Non-outbreak Strains Research Articles

Research Note: Evaluating the vertical transmission potential of Salmonella Reading in broiler breeders

Salmonella Reading (S. Reading) recently emerged as a foodborne pathogen causing extensive human outbreaks in North America from consuming contaminated poultry products, mostly from turkeys. Understanding the transmission dynamics of this pathogen is crucial for preventing future outbreaks. This study investigated the ability of S. Reading to colonize the tissues and contaminate eggs of broiler breeders. We utilized 2 S. Reading strains, marked with bioluminescence gene: the outbreak strain RS330 and a reference strain RS326. We used 32 commercially sourced broiler breeder hens, 34 wk of age, randomly assigned to the 2 treatments (16 hens per strain). Each hen was intravaginally inoculated with 108 CFU of the respective strain on d 1 and was rechallenged on d 4. Eggs were collected daily postchallenge to recover bioluminescent S. Reading strains from the external eggshell surface and internal egg contents. On d 7 postchallenge, 10 hens from each treatment group were euthanized. Ovaries, oviducts, and ceca were aseptically collected to detect S. Reading colonization. Results showed that 70.5% (36 of 51) and 34.5% (19 of 55) of external eggshell surfaces, and 4.0% (2 of 50) and 1.8% (1 of 54) of the internal egg contents tested positive for the outbreak and nonoutbreak strains. Additionally, 40.0% of ovaries, 70.0% of oviduct, and 70.0% of ceca samples from the outbreak strain group, and 20.0% of ovaries, 70.0% of oviduct, and 80.0% of ceca samples from nonoutbreak strain group were positive. No significant difference (P = 0.05) was observed in all the findings among the strains except for the eggshell surface contamination. These findings suggest that S. Reading can effectively colonize reproductive tissues, translocate to the ceca, and contaminate the eggs of hens. Future research is needed to determine whether S. Reading can remain viable within the eggs throughout incubation and until hatching.

Epidemiological and molecular investigations of sequential outbreaks of Campylobacter jejuni infecting adults and schoolchildren in southeastern China, 2021-2022

ObjectivesTo investigate cases of five Campylobacter jejuni outbreaks and describe laboratory characteristics of these infections. MethodsWhole-genome sequencing and conventional methods were combined to thoroughly investigate the outbreaks, and data of contemporaneous sporadic cases was included for comparison. ResultsSeven sequence types (STs) of C. jejuni caused 83 cases, including ST9079 which recurred across 2 years. Trace-back investigation could not identify any food items of infection but detected identical campylobacters from food contacts. Phylogenetic analysis unveiled genetic closeness between outbreak strains and some concurrent sporadic strains, indicating local campylobacteriosis may not be wholly sporadic but rather a series of linked cases. Virulence genes disclosed species/case-specific signatures to differentiate outbreaks from truly non-outbreak strains. Resistance to fluoroquinolones and/or macrolides was prevalent (90.8%, 108/119), with a noteworthy portion exhibiting multidrug resistance (31.1%, 37/119). Five types of plasmids were harbored among outbreak isolates, of which one plasmid harboring anti-stress and resistant genes was rarely found in C. jejuni. ConclusionsThis is the first reported sequential outbreak of C. jejuni in China. Our observations help to define the genomic landscape and antimicrobial resistance patterns of Campylobacter, emphasizing the need for a broader ‘One Health’ perspective to combat the threats posed by campylobacteriosis.

A Persistent Tuberculosis Outbreak in the UK Is Characterized by Hydrophobic fadB4-Deficient Mycobacterium tuberculosis That Replicates Rapidly in Macrophages.

The genetic diversity of Mycobacterium tuberculosis can influence disease severity and transmissibility. To better understand how this diversity influences individuals and communities, we phenotyped M. tuberculosis that was causing a persistent outbreak in the East Midlands, United Kingdom. Compared to nonoutbreak isolates, bacilli had higher lipid contents and more hydrophobic cell surfaces. In macrophage infection models, the bacteria increased more rapidly, provoked the enhanced accumulation of macrophage lipid droplets and enhanced the secretion of IL-1β. Natural deletions in fadB4, nrdB, and plcC distinguished the outbreak isolates from other lineage 3 isolates in the region. fadB4 is annotated with a putative role in cell envelope biosynthesis, so the loss of this gene has the potential to alter the interactions of bacteria with immune cells. Reintroduction of fadB4 to the outbreak strain led to a phenotype that more closely resembled those of nonoutbreak strains. The improved understanding of the microbiological characteristics and the corresponding genetic polymorphisms that associate with outbreaks have the potential to inform tuberculosis control. IMPORTANCE Tuberculosis (TB) killed 1.5 million people in 2020 and affects every country. The extent to which the natural genetic diversity of Mycobacterium tuberculosis influences disease manifestation at both the individual and epidemiological levels remains poorly understood. Insights into how pathogen polymorphisms affect patterns of TB have the potential to translate into clinical and public health practice. Two distinct lineage 3 strains isolated from local TB outbreaks, one of which (CH) was rapidly terminated and the other of which (Lro) persistently transmitted for over a decade, provided us with an opportunity to study these issues. We compared genome sequences, microbiological characteristics, and early immune responses that were evoked upon infection. Our results indicate that the natural lack of fadB4 in the Lro strain contributes to its unique features.

Increase of vancomycin-resistant Enterococcus faecium strain type ST117 CT71 at Charit\xe9 - Universit\xe4tsmedizin Berlin, 2008 to 2018

BackgroundIn addition to an overall rise in vancomycin-resistant Enterococcus faecium (VREfm), an increase in certain strain types marked by sequence type (ST) and cluster type (CT) has been reported in Germany over the past few years. Outbreak analyses at Charité - Universitätsmedizin Berlin revealed the frequent occurrence of VREfm ST117 CT71 isolates in 2017 and 2018. To investigate whether ST117 CT71 have emerged in recent years or whether these strains have been circulating for a longer time, we retrospectively analyzed non-outbreak strains that occurred between 2008 and 2018 to identify frequent STs and CTs.MethodsIn total, 120 VREfm isolates obtained from clinical and surveillance cultures from the years 2008, 2013, 2015, and 2018 were analyzed. Thirty isolates per year comprising the first 7–8 non-outbreak isolates of each quarter of the respective year were sequenced using whole genome sequencing. MLST and cgMLST were determined as well as resistance genes and virulence factors. Risk factors for VREfm ST117 were analyzed in a multivariable analysis with patient characteristics as possible confounders.ResultsThe percentage of VREfm of type ST117 increased from 17% in 2008 to 57% in 2018 (p = 0.012). In 2008, vanA genotype accounted for 80% of all ST117 isolates compared to 6% in 2018. VanB CT71 first appeared in 2018 and predominated over all other ST117 at 43% (p < 0.0001).The set of resistance genes (msrC, efmA, erm(B), dfrG, aac(6′)-Ii, gyrA, parC and pbp5) and virulence factors (acm, esp, hylEfm, ecbA and sgrA) in CT71 was also found in other ST117 non-CT71 strains, mainly in CT36. The study population did not differ among the different calendar years analyzed in terms of age, gender, length of stay, or ward type (each p > 0.2).ConclusionThis study revealed an increase in ST117 strains from 2008 to 2018, accompanied by a shift toward CT71 strains with the vanB genotype in 2018. We did not detect resistance or virulence traits in CT71 that could confer survival advantage compared to other CTs among ST117 strains. To date, it is not clear why ST117 and in particular strain type ST117 CT71 predominates over other strains.

Large scale automated phylogenomic analysis of bacterial isolates and the Evergreen Online platform

Public health authorities whole-genome sequence thousands of isolates each month for microbial diagnostics and surveillance of pathogenic bacteria. The computational methods have not kept up with the deluge of data and the need for real-time results. We have therefore created a bioinformatics pipeline for rapid subtyping and continuous phylogenomic analysis of bacterial samples, suited for large-scale surveillance. The data is divided into sets by mapping to reference genomes, then consensus sequences are generated. Nucleotide based genetic distance is calculated between the sequences in each set, and isolates are clustered together at 10 single-nucleotide polymorphisms. Phylogenetic trees are inferred from the non-redundant sequences and the clustered isolates are added back. The method is accurate at grouping outbreak strains together, while discriminating them from non-outbreak strains. The pipeline is applied in Evergreen Online, which processes publicly available sequencing data from foodborne bacterial pathogens on a daily basis, updating phylogenetic trees as needed.

Survival of outbreak, food, and environmental strains of Listeria monocytogenes on whole apples as affected by cultivar and wax coating

The 2014–2015 U.S. nationwide outbreak of listeriosis linked to apples used in commercially produced, prepackaged caramel apples was the first implication of whole apples in outbreaks of foodborne illnesses. Two case patients of this outbreak didn’t consume caramel apples but did eat whole apples, suggesting that contaminated whole apple may serve as a vehicle for foodborne listeriosis. The current study evaluated the effect of conventional fruit coating with wax and that of apple cultivar on the survival of outbreak-associated and non-outbreak Listeria monocytogenes strains on Red Delicious, Granny Smith and Fuji apples during 160 days under simulated commercial storage. L. monocytogenes survived in calyxes and stem ends of apples of all 3 cultivars through the duration of the experiment. After 2 months of storage, significantly (p < 0.05) larger L. monocytogenes populations were recovered from apples coated with wax than those un-waxed, regardless of the cultivar. No differences in survival amongst L. monocytogenes strains (serotypes 1/2a and 4b) from clinical, food, and environmental sources were observed. The observation that coating with wax facilitates prolonged survival of L. monocytogenes on whole apples is novel and reveals gaps in understanding of microbiological risks associated with postharvest practices of tree fruit production.

Genomic Epidemiology and Phenotyping Reveal on-Farm Persistence and Cold Adaptation of Raw Milk Outbreak-Associated Yersinia pseudotuberculosis.

Packaged raw milk contaminated with Yersinia pseudotuberculosis mediated a large yersiniosis outbreak in southern Finland in 2014. The outbreak was traced back to a single dairy farm in southern Finland. Here we explore risk factors leading to the outbreak through epidemiologic investigation of the outbreak farm and through genomic and phenotypic characterization of the farm’s outbreak and non-outbreak associated Y. pseudotuberculosis strains. We show that the outbreak strain persisted on the farm throughout the 7-month study, whereas the non-outbreak strains occurred sporadically. Phylogenomic analysis illustrated that the outbreak strain was related to previously published genomes of wild animal isolates from Finland, implying that wild animals were a potential source of the outbreak strain to the farm. We observed allelic differences between the farm’s outbreak and non-outbreak strains in several genes associated with virulence, stress response and biofilm formation, and found that the outbreak strain formed biofilm in vitro and maintained better growth fitness during cold stress than the non-outbreak strains. Finally, we demonstrate the rapid growth of the outbreak strain in packaged raw milk during refrigerated storage. This study provides insight of the risk factors leading to the Y. pseudotuberculosis outbreak, highlights the importance of pest control to avoid the spread of pathogens from wild to domestic animals, and demonstrates that the cold chain is insufficient as the sole risk management strategy to control Y. pseudotuberculosis risk associated with raw drinking milk.

In Vitro Evaluation of Povidone-Iodine and Chlorhexidine against Outbreak and Nonoutbreak Strains of Mycobacterium abscessus Using Standard Quantitative Suspension and Carrier Testing.

Povidone-iodine (PI) and chlorhexidine (CHX) are widely used antiseptics active against conventional Staphylococcus aureus, Enterobacteriaceae, Candida species, and viruses, but their efficacy against Mycobacterium abscessus remains unproven. We determined the in vitro potency of alcoholic PI and CHX against M. abscessus subsp. abscessus (ATCC 19977), M. abscessus subsp. bolletii (BCRC 16915), and our outbreak strain of M. abscessus subsp. massiliense (TPE 101) in reference to Staphylococcus aureus (ATCC 29213) by standard quantitative suspension and carrier methods (EN 14563). By suspension, all mycobacterial strains compared to S. aureus were significantly more resistant to CHX, but not PI. By carrier, the mean logarithmic reductions (LR) achieved by PI under clean (dirty) conditions were 6.575 (2.482), 5.540 (2.298), 4.595 (1.967), and 1.173 (0.889), while those achieved by CHX under clean (dirty) conditions were 3.164 (5.445), 5.307 (2.564), 3.844 (2.232), and 0.863 (0.389) for S. aureus, M. abscessus subsp. bolletii, M. abscessus subsp. abscessus, and M. abscessus subsp. massiliense, respectively. M. abscessus subsp. massiliense (outbreak strain) was significantly more resistant than the other tested strains to PI and CHX. By both methods, the mean LR achieved by PI was higher than for CHX for all mycobacterial strains, but under dirty conditions, neither antiseptic was effectively mycobactericidal (LR < 5). These preliminary findings caution against the universal replacement of PI with CHX as the first-line skin antiseptic, since all M. abscessus isolates were resistant to CHX. More studies are needed to establish the best practice for skin antisepsis if mycobacterial infections are also to be prevented.

Complete Genome Sequence of Mycobacterium chimaera SJ42, a Nonoutbreak Strain from an Immunocompromised Patient with Pulmonary Disease.

ABSTRACTMycobacterium chimaera, a nontuberculous mycobacterium (NTM) belonging to the Mycobacterium avium complex (MAC), is an opportunistic pathogen that can cause respiratory and disseminated disease. We report the complete genome sequence of a strain, SJ42, isolated from an immunocompromised male presenting with MAC pneumonia, assembled from Illumina and Oxford Nanopore data.

Host factors are more important in predicting recurrent Clostridium difficile infection than ribotype and use of antibiotics

ObjectiveA frequent complication of Clostridium difficile infection (CDI) is recurrent disease. The aim of this study was to determine whether early recurrence risk was higher after infection with ribotype 027 (outbreak strain) compared with infection with endemic strain types of C. difficile. MethodsConsecutive patients diagnosed with CDI between May 2013 and March 2014 were included (outbreak strain, and non-outbreak strains). Patients who developed recurrent CDI within 30 days after completion of CDI treatment, were compared with patients without a recurrence. Medical charts were reviewed for demographic and clinical characteristics. General practitioners were contacted to complete data about the occurrence of recurrent CDI, and the use of medication after hospital discharge. ResultsIn total, 135 patients were at risk for the development of recurrent CDI; 74 patients were infected by ribotype 027, and 61 patients by other ribotypes. Thirty-nine patients (29%) developed recurrent CDI within 30 days after completion of CDI treatment. In multivariable analysis, age ≥70 years (HR 3.05, 95% CI 1.54–6.03), and a duration of CDI treatment ≥11 days (HR 1.92, 95% CI 1.00–3.69) were clearly associated with recurrence; infection with ribotype 027 showed a HR of 1.72 (95% CI 0.88–3.33). ConclusionDuring this outbreak of C. difficile in a tertiary care centre, age and a prolonged duration of CDI therapy (which is most likely a marker of underlying disease severity) were the main risk factors for recurrent CDI. This points to host factors as more important predictors for recurrent CDI than strain type or antibiotic use.

External Validation of Three Prediction Tools for Patients at Risk of a Complicated Course of Clostridium difficile Infection: Disappointing in an Outbreak Setting.

OBJECTIVE Estimating the risk of a complicated course of Clostridium difficile infection (CDI) might help doctors guide treatment. We aimed to validate 3 published prediction models: Hensgens (2014), Na (2015), and Welfare (2011). METHODS The validation cohort comprised 148 patients diagnosed with CDI between May 2013 and March 2014. During this period, 70 endemic cases of CDI occurred as well as 78 cases of CDI related to an outbreak of C. difficile ribotype 027. Model calibration and discrimination were assessed for the 3 prediction rules. RESULTS A complicated course (ie, death, colectomy, or ICU admission due to CDI) was observed in 31 patients (21%), and 23 patients (16%) died within 30 days of CDI diagnosis. The performance of all 3 prediction models was poor when applied to the total validation cohort with an estimated area under the curve (AUC) of 0.68 for the Hensgens model, 0.54 for the Na model, and 0.61 for the Welfare model. For those patients diagnosed with CDI due to non-outbreak strains, the prediction model developed by Hensgens performed the best, with an AUC of 0.78. CONCLUSION All 3 prediction models performed poorly when using our total cohort, which included CDI cases from an outbreak as well as endemic cases. The prediction model of Hensgens performed relatively well for patients diagnosed with CDI due to non-outbreak strains, and this model may be useful in endemic settings. Infect Control Hosp Epidemiol 2017;38:897-905.

Thermal inactivation and growth of Listeria monocytogenes during production and storage of caramel apples

During the fall of 2014, commercially produced pre-packaged caramel apples were linked to 35 cases of listeriosis in 12 states. In response, this study aimed to assess 1) the reduction of different outbreak and non-outbreak strains of Listeria monocytogenes during caramel dipping of apples, and 2) subsequent growth of the apple outbreak strains within caramel apples during storage at 22 and 4 °C. In aim 1, three unwaxed Jonathan apples were dip-inoculated with three different 4-strain L. monocytogenes cocktails (apple outbreak, unrelated outbreak or unrelated environmental) at ∼8 log CFU/apple, dried for 1 h, dipped for 5 s in caramel at 82, 88, 93 or 99 °C, cooled for 1 h at room temperature and assessed for survivors. In aim 2, Jonathan apples were spot-inoculated with the apple outbreak cocktail (∼3 log CFU/apple) at the stem juncture, dried for 1 h, pushed onto wooden sticks, and dipped in caramel at 82 °C. During storage at 4 and 22 °C for 28 and 14 days, respectively, four different apple sections (top, middle, bottom and core) were cut from three apples, homogenized and plated for Listeria. After dipping apples in caramel at 82 and 99 °C, the apple outbreak, unrelated outbreak and environmental Listeria strains decreased 2.0 ± 0.6 and 2.7 ± 0.1, 1.8 ± 0.3 and 2.6 + 0.1, and 1.7 ± 0.1 and 2.9 ± 0.2 logs, respectively, with the environmental cocktail significantly less heat resistant (P < 0.05) at 99 °C compared to the other two cocktails. After 14 days of storage at 22 °C, Listeria populations were significantly higher (P < 0.05) in the core (7.4 ± 0.6 log CFU/g) compared to the other three sections (4.9–5.4 log CFU/g). The same trend was seen for the core (7.7 ± 0.6 log CFU/g) and the other three sections (5.0–5.4 log CFU/g) after 28 days of storage at 4 °C. Since dipping in hot caramel cannot ensure pathogen elimination, producers of caramel apples should implement good agricultural practices, post-harvest preventive controls and refrigeration of the final product to minimize the risks from Listeria.

In-vitro Evaluation of Povidone–Iodine and Chlorhexidine Against Outbreak and Non-outbreak Strains of Mycobacterium abscessus Using Standard Quantitative Suspension and Carrier Testing

In-vitro Evaluation of Povidone–Iodine and Chlorhexidine Against Outbreak and Non-outbreak Strains of Mycobacterium abscessus Using Standard Quantitative Suspension and Carrier Testing

Whole-Genome Sequencing for the Investigation of a Hospital Outbreak of MRSA in China.

Staphylococcus aureus is a globally disseminated drug-resistant bacterial species. It remains a leading cause of hospital-acquired infection, primarily among immunocompromised patients. In 2012, the Affiliated People’s Hospital of Jiangsu University experienced a putative outbreak of methicillin-resistant S. aureus (MRSA) that affected 12 patients in the Neurosurgery Department. In this study, whole-genome sequencing (WGS) was used to gain insight into the epidemiology of the outbreak caused by MRSA, and traditional bacterial genotyping approaches were also applied to provide supportive evidence for WGS. We sequenced the DNA from 6 isolates associated with the outbreak. Phylogenetic analysis was constructed by comparing single-nucleotide polymorphisms (SNPs) in the core genome of 6 isolates in the present study and another 3 referenced isolates from GenBank. Of the 6 MRSA sequences in the current study, 5 belonged to the same group, clustering with T0131, while the other one clustered closely with TW20. All of the isolates were identified as ST239-SCCmecIII clones. Whole-genome analysis revealed that four of the outbreak isolates were more tightly clustered into a group and SA13002 together with SA13009 were distinct from the outbreak strains, which were considered non-outbreak strains. Based on the sequencing results, the antibiotic-resistance gene status (present or absent) was almost perfectly concordant with the results of phenotypic susceptibility testing. Various toxin genes were also analyzed successfully. Our analysis demonstrates that using traditional molecular methods and WGS can facilitate the identification of outbreaks and help to control nosocomial transmission.

Complete Genome Sequences of Salmonella enterica Serovar Heidelberg Strains Associated with a Multistate Food-Borne Illness Investigation.

Next-generation sequencing is being evaluated for use with food-borne illness investigations, especially when the outbreak strains produce patterns that cannot be discriminated from non-outbreak strains using conventional procedures. Here we report complete genome assemblies of two Salmonella enterica serovar Heidelberg strains with a common pulsed-field gel electrophoresis pattern isolated during an outbreak investigation.

Distinguishing highly-related outbreak-associated Clostridium botulinum type A(B) strains.

BackgroundIn the United States, most Clostridium botulinum type A strains isolated during laboratory investigations of human botulism demonstrate the presence of an expressed type A botulinum neurotoxin (BoNT/A) gene and an unexpressed BoNT/B gene. These strains are designated type A(B). The most common pulsed-field gel electrophoresis (PFGE) pattern in the C. botulinum PulseNet database is composed of A(B) strains. The purpose of this study was to evaluate the ability of genome sequencing and multi-loci variable number of tandem repeat analysis (MLVA) to differentiate such strains.ResultsThe genome sequences of type A(B) strains evaluated in this study are closely related and cluster together compared to other available C. botulinum Group I genomes. In silico multilocus sequence typing (MLST) analysis (7-loci) was unable to differentiate any of the type A(B) strains isolated from seven different outbreak investigations evaluated in this study. A 15-locus MLVA scheme demonstrated an improved ability to differentiate these strains, however, repeat unit variation among the strains was restricted to only two loci. Reference-free single nucleotide polymorphism (SNP) analysis demonstrated the ability to differentiate strains from all of the outbreaks examined and a non-outbreak associated strain.ConclusionsThis study confirms that type A(B) strains that share the same PFGE pattern also share closely-related genome sequences. The lack of a complete type A(B) strain representative genome sequence hinders the ability to assemble genomes by reference mapping and analysis of SNPs at pre-identified sites. However, compared to other methods evaluated in this study, a reference-free SNP analysis demonstrated optimal subtyping utility for type A(B) strains using de novo assembled genome sequences.

Genetic Relatedness among Hepatitis A Virus Strains Associated with Food-Borne Outbreaks

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2–3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association.

Cryptococcus gattiiInfections in Multiple States Outside the US Pacific Northwest

Clonal VGII subtypes (outbreak strains) of Cryptococcus gattii have caused an outbreak in the US Pacific Northwest since 2004. Outbreak-associated infections occur equally in male and female patients (median age 56 years) and usually cause pulmonary disease in persons with underlying medical conditions. Since 2009, a total of 25 C. gattii infections, 23 (92%) caused by non–outbreak strain C. gattii, have been reported from 8 non–Pacific Northwest states. Sixteen (64%) patients were previously healthy, and 21 (84%) were male; median age was 43 years (range 15–83 years). Ten patients who provided information reported no past-year travel to areas where C. gattii is known to be endemic. Nineteen (76%) patients had central nervous system infections; 6 (24%) died. C. gattii infection in persons without exposure to known disease-endemic areas suggests possible endemicity in the United States outside the outbreak-affected region; these infections appear to differ in clinical and demographic characteristics from outbreak-associated C. gattii. Clinicians outside the outbreak-affected areas should be aware of locally acquired C. gattii infection and its varied signs and symptoms.

Virulence of Vancomycin-Resistant Enterococcus faecium According to Linezolid Resistance and Clinical Outbreak Status

Assessing clinical virulence differences between vancomycin-resistant Enterococcus faecium (VREF) strains resistant to linezolid (LRVRE) and linezolid-susceptible VRE (LSVRE) strains is difficult due to confounding patient variables. Galleria mellonella is a validated host interaction model allowing straightforward organism virulence assessment. The objective of this study was to assess the virulence of VREF in G. mellonella according to linezolid resistance and clinical outbreak status. A genetically related pair of VREF strains with and without genotypically confirmed linezolid resistance was selected for analysis. Additionally, six strains of LSVRE and two strains of LRVRE were selected according to epidemiologic outbreak status. Mortality of G. mellonella was assessed daily over a 5-day period and analyzed using Kaplan-Meier survival curves and log rank tests. Linezolid resistance did not have a significant effect on G. mellonella mortality in the genetically related pair (P = 0.93). There was no significant difference in mortality over time between strains (non-outbreak [i.e., no patient transmissions were recorded] [n = 2] versus outbreak [i.e., transmission occurred between 3 or more patients in a period of 30 days] [n = 6], P = 0.84; extensive transmission [i.e., the isolate was transmitted between at least 80 patients] [n = 2] versus limited transmission [i.e., the isolate was transmitted between fewer than 10 patients] [n = 4], P = 0.78). These results suggest that patients infected with LRVRE or outbreak strains of VREF are at no greater risk of poor outcomes mediated by organism virulence than those infected with LSVRE or non-outbreak strains.

Congenic Strains for Genetic Analysis of Virulence Traits in Cryptococcus gattii

Cryptococcus gattii is responsible for a large outbreak of potentially fatal disease that started in the late 1990s on Vancouver Island, Canada. How this fungus and the outbreak isolates in particular cause disease in immunocompetent people is unknown, with differing hypotheses. To explore genetic contributions, a pair of congenic a and α mating type strains was generated by a series of 11 backcrosses to introgress the MAT locus from a nonoutbreak strain into the background of strain R265, isolated from a Vancouver Island patient. The congenic pair was used to investigate three traits: mitochondrial inheritance, the effect of the MAT alleles on virulence, and the impact of a predicted virulence factor on pathogenicity. The two congenic strains show the same virulence in different models of cryptococcosis and equivalent levels of competition in coinfection assays. These results rule out a role of the MAT locus and mitochondrial genotype as major virulence factors in the outbreak strains. Disruption of Bwc2, a light-dependent transcription factor, resulted in reduced virulence, consistent with a similar function in the related species Cryptococcus neoformans. The C. gattii congenic strains represent a new resource for exploring the evolution of virulence in the C. neoformans-C. gattii clade.