Staphylococcus epidermidis joint isolates: Whole-genome sequencing demonstrates evidence of hospital transmission and common antimicrobial resistance.

OBJECTIVE: We investigated genetic, epidemiologic, and environmental factors contributing to positive Staphylococcus epidermidis joint cultures. DESIGN: Retrospective cohort study with whole-genome sequencing (WGS). PATIENTS: We identified S. epidermidis isolates from hip or knee cultures in patients with 1 or more prior corresponding intra-articular procedure at our hospital. METHODS: WGS and single-nucleotide polymorphism-based clonality analyses were performed, including species identification, in silico multilocus sequence typing (MLST), phylogenomic analysis, and genotypic assessment of the prevalence of specific antibiotic resistance and virulence genes. Epidemiologic review was performed to compare cluster and noncluster cases. RESULTS: In total, 60 phenotypically distinct S. epidermidis isolates were identified. After removal of duplicates and impure samples, 48 isolates were used for the phylogenomic analysis, and 45 (93.7%) isolates were included in the clonality analysis. Notably, 5 S. epidermidis strains (10.4%) showed phenotypic susceptibility to oxacillin yet harbored mecA, and 3 (6.2%) strains showed phenotypic resistance despite not having mecA. Smr was found in all isolates, and mupA positivity was not observed. We also identified 6 clonal clusters from the clonality analysis, which accounted for 14 (31.1%) of the 45 S. epidermidis isolates. Our epidemiologic investigation revealed ties to common aspirations or operative procedures, although no specific common source was identified. CONCLUSIONS: Most S. epidermidis isolates from clinical joint samples are diverse in origin, but we identified an important subset of 31.1% that belonged to subclinical healthcare-associated clusters. Clusters appeared to resolve spontaneously over time, suggesting the benefit of routine hospital infection control and disinfection practices.

Bayesian modeling of the impact of antibiotic resistance on the efficiency of MRSA decolonization.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality. Colonization by MRSA increases the risk of infection and transmission, underscoring the importance of decolonization efforts. However, success of these decolonization protocols varies, raising the possibility that some MRSA strains may be more persistent than others. Here, we studied how the persistence of MRSA colonization correlates with genomic presence of antibiotic resistance genes. Our analysis using a Bayesian mixed effects survival model found that genetic determinants of high-level resistance to mupirocin was strongly associated with failure of the decolonization protocol. However, we did not see a similar effect with genetic resistance to chlorhexidine or other antibiotics. Including strain-specific random effects improved the predictive performance, indicating that some strain characteristics other than resistance also contributed to persistence. Study subject-specific random effects did not improve the model. Our results highlight the need to consider the properties of the colonizing MRSA strain when deciding which treatments to include in the decolonization protocol.

Post-discharge decolonization of patients harboring methicillin-resistant Staphylococcus aureus (MRSA) USA300 strains: secondary analysis of the CLEAR Trial.

The CLEAR Trial recently found that decolonization reduced infections and hospitalizations in MRSA carriers in the year following hospital discharge. In this secondary analysis, we explored whether decolonization had a similar benefit in the subgroup of trial participants who harbored USA300, using two different definitions for the USA300 strain-type.

Epidemiology and genomics of a slow outbreak of methicillin-resistant Staphyloccus aureus (MRSA) in a neonatal intensive care unit: Successful chronic decolonization of MRSA-positive healthcare personnel.

OBJECTIVE: To describe the genomic analysis and epidemiologic response related to a slow and prolonged methicillin-resistant Staphylococcus aureus (MRSA) outbreak. DESIGN: Prospective observational study. SETTING: Neonatal intensive care unit (NICU). METHODS: We conducted an epidemiologic investigation of a NICU MRSA outbreak involving serial baby and staff screening to identify opportunities for decolonization. Whole-genome sequencing was performed on MRSA isolates. RESULTS: A NICU with excellent hand hygiene compliance and longstanding minimal healthcare-associated infections experienced an MRSA outbreak involving 15 babies and 6 healthcare personnel (HCP). In total, 12 cases occurred slowly over a 1-year period (mean, 30.7 days apart) followed by 3 additional cases 7 months later. Multiple progressive infection prevention interventions were implemented, including contact precautions and cohorting of MRSA-positive babies, hand hygiene observers, enhanced environmental cleaning, screening of babies and staff, and decolonization of carriers. Only decolonization of HCP found to be persistent carriers of MRSA was successful in stopping transmission and ending the outbreak. Genomic analyses identified bidirectional transmission between babies and HCP during the outbreak. CONCLUSIONS: In comparison to fast outbreaks, outbreaks that are "slow and sustained" may be more common to units with strong existing infection prevention practices such that a series of breaches have to align to result in a case. We identified a slow outbreak that persisted among staff and babies and was only stopped by identifying and decolonizing persistent MRSA carriage among staff. A repeated decolonization regimen was successful in allowing previously persistent carriers to safely continue work duties.

Erratum: Whole-genome sequencing to assess clonality in a series of prosthetic joint Staphylococcus epidermidis isolates - WITHDRAWAL.

[This corrects the article DOI: 10.1017/ash.2022.160.].

Modelling methicillin-resistant Staphylococcus aureus decolonization: interactions between body sites and the impact of site-specific clearance.

Methicillin-resistant Staphylococcus aureus (MRSA) can colonize multiple body sites, and carriage is a risk factor for infection. Successful decolonization protocols reduce disease incidence; however, multiple protocols exist, comprising diverse therapies targeting multiple body sites, and the optimal protocol is unclear. Standard methods cannot infer the impact of site-specific components on successful decolonization. Here, we formulate a Bayesian coupled hidden Markov model, which estimates interactions between body sites, quantifies the contribution of each therapy to successful decolonization, and enables predictions of the efficacy of therapy combinations. We applied the model to longitudinal data from a randomized controlled trial (RCT) of an MRSA decolonization protocol consisting of chlorhexidine body and mouthwash and nasal mupirocin. Our findings (i) confirmed nares as a central hub for MRSA colonization and nasal mupirocin as the most crucial therapy and (ii) demonstrated all components contributed significantly to the efficacy of the protocol and the protocol reduced self-inoculation. Finally, we assessed the impact of hypothetical therapy improvements in silico and found that enhancing MRSA clearance at the skin would yield the largest gains. This study demonstrates the use of advanced modelling to go beyond what is typically achieved by RCTs, enabling evidence-based decision-making to streamline clinical protocols.

Ralstonia pickettii and Pseudomonas aeruginosa Bloodstream Infections Associated With Contaminated Extracorporeal Membrane Oxygenation Water Heater Devices.

We report on probable factory-based contamination of portable water heaters with waterborne pathogens and 2 bloodstream infections potentially attributable to off-label use of these water heaters to warm extracorporeal membrane oxygenation circuits. Great caution is warranted when using water-based devices to care for critically ill patients.

Cluster of Burkholderia cepacia Complex Infections Associated With Extracorporeal Membrane Oxygenation Water Heater Devices.

BACKGROUND: Burkholderia cepacia complex is a group of potential nosocomial pathogens often linked to contaminated water. We report on a cluster of 8 B. cepacia complex infections in cardiothoracic intensive care unit patients, which were attributed to contaminated extracorporeal membrane oxygenation (ECMO) water heaters. METHODS: In December 2020, we identified an increase in B. cepacia complex infections in the cardiothoracic intensive care unit at Brigham and Women's Hospital. We sought commonalities, sequenced isolates, obtained environmental specimens, and enacted mitigation measures. RESULTS: Whole-genome sequencing of 13 B. cepacia complex clinical specimens between November 2020 and February 2021 identified 6 clonally related isolates, speciated as Burkholderia contaminans. All 6 occurred in patients on ECMO. Microbiology review identified 2 additional B. contaminans cases from June 2020 that may have also been cluster related, including 1 in a patient receiving ECMO. All 8 definite or probable cluster cases required treatment; 3 patients died, and 3 experienced recurrent infections. After ECMO was identified as the major commonality, all 9 of the hospital's ECMO water heaters were cultured, and B. contaminans grew in all cultures. Cultures from air sampled adjacent to the water heaters were negative. Water heater touch screens were culture positive for B. contaminans, and the sink drain in the ECMO heater reprocessing room also grew clonal B. contaminans. Observations of reprocessing revealed opportunities for cross-contamination between devices through splashing from the contaminated sink. The cluster was aborted by removing all water heaters from clinical service. CONCLUSIONS: We identified a cluster of 8 B. cepacia complex infections associated with contaminated ECMO water heaters. This cluster underscores the potential risks associated with water-based ECMO heaters and, more broadly, water-based care for vulnerable patients.

Shiga Toxin-Producing Escherichia coli Transmission via Fecal Microbiota Transplant.

Fecal microbiota transplantation (FMT) is recommended therapy for multiply recurrent Clostridioides difficile infection. We report adverse events in 7 patients who received FMT from a stool donor who was colonized with Shiga toxin-producing Escherichia coli (STEC). No patients died of FMT-transmitted STEC. Improved screening can likely avoid future transmission.

Plasmids and genes contributing to high-level quinolone resistance in Escherichia coli.

INTRODUCTION: The importance of plasmid-mediated quinolone resistance (PMQR) in Enterobacterales and its high incidence has been emphasised many times. However, a clinical strain carrying more than two PMQR genes is rare. This study sequenced plasmid transconjugants from a donor strain carrying four different PMQR genes to establish their genetic locations. METHODS: An Escherichia coli clinical strain displayed remarkable quinolone resistance with a ciprofloxacin MIC of 1024 mg/L carrying four PMQR genes: qnrA1, qepA1, aac(6')1b-cr and oqxAB. When outcrossed to Escherichia coli J53 AziR, different PMQR genes were transferred and the resulting strains 7C and 8C were chosen for further characterisation. Plasmids were extracted and sequenced by the Illumina and Oxford Nanopore Technologies platforms. S1 nuclease-PFGE was used to estimate the number and size of plasmids. RESULTS: The parental strain had three plasmid bands, as determined by PFGE. Transconjugant 8C obtained three plasmids: pMG336 (162 647 bp, F18:A-:B1:C4) carrying oqxAB; pMG335 carrying qepA1 (73 874 bp, F2:A-:B-); and pMG334 (59 724 bp, IncN (ST5)) with qnrA1 and aac(6')1b-cr. Interestingly, strain 7C obtained plasmid pMG333 (134 435 bp), which was not present in the parental strain but was an IncN-IncF cointegrate of plasmids pMG334 and pMG335 linked via insertion sequence IS26. CONCLUSION: This study described the complete nucleotide sequence of three plasmids carrying four PMQR genes in a single strain and the plasmid profile obtained after outcrosses. In addition, it described a cointegrate of two plasmids formed with flanking insertion sequences.

Community-acquired in name only: A cluster of carbapenem-resistant Acinetobacter baumannii in a burn intensive care unit and beyond.

OBJECTIVE: To describe an investigation into 5 clinical cases of carbapenem-resistant Acinetobacter baumannii (CRAB). DESIGN: Epidemiological investigation supplemented by whole-genome sequencing (WGS) of clinical and environmental isolates. SETTING: A tertiary-care academic health center in Boston, Massachusetts. PATIENTS OR PARTICIPANTS: Individuals identified with CRAB clinical infections. METHODS: A detailed review of patient demographic and clinical data was conducted. Clinical isolates underwent phenotypic antimicrobial susceptibility testing and WGS. Infection control practices were evaluated, and CRAB isolates obtained through environmental sampling were assessed by WGS. Genomic relatedness was measured by single-nucleotide polymorphism (SNP) analysis. RESULTS: Four clinical cases spanning 4 months were linked to a single index case; isolates differed by 1-7 SNPs and belonged to a single cluster. The index patient and 3 case patients were admitted to the same room prior to their development of CRAB infection, and 2 case patients were admitted to the same room within 48 hours of admission. A fourth case patient was admitted to a different unit. Environmental sampling identified highly contaminated areas, and WGS of 5 environmental isolates revealed that they were highly related to the clinical cluster. CONCLUSIONS: We report a cluster of highly resistant Acinetobacter baumannii that occurred in a burn ICU over 5 months and then spread to a separate ICU. Two case patients developed infections classified as community acquired under standard epidemiological definitions, but WGS revealed clonality, highlighting the risk of burn patients for early-onset nosocomial infections. An extensive investigation identified the role of environmental reservoirs.

Drug-Resistant E. coli Bacteremia Transmitted by Fecal Microbiota Transplant.

Fecal microbiota transplantation (FMT) is an emerging therapy for recurrent or refractory Clostridioides difficile infection and is being actively investigated for other conditions. We describe two patients in whom extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli bacteremia occurred after they had undergone FMT in two independent clinical trials; both cases were linked to the same stool donor by means of genomic sequencing. One of the patients died. Enhanced donor screening to limit the transmission of microorganisms that could lead to adverse infectious events and continued vigilance to define the benefits and risks of FMT across different patient populations are warranted.

Multiple Copies of qnrA1 on an IncA/C2 Plasmid Explain Enhanced Quinolone Resistance in an Escherichia coli Mutant.

In a previous study, mutants with enhanced ciprofloxacin resistance (Cipr) were selected from Escherichia coli J53/pMG252 carrying qnrA1 Strain J53 Cipr 8-2 showed an increase in the copy number and transcription level of qnrA1 We sequenced the plasmids on Illumina and MinION platforms. Parental plasmid pMG252 and plasmid pMG252A from strain J53 Cipr 8-2 were almost identical, except for the region containing qnrA1 that in pMG252A contained 4 additional copies of the qnrA1-qacEΔ1-sul1-ISCR1 region.

Impact of Species Diversity on the Design of RNA-Based Diagnostics for Antibiotic Resistance in Neisseria gonorrhoeae.

Quantitative assessment of antibiotic-responsive RNA transcripts holds promise for a rapid point-of-care (POC) diagnostic tool for antimicrobial susceptibility testing. These assays aim to distinguish susceptible and resistant isolates by transcriptional differences upon drug exposure. However, an often-overlooked dimension of designing these tests is that the genetic diversity within a species may yield differential transcriptional regulation independent of resistance phenotype. Here, we use a phylogenetically diverse panel of Neisseria gonorrhoeae and transcriptome profiling coupled with reverse transcription-quantitative PCR to test this hypothesis, to identify azithromycin responsive transcripts and evaluate their potential diagnostic value, and to evaluate previously reported diagnostic markers for ciprofloxacin resistance (porB and rpmB). Transcriptome profiling confirmed evidence of genetic distance and population structure impacting transcriptional response to azithromycin. Taking this into account, we found azithromycin-responsive transcripts overrepresented in susceptible strains compared to resistant strains and selected four candidate diagnostic transcripts (rpsO, rplN, omp3, and NGO1079) that were the most significantly differentially regulated between phenotypes across drug exposure. RNA signatures for these markers categorically predicted resistance in 19/20 cases, with the one incorrect categorical assignment for an isolate at the threshold of reduced susceptibility. Finally, we found that porB and rpmB expression were not uniformly diagnostic of ciprofloxacin resistance in a panel of isolates with unbiased phylogenetic sampling. Overall, our results suggest that RNA signatures as a diagnostic tool are promising for future POC diagnostics; however, development and testing should consider representative genetic diversity of the target pathogen.

A Bayesian model of acquisition and clearance of bacterial colonization incorporating within-host variation.

Bacterial populations that colonize a host can play important roles in host health, including serving as a reservoir that transmits to other hosts and from which invasive strains emerge, thus emphasizing the importance of understanding rates of acquisition and clearance of colonizing populations. Studies of colonization dynamics have been based on assessment of whether serial samples represent a single population or distinct colonization events. With the use of whole genome sequencing to determine genetic distance between isolates, a common solution to estimate acquisition and clearance rates has been to assume a fixed genetic distance threshold below which isolates are considered to represent the same strain. However, this approach is often inadequate to account for the diversity of the underlying within-host evolving population, the time intervals between consecutive measurements, and the uncertainty in the estimated acquisition and clearance rates. Here, we present a fully Bayesian model that provides probabilities of whether two strains should be considered the same, allowing us to determine bacterial clearance and acquisition from genomes sampled over time. Our method explicitly models the within-host variation using population genetic simulation, and the inference is done using a combination of Approximate Bayesian Computation (ABC) and Markov Chain Monte Carlo (MCMC). We validate the method with multiple carefully conducted simulations and demonstrate its use in practice by analyzing a collection of methicillin resistant Staphylococcus aureus (MRSA) isolates from a large recently completed longitudinal clinical study. An R-code implementation of the method is freely available at: https://github.com/mjarvenpaa/bacterial-colonization-model.

Azithromycin Resistance through Interspecific Acquisition of an Epistasis-Dependent Efflux Pump Component and Transcriptional Regulator in Neisseria gonorrhoeae.

Mosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with increased resistance to azithromycin in Neisseria gonorrhoeae in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hot spot of interspecific recombination introducing alleles from Neisseria meningitidis and Neisseria lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrate that resistance to azithromycin, as well as to other antimicrobial compounds such as polymyxin B and crystal violet, is mediated through epistasis between these two loci and that the full-length mosaic mtrD allele is required. Gene expression profiling reveals the mechanism of resistance in mosaics couples novel mtrD alleles with promoter mutations that increase expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple neisserial species has contributed to increased gonococcal resistance to diverse antimicrobial agents.IMPORTANCENeisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for more than 100 million cases of gonorrhea worldwide each year. The incidence of resistance to the macrolide azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance remains unexplained. This study is the first to conclusively demonstrate the acquisition of macrolide resistance through mtr alleles from other Neisseria species, demonstrating that commensal Neisseria bacteria are a reservoir for antibiotic resistance to macrolides, extending the role of interspecies mosaicism in resistance beyond what has been previously described for cephalosporins. Ultimately, our results emphasize that future fine-mapping of genome-wide interspecies mosaicism may be valuable in understanding the pathways to antimicrobial resistance. Our results also have implications for diagnostics and public health surveillance and control, as they can be used to inform the development of sequence-based tools to monitor and control the spread of antibiotic-resistant gonorrhea.

Trends in Antibiotic Susceptibility in Staphylococcus aureus in Boston, Massachusetts, from 2000 to 2014.

The rate of infection by methicillin-resistant Staphylococcus aureus (MRSA) has declined over the past decade, but it is unclear whether this represents a decline in S. aureus infections overall. To evaluate the trends in the annual rates of infection by S. aureus subtypes and mean antibiotic resistance, we conducted a 15-year retrospective observational study at two tertiary care institutions in Boston, MA, of 31,753 adult inpatients with S. aureus isolated from clinical specimens. We inferred the gain and loss of methicillin resistance through genome sequencing of 180 isolates from 2016. The annual rates of infection by S. aureus declined from 2003 to 2014 by 4.2% (2.7% to 5.6%), attributable to an annual decline in MRSA of 10.9% (9.3% to 12.6%). Penicillin-susceptible S. aureus (PSSA) increased by 6.1% (4.2% to 8.1%) annually, and rates of methicillin-susceptible penicillin-resistant S. aureus (MSSA) did not change. Resistance in S. aureus decreased from 2000 to 2014 by 0.8 antibiotics (0.7 to 0.8). Within common MRSA clonal complexes, 3/14 MSSA and 2/21 PSSA isolates arose from the loss of resistance-conferring genes. Overall, in two tertiary care institutions in Boston, MA, a decline in S. aureus infections has been accompanied by a shift toward increased antibiotic susceptibility. The rise in PSSA makes penicillin an increasingly viable treatment option.

Emergence and genomic diversification of a virulent serogroup W:ST-2881(CC175) Neisseria meningitidis clone in the African meningitis belt.

Countries of the African 'meningitis belt' are susceptible to meningococcal meningitis outbreaks. While in the past major epidemics have been primarily caused by serogroup A meningococci, W strains are currently responsible for most of the cases. After an epidemic in Mecca in 2000, W:ST-11 strains have caused many outbreaks worldwide. An unrelated W:ST-2881 clone was described for the first time in 2002, with the first meningitis cases caused by these bacteria reported in 2003. Here we describe results of a comparative whole-genome analysis of 74 W:ST-2881 strains isolated within the framework of two longitudinal colonization and disease studies conducted in Ghana and Burkina Faso. Genomic data indicate that the W:ST-2881 clone has emerged from Y:ST-175(CC175) bacteria by capsule switching. The circulating W:ST-2881 populations were composed of a variety of closely related but distinct genomic variants with no systematic differences between colonization and disease isolates. Two distinct and geographically clustered phylogenetic clonal variants were identified in Burkina Faso and a third in Ghana. On the basis of the presence or absence of 17 recombination fragments, the Ghanaian variant could be differentiated into five clusters. All 25 Ghanaian disease isolates clustered together with 23 out of 40 Ghanaian isolates associated with carriage within one cluster, indicating that W:ST-2881 clusters differ in virulence. More than half of the genes affected by horizontal gene transfer encoded proteins of the 'cell envelope' and the 'transport/binding protein' categories, which indicates that exchange of non-capsular antigens plays an important role in immune evasion.

ELF-MF exposure affects the robustness of epigenetic programming during granulopoiesis.

Extremely-low-frequency magnetic fields (ELF-MF) have been classified as "possibly carcinogenic" to humans on the grounds of an epidemiological association of ELF-MF exposure with an increased risk of childhood leukaemia. Yet, underlying mechanisms have remained obscure. Genome instability seems an unlikely reason as the energy transmitted by ELF-MF is too low to damage DNA and induce cancer-promoting mutations. ELF-MF, however, may perturb the epigenetic code of genomes, which is well-known to be sensitive to environmental conditions and generally deranged in cancers, including leukaemia. We examined the potential of ELF-MF to influence key epigenetic modifications in leukaemic Jurkat cells and in human CD34+ haematopoietic stem cells undergoing in vitro differentiation into the neutrophilic lineage. During granulopoiesis, sensitive genome-wide profiling of multiple replicate experiments did not reveal any statistically significant, ELF-MF-dependent alterations in the patterns of active (H3K4me2) and repressive (H3K27me3) histone marks nor in DNA methylation. However, ELF-MF exposure showed consistent effects on the reproducibility of these histone and DNA modification profiles (replicate variability), which appear to be of a stochastic nature but show preferences for the genomic context. The data indicate that ELF-MF exposure stabilizes active chromatin, particularly during the transition from a repressive to an active state during cell differentiation.

DNA Methylation Assessed by SMRT Sequencing Is Linked to Mutations in Neisseria meningitidis Isolates.

The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.