Multiplex PCR reveals unique trends in pathogen and parasitoid infestations of alfalfa leafcutting brood cells.

The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. Megachile rotundata is a solitary bee that forms brood cocoons where its larvae can develop. During the developmental stages of growth, broods can be preyed upon by multiple different fungal and bacterial pathogens and insect predators and parasitoids, resulting in the loss of the developing larvae. Larval loss is a major concern for alfalfa (Medicago sativa L.) seed producers because they rely on pollinator services provided by Megachile rotundata and reduced pollination rates result in lower yields and increased production costs. In the present study, we examined the taxonomic composition of organisms found within M. rotundata brood cells using a multiplex PCR assay which was developed for the detection of the most common bacterial, fungal, and invertebrate pests and pathogens of M. rotundata larvae. Known pests of M. rotundata were detected, including members of the fungal genus Ascosphaera, the causative agent of chalkbrood. Co-infection of single brood cells by multiple Ascosphaera species was confirmed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species including multiple new predators and pathogenetic species not previously documented in associated with M. rotundata brood cells.

Predominance of Acinetobacter spp., Harboring the blaIMP Gene, Contaminating the Hospital Environment in a Tertiary Hospital in Mwanza, Tanzania: A Cross-Sectional Laboratory-Based Study

Data on colonization and hospital contamination of carbapenem-resistant Gram-negative bacteria (CR-GNB) are limited in low- and middle-income countries. We designed this study to determine the prevalence and co-existence of carbapenemase genes among CR-GNB isolated from clinical, colonization, and hospital environmental samples at a tertiary hospital in Mwanza, Tanzania. The modified Hodge test (MHT), the combined disk test (CDT), and the double-disk synergy test (DDST) were used for the phenotypic detection of carbapenemases. A multiplex PCR assay was used to detect blaIMP and blaKPC, and a singleplex PCR assay was used to detect blaOXA-48. Data were analyzed by STATA version 13.0. Overall, 68.8% (44/64) of the CR-GNB had at least one phenotype by phenotypic methods, whereby 60.9% (39/64) were both CDT and DDST positive and 31.3% (20/64) were MHT positive. A total of 23/64 (35.9%) had at least one of the genes tested with the predominance of blaIMP (91.3%; 21/23). In addition, 47.7% (21/44) of the CR-GNB phenotypes had at least one gene. Around 47.8% (11/23) of the CR-GNB carried multiple genes encoding for carbapenem resistance, with the maximum co-existence of blaIMP/blaKPC/blaOXA-48 (45.5%; 5/11). The majority of carbapenem-resistant genes were detected in Acinetobacter spp. (82.6%; 19/23) and isolated from bed swabs (69.6%; 16/23). Acinetobacter spp. carrying the blaIMP gene predominantly contaminated the hospital environment. Therefore, we recommend routine decontamination of inanimate hospital surfaces, including patient beds.

Mannheimia haemolytica serovars associated with respiratory disease in cattle in Great Britain

Background Mannheimia haemolytica is commonly associated with respiratory disease in cattle worldwide as a cause of fibrinous pneumonia, bronchopneumonia and pleuritis. M. haemolytica is further subdivided into 12 serovars, however not all are considered to be pathogenic in cattle. The study aim was to determine the most common serovars of M. haemolytica associated with respiratory disease in cattle in Great Britain, which is currently unknown and could be useful information for clinicians when considering preventative strategies. Results One hundred four M. haemolytica isolates isolated from bovine clinical pathology and post-mortem samples from pneumonia cases between 2016 and 2018 were tested using a multiplex PCR assay to identify M. haemolytica serovars A1, A2 and A6. 46 isolates (44.2%) typed as M. haemolytica serovar A1, 31 (29.8%) as M. haemolytica serovar A2 and 18 isolates (17.3%) as M. haemolytica serovar A6. Nine isolates (8.7%) were not A1, A2 or A6 so were considered to belong to other serovars or were not typable. Conclusion This study highlights the importance of M. haemolytica serovars other than A1 which may be responsible for respiratory disease in cattle and could help guide the veterinarian when making choices on preventative vaccination programmes.

A simple RT-multiplex PCR method for diagnosis of L-A and M totiviruses in Saccharomyces cerevisiae

Killer yeasts and their toxins have many potential applications in environmental, medical and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait is usually performed phenotypically, on the basis of their toxin production and immunity. In this study, we describe a simple and specific RT-multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated to the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primers addition to the RT reaction. Using a mixture of specific primers at the PCR step, this RT-multiplex PCR protocol provides accurate diagnosis of both L-A and M totivirus in all its known variants L-A-1/M1, L-A-2/M2, L-A-28/M28 and L-A-lus/Mlus to be found in infected killer yeasts. By means of this method, expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified, but importantly, asymptomatic L-A-2/M2 infected cells, as well as unexpected L-A-lus/M2 totiviral associations, were also found. Importance The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, development of simple methods for their detection significantly facilitates their study. The simplified RT-multiplex PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depends on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provides valuable insights into L-A/M viral associations and their coadaptional events in nature.

Monitoring Sexually Transmitted Infections in Cervicovaginal Exfoliative Samples in Mexican Women

Background. Globally, Sexually Transmitted Infections (STIs) are a major cause of morbidity in sexually active individuals, having complications in reproduction health and quality of life. In concordance with the Sustainable Development Goals (SDG), the study aimed to investigate the prevalence of Candida spp., Ureaplasma spp., Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, HSV, and Mycoplasma spp. from cervicovaginal samples and to correlate them with the gynecological history of the patients. Methods. Our analytical, prospective, and cross-sectional study included 377 women who participated in a reproductive health campaign during 2015–2016. Anthropometric and gynecological variables were obtained. Cervicovaginal specimens were collected and analyzed with a multiplex in-house PCR to detect Candida spp., Ureaplasma spp., Trichomonas vaginalis, Neisseria gonorrhoeae, HSV, Mycoplasma spp., and Chlamydia trachomatis. Results. The positive cases were 175/377 (46.4%) to at least one of the microorganisms. The most frequent pathogen detected in this population was Ureaplasma spp. (n = 111, 29.4%), followed by Mycoplasma spp. (n = 56, 14.9%) and Candida spp. (n = 47, 12.5%); 33.7% of the positive cases were single infections, whereas 12.7% had coinfection. The multiplex PCR assay was designed targeting nucleotide sequences. Conclusions. Our data demonstrated that monitoring STIs among asymptomatic patients will encourage target programs to be more precisely and effectively implemented, as well as make these programs more affordable, to benefit society by decreasing the prevalence of STIs.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.