SSR MARKERS REVEALED GENETIC DIVERGENCE OF RICE BROWN PLANTHOPPER POPULATIONS MAINTAINED ON TWO SETS OF DIFFERENTIAL HOST VARIETIES

<p>Resistance screening of promising rice lines in Indonesia requires the use of brown planthopper (BPH) biotypes 1, 2, and 3. Three BPH populations have been raised as biotypes 1, 2, and 3 on differential rice host of improved varieties Pelita I-1 (no <em>Bph </em>gene), IR26 (<em>Bph1</em>), and IR42 (<em>bph2</em>), respectively. Three alternative populations have also been developed on the respective traditional varieties TN1 (no <em>Bph </em>gene), Mudgo (<em>Bph1</em>), and ASD7 (<em>bph2</em>). Although these populations displayed two virulent patterns other than biotype 1 to 3 phenotypes, they were expected to be discriminated into two virulence groups by SSR analysis. The study aimed to investigate the level of genetic variation among the six BPH populations using SSR markers and to relate it with the observed virulence patterns. Genotyping of 30 females with 29 polymorphic SSR markers revealed higher genetic parameter values in populations reared on improved varieties than those on traditional varieties. This difference was marked as two population clusters in PCoA plots corresponding to the host variety type, in contrast to the previous assumption that clustering would be based on virulence patterns. The presence of individuals with unwanted virulence allele, either resulting from contamination during the long period of rearing or lack of host adaptation period, is suspected. The result of this study indicates that the six populations are not suitable for resistance screening. Virulence selection must be performed until they attain biotype 1 to 3 phenotypes which can be genetically separated by DNA markers.</p>

Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test

The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the mecA gene, which encodes for the PBP2a protein and lends the resistance to β-lactams. Verifying the correspondence between gene harboring and protein expression and accelerating methicillin resistance diagnosis is critical to improve the management of antimicrobial administration and to reduce the spread of drug resistances. We tested the applicability of immunofluorescence targeting PBP2a protein to identify a new potential methicillin resistance screening test, ancillary to conventional culture methods. We collected 26 clinical Staphylococcus pseudintermedius (SP) isolates: 25 from canine pyoderma and 1 from dermatitis in a dog owner. SP is one of the most important etiological agents in canine pyoderma and can harbor the mecA gene. We performed PCR for mecA gene detection, broth microdilution (BMD) for phenotypic methicillin resistance, and immunofluorescence targeting PBP2a protein. Compared to the PCR as the gold standard, immunofluorescence showed an apparent prevalence of 34.6% vs. a true prevalence of 53.8%, with 100% specificity, 64.3% sensitivity, and 80.8% diagnostic accuracy. PBP2a expression showed isolate-dependent variability: in some isolates, most of the bacterial cells showed an intense and clearly membranous pattern, while in others only a few of them could be detected. Performing the assay in duplicate improved the diagnostic accuracy. Since the mecA gene is shared among the members of the Staphylococcus genus, the test can be applied to identify methicillin resistance independently from the staphylococcal species, both in human and animal samples. Being a rapid and easy method and providing the unique possibility to study the expression of PBP2a by directly visualizing the morphology, it could represent a new interesting tool for both research and diagnostics. To accelerate methicillin resistance diagnosis, it would be worth further testing of its performance on cytological samples.

Defining Fluoroquinolone Resistance-Mediating Mutations from Non-Resistance Polymorphisms in Mycoplasma hominis Topoisomerases

Often dismissed as a commensal, Mycoplasma hominis is an increasingly prominent target of research due to its role in septic arthritis and organ transplant failure in immunosuppressed patients, particularly lung transplantation. As a mollicute, its highly reductive genome and structure render it refractile to most forms of treatment and growing levels of resistance to the few sources of treatment left, such as fluoroquinolones. We examined antimicrobial susceptibility (AST) to fluoroquinolones on 72 isolates and observed resistance in three (4.1%), with corresponding mutations in the quinolone resistance-determining region (QRDR) of S83L or E87G in gyrA and S81I or E85V in parC. However, there were high levels of polymorphism identified between all isolates outside of the QRDR, indicating caution for a genomics-led approach for resistance screening, particularly as we observed a further two quinolone-susceptible isolates solely containing gyrA mutation S83L. However, both isolates spontaneously developed a second spontaneous E85K parC mutation and resistance following prolonged incubation in 4 mg/L levofloxacin for an extra 24–48 h. Continued AST surveillance and investigation is required to understand how gyrA QRDR mutations predispose M. hominis to rapid spontaneous mutation and fluoroquinolone resistance, absent from other susceptible isolates. The unusually high prevalence of polymorphisms in M. hominis also warrants increased genomics’ surveillance.

Virulence and SSR Diversity of Brown Planthopper (Nilaparvata lugens) Adapted on Differential Rice Host Varieties

Brown planthopper biotype 1, 2, 3 and a representative field population are required for resistance screening of promising rice lines in Indonesia, but the current biotype stocks has shown deviation in virulence patterns. The objectives of this study were to develop a set of brown planthopper populations with differential virulence and to investigate their genetic variability using SSR marker. Females originated from two field populations were selected on variety Mudgo (carries Bph1 gene) or ASD7 (bph2 gene) using honeydew excretion as the virulence parameter. Selection cycles resulted in population T, M, A, and R, which was raised and adapted on variety TN1 (carries no Bph gene), Mudgo, ASD7, and Rathu Heenathi (Bph3, Bph17), respectively. Population R was the most virulent as expected and can be used to represent a field population, but the remaining populations still showed high virulence level. AMOVA and PCoA results based on analysis with 38 SSR primer pairs revealed partial genetic separation among populations, with population R was the most genetically distant from the remaining populations. The desired virulence character of the remaining populations is expected could be achieved after further selection and prolonged adaptation on their respective hosts.

Dressed chicken as potential vehicle for spread of methicillin-resistant Staphylococcus aureus in Sokoto, Nigeria

Aim: To evaluate the role of dressed chicken in the spread of methicillin-resistant Staphylococcus aureus (MRSA) in Sokoto, Nigeria. Materials and methods: 190 chicken carcass rinsates were subjected to culture and biochemical analyses to isolate and identify MRSA. PCR was used to amplify mecA gene that is responsible for methicillin resistance. Results & conclusion: Culture and molecular analysis showed 19.5% (37/190) of the rinse had MRSA on oxacillin-resistance screening agar base (ORSAB) with 7.9% (15/190) possessing the mecA gene. Significant association (p = 0.044) exist between local-chicken and presence of MRSA, being twice more likely to have MRSA compared to exotic-chickens (odds ratio [OR] = 2.132). Results indicate possible role of dressed-chicken in the spread of MRSA. Authorities should regulate the sale and use of antibiotics by farmers, and enhance hygienic practices at slaughterhouses.

Screening for Sarawak Paddy Landraces with Resistance to Yellow Rice Stem Borer, Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae)

The yellow rice stem borer, Scirpophaga incertulas (Walker) is a prevalent pest in paddy fields worldwide. In Sarawak, a survey on pest of paddy carried out from 2009 to 2011 covering 166 paddy fields revealed that rice stem borers caused 11.4% of total paddy damage. In order to reduce the damage, identifying resistance paddy variety is crucial. The objective of this study was to screen Sarawak paddy landraces with resistance to S. incertulas. Twelve Sarawak paddy landraces were selected randomly for this study. Antixenosis resistance screening was performed in aquariums (60 x 28 x 33 cm). Three replications of one-month-old seedlings were randomly arranged in aquarium and exposed to adult S. incertulas. The number and position of egg mass on each plant were recorded. Egg mass abnormalities were also observed. For antibiosis, rice culms of two-month old seedlings from each landrace were infested with larvae. The length of surviving larvae from five rice culms was measured. The experiment revealed variations in landrace of preference for ovipositing. Among the twelve Sarawak paddy landraces, Kanowit was more preferred by S. incertulas for ovipositing in comparison to other paddy landraces suggesting susceptibility towards the pest. Abaxial leaf surface was the preferred oviposition site. There was no clear antibiosis response of the paddy landraces towards S. incertulas larvae in this study.

Settleable Dust and Bioburden in Portuguese Dwellings

Monitoring campaigns in several buildings have shown that occupants exposed to contaminated indoor air generally exhibit diverse health symptoms. This study intends to assess settleable dust loading rates and bioburden in Portuguese dwellings by passive sampling onto quartz fiber filters and electrostatic dust cloths (EDCs), respectively. Settled dust collected by EDCs was analyzed by culture-based methods (including azole-resistance screening) and qPCR, targeting four different toxigenic Aspergillus sections (Flavi, Fumigati, Circumdati, and Nidulantes). Dust loading rates and bioburden showed higher variability in the summer season. In both seasons, Penicillium sp. was the one with the highest prevalence (59.1% winter; 58.1% summer), followed by Aspergillus sp. in winter (13.0%). Fungal contamination increased in the winter period, while bacterial counts decreased. Aspergillus sections Circumdati and Nidulantes, detected in voriconazole supplemented media, and Aspergillus sections Fumigati and Nidulantes, detected by molecular tools, were found in the winter samples. This study reinforces the importance of applying: (a) Passive sampling methods in campaigns in dwellings; (b) two different culture media (MEA and DG18) to assess fungi; (c) in parallel, molecular tools targeting the most suitable indicators of fungal contamination; and (d) azole resistance screening to unveil azole resistance detection in fungal species.