Development of a novel real-time PCR multiplex assay for detection of Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus.

Strangles is a contagious bacterial disease of horses caused by Streptococcus equi subspecies equi (SEE) that occurs globally. Rapid and accurate identification of infected horses is essential for controlling strangles. Because of limitations of existing PCR assays for SEE, we sought to identify novel primers and probes that enable simultaneous detection and differentiation of infection with SEE and S. equi subsp. zooepidemicus (SEZ). Comparative genomics of U.S. strains of SEE and SEZ (n = 50 each) identified SE00768 from SEE and comB from SEZ as target genes. Primers and probes for real-time PCR (rtPCR) were designed for these genes and then aligned in silico with the genomes of strains of SEE (n = 725) and SEZ (n = 343). Additionally, the sensitivity and specificity relative to microbiologic culture were compared between 85 samples submitted to an accredited veterinary medical diagnostic laboratory. The respective primer and probe sets aligned with 99.7 % (723/725) isolates of SEE and 97.1 % (333/343) of SEZ. Of 85 diagnostic samples, 20 of 21 (95.2 %) SEE and 22 of 23 SEZ (95.6 %) culture-positive samples were positive by rtPCR for SEE and SEZ, respectively. Both SEE (n = 2) and SEZ (n = 3) were identified by rtPCR among 32 culture-negative samples. Results were rtPCR-positive for both SEE and SEZ in 21 of 44 (47.7 %) samples that were culture-positive for SEE or SEZ. The primers and probe sets reported here reliably detect SEE and SEZ from Europe and the U.S., and permit detection of concurrent infection with both subspecies.

Conservation of vaccine antigen sequences encoded by sequenced strains of Streptococcus equi subsp. equi.

BACKGROUND: Streptococcus equi subspecies equi (S equi) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight-component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. OBJECTIVE: To define the diversity of the eight Strangvac antigens across a diverse S equi population. STUDY DESIGN: Genomic description. METHODS: Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 assembled genomes and compared. RESULTS: The predicted amino acid sequences of SclC, SclI and IdeE were identical across all 759 genomes. CNE was truncated in the genome of five (0.7%) isolates. SclF was absent from one genome and another encoded a single amino acid substitution. EAG was truncated in two genomes. Eq5 was truncated in four genomes and 123 genomes encoded a single amino acid substitution. Eq8 was truncated in three genomes, one genome encoded four amino acid substitutions and 398 genomes encoded a single amino acid substitution at the final amino acid of the Eq8 antigen fragment. Therefore, at least 1579 (99.9%) of 1580 amino acids in Strangvac were identical in 743 (97.9%) genomes, and all genomes encoded identical amino acid sequences for at least six of the eight Strangvac antigens. MAIN LIMITATIONS: Three hundred and seven (40.4%) isolates in this study were recovered from horses in the UK. CONCLUSIONS: The predicted amino acid sequences of antigens in Strangvac were highly conserved across this collection of S equi.

Differences in the Accessory Genomes and Methylomes of Strains of Streptococcus equi subsp. equi and of Streptococcus equi subsp. zooepidemicus Obtained from the Respiratory Tract of Horses from Texas.

Streptococcus equi subsp. equi (SEE) is a host-restricted equine pathogen considered to have evolved from Streptococcus equi subsp. zooepidemicus (SEZ). SEZ is promiscuous in host range and is commonly recovered from horses as a commensal. Comparison of a single strain each of SEE and SEZ using whole-genome sequencing, supplemented by PCR of selected genes in additional SEE and SEZ strains, was used to characterize the evolution of SEE. But the known genetic variability of SEZ warrants comparison of the whole genomes of multiple SEE and SEZ strains. To fill this knowledge gap, we utilized whole-genome sequencing to characterize the accessory genome elements (AGEs; i.e., elements present in some SEE strains but absent in SEZ or vice versa) and methylomes of 50 SEE and 50 SEZ isolates from Texas. Consistent with previous findings, AGEs consistently found in all SEE isolates were primarily from mobile genetic elements that might contribute to host restriction or pathogenesis of SEE. Fewer AGEs were identified in SEZ because of the greater genomic variability among these isolates. The global methylation patterns of SEE isolates were more consistent than those of the SEZ isolates. Among homologous genes of SEE and SEZ, differential methylation was identified only in genes of SEE encoding proteins with functions of quorum sensing, exopeptidase activity, and transitional metal ion binding. Our results indicate that effects of genetic mobile elements in SEE and differential methylation of genes shared by SEE and SEZ might contribute to the host specificity of SEE. IMPORTANCE Strangles, caused by the host-specific bacterium Streptococcus equi subsp. equi (SEE), is the most commonly diagnosed infectious disease of horses worldwide. Its ancestor, Streptococcus equi subsp. zooepidemicus (SEZ), is frequently isolated from a wide array of hosts, including horses and humans. A comparison of the genomes of a single strain of SEE and SEZ has been reported, but sequencing of further isolates has revealed variability among SEZ strains. Thus, the importance of this study is that it characterizes genomic and methylomic differences of multiple SEE and SEZ isolates from a common geographic region (viz., Texas). Our results affirm many of the previously described differences between the genomes of SEE and SEZ, including the role of mobile genetic elements in contributing to host restriction. We also provide the first characterization of the global methylome of Streptococcus equi and evidence that differential methylation might contribute to the host restriction of SEE.

Differences in the genome, methylome, and transcriptome do not differentiate isolates of Streptococcus equi subsp. equi from horses with acute clinical signs from isolates of inapparent carriers.

Streptococcus equi subsp. equi (SEE) is a host-restricted bacterium that causes the common infectious upper respiratory disease known as strangles in horses. Perpetuation of SEE infection appears attributable to inapparent carrier horses because it neither persists long-term in the environment nor infects other host mammals or vectors, and infection results in short-lived immunity. Whether pathogen factors enable SEE to remain in horses without causing clinical signs remains poorly understood. Thus, our objective was to use next-generation sequencing technologies to characterize the genome, methylome, and transcriptome of isolates of SEE from horses with acute clinical strangles and inapparent carrier horses-including isolates recovered from individual horses sampled repeatedly-to assess pathogen-associated changes that might reflect specific adaptions of SEE to the host that contribute to inapparent carriage. The accessory genome elements and methylome of SEE isolates from Sweden and Pennsylvania revealed no significant or consistent differences between acute clinical and inapparent carrier isolates of SEE. RNA sequencing of SEE isolates from Pennsylvania demonstrated no genes that were differentially expressed between acute clinical and inapparent carrier isolates of SEE. The absence of specific, consistent changes in the accessory genomes, methylomes, and transcriptomes of acute clinical and inapparent carrier isolates of SEE indicates that adaptations of SEE to the host are unlikely to explain the carrier state of SEE. Efforts to understand the carrier state of SEE should instead focus on host factors.

Correction: Detection of vapN in Rhodococcus equi isolates cultured from humans.

[This corrects the article DOI: 10.1371/journal.pone.0190829.].

The novel and transferable erm(51) gene confers macrolides, lincosamides and streptogramins B (MLSB ) resistance to clonal Rhodococcus equi in the environment.

The use of mass antimicrobial treatment has been linked to the emergence of antimicrobial resistance in human and animal pathogens. Using whole-genome single-molecule real-time (SMRT) sequencing, we characterized genomic variability of multidrug-resistant Rhodococcus equi isolated from soil samples from 100 farms endemic for R. equi infections in Kentucky. We discovered the novel erm(51)-encoding resistance to MLSB in R. equi isolates from soil of horse-breeding farms. Erm(51) is inserted in a transposon (TnErm51) that is associated with a putative conjugative plasmid (pRErm51), a mobilizable plasmid (pMobErm51), or both enabling horizontal gene transfer to susceptible organisms and conferring high levels of resistance against MLSB in vitro. This new resistant genotype also carries a previously unidentified rpoB mutation conferring resistance to rifampicin. Isolates carrying both vapA and erm(51) were rarely found, indicating either a recent acquisition of erm(51) and/or impaired survival when isolates carry both genes. Isolates carrying erm(51) are closely related genetically and were likely selected by antimicrobial exposure in the environment.

Comparison of whole genome sequences of Streptococcus equi subsp. equi from an outbreak in Texas with isolates from within the region, Kentucky, USA, and other countries.

Strangles, caused by Streptococcus equi subspecies equi (S. equi) is an infectious disease of horses with worldwide distribution, but there are limited data available regarding strain variation using whole genome sequencing among and within outbreaks in the United States (US), and how US isolates compare with S. equi isolated globally. To address this knowledge-gap, we compared the whole genomes of 54 S. equi isolates from Texas and Kentucky and those of 230 publicly available sequences of S. equi isolates collected from other countries. Our results show that despite minimal variation among isolates within an outbreak some mutations do occur among individual outbreak isolates. Some S. equi strains from the US are closely related to S. equi isolates from other countries, likely reflecting international dissemination of isolates. Collectively, these data improve our understanding of phenotypic and genotypic variation of isolates within an outbreak, and the international distribution of S. equi. We also identify a novel variant of the S. equi M-protein, and observed cases of strangles that were caused by the modified-live vaccine but that were not recognized as vaccine-associated at the time of clinical sample submission.

A Conserved Streptococcal Virulence Regulator Controls the Expression of a Distinct Class of M-Like Proteins.

Streptococcus equi subspecies zooepidemicus (SEZ) are group C streptococci that are important pathogens of economically valuable animals such as horses and pigs. Here, we found that many SEZ isolates bind to a monoclonal antibody that recognizes poly-N-acetylglucosamine (PNAG), a polymer that is found as a surface capsule-like structure on diverse microbes. A fluorescence-activated cell sorting-based transposon insertion sequencing (Tn-seq) screen, coupled with whole-genome sequencing, was used to search for genes for PNAG biosynthesis. Surprisingly, mutations in a gene encoding an M-like protein, szM, and the adjacent transcription factor, designated sezV, rendered strains PNAG negative. SezV was required for szM expression and transcriptome analysis showed that SezV has a small regulon. SEZ strains with inactivating mutations in either sezV or szM were highly attenuated in a mouse model of infection. Comparative genomic analyses revealed that linked sezV and szM homologues are present in all SEZ, S. equi subspecies equi (SEE), and M18 group A streptococcal (GAS) genomes in the database, but not in other streptococci. The antibody to PNAG bound to a wide range of SEZ, SEE, and M18 GAS strains. Immunochemical studies suggest that the SzM protein may be decorated with a PNAG-like oligosaccharide although an intact oligosaccharide substituent could not be isolated. Collectively, our findings suggest that the szM and sezV loci define a subtype of virulent streptococci and that an antibody to PNAG may have therapeutic applications in animal and human diseases caused by streptococci bearing SzM-like proteins.IMPORTANCE M proteins are surface-anchored virulence factors in group A streptococci, human pathogens. Here, we identified an M-like protein, SzM, and its positive regulator, SezV, in Streptococcus equi subspecies zooepidemicus (SEZ), an important group of pathogens for domesticated animals, including horses and pigs. SzM and SezV homologues were found in the genomes of all SEZ and S. equi subspecies equi and M18 group A streptococcal strains analyzed but not in other streptococci. Mutant SEZ strains lacking either sezV or szM were highly attenuated in a mouse model of infection. Collectively, our findings suggest that SezV-related regulators and the linked SzM family of M-like proteins define a new subset of virulent streptococci.

Bactericidal activity of 3D-printed hydrogel dressing loaded with gallium maltolate.

Chronic wounds are projected to reach epidemic proportions worldwide because of the aging population and the increasing incidence of diabetes. Despite extensive research, infection remains one of the leading sources of complications in chronic wounds, resulting in improper healing, biofilm formation, and lower extremity amputation. To address the limitations of standard treatments, we have developed a hydrogel wound dressing with self-tuning moisture control that incorporates a novel antimicrobial agent to eliminate and prevent infection. 3D-printing of a hydrogel dressing with dual porosity resulted in a new dressing with greater flexibility, increased water uptake, and more rapid swelling than bulk hydrogel dressings. Additionally, gallium maltolate (GaM) was incorporated into the dressing to investigate the efficacy of this antimicrobial agent. Loading profiles, release kinetics, and the bactericidal activity against Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus) of GaM were investigated in vitro to identify target profiles that supported infection control. Finally, GaM-loaded hydrogel dressings were evaluated in vivo, utilizing a murine splinted-wound model that was inoculated with S. aureus. In comparison to an untreated control, GaM dressings markedly reduced the wound bacterial load without compromising wound closure rates. Overall, this work demonstrates the utility of a 3D-printed hydrogel dressing as an antimicrobial dressing to control infection in chronic wounds.

Guinea pig infection with the intracellular pathogen Rhodococcus equi.

Rhodococcus equi is an opportunistic, intracellular pathogen that causes pyogranulomatous pneumonia in foals and immunocompromised people. Currently, there is no experimental model of R. equi pneumonia other than intra-bronchial experimental infection of foals with R. equi, which is labor-intensive and costly. This study's objective was to develop a guinea pig (GP) model of R. equi pneumonia that would facilitate development of novel approaches for controlling and preventing this disease. Guinea pigs were infected with either 101, 102, 103, or 104 colony forming units (CFUs) of a virulent strain of R. equi using a Madison aerosol chamber, or 106 or 107 CFUs of this strain intratracheally. Animals were monitored daily for clinical signs of pneumonia, and were euthanized and necropsied on days 1, 3, 7, or 35 post-infection (PI). Lung homogenates were plated onto selective agar to determine bacterial load. No clinical signs of disease were observed regardless of the inoculum dose or infection method. No bacteria were recovered from GPs euthanized at 35 days PI. Histology and immunostaining of T-cells, B-cells, and macrophages in lungs showed that inflammatory responses in infected GPs were similarly unremarkable irrespective of dose or route of infection. Guinea pigs appear to be resistant to pulmonary infection with virulent R. equi even at doses that reliably produce clinical pneumonia in foals.

Detection of vapN in Rhodococcus equi isolates cultured from humans.

Rhodococcus equi can cause severe infections in people, particularly in immunocompromised individuals. The R. equi virulence plasmids (vap) encoding vapA and vapB are linked to development of infections in domestic animals. Recently, a novel virulence plasmid, vapN, was identified in isolates cultured from cattle, but its prevalence or significance in human R. equi infections has not been extensively studied. To determine the prevalence of vapN in a diverse collection of human-derived isolates from different countries, 65 R. equi isolates collected by various institutions from 1984 to 2002 were screened for the presence of vapN and other virulence plasmids through polymerase chain reaction (PCR) using redesigned primer sets. Of the isolates that carried plasmids, 43% (16/37) were vapN-positive and fewer were vapB or vapA-positive (30 and 16%, respectively). This is the first report of vapN carriage in R. equi isolated from human infections in the United States. One isolate (H-30) carried vapN but did not amplify the conjugal plasmid transfer gene traA associated with carriage of vap, which could be explained by sequence variation within the traA gene. Another isolate (H-55) amplified traA, but did not amplify vapA, B, or N (traA+ vapABN-) with previously described primer sets or those developed for this study. The H-55 traA sequence had 98% identity to traA sequences in vapA plasmids, which suggests that it may carry a variant of previously characterized virulence plasmids or a novel virulence plasmid. Carriage of vapN in R. equi isolates derived from people is not uncommon and more research is needed to determine its significance in the epidemiology and pathogenesis of human R. equi infections.

Metformin-Associated Lactic Acidosis in a Patient with Normal Renal Function.

We report a case of metformin-associated lactic acidosis (MALA) in the setting of normal renal function and review the relevant medical literature. A 77-year-old female diagnosed with type 2 diabetes mellitus previously treated with insulin and gliclazide MR was started on metformin. A few weeks later, she was found to have lactic acidosis. Renal function was normal, and no severe underlying illness was identified. Metformin was discontinued, and lactate levels normalized within 4 days, suggesting metformin was a reversible precipitant of the lactic acidosis. MALA can occur in the absence of renal impairment, systemic hypoperfusion or severe liver disease. A possible mechanism is a genetically determined alteration in metformin pharmacokinetics. Metformin is beneficial and safe in patients with normal renal function, but the development of MALA, although rare, should be kept in mind to prevent potentially life-threatening toxicity.

Motivation and experience of nulliparous women using intrauterine contraceptive devices.

OBJECTIVES: Use of an intrauterine contraceptive device (IUD) has not been recommended to nulliparous women in the past. There is now good evidence that there is no increased risk of pelvic inflammatory disease or infertility in nulliparas who use IUDs and the recommendations have changed. Our objective was to understand more about the motivations and experience of nulliparous women using IUDs. METHODS: This was a mixed method study. First, we asked 44 nulliparous women who had had an IUD inserted within the previous six months about their reasons for seeking the IUD, their history with other forms of contraception, their perception of the insertion experience, and their feelings after insertion. Questionnaires were then distributed to 154 nulliparous women presenting for IUDs, asking about their past experience with hormonal contraception. RESULTS: The main theme arising from the interviews was a desire to avoid hormonal contraception. Other reasons for choosing the IUD were greater contraceptive effectiveness than other methods, convenience of use, and lower cost. Responses to the questionnaire indicated that 138 women (89.7%) had used hormonal contraception in the past and, of those, 98 (63.0%) complained of mood side effects, 64 (41.6%) of sexual side effects, and 64 (41.6%) of physical side effects. CONCLUSION: The most important motivation for nulliparous women in this study to choose IUDs was to avoid the potential or actual side effects of hormonal contraception. Despite experiencing some discomfort at the time of insertion, this group of nulliparous women was very positive about using IUDs for contraception.

Systematic review of motor vehicle crash risk in persons with sleep apnea.

STUDY OBJECTIVES: To determine whether drivers with sleep apnea are at increased risk of motor vehicle crash; whether disease severity, daytime sleepiness, or both disease severity and daytime sleepiness affect this risk, and whether treatment of sleep apnea reduces crash risk. DESIGN: Systematic review of published literature. SETTING: N/A. PATIENTS/PARTICIPANTS: Patients with sleep apnea. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Forty pertinent studies were identified. For studies investigating whether noncommercial drivers with sleep apnea have increased crash rates, the majority (23 of 27 studies and 18 of 19 studies with control groups) found a statistically significant increased risk, with many of the studies finding a 2 to 3 times increased risk. Methodologic quality of the studies did not influence this relationship (p = .22). For commercial drivers, only 1 of 3 studies found an increased crash rate, with this association being weak (odds ratio of 1.3). The evidence was mixed regarding whether the risk of crash involvement is proportional to the severity of the sleep apnea, with about half of the studies finding a statistically significant increased risk with increased severity. Correlation with subjective daytime sleepiness and crash risk was also found in only half of the studies reviewed. Treatment of sleep apnea consistently improved driver performance (including crashes) across all studies. CONCLUSIONS: Noncommercial drivers with sleep apnea are at a statistically significant increased risk of involvement in motor vehicle crashes. Studies did not consistently find that daytime sleepiness and the severity of sleep apnea were correlated with crash risk. Successful treatment of sleep apnea improves driver performance. Clinicians should educate their patients with sleep apnea about the importance of treatment adherence for driving safety.

Thromboelastography: a means to transfusion reduction.

Consider using thromboelastography to improve care outcomes for cardiac surgery patients.