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.
Although AmpC β-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 bla
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 β-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 bla
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.
Since the year 2000 there has been a sharp increase in the prevalence of healthcare-related infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. However, the high ...community prevalence of ESBL-producing E. coli isolates means that many E. coli typing techniques may not be suitable for detecting E. coli transmission events. Therefore, we investigated if High-throughput MultiLocus Sequence Typing (HiMLST) and/or Raman spectroscopy were suitable techniques for detecting recent E. coli transmission events.
This study was conducted from January until December 2010 at Erasmus University Medical Center, Rotterdam, the Netherlands. Isolates were typed using HiMLST and Raman spectroscopy. A genetic cluster was defined as two or more patients carrying identical isolates. We used predefined definitions for epidemiological relatedness to assess healthcare-related transmission.
We included 194 patients; strains of 112 patients were typed using HiMLST and strains of 194 patients were typed using Raman spectroscopy. Raman spectroscopy identified 16 clusters while HiMLST identified 10 clusters. However, no healthcare-related transmission events were detected. When combining data from both typing techniques, we identified eight clusters (n = 34 patients), as well as 78 patients with a non-cluster isolate. However, we could not detect any healthcare-related transmission in these 8 clusters.
Although clusters were genetically detected using HiMLST and Raman spectroscopy, no definite epidemiological relationships could be demonstrated which makes the possibility of healthcare-related transmission events highly unlikely. Our results suggest that typing of ESBL-producing E. coli using HiMLST and/or Raman spectroscopy is not helpful in detecting E. coli healthcare-related transmission events.
Pneumonia caused by multi-drug-resistant
(MDR-
) 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-
. Human bronchial epithelial (HBE) cells, grown on an air-liquid interface, were inoculated with MDR-
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-
-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-
. Flagellin enhances antimicrobial gene expression and chemokine release by the MDR-
-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.
Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in neonatal intensive care units (NICU) worldwide. Infecting strains of these commensal bacteria may originate from NICU personnel. ...Therefore, we studied the characteristics of CoNS isolates from NICU personnel and compared them to those of isolates from the general population and from sepsis patients. Furthermore, we studied the epidemiological effect on CoNS carriage of NICU personnel after a period of absence. In our study, we isolated CoNS from the thumbs of NICU personnel every 2 weeks during the summer of 2005 and sampled personnel returning from vacation and a control group from the general population. Furthermore, we collected sepsis isolates from this period. Isolates were tested for antibiotic resistance, mecA and icaA carriage, biofilm production, and genetic relatedness. We found that mecA and icaA carriage as well as penicillin, oxacillin, and gentamicin resistance were significantly more prevalent in CoNS strains from NICU personnel than in community isolates. Similar trends were observed when postvacation strains were compared to prevacation strains. Furthermore, genetic analysis showed that 90% of the blood isolates were closely related to strains found on the hands of NICU personnel. Our findings revealed that CoNS carried by NICU personnel differ from those in the general population. Hospital strains are replaced by community CoNS after a period of absence. NICU personnel are a likely cause for the cross-contamination of virulent CoNS that originate from the NICU to patients.
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.
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 (
= 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.
Resistance to carbapenem antibiotics through the production of New Delhi metallo-β-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
gene, compared to 5 of 41 (12.1%) samples from COM samples (
< 0.001). All tap water samples were negative for NDM-1-producing bacteria.
(44%) was the predominant bacterial species among
-positive isolates, followed by
(29%),
spp. (15%), and
spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes, including
(80%),
(63%),
(76%),
(33%),
(16%),
(2%),
(53%), and
(60%) genes. Around 40% of the isolates contained a
gene, while 50% had 16S rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30 to 50 MDa carrying
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.
Infections caused by carbapenemase-producing
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
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.
The emergence of carbapenem-resistant
represents a worldwide problem. To understand the carbapenem-resistance mechanisms and their spreading among
strains, whole genome sequences were determined of ...two extensively drug-resistant strains that are endemic in Dutch hospitals. Strain Carb01 63 is of O-antigen serotype O12 and of sequence type ST111, whilst S04 90 is a serotype O11 strain of ST446. Both strains carry a gene for metallo-β-lactamase VIM-2 flanked by two
genes encoding aminoglycoside acetyltransferases on a class 1 integron. The integron is located on the chromosome in strain Carb01 63 and on a plasmid in strain S04 90. The backbone of the 159-kb plasmid, designated pS04 90, is similar to a previously described plasmid, pND6-2, from
. Analysis of the context of the integron showed that it is present in both strains on a ∼30-kb mosaic DNA segment composed of four different transposons that can presumably act together as a novel, active, composite transposon. Apart from the presence of a 1237-bp insertion sequence element in the composite transposon on pS04 90, these transposons show > 99% sequence identity indicating that transposition between plasmid and chromosome could have occurred only very recently. The pS04 90 plasmid could be transferred by conjugation to a susceptible
strain. A second class 1 integron containing a gene for a CARB-2 β-lactamase flanked by an
and an
gene, encoding an aminoglycoside acetyltransferase and adenylyltransferase, respectively, was present only in strain Carb01 63. This integron is located also on a composite transposon that is inserted in an integrative and conjugative element on the chromosome. Additionally, this strain contains a frameshift mutation in the
gene encoding a porin involved in the transport of carbapenems across the outer membrane. Together, the results demonstrate that integron-encoded carbapenem and carbapenicillin resistance can easily be disseminated by transposition and conjugation among
strains.
PDF
