Arbutus pavarii Pamp is a medicinal plant commonly used by local tribes in East Libya

for the treatment of many diseases, such as gastritis, renal infections, cancer and kidney diseases.

In this study, the antibacterial activity of the leaf and stem bark extracts of the plant against

methicillin-resistant Staphylococcus aureus (MRSA), as well as the metabolite profiles of the bioactive

fractions, was investigated. The antibacterial activity was determined by disc diffusion method,

minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), while the

microbial reduction by the bioactive fraction was evaluated using time–kill test. The bioactive

fraction was further subjected to ultrahigh-performance liquid chromatography–mass spectrometry

(UHPLC-ESI-MS/MS) analysis to putatively identify the chemical constituents contained therein.

All the extracts and fractions showed different levels of antibacterial activity on the tested MRSA

strains. The highest total antibacterial activity, i.e., 4007.6 mL/g, was exhibited by the crude leaf

methanolic extract. However, the ethyl acetate fraction of the leaf showed moderate to significant

antibacterial activity against MRSA at low MIC (0.08–1.25 mg/mL). Metabolite profiling of this fraction

using UHPLC-ESI-MS/MS resulted in the putative identification of 28 compounds, which included

phenolic acids, flavan-3-ols and flavonols. The results of this study showed that the ethyl acetate

fraction of Arbutus pavarii leaf possessed potential antibacterial activity against MRSA and hence can

be further explored for pharmaceutical applications as a natural antibacterial agent.

Plasmids play a major role in the bacterial adaptation to changing and stressful environmental conditions caused by antibiotics, heavy metals, and disinfectants. However, the investigation of the ecology and diversity of environmental plasmids is challenging due to their typically low abundance in soil bacterial communities and the low cultivability of their hosts. Here we discuss the potentials and limitations of cultivation-dependent and cultivation-independent approaches for detecting and quantifying plasmids in total community DNA from environmental samples. Protocols for PCR-based detection of plasmid-specific sequences in total community DNA are presented. Furthermore, protocols to obtain and characterize plasmids either from isolates (endogenous plasmid isolation) or by capturing into a recipient strain by biparental and triparental mating will be provided.

Due to the high prevalence of colistin-resistant Enterobacteriaceae in poultry and pigs, process waters and wastewater from slaughterhouses were considered as a hotspot for isolates carrying plasmid-encoded, mobilizable colistin resistances (mcr genes). Thus, questions on the effectiveness of wastewater treatment in in-house and municipal wastewater treatment plants (WWTPs) as well as on the diversity of the prevailing isolates, plasmid types, and their transmissibility arise. Process waters and wastewater accruing in the delivery and unclean areas of two poultry and two pig slaughterhouses were screened for the presence of target colistin-resistant bacteria (i.e., Escherichia coli, Klebsiella spp., Enterobacter cloacae complex). In-house and municipal WWTPs (mWWTPs) including receiving waterbodies were investigated as well. Samples taken in the poultry slaughterhouses yielded the highest occurrence of target colistin-resistant Enterobacteriaceae (40.2%, 33/82), followed by mWWTPs (25.0%, 9/36) and pig slaughterhouses (14.9%, 10/67). Recovered isolates exhibited various resistance patterns. The resistance rates using epidemiological cut-off values were higher in comparison to those obtained with clinical breakpoints. Noteworthy, MCR-1-producing Klebsiella pneumoniae and E. coli were detected in scalding waters and preflooders of mWWTPs. A total of 70.8% (46/65) of E. coli and 20.6% (7/34) of K. pneumoniae isolates carried mcr-1 on a variety of transferable plasmids with incompatibility groups IncI1, IncHI2, IncX4, IncF, and IncI2 ranging between 30 and 360 kb. The analyzed isolates carrying mcr-1 on transferable plasmids (n = 53) exhibited a broad diversity, as they were assigned to 25 different XbaI profiles. Interestingly, in the majority of colistin-resistant mcr-negative E. coli and K. pneumoniae isolates non-synonymous polymorphisms in pmrAB were detected. Our findings demonstrated high occurrence of colistin-resistant E. coli and K. pneumoniae carrying mcr-1 on transferrable plasmids in poultry and pig slaughterhouses and indicate their dissemination into surface water.