Structural and functional characterization of genes PYL-PP2C-SnRK2s in the ABA signalling pathway of Cucurbita pepo.

BACKGROUND: The core regulation of the abscisic acid (ABA) signalling pathway comprises the multigenic families PYL, PP2C, and SnRK2. In this work, we conducted a genome-wide study of the components of these families in Cucurbita pepo. RESULTS: The bioinformatic analysis of the C. pepo genome resulted in the identification of 19 CpPYL, 102 CpPP2C and 10 CpSnRK2 genes. The investigation of gene structure and protein motifs allowed to define 4 PYL, 13 PP2C and 3 SnRK2 subfamilies. RNA-seq analysis was used to determine the expression of these gene families in different plant organs, as well as to detect their differential gene expression during germination, and in response to ABA and cold stress in leaves. The specific tissue expression of some gene members indicated the relevant role of some ABA signalling genes in plant development. Moreover, their differential expression under ABA treatment or cold stress revealed those ABA signalling genes that responded to ABA, and those that were up- or down-regulated in response to cold stress. A reduced number of genes responded to both treatments. Specific PYL-PP2C-SnRK2 genes that had potential roles in germination were also detected, including those regulated early during the imbibition phase, those regulated later during the embryo extension and radicle emergence phase, and those induced or repressed during the whole germination process. CONCLUSIONS: The outcomes of this research open new research lines for agriculture and for assessing gene function in future studies.

A miR164 resistant mutation in the transcription factor gene CpCUC2B enhances carpel arrest and ectopic boundary specification in Cucurbita pepo flower development.

Sex determination process in cucurbits involves the control of stamen or carpel development during the specification of male or female flowers from a bisexual floral meristem, a function coordinated by ethylene. A gain-of-function mutation in the miR164-binding site of CpCUC2B, ortholog of the Arabidopsis transcription factor gene CUC2, not only produced ectopic floral meristems and organs, but also suppressed the development of carpels and promotes the development of stamens. The cuc2b mutation induced the transcription of CpCUC2B in the apical shoots of plants after female flowering but repressed other CUC genes regulated by miR164, suggesting a conserved functional redundancy of these genes in the development of squash flowers. The synergistic androecious phenotype of the double mutant between cuc2b and etr2b, an ethylene insensitive mutation that enhances the production of male flowers, demonstrated that CpCUC2B arrests the development of carpels independently of ethylene and CpWIP1B. The transcriptional regulation of CpCUC1, CpCUC2, and ethylene genes in cuc2b and ethylene mutants also confirms this conclusion. However, the epistasis of cuc2b over aco1a, a mutation that suppresses stamen arrest in female flowers, and the down-regulation of CpACS27A in cuc2b female apical shoots, indicated that CpCUC2B promotes stamen development by suppressing the late ethylene production.

Comparative RNA-Seq Analysis between Monoecious and Androecious Plants Reveals Regulatory Mechanisms Controlling Female Flowering in Cucurbita pepo.

In the monoecious Cucurbita pepo, the transition to female flowering is the time at which the plant starts the production of female flowers after an initial male phase of development. Ethylene plays an essential role in this process since some ethylene deficient and ethylene-insensitive mutants are androecious and only produce male flowers. To gain insight into the molecular mechanisms regulating the specification and early development of female flowers, we have compared the transcriptomic changes occurring in the shoot apices of WT and androecious ethylene-insensitive etr1b mutant plants upon female flowering transition. There were 1160 female flowering-specific DEGs identified in WT plants upon female flowering, and 284 of them were found to be modulated by the ethylene-insensitive etr1b mutation. The function of these DEGs indicated that female flower specification depends on the adoption of a transcriptional program that includes previously identified sex-determining genes in the ethylene pathway, but also genes controlling the biosynthesis and signaling pathways of other phytohormones, and those encoding for many different transcription factors. The transcriptomic changes suggested that gibberellins play a negative role in female flowering, while ethylene, auxins, ABA and cytokinins are positive regulators. Transcription factors from 34 families, including NAC, ERF, bHLH, bZIP, MYB and C2H2/CH3, were found to be regulating female flowering in an ethylene-dependent or -independent manner. Our data open a new perspective of the molecular mechanisms that control the specification and development of female flowers in C. pepo.

Genomic analysis of Chlamydia psittaci from a multistate zoonotic outbreak in two chicken processing plants.

Four Chlamydia psittaci isolates were recovered from clinical specimens from ill workers during a multistate outbreak at two chicken processing plants. Whole genome sequencing analyses revealed high similarity to C. psittaci genotype D. The isolates differed from each other by only two single nucleotide polymorphisms, indicating a common source.

Multiplex Real-Time Reverse Transcription PCR for Influenza A Virus, Influenza B Virus, and Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, and the outbreak rapidly evolved into the current coronavirus disease pandemic. SARS-CoV-2 is a respiratory virus that causes symptoms similar to those caused by influenza A and B viruses. On July 2, 2020, the US Food and Drug Administration granted emergency use authorization for in vitro diagnostic use of the Influenza SARS-CoV-2 Multiplex Assay. This assay detects influenza A virus at 102.0, influenza B virus at 102.2, and SARS-CoV-2 at 100.3 50% tissue culture or egg infectious dose, or as few as 5 RNA copies/reaction. The simultaneous detection and differentiation of these 3 major pathogens increases overall testing capacity, conserves resources, identifies co-infections, and enables efficient surveillance of influenza viruses and SARS-CoV-2.

Use of Real-Time PCR for Chlamydia psittaci Detection in Human Specimens During an Outbreak of Psittacosis - Georgia and Virginia, 2018.

Psittacosis is typically a mild febrile respiratory illness caused by infection with the bacterium Chlamydia psittaci and usually transmitted to humans by infected birds (1). On average, 11 psittacosis cases per year were reported in the United States during 2000-2017. During August-October 2018, the largest U.S. psittacosis outbreak in 30 years (82 cases identified*) occurred in two poultry slaughter plants, one each in Virginia and Georgia, that shared source farms (2). CDC used C. psittaci real-time polymerase chain reaction (PCR) to test 54 human specimens from this outbreak. This was the largest number of human specimens from a single outbreak ever tested for C. psittaci using real-time PCR, which is faster and more sensitive than commercially available serologic tests. This represented a rare opportunity to assess the utility of multiple specimen types for real-time PCR detection of C. psittaci. C. psittaci was detected more frequently in lower respiratory specimens (59% [10 of 17]) and stool (four of five) than in upper respiratory specimens (7% [two of 28]). Among six patients with sputum and nasopharyngeal swabs tested, C. psittaci was detected only in sputum in five patients. Cycle threshold (Ct) values suggested bacterial load was higher in lower respiratory specimens than in nasopharyngeal swabs. These findings support prioritizing lower respiratory specimens for real-time PCR detection of C. psittaci. Stool specimens might also have utility for diagnosis of psittacosis.

Concurrent tonic pupil and trochlear nerve palsy in COVID-19.

Since COVID-19 was first reported, different neurological complications have been acknowledged, but their description is constantly evolving. We report a case of concurrent tonic pupil and trochlear nerve palsy in this context. A 62-year-old man reported a 5-day history of binocular vertical diplopia and blurred vision in his left eye, noticing that his left pupil was dilated. He had suffered a flu-like syndrome 2 weeks before. Clinical exam showed a right trochlear nerve palsy and a left mydriatic pupil. MRI, X chest ray, and analytical results were normal. Antibodies for SARS-CoV-2 were positive (low IgM and high IgG titers). Antiganglioside antibodies were negative. A 0.125% pilocarpine test confirmed Adie's pupil diagnosis. The patient was treated with a tapered prednisone dose with resolution of his diplopia but no change in Adie's pupil. This is the first case reporting Adie's pupil as a postinfectious manifestation of COVID-19. An immune-mediated mechanism is presumed.

Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae.

We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, the BioFire FilmArray respiratory panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex respiratory pathogen panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB research use only (RUO) PCR, and the SpeeDx Resistance Plus MP assays. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed positives for M. pneumoniae The highest clinical sensitivities were found with the InGenius PCR (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6, 64.6, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (P < 0.05). Specificities of all assays were 99.5 to 100%. The Resistance Plus MP assay detected macrolide resistance in 27/33 specimens, resulting in a sensitivity of 81.8%. This study provides the first large-scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of various test performances on patient outcome.

Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30-45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.

Epidemiology and Molecular Identification and Characterization of Mycoplasma pneumoniae, South Africa, 2012-2015.

During 2012-2015, we tested respiratory specimens from patients with severe respiratory illness (SRI), patients with influenza-like illness (ILI), and controls in South Africa by real-time PCR for Mycoplasma pneumoniae, followed by culture and molecular characterization of positive samples. M. pneumoniae prevalence was 1.6% among SRI patients, 0.7% among ILI patients, and 0.2% among controls (p<0.001). Age <5 years (adjusted odd ratio 7.1; 95% CI 1.7-28.7) and HIV infection (adjusted odds ratio 23.8; 95% CI 4.1-138.2) among M. pneumonia-positive persons were associated with severe disease. The detection rate attributable to illness was 93.9% (95% CI 74.4%-98.5%) in SRI patients and 80.7% (95% CI 16.7%-95.6%) in ILI patients. The hospitalization rate was 28 cases/100,000 population. We observed the macrolide-susceptible M. pneumoniae genotype in all cases and found P1 types 1, 2, and a type 2 variant with multilocus variable number tandem repeat types 3/6/6/2, 3/5/6/2, and 4/5/7/2.

Correction: Comprehensive bioinformatics analysis of Mycoplasma pneumoniae genomes to investigate underlying population structure and type-specific determinants.

[This corrects the article DOI: 10.1371/journal.pone.0174701.].

Mycoplasma hominis Infections Transmitted Through Amniotic Tissue Product.

Background: Mycoplasma hominis is a commensal genitourinary tract organism that can cause infections outside the genitourinary tract. We investigated a cluster of M. hominis surgical site infections in patients who underwent spine surgery, all associated with amniotic tissue linked to a common donor. Methods: Laboratory tests of tissue product from the donor, including culture, quantitative real-time polymerase chain reaction (qPCR), and whole-genome sequencing were performed. Use of this amniotic tissue product was reviewed. A multistate investigation to identify additional cases and locate any unused products was conducted. Results: Twenty-seven tissue product vials from a donor were distributed to facilities in 7 states; at least 20 vials from this donor were used in 14 patients. Of these, 4 of 14 (29%) developed surgical site infections, including 2 M. hominis infections. Mycoplasma hominis was detected by culture and qPCR in 2 unused vials from the donor. Sequencing indicated >99% similarity between patient and unopened vial isolates. For 5 of 27 (19%) vials, the final disposition could not be confirmed. Conclusions: Mycoplasma hominis was transmitted through amniotic tissue from a single donor to 2 recipients. Current routine donor screening and product testing does not detect all potential pathogens. Clinicians should be aware that M. hominis can cause surgical site infections, and may not be detected by routine clinical cultures. The lack of a standardized system to track tissue products in healthcare facilities limits the ability of public health agencies to respond to outbreaks and investigate other adverse events associated with these products.

Molecular Characterization of Mycoplasma pneumoniae Infections in Two Rural Populations of Thailand from 2009 to 2012.

Studies on Mycoplasma pneumoniae in Thailand have focused on urban centers and have not included molecular characterization. In an attempt to provide a more comprehensive understanding of this organism, we conducted a systematic random sampling to identify 3,000 nasopharyngeal swab specimens collected from January 2009 through July 2012 during population-based surveillance for influenza-like illness in two rural provinces. M. pneumoniae was detected by real-time PCR in 175 (5.8%) specimens. Genotyping was performed using the major adhesion protein (P1) and multilocus variable-number tandem-repeat analysis (MLVA). Of the 157 specimens typed, 97 were P1 type 1 and 60 were P1 type 2. Six different MLVA profiles were identified in 149 specimens, with 4/5/7/2 (40%) and 3/5/6/2 (26%) predominating. There was no discrete seasonality to M. pneumoniae infections. Examination of the 23S rRNA sequence for known polymorphisms conferring macrolide resistance revealed that all 141 tested to possess the genotype associated with macrolide susceptibility.

Comprehensive bioinformatics analysis of Mycoplasma pneumoniae genomes to investigate underlying population structure and type-specific determinants.

Mycoplasma pneumoniae is a significant cause of respiratory illness worldwide. Despite a minimal and highly conserved genome, genetic diversity within the species may impact disease. We performed whole genome sequencing (WGS) analysis of 107 M. pneumoniae isolates, including 67 newly sequenced using the Pacific BioSciences RS II and/or Illumina MiSeq sequencing platforms. Comparative genomic analysis of 107 genomes revealed >3,000 single nucleotide polymorphisms (SNPs) in total, including 520 type-specific SNPs. Population structure analysis supported the existence of six distinct subgroups, three within each type. We developed a predictive model to classify an isolate based on whole genome SNPs called against the reference genome into the identified subtypes, obviating the need for genome assembly. This study is the most comprehensive WGS analysis for M. pneumoniae to date, underscoring the power of combining complementary sequencing technologies to overcome difficult-to-sequence regions and highlighting potential differential genomic signatures in M. pneumoniae.

Multi-center evaluation of one commercial and 12 in-house real-time PCR assays for detection of Mycoplasma pneumoniae.

Detection of Mycoplasma pneumoniae by real-time PCR is not yet standardized across laboratories. We have implemented a standardization protocol to compare the performance of thirteen commercial and in-house approaches. Despite differences on threshold values of samples, all assays were able to detect at least 20M. pneumoniae genomes per reaction.

Complete Genome Sequence of Mycoplasma pneumoniae Type 2 Reference Strain FH Using Single-Molecule Real-Time Sequencing Technology.

Mycoplasma pneumoniae type 2 strain FH was previously sequenced with Illumina (FH-Illumina) and 454 (FH-454) technologies according to Xiao et al. (2015) and Krishnakumar et al. (2010). Comparative analyses revealed differences in genomic content between these sequences, including a 6-kb region absent from the FH-454 submission. Here, we present a complete genome sequence of FH sequenced with the Pacific Biosciences RSII platform.

Epidemiology and Molecular Characteristics of Mycoplasma pneumoniae During an Outbreak of M. pneumoniae-associated Stevens-Johnson Syndrome.

BACKGROUND: An increase in Mycoplasma pneumoniae-associated Stevens-Johnson syndrome (SJS) cases at a Colorado pediatric hospital led to an outbreak investigation. We describe the epidemiologic and molecular characteristics of M. pneumoniae among SJS case-patients and surrounding community members during the outbreak. METHODS: M. pneumoniae polymerase chain reaction-positive respiratory specimens from 5 Colorado hospitals and 4 referral laboratories underwent confirmatory polymerase chain reaction testing; positive specimens then underwent multilocus variable-number tandem-repeat analysis (MLVA) and macrolide resistance testing. Three SJS-M. pneumoniae case-patient households were surveyed using a standardized questionnaire, and nasopharyngeal/oropharyngeal swabs were obtained from all consenting/assenting household contacts. International Classification of Diseases, 9th revision codes were used to identify pneumonia cases among Colorado patients 5-21 years of age from January 2009 to March 2014. RESULTS: Three different M. pneumoniae MLVA types were identified among the 5 SJS case-patients with confirmed infection; MLVA type 3-X-6-2 was seen more commonly in SJS case-patients (60%) than in 69 non-SJS community specimens (29%). Macrolide resistance was identified in 7% of community specimens but not among SJS case-patients. Of 15 household contacts, 5 (33%) were M. pneumoniae positive; all MLVA types were identical to those of the corresponding SJS case-patient, although the specimen from 1 contact was macrolide resistant. Overall pneumonia cases as well as those caused by M. pneumoniae specifically peaked in October 2013, coinciding with the SJS outbreak. CONCLUSIONS: The outbreak of M. pneumoniae-associated SJS may have been associated with a community outbreak of M. pneumoniae; clinicians should be aware of the M. pneumoniae-SJS relationship. Household transmission of M. pneumoniae was common within the households investigated.

Development of a multiplex taqMan real-time PCR assay for typing of Mycoplasma pneumoniae based on type-specific indels identified through whole genome sequencing.

We developed a multiplex real-time PCR assay for simultaneously detecting M. pneumoniae and typing into historically-defined P1 types. Typing was achieved based on the presence of short type-specific indels identified through whole genome sequencing. This assay was 100% specific compared to existing methods and may be useful during epidemiologic investigations.

Identification of Bacterial and Viral Codetections With Mycoplasma pneumoniae Using the TaqMan Array Card in Patients Hospitalized With Community-Acquired Pneumonia.

Mycoplasma pneumoniae was detected in a number of patients with community-acquired pneumonia in a recent prospective study. To assess whether other pathogens were also detected in these patients, TaqMan Array Cards were used to test 216 M pneumoniae-positive respiratory specimens for 25 additional viral and bacterial respiratory pathogens. It is interesting to note that 1 or more codetections, predominantly bacterial, were identified in approximately 60% of specimens, with codetections being more common in children.