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Whole-genome sequencing for outbreak investigations of methicillin-resistant Staphylococcus aureus in the neonatal intensive care unit: time for routine practice?

ABSTRACT: BACKGROUND Infants in the neonatal intensive care unit (NICU) are at increased risk for methicillin-resistant Staphylococcus aureus (MRSA) acquisition. Outbreaks may be difficult to identify due in part to limitations in current molecular genotyping available in clinical practice. Comparison of genome-wide single nucleotide polymorphisms (SNPs) may identify epidemiologically distinct isolates among a population sample that appears homogenous when evaluated using conventional typing methods. OBJECTIVE To investigate a putative MRSA outbreak in a NICU utilizing whole-genome sequencing and phylogenetic analysis to identify recent transmission events. DESIGN Clinical and surveillance specimens collected during clinical care and outbreak investigation. PATIENTS A total of 17 neonates hospitalized in a 43-bed level III NICU in northeastern Florida from December 2010 to October 2011 were included in this study. METHODS We assessed epidemiological data in conjunction with 4 typing methods: antibiograms, PFGE, spa types, and phylogenetic analysis of genome-wide SNPs. RESULTS Among the 17 type USA300 isolates, 4 different spa types were identified using pulsed-field gel electrophoresis. Phylogenetic analysis identified 5 infants as belonging to 2 clusters of epidemiologically linked cases and excluded 10 unlinked cases from putative transmission events. The availability of these results during the initial investigation would have improved infection control interventions. CONCLUSION Whole-genome sequencing and phylogenetic analysis are invaluable tools for epidemic investigation; they identify transmission events and exclude cases mistakenly implicated by traditional typing methods. When routinely applied to surveillance and investigation in the clinical setting, this approach may provide actionable intelligence for measured, appropriate, and effective interventions.

SUBMITTER: Azarian T

PROVIDER: S-EPMC4507300 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Project description:Despite infection prevention efforts, neonatal intensive care unit (NICU) patients remain at risk of Methicillin-resistant Staphylococcus aureus (MRSA) infection. Modes of transmission for healthcare-associated (HA) and community-associated (CA) MRSA remain poorly understood and may vary by genotype, hindering the development of effective prevention and control strategies. From 2008-2010, all patients admitted to a level III NICU were screened for MRSA colonization, and all available isolates were spa-typed. Spa-type t008, the most prevalent CA- genotype in the United States, spa-type t045, a HA- related genotype, and a convenience sample of strains isolated from 2003-2011, underwent whole-genome sequencing and phylodynamic analysis. Patient risk factors were compared between colonized and noncolonized infants, and virulence and resistance genes compared between spa-type t008 and non-t008 strains. Epidemiological and genomic data were used to estimate MRSA importations and acquisitions through transmission reconstruction. MRSA colonization was identified in 9.1% (177/1940) of hospitalized infants and associated with low gestational age and birth weight. Among colonized infants, low gestational age was more common among those colonized with t008 strains. Our data suggest that approximately 70% of colonizations were the result of transmission events within the NICU, with the remainder likely to reflect importations of "outside" strains. While risk of transmission within the NICU was not affected by spa-type, patterns of acquisition and importation differed between t008 and t045 strains. Phylodynamic analysis showed the effective population size of spa-type t008 has been exponentially increasing in both community and hospital, with spa-type t008 strains possessed virulence genes not found among t045 strains; t045 strains, in contrast, appeared to be of more recent origin, with a possible hospital source. Our data highlight the importance of both intra-NICU transmission and recurrent introductions in maintenance of MRSA colonization within the NICU environment, as well as spa-type-specific differences in epidemiology.

Project description:Active surveillance for methicillin-resistant Staphylococcus aureus (MRSA) is a component of our neonatal intensive care unit (NICU) infection prevention efforts. Recent atypical trends prompted review of 42 suspected MRSA isolates. Species identification was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and methicillin resistance was reevaluated by PBP2a lateral flow assay, cefoxitin/oxacillin susceptibility testing, mecA and mecC PCR, and six commercially available MRSA detection agars. All isolates were confirmed S. aureus, but only eight were MRSA (cefoxitin resistant, PBP2a positive, mecA positive, growth on all MRSA screening agars). One MRSA isolate was cefoxitin susceptible but PBP2a and mecA positive, and the remaining 33 were cefoxitin susceptible, PBP2a negative, and mecA negative; interestingly, these isolates grew inconsistently across MRSA screening agars and had susceptibility profiles consistent with that of borderline oxacillin-resistant S. aureus (BORSA). Comparative genomic analyses found these BORSA isolates to be phylogenetically diverse and not representative of clonal expansion or shared gene content, though clones of two NICU strains were infrequently observed over 8 months. We identified 6 features-substitutions and truncations in PBP2, PBP4, and GdpP and beta-lactamase hyperproduction-that were used to generate a random forest classifier to distinguish BORSA from methicillin-susceptible S. aureus (MSSA) in our cohort. Our model demonstrated a robust ability to predict the BORSA phenotype among isolates collected across two continents (validation area under the curve [AUC], 0.902). Taking these findings together, we observed an unexpected prevalence of BORSA in our NICU, BORSA misclassification by existing MRSA screening methods, and markers that are together discriminatory for BORSA and MSSA within our cohort. This work has implications for epidemiological reporting of MRSA rates for centers using different screening methods. IMPORTANCE In this study, we found a high prevalence of Staphylococcus aureus isolates exhibiting a borderline oxacillin resistance phenotype (BORSA) in our neonatal intensive care unit (NICU) serendipitously due to the type of MRSA screening agar used by our laboratory for active surveillance cultures. Subsequent phenotypic and molecular characterization highlighted an unexpected prevalence and variability of BORSA isolates. Through whole-genome sequencing, we interrogated core and accessory genome content and generated a random forest classification model to identify mutations and truncations in the PBP2, PBP4, and GdpP proteins and beta-lactamase hyperproduction, which correlated with BORSA and MSSA phenotypes among S. aureus clinical isolates collected across two continents. In consideration of these findings, this work will help clinical microbiology laboratories and clinicians identify MRSA screening shortfalls and draw attention to the non-mecA-mediated BORSA phenotype.

Project description:ImportanceStaphylococcus aureus is one of the leading causes of infections in neonatal intensive care units (NICUs). Most studies in this patient group focus on methicillin-resistant S aureus or the outbreak setting, whereas data for methicillin-susceptible S aureus are limited.ObjectivesTo identify risk factors for S aureus colonization and infections in hospitalized newborns and to investigate S aureus transmission and its dynamics in a nonoutbreak setting.Design, setting, and participantsThis monocentric cohort study in a tertiary NICU in Heidelberg, Germany, enrolled all hospitalized neonates (n = 590) with at least 1 nasal screening swab positive for S aureus. Data were collected from January 1, 2018, to December 31, 2019.ExposuresWeekly screening for S aureus colonization was performed for all newborns until discharge.Main outcomes and measuresThe primary end point was any S aureus infection until hospital discharge. Transmission of S aureus and performance of routine typing to detect transmissions were defined as the secondary outcomes of the study.ResultsIn total, 590 newborns were enrolled (276 [46.8%] female and 314 [53.2%] male; 220 [37.3%] with birthweight <1500 g; 477 [80.8%] preterm; 449 [76.1%] singletons; 419 [71.5%] delivered via cesarean section). The median length of stay was 26 (range, 10-62) days. Overall, 135 infants (22.9%) were colonized by S aureus at some time during their hospital stay. The median time to first detection was 17 (interquartile range, 11-37) days. The overall incidence of S aureus infection was 1.7% (10 of 590). Low birth weight (<1500 g [odds ratio, 9.3; 95% CI, 5.9-14.6; P < .001]) and longer hospital stay (odds ratio, 2.3; 95% CI, 1.9-2.7; P < .001) were associated with colonization. Nasal carriage was significantly associated with S aureus infection (odds ratio, 8.2; 95% CI, 2.1-32.3; P = .002). A total of 123 of 135 colonization isolates were sequenced. All recoverable infection isolates (4 of 7) of newborns with colonization were genetically identical to the colonizing isolate. Whole-genome sequencing indicated 23 potential transmission clusters.Conclusions and relevanceThe findings of this cohort study suggest that nasal colonization is a relevant risk factor for S aureus infection in a nonoutbreak NICU setting. In colonized newborns, infection and colonization isolates were genetically identical, suggesting that eradication of colonization may be a useful measure to prevent infection. Further investigations are necessary to validate and assess the generalizability of our findings.

Project description:Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) are well described in the neonatal intensive care unit (NICU) setting. Genomics has revolutionized the investigation of such outbreaks; however, to date, this has largely been completed retrospectively and has typically relied on short-read platforms. In 2022, our laboratory established a prospective genomic surveillance system using Oxford Nanopore Technologies sequencing for rapid outbreak detection. Herein, using this system, we describe the detection and control of an outbreak of sequence-type (ST)97 MRSA in our NICU. The outbreak was identified 13 days after the first MRSA-positive culture and at a point where there were only two known cases. Ward screening rapidly defined the extent of the outbreak, with six other infants found to be colonized. There was minimal transmission once the outbreak had been detected and appropriate infection control measures had been instituted; only two further ST97 cases were detected, along with three unrelated non-ST97 MRSA cases. To contextualize the outbreak, core-genome single-nucleotide variants were identified for phylogenetic analysis after de novo assembly of nanopore data. Comparisons with global (n=45) and national surveillance (n=35) ST97 genomes revealed the stepwise evolution of methicillin resistance within this ST97 subset. A distinct cluster comprising nine of the ten ST97-IVa genomes from the NICU was identified, with strains from 2020 to 2022 national surveillance serving as outgroups to this cluster. One ST97-IVa genome presumed to be part of the outbreak formed an outgroup and was retrospectively excluded. A second phylogeny was created using Illumina sequencing, which considerably reduced the branch lengths of the NICU isolates on the phylogenetic tree. However, the overall tree topology and conclusions were unchanged, with the exception of the NICU outbreak cluster, where differences in branch lengths were observed. This analysis demonstrated the ability of a nanopore-only prospective genomic surveillance system to rapidly identify and contextualize an outbreak of MRSA in a NICU.

Project description:Methicillin-susceptible Staphylococcus aureus (MSSA) is a more prevalent neonatal intensive care unit (NICU) pathogen than methicillin-resistant S. aureus (MRSA). However, the introduction and spread of MSSA, the role of systematic decolonization, and optimal infection prevention and control strategies remain incompletely understood. We previously screened infants hospitalized in a university-affiliated level III to IV NICU twice monthly over 18 months for S. aureus colonization and identified several prevalent staphylococcal protein A (spa) types. Here, we performed whole-genome sequencing (WGS) and phylogenetic comparisons of 140 isolates from predominant spa types t279, t1451, and t571 to examine possible transmission routes and identify genomic and epidemiologic features associated with the spread of dominant clones. We identified two major MSSA clones: sequence type 398 (ST398), common in the local community, and ST1898, not previously encountered in the region. ST398 NICU isolates formed distinct clusters with closely related community isolates from previously published data sets, suggesting multiple sources of acquisition, such as family members or staff, including residents of the local community. In contrast, ST1898 isolates were nearly identical, pointing to clonal expansion within the NICU. Almost all ST1898 isolates harbored plasmids encoding mupirocin resistance (mupA), suggesting an association between the proliferation of this clone and decolonization efforts with mupirocin. Comparative genomics indicated genotype-specific pathways of introduction and spread of MSSA via community-associated (ST398) or health care-associated (ST1898) sources and the potential role of mupirocin resistance in dissemination of ST1898. Future surveillance efforts could benefit from routine genotyping to inform clone-specific infection prevention strategies. IMPORTANCE Methicillin-susceptible Staphylococcus aureus (MSSA) is a significant pathogen in neonates. However, surveillance efforts in neonatal intensive care units (NICUs) have focused primarily on methicillin-resistant S. aureus (MRSA), limiting our understanding of colonizing and infectious MSSA clones which are prevalent in the NICU. Here, we identify two dominant colonizing MSSA clones during an 18-month surveillance effort in a level III to IV NICU, ST398 and ST1898. Using genomic surveillance and phylogenetic analysis, coupled with epidemiological investigation, we found that these two sequence types had distinct modes of spread, namely the suggested exchange with community reservoirs for ST398 and the contribution of antibiotic resistance to dissemination of ST1898 in the health care setting. This study highlights the additional benefits of whole-genome surveillance for colonizing pathogens, beyond routine species identification and genotyping, to inform targeted infection prevention strategies.

Project description:The French National Reference Center for Staphylococci currently uses DNA arrays and spa typing for the initial epidemiological characterization of Staphylococcus aureus strains. We here describe the use of whole-genome sequencing (WGS) to investigate retrospectively four distinct and virulent S. aureus lineages [clonal complexes (CCs): CC1, CC5, CC8, CC30] involved in hospital and community outbreaks or sporadic infections in France. We used a WGS bioinformatics pipeline based on de novo assembly (reference-free approach), single nucleotide polymorphism analysis, and on the inclusion of epidemiological markers. We examined the phylogeographic diversity of the French dominant hospital-acquired CC8-MRSA (methicillin-resistant S. aureus) Lyon clone through WGS analysis which did not demonstrate evidence of large-scale geographic clustering. We analyzed sporadic cases along with two outbreaks of a CC1-MSSA (methicillin-susceptible S. aureus) clone containing the Panton-Valentine leukocidin (PVL) and results showed that two sporadic cases were closely related. We investigated an outbreak of PVL-positive CC30-MSSA in a school environment and were able to reconstruct the transmission history between eight families. We explored different outbreaks among newborns due to the CC5-MRSA Geraldine clone and we found evidence of an unsuspected link between two otherwise distinct outbreaks. Here, WGS provides the resolving power to disprove transmission events indicated by conventional methods (same sequence type, spa type, toxin profile, and antibiotic resistance profile) and, most importantly, WGS can reveal unsuspected transmission events. Therefore, WGS allows to better describe and understand outbreaks and (inter-)national dissemination of S. aureus lineages. Our findings underscore the importance of adding WGS for (inter-)national surveillance of infections caused by virulent clones of S. aureus but also substantiate the fact that technological optimization at the bioinformatics level is still urgently needed for routine use. However, the greatest limitation of WGS analysis is the completeness and the correctness of the reference database being used and the conversion of floods of data into actionable results. The WGS bioinformatics pipeline (EpiSeqTM) we used here can easily generate a uniform database and associated metadata for epidemiological applications.

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Whole-genome sequencing for outbreak investigations of methicillin-resistant Staphylococcus aureus in the neonatal intensive care unit: time for routine practice?

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