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

An outbreak of ST307 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in a rehabilitation center: An unusual source and route of transmission

Objective: Nosocomial outbreaks due to multidrug-resistant microorganisms in rehabilitation centers have rarely been reported. We report an outbreak of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) on a single ward in a rehabilitation center in Rotterdam, The Netherlands.Design: Outbreak description.Setting: A 40-bed ward of a rehabilitation center in the Netherlands.Methods: In October 2016, 2 patients were found to be colonized by genetically indistinguishable ESBL-K. pneumoniae isolates. Therefore, an outbreak management team was installed, by whom a contact tracing plan was made. In addition to general outbreak measures, specific measures were formulated to allow continuation of the rehabilitation process. Also, environmental cultures were taken. Multiple-locus variable-number tandem-repeat analysis and amplification fragment-length polymorphism were used to determine strain relatedness. Selected isolates were subjected to whole-genome multilocus sequence typing.Results: The outbreak lasted 8 weeks. In total, 14 patients were colonized with an ESBL-K. pneumoniae, of whom 11 patients had an isolate belonging to

Improving hand hygiene compliance in nursing homes: Protocol for a cluster randomized controlled trial (HANDSOME Study)

Background: Hand hygiene compliance is considered the most (cost-)effective measure for preventing health care-associated infections. While hand hygiene interventions have frequently been implemented and assessed in hospitals, there is limited knowledge about hand hygiene compliance in other health care settings and which interventions and implementation methods are effective. Objective: This study aims to evaluate the effect of a multimodal intervention to increase hand hygiene compliance of nurses in nursing homes through a cluster randomized controlled trial (HANDSOME study). Methods: Nursing homes were randomly allocated to 1 of 3 trial arms: Receiving the intervention at a predetermined date, receiving the identical intervention after an infectious disease outbreak, or serving as a control arm. Hand hygiene was evaluated in nursing homes by direct observation at 4 timepoints. We documented compliance with the World Health Organization's 5 moments of hand hygiene, specifically before touching a patient, before a clean/aseptic procedure, after body fluid exposure risk, after touching a patient, and after touching patient surroundings. The primary outcome is hand hygiene compliance of the nurses to the standards of the World Health Organization. The secondary outcome is infectious disease incidence among residents. Infectious disease incidence was documented by a staff member at each nursing home unit. Outcomes will be compared with the presence of norovirus, rhinovirus, and Escherichia coli on surfaces in the nursing homes, as measured using quantitative polymerase chain reaction. Results: The study was funded in September 2015. Data collection started in October 2016 and was completed in October 2017. Data analysis will be completed in 2020. Conclusions: HANDSOME studies the effectiveness of a hand hygiene intervention specifically for the nursing hom

Prevalence and risk factors for carriage of ESBL-producing Enterobacteriaceae in a population of Dutch travellers: A cross-sectional study

Background: We investigated prevalence and predictive factors for ESBL-E carriage in a population of mostly travellers prior to their travel (n = 2216). In addition, we examined ESBL genotype before travel and compared these to returning travellers. Method: A questionnaire and faecal sample were collected before travel, and a second faecal sample was collected immediately after travel. Faecal samples were analysed for ESBL-E, with genotypic characterization by PCR and sequencing. Risk factors for ESBL-E carriage prior to travel were identified by logistic regression analyses. Results: Before travel, 136 participants (6.1%) were colonized with ESBL-E. Antibiotic use in the past three months (ORadjusted 2.57; 95% CI 1.59–4.16) and travel outside of Europe in the past year (1.92, 1.28–2.87) were risk factors for ESBL-E colonisation prior to travel. Travel outside of Europe carried the largest attributable risk (39.8%). Prior to travel 31.3% (40/128) of participants carried blaCTX-M 15 and 21.9% (28/128) blaCTX-M 14/18. In returning travellers 633 acquired ESBL-E of who 53.4% (338/633) acquired blaCTX-M 15 and 17.7% (112/633) blaCTX-M 14/18. Conclusion: In our population of Dutch travellers we found a pre-travel ESBL-E prevalence of 6.1%. Prior to travel, previous antibiotic use and travel outside of Europe were the strongest independent predictors

Investigation of possible transmission of a susceptible microorganism through a contaminated duodenoscope

Background: Despite compliance to extensive reprocessing protocols, duodenoscopes have been linked to outbreaks of susceptible and multi-drug resistant organisms (MDRO) due to persistent duodenoscope contamination. Duodenoscope-associated infections (DAIs) based on transmission of susceptible microorganisms are likely to be underreported due to detection bias. Case presentation: We describe the retrospective detection of a DAI case caused by a susceptible microorganism which at the time of clinical infection was not recognized as such. During 2017 and 2018, duodenoscopes were cultured on a daily basis due to research activities. While analyzing this data, it was found that a duodenoscope had been contaminated with Enterobacter cloacae complex over a period of 3 months. We checked whether patients treated wit

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics

Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography–mass spectrometry (LC–MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC–MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC–MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC–MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.</p

Genetic Analysis of mcr-1-Carrying Plasmids From Gram-Negative Bacteria in a Dutch Tertiary Care Hospital

The role of plasmids in the complex pandemic of antimicrobial resistance is increasingly being recognized. In this respect, multiple mobile colistin resistance (mcr) gene-carrying plasmids have been described. However, the characteristics and epidemiology of these plasmids within local healthcare settings are largely unknown. We retrospectively characterized the genetic composition and epidemiology of plasmids from mcr-1-positive bacterial isolates identified from patients from a large academic hospital in the Netherlands. Clinical Gram-negative bacteria with an MIC &gt; 2 μg/mL for colistin, obtained from patients hospitalized at the Erasmus MC University Medical Center Rotterdam during the years 2010–2018, were screened for presence of the mcr-1 gene. Extracted plasmids from mcr-1-positive isolates were sequenced using a combination of short- and long-read sequencing platforms, characterized by incompatibility type and genetic composition and compared to publicly available mcr-1-carrying plasmid sequences. In 21 isolates from 14 patients, mcr-1 was located on a plasmid. These plasmids were of diverse genetic background involving Inc types IncX4, IncI2(delta), IncHI2, as well as double Inc types IncHI2/IncN and IncHI2/IncQ. mcr-1-carrying plasmids were found in Escherichia coli, Klebsiella pneumoniae, and Kluyvera georgiana, and within the chromosome of an ST147 K. pneumoniae isolate. In depth analysis indicated intrapatient, interpatient, and interspecies transmission events of mcr-1-carrying plasmids. In addition, our results show that the mcr-1 gene resides in a rich environment full of other (mcr-1 negative) plasmids and of many different Inc types, enabling interplasmidal transfer events and facilitating widespread dissemination of the mcr-1 gene. Multiple mcr-1-carrying plasmid transmission events had likely occurred among isolates from hospitalized patients. Recognition and identification of plasmid transmission events within hospitals is necessary in order to design and implement effective infection control measures.</p