Nicola K. Beck

A novel transposon, Tn6009, composed of a Tn916 element linked with a Staphylococcus aureus mer operon

OBJECTIVES The aim of this study was to characterize a novel conjugative transposon Tn6009 composed of a Tn916 linked to a Staphylococcus aureus mer operon in representative Gram-positive and Gram-negative bacteria isolated in Nigeria and Portugal. METHODS Eighty-three Gram-positive and 34 Gram-negative bacteria were screened for the presence of the Tn6009 using DNA-DNA hybridization, PCR, hybridization of PCR products, sequencing and mating experiments by established procedures. RESULTS Forty-three oral and 23 urine Gram-negative and Gram-positive isolates carried the Tn6009. Sequencing was performed to verify the direct linkage between the mer resistance genes and the tet(M) gene. A Nigerian Klebsiella pneumoniae, isolated from a urinary tract infection patient, and one commensal isolate from each of the other Tn6009-positive genera, Serratia liquefaciens, Pseudomonas sp., Enterococcus sp. and Streptococcus sp. isolated from the oral and urine samples of healthy Portuguese children, were able to act as donors and conjugally transfer the Tn6009 to the Enterococcus faecalis JH2-2 recipient, resulting in tetracycline- and mercury-resistant E. faecalis transconjugants. CONCLUSIONS This study reports a novel non-composite conjugative transposon Tn6009 containing a Tn916 element linked to an S. aureus mer operon carrying genes coding for inorganic mercury resistance (merA), an organic mercury resistance (merB), a regulatory protein (merR) and a mercury transporter (merT). This transposon was identified in 66 isolates from two Gram-positive and three Gram-negative genera and is the first transposon in the Tn916 family to carry the Gram-positive mer genes directly linked to the tet(M) gene.

Development of a novel bag-mediated filtration system for environmental recovery of poliovirus

Poliovirus (PV) is on the verge of global eradication. Due to asymptomatic shedding, eradication certification requires environmental and clinical surveillance. Current environmental surveillance methods involve collection and processing of 400-mL to 1-L grab samples by a two-phase separation method, where sample volume limits detection sensitivity. Filtration of larger sample volumes facilitates increased detection sensitivity. This study describes development of a pumpless in-field filtration system for poliovirus recovery from environmental waters. Recovery of PV types 1, 2, and 3 were compared for glass wool, ViroCap, and NanoCeram (PV1 only) filters. Seeded experiments were performed using 10(5) plaque forming units of PV inoculated into 10-L volumes of secondary effluent, surface water, or a 50:50 mixture of each at pH 7.0. Filter eluates were plated onto buffalo green monkey kidney cells for virus enumeration by plaque assay. Across all water types, recovery from glass wool filters for PV1, PV2, and PV3 averaged 17%, 28%, and 6%, respectively. Recovery from ViroCaps for PV1, PV2, and PV3 averaged 44%, 70%, and 81%, respectively. 10-L samples of moderate turbidity water were processed through ViroCap filters in less than 30 minutes using a pumpless, bag-mediated filtration system. Bag-mediated filtration offers a simple, compact, and efficient method for enhanced environmental PV surveillance.

Inactivation of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) on various environmental surfaces by mist application of a stabilized chlorine dioxide and quaternary ammonium compound-based disinfectant.

Contaminated environmental surfaces are important reservoirs in the transmission of many human pathogens. Although several options exist for disinfecting contaminated environmental surfaces, few are compatible with use on both hard smooth non-porous (hard) and soft porous surfaces (soft) while still offering significant disinfection of the contaminating organisms. This study evaluated the efficacy of mist application of a stabilized chlorine dioxide and quaternary ammonium compound-based disinfectant (Cryocide20) for inactivation of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) on various environmental surfaces. MRSA and VRE were applied to hard and soft surfaces (glass, steel, tile, carpet, and cotton fabric), allowed to dry, and exposed to a uniform mist application of the disinfectant solution. After 1 hr of contact time, the residual disinfectant was neutralized, and the bacteria were recovered and enumerated on brain heart infusion (BHI) agar. Reduction of both test bacteria was observed on most of the hard and soft surfaces tested. Log10 reduction of the organisms tended to be higher on steel, tile, and carpet than glass or cotton. Overall, these results suggest that mist application of Cryocide20 disinfectant may be an effective option for reduction of low levels of infectious bacterial pathogens from contaminated environmental surfaces.

Extraction of MS2 Phage RNA from Upper Respiratory Tract Specimens by Use of Flat Glass Devices

ABSTRACT The isolation of pure nucleic acids from clinical samples is a crucial step in the molecular diagnosis of viral infections by nucleic acid testing (NAT). In this study, novel flat glass devices (cards) were demonstrated to support the rapid and efficient extraction of nucleic acids from upper respiratory tract specimens (nasal washes and swabs). The performance of the nucleic acid extraction cards was directly compared to an existing standardized and automated platform for viral extraction from these types of specimens. The flowthrough card method improved the speed of nucleic acid purification and accommodated larger sample volumes in extraction of bacteriophage MS2 RNA from the various specimen matrices. The dynamic range and estimated sensitivity of the card extraction method for reverse transcriptase quantitative real-time PCR (RT-qPCR)-based detection approximate those of the standardized magnetic glass bead extraction method used in this study.

Evaluation of the bag-mediated filtration system as a novel tool for poliovirus environmental surveillance: Results from a comparative field study in Pakistan

Poliovirus (PV) environmental surveillance (ES) plays an important role in the global eradication program and is crucial for monitoring silent PV circulation especially as clinical cases decrease. This study compared ES results using the novel bag-mediated filtration system (BMFS) with the current two-phase separation method. From February to November 2016, BMFS and two-phase samples were collected concurrently from twelve sites in Pakistan (n = 117). Detection was higher in BMFS than two-phase samples for each Sabin-like (SL) PV serotype (p<0.001) and wild PV type 1 (WPV1) (p = 0.065). Seventeen sampling events were positive for WPV1, with eight discordant in favor of BMFS and two in favor of two-phase. A vaccine-derived PV type 2 was detected in one BMFS sample but not the matched two-phase. After the removal of SL PV type 2 (SL2) from the oral polio vaccine in April 2016, BMFS samples detected SL2 more frequently than two-phase (p = 0.016), with the last detection by either method occurring June 12, 2016. More frequent PV detection in BMFS compared to two-phase samples is likely due to the greater effective volume assayed (1620 mL vs. 150 mL). This study demonstrated that the BMFS achieves enhanced ES for all PV serotypes in an endemic country.

Evaluation of micro-well collector for capture and analysis of aerosolized Bacillus subtilis spores

Bioaerosol sampling and identification are vital for the assessment and control of airborne pathogens, allergens, and toxins. In-situ analysis of chemical and biological particulate matter can significantly reduce the costs associated with sample preservation, transport, and analysis. The analysis of conventional filters is challenging, due to dilute samples in large collection regions. A low-cost cartridge for collection and analysis of aerosols is developed for use in epidemiological studies and personal exposure assessments. The cartridge collects aerosol samples in a micro-well which reduces particles losses due to the bounce and does not require any coating. The confined particle collection area (dwell~1.4 mm) allows reducing the elution volume for subsequent analysis. The performance of the cartridge is validated in laboratory studies using aerosolized bacterial spores (Bacillus subtilis). Colony forming unit analysis is used for bacterial spore enumeration. Cartridge collection efficiency is evaluated by comparison with the reference filters and found to be consistent with tested flow rates. Sample recovery for the pipette elution is ~80%. Due to the high density of the collected sample, the cartridge is compatible with in-situ spectroscopic analysis and sample elution into the 10–20 μl liquid volume providing a significant increase in sample concentration for subsequent analysis.