Outbreak of Vancomycin-resistant Enterococcus faecium in Interventional Radiology: Detection Through Whole-genome Sequencing-based Surveillance.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are a major cause of hospital-acquired infections. The risk of infection from interventional radiology (IR) procedures is not well documented. Whole-genome sequencing (WGS) surveillance of clinical bacterial isolates among hospitalized patients can identify previously unrecognized outbreaks. METHODS: We analyzed WGS surveillance data from November 2016 to November 2017 for evidence of VRE transmission. A previously unrecognized cluster of 10 genetically related VRE (Enterococcus faecium) infections was discovered. Electronic health record review identified IR procedures as a potential source. An outbreak investigation was conducted. RESULTS: Of the 10 outbreak patients, 9 had undergone an IR procedure with intravenous (IV) contrast ≤22 days before infection. In a matched case-control study, preceding IR procedure and IR procedure with contrast were associated with VRE infection (matched odds ratio [MOR], 16.72; 95% confidence interval [CI], 2.01 to 138.73; P = .009 and MOR, 39.35; 95% CI, 7.85 to infinity; P < .001, respectively). Investigation of IR practices and review of the manufacturer's training video revealed sterility breaches in contrast preparation. Our investigation also supported possible transmission from an IR technician. Infection prevention interventions were implemented, and no further IR-associated VRE transmissions have been observed. CONCLUSIONS: A prolonged outbreak of VRE infections related to IR procedures with IV contrast resulted from nonsterile preparation of injectable contrast. The fact that our VRE outbreak was discovered through WGS surveillance and the manufacturer's training video that demonstrated nonsterile technique raise the possibility that infections following invasive IR procedures may be more common than previously recognized.

Use of online tools for antimicrobial resistance prediction by whole-genome sequencing in methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).

OBJECTIVES: The antimicrobial resistance (AMR) crisis represents a serious threat to public health and has resulted in concentrated efforts to accelerate development of rapid molecular diagnostics for AMR. In combination with publicly available web-based AMR databases, whole-genome sequencing (WGS) offers the capacity for rapid detection of AMR genes. Here we studied the concordance between WGS-based resistance prediction and phenotypic susceptibility test results for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) clinical isolates using publicly available tools and databases. METHODS: Clinical isolates prospectively collected at the University of Pittsburgh Medical Center between December 2016 and December 2017 underwent WGS. The AMR gene content was assessed from assembled genomes by BLASTn search of online databases. Concordance between the WGS-predicted resistance profile and phenotypic susceptibility as well as the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for each antibiotic/organism combination, using the phenotypic results as gold standard. RESULTS: Phenotypic susceptibility testing and WGS results were available for 1242 isolate/antibiotic combinations. Overall concordance was 99.3%, with a sensitivity, specificity, PPV and NPV of 98.7% (95% CI 97.2-99.5%), 99.6% (95% CI 98.8-99.9%), 99.3% (95% CI 98.0-99.8%) and 99.2% (95% CI 98.3-99.7%), respectively. Additional identification of point mutations in housekeeping genes increased the concordance to 99.4%, sensitivity to 99.3% (95% CI 98.2-99.8%) and NPV to 99.4% (95% CI 98.4-99.8%). CONCLUSION: WGS can be used as a reliable predicator of phenotypic resistance both for MRSA and VRE using readily available online tools.

Phylogenomics of colistin-susceptible and resistant XDR Acinetobacter baumannii.

Background: Acinetobacter baumannii is a healthcare-associated pathogen with high rates of carbapenem resistance. Colistin is now routinely used for treatment of infections by this pathogen. However, colistin use has been associated with development of resistance to this agent. Objectives: To elucidate the phylogenomics of colistin-susceptible and -resistant A. baumannii strain pairs from a cohort of hospitalized patients at a tertiary medical centre in the USA. Methods: WGS data from 21 pairs of colistin-susceptible and -resistant, XDR clinical strains were obtained and compared using phylogeny of aligned genome sequences, assessment of pairwise SNP differences and gene content. Results: Fourteen patients had colistin-resistant strains that were highly genetically related to their own original susceptible strain with a median pairwise SNP distance of 5.5 (range 1-40 SNPs), while seven other strain pairs were divergent with ≥84 SNP differences. In addition, several strains from different patients formed distinct clusters on the phylogeny in keeping with closely linked transmission chains. The majority of colistin-resistant strains contained non-synonymous mutations within the pmrAB locus suggesting a central role for pmrAB mutations in colistin resistance. Excellent genotype-phenotype correlation was also observed for carbapenems, aminoglycosides and tetracyclines. Conclusions: The findings suggest that colistin resistance in the clinical setting arises through both in vivo evolution from colistin-susceptible strains and reinfection by unrelated colistin-resistant strains, the latter of which may involve patient-to-patient transmission.

Origin of the plasmid-mediated fosfomycin resistance gene fosA3.

Background: fosA3 is the most commonly reported plasmid-mediated fosfomycin resistance gene among Enterobacteriaceae. Objectives: To identify the origin of fosA3. Methods: The chromosome of Kluyvera georgiana clinical strain YDC799 was fully sequenced with single-molecule real-time sequencing. Comparative genetic analysis was performed for K. georgiana YDC799, K. georgiana type strain ATCC 51603 and representative fosA3-carrying plasmids. fosA genes were cloned in Escherichia coli to confirm function. Results: K. georgiana YDC799 harboured fosA (designated fosAKG) and blaCTX-M-8 on the chromosome. The genetic environments surrounding fosA3 and bounded by IS26 were nearly identical with the corresponding regions of K. georgiana YDC799 and ATCC 51603. The amino acid sequence of FosAKG from YDC799 and K. georgiana ATCC 51603 shared 99% and 94% identity with FosA3, respectively. Cloned FosAKG conferred fosfomycin resistance with an MIC of >1024 mg/L for E. coli. Conclusions: The plasmid-mediated fosA3 gene was likely mobilized from the chromosome of K. georgiana by an IS26-mediated event.

Frequency and Mechanisms of Spontaneous Fosfomycin Nonsusceptibility Observed upon Disk Diffusion Testing of Escherichia coli.

Fosfomycin maintains activity against most Escherichia coli clinical isolates, but the growth of E. coli colonies within the zone of inhibition around the fosfomycin disk is occasionally observed upon susceptibility testing. We aimed to estimate the frequency of such nonsusceptible inner colony mutants and identify the underlying resistance mechanisms. Disk diffusion testing of fosfomycin was performed on 649 multidrug-resistant E. coli clinical isolates collected between 2011 and 2015. For those producing inner colonies inside the susceptible range, the parental strains and their representative inner colony mutants were subjected to MIC testing, whole-genome sequencing, reverse transcription-quantitative PCR (qRT-PCR), and carbohydrate utilization studies. Of the 649 E. coli clinical isolates, 5 (0.8%) consistently produced nonsusceptible inner colonies. Whole-genome sequencing revealed the deletion of uhpT encoding hexose-6-phosphate antiporter in 4 of the E. coli inner colony mutants, while the remaining mutant contained a nonsense mutation in uhpA The expression of uhpT was absent in the mutant strains with uhpT deletion and was not inducible in the strain with the uhpA mutation, unlike in its parental strain. All 5 inner colony mutants had reduced growth on minimal medium supplemented with glucose-6-phosphate. In conclusion, fosfomycin-nonsusceptible inner colony mutants can occur due to the loss of function or induction of UhpT but are rare among multidrug-resistant E. coli clinical strains. Considering that these mutants carry high biological costs, we suggest that fosfomycin susceptibility of strains that generate inner colony mutants can be interpreted on the basis of the zone of inhibition without accounting for the inner colonies.

Structural modification of LPS in colistin-resistant, KPC-producing Klebsiella pneumoniae.

BACKGROUND: Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited. OBJECTIVES: To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains. METHODS: The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS. RESULTS: All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype. CONCLUSIONS: The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings.

Chromosomal 16S Ribosomal RNA Methyltransferase RmtE1 in Escherichia coli Sequence Type 448.

We identified rmtE1, an uncommon 16S ribosomal methyltransferase gene, in an aminoglycoside- and cephalosporin-resistant Escherichia coli sequence type 448 clinical strain co-harboring blaCMY-2. Long-read sequencing revealed insertion of a 101,257-bp fragment carrying both resistance genes to the chromosome. Our findings underscore E. coli sequence type 448 as a potential high-risk multidrug-resistant clone.

Draft Genome Sequences of Four Hospital-Associated Pseudomonas putida Isolates.

We present here the draft genome sequences of four Pseudomonas putida isolates belonging to a single clone suspected for nosocomial transmission between patients and a bronchoscope in a tertiary hospital. The four genome sequences belong to a single lineage but contain differences in their mobile genetic elements.