 patients were analyzed. We identified 17 clusters, with 17 primary and 56 secondary patients. Fifty-nine patients had a unique isolate. Patients (n = 15) in four out of the 17 clusters were epidemiologically related. Ten of these 15 patients developed an infection.
Clonal outbreaks of ESBL-producing Klebsiella species were detected in our hospital. Theoretically, after Raman spectroscopy had detected a cluster of n ≥ 2, six infections in secondary patients could have been prevented. These findings demonstrate that spread of ESBL-producing Klebsiella species occurs, even in a non-outbreak setting, and underscore the need for routine rapid typing of these MDR bacteria.
•Susceptibility to AA139 and SET-M33 did not differ among clinically relevant Klebsiella pneumoniae isolates.•AA139 and SET-M33 showed a bactericidal effect irrespective of colistin ...susceptibility.•Exposure to low concentrations of colistin resulted in development of colistin resistance.•Susceptibility to AA139 or SET-M33 was maintained after exposure to the respective antimicrobial peptides.
Colistin is an antimicrobial peptide (AMP) used as a drug of last resort, although plasmid-mediated colistin resistance (MCR) has been reported. AA139 and SET-M33 are novel AMPs currently in development for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. As many AMPs have a similar mode of action to colistin, potentially leading to cross-resistance, the antimicrobial activity of AA139 and SET-M33 was investigated against a collection of 50 clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles, including colistin-resistant strains. The collection was genotypically characterised and susceptibility to clinically relevant antibiotics was determined. Susceptibility to AA139 and SET-M33 did not differ among the collection despite differences in underlying mechanisms of resistance or susceptibility to colistin. For three colistin-susceptible and three colistin-resistant strains with distinct MDR profiles as well as an additional MCR-producing strain, the bactericidal activity of AA139, SET-M33 and colistin during 24 h of exposure was examined. Following 24 h of exposure to AA139, SET-M33 or colistin, the seven strains were tested for changes in susceptibility to the respective AMPs. AA139 and SET-M33 showed a concentration-dependent bactericidal effect irrespective of bacterial susceptibility to colistin. Exposure to low colistin concentrations resulted in the development of colistin resistance in colistin-susceptible strains, whereas susceptibility to AA139 and SET-M33 following exposure to the respective AMPs was maintained. The two novel AMPs remained effective against colistin-resistant strains and may be promising novel drugs for the treatment of clinically and genotypically diverse MDR K. pneumoniae infections, including infections associated with colistin-resistant bacteria.
Respiratory tract infections are one of the most frequent infections worldwide, with an increasing number being associated with (multiple) antibiotic-resistant pathogens. Improved treatment requires ...the development of new therapeutic strategies, including the possible development of antibiotic-nanomedicines. Antibiotic-nanomedicines comprise antibiotic molecules coupled to nanocarriers via surface adsorption, surface attachment, entrapment or conjugation and can be administered via aerosolization. The efficacy and tolerability of this approach has been shown in clinical studies, with amikacin liposome inhalation suspension being the first inhalatory antibiotic-nanomedicine approved by the US FDA. In this special report, we summarize and discuss the potential value and the clinical status of antibiotic-nanomedicines for the treatment of (antibiotic-resistant) respiratory tract infections.
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.
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 β-lactam common structural motif, which can be detected using MALDI-TOF MS.
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.
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.
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.
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•SET-M33, an antimicrobial branched peptide, is active against colistin-resistant Gram negative bacteria.•It adopts a α-helix conformation in a membrane mimicking environment.•It shows little toxicity against eukaryotic cells and acts without selection of resistant clones within 24h.
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