Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters.

Waterborne diseases are known as a leading cause of illness and death in both developing and developed countries. Several pathogens can be present in contaminated water, particularly waters containing faecal material; however, routine monitoring of all pathogens is not currently possible. Enterococcus faecalis, which is present in the microflora of human and animals has been used as a faecal indicator in water due to its abundance in surface water and soil. Accurate and fast detection methods are critical for the effective monitoring of E. faecalis in the environment. Although conventional and current molecular detection techniques provide sufficient sensitivity, specificity and throughput, their use is hampered by the long waiting period (1-6 days) to obtain results, the need for expensive laboratory equipment, skilled personnel, and cold-chain storage. Therefore, this study aimed to develop a detection system for E. faecalis that would be simple, rapid, and low-cost, using an isothermal DNA amplification assay called recombinase polymerase amplification (RPA), integrated with a lateral flow assay (LFA). The assay was found to be 100% selective for E. faecalis and capable of detecting rates as low as 2.8 × 103 cells per 100 mL from water and wastewater, and 2.8 × 104 cells per 100 mL from saline water. The assay was completed in approximately 30 min using one constant temperature (38 °C). In addition, this study demonstrated the quantitation of E. faecalis using a lateral flow strip reader for the first time, enhancing the potential use of RPA assay for the enumeration of E. faecalis in wastewater and heavily contaminated environmental waters, surface water, and wastewater. However, the sensitivity of the RPA-LFA assay for the detection of E. faecalis in tap water, saline water and in wastewater was 10-1000 times lower than that of the Enterolert-E test, depending on the water quality. Nevertheless, with further improvements, this low-cost RPA-LFA may be suitable to be used at the point-of-need (PON) if conjugated with a rapid field-deployable DNA extraction method.

The Mechanisms of Disease Caused by Acinetobacter baumannii.

Acinetobacter baumannii is a Gram negative opportunistic pathogen that has demonstrated a significant insurgence in the prevalence of infections over recent decades. With only a limited number of "traditional" virulence factors, the mechanisms underlying the success of this pathogen remain of great interest. Major advances have been made in the tools, reagents, and models to study A. baumannii pathogenesis, and this has resulted in a substantial increase in knowledge. This article provides a comprehensive review of the bacterial virulence factors, the host immune responses, and animal models applicable for the study of this important human pathogen. Collating the most recent evidence characterizing bacterial virulence factors, their cellular targets and genetic regulation, we have encompassed numerous aspects important to the success of this pathogen, including membrane proteins and cell surface adaptations promoting immune evasion, mechanisms for nutrient acquisition and community interactions. The role of innate and adaptive immune responses is reviewed and areas of paucity in our understanding are highlighted. Finally, with the vast expansion of available animal models over recent years, we have evaluated those suitable for use in the study of Acinetobacter disease, discussing their advantages and limitations.

Evolution of Daptomycin Resistance in Coagulase-Negative Staphylococci Involves Mutations of the Essential Two-Component Regulator WalKR.

Coagulase-negative staphylococci (CoNS) represent one of the major causes of health care- and medical device-associated infections. Emerging antimicrobial resistance has complicated the treatment of systemic infections caused by CoNS. Here, we describe the prevalence of antimicrobial resistance in clinical CoNS strains from a tertiary care hospital over a 4-year period, and we observed a significant increase in resistance to daptomycin. Notably, Staphylococcus capitis accounted for the majority of these daptomycin-resistant (DAP-R) CoNS. To further investigate the mechanisms of daptomycin resistance in CoNS, daptomycin-susceptible clinical strains of S. capitis and Staphylococcus epidermidis underwent in vitro daptomycin exposure to generate DAP-R CoNS mutants. Unlike that seen with Staphylococcus aureus, alteration of cell surface charge was not observed in the DAP-R CoNS strains, but biofilm formation was compromised. Whole-genome sequencing analysis of the DAP-R CoNS strains identified single nucleotide polymorphisms (SNPs) in walKR, the essential two-component regulatory system controlling cell wall biogenesis. PCR and sequencing of walK and walR from 17 DAP-R CoNS clinical isolates identified seven nonsynonymous mutations. The results were confirmed by the recreation of the walK SNP in S. epidermidis, which resulted in reduced susceptibility to daptomycin and vancomycin. This study highlights the significance of CoNS in evolving daptomycin resistance and showed that walKR is shared among the staphylococcal species and is involved in antibiotic resistance development. Notably, we did not observe mutations in genes responsible for phospholipid biosynthesis or an altered cell surface charge, suggesting that reduced daptomycin susceptibility in CoNS may emerge in a fashion distinct from that in S. aureus.

Community-acquired Acinetobacter baumannii: clinical characteristics, epidemiology and pathogenesis.

Community-acquired Acinetobacter baumannii (CA-Ab) is a rare but serious cause of community-acquired pneumonia in tropical regions of the world. CA-Ab infections predominantly affect individuals with risk factors, which include excess alcohol consumption, diabetes mellitus, smoking and chronic lung disease. CA-Ab pneumonia presents as a surprisingly fulminant course and is characterized by a rapid onset of fever, severe respiratory symptoms and multi-organ dysfunction, with a mortality rate reported as high as 64%. It is unclear whether the distinct clinical syndrome caused by CA-Ab is because of host predisposing factors or unique bacterial characteristics, or a combination of both. Deepening our understanding of the drivers of overwhelming CA-Ab infection will provide important insights into preventative and therapeutic strategies.