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.

A floristic survey of Sedrores Al-Qawasim mountains in Gharyan district - Libya was conducted in two consecutive growing seasons from 01/04/2018 to 01/6/2019. In this study area, a total number of 320 plant species belonging to 214 genera were collected representing 44 families, of which 39 families and 276 species belonging to dicotyledons, and 5 families and 44 species are belonging to monocotyledons. The results showed the dominance of the family Asteraceae with 60 species, followed by the family Fabaceae with 32 species, then the family Poaceae with 29 species. The results showed the dominance of the (Euphorbia genus) with 7 species, followed by Helianthenum, Plantago and Erodium with 6 species each. Moreover, life-form spectrum analysis showed the predominance of therophytes with 210, followed by Hemicryptophytes with 59 species, while chorotype spectrum analysis showed the dominance of Mediterranian species with 151 species, followed by Mediterrean/Iranu-Turanean with 72 species. 

The role of agriculture in the transfer of drug resistant pathogens to humans is widely debated and poorly understood. Escherichia coli is a valuable indicator organism for contamination and carriage of antimicrobial resistance (AMR) in foods. Whilst whole genome sequences for E. coli from animals and associated meats are common, sequences from produce are scarce. Produce may acquire drug resistant E. coli from animal manure fertilizers, contaminated irrigation water and wildlife, particularly birds. Whole genome sequencing was used to characterize 120 tetracycline (TET) resistant E. coli from store-bought, ready-to-eat cilantro, arugula and mixed salad from two German cities. E. coli were recovered on the day of purchase and after 7 days of refrigeration. Cilantro was far more frequently contaminated with TET-resistant E. coli providing 102 (85%) sequenced strains. Phylogroup B1 dominated the collection (n = 84, 70%) with multi-locus sequence types B1-ST6186 (n = 37, 31%), C-ST165 (n = 17, 14%), B1-ST58 (n = 14, 12%), B1-ST641 (n = 8, 7%), and C-ST88 (n = 5, 4%) frequently identified. Notably, seven strains of diverse sequence type (ST) carried genetic indicators of ColV virulence plasmid carriage. A number of previously identified and novel integrons associated with insertion elements including IS26 were also identified. Storage may affect the lineages of E. coli isolated, however further studies are needed. Our study indicates produce predominantly carry E. coli with a commensal phylogroup and a variety of AMR and virulence-associated traits. Genomic surveillance of bacteria that contaminate produce should be a matter of public health importance in order to develop a holistic understanding of the environmental dimensions of AMR.

A new record for Abutilon theophrasti is recorded for the first time in the flora of Libya. This widespread agricultural weed was collected from El-Hamra region (El-Qabel) in the Nafusa Mountain south of Tripoli. To facilitate further identification and future detection, a full description, habitat information, distribution map and distribution data are provided. A brief discussion about the most important threats posed by this species is presented.

Manure application to agricultural soil introduces antibiotic residues and increases the abundance of antibiotic-resistant bacteria (ARB) carrying antibiotic resistance genes (ARGs), often located on mobile genetic elements (MGEs). The rhizosphere is regarded as a hotspot of microbial activity and gene transfer, which can alter and prolong the effects of organic fertilizers containing antibiotics. However, not much is known about the influence of plants on the effects of doxycycline applied to soil via manure. In this study, the effects of manure spiked with or without doxycycline on the prokaryotic community composition as well as on the relative abundance of ARGs and MGEs in lettuce rhizosphere and bulk soil were investigated by means of a polyphasic cultivation-independent approach. Samples were taken 42 days after manure application, and total community DNA was extracted. Besides a pronounced manure effect, doxycycline spiking caused an additional enrichment of ARGs and MGEs. High-throughput quantitative PCR revealed an increase in tetracycline, aminoglycoside, and macrolide–lincosamide–streptogramin B (MLS_B) resistance genes associated with the application of manure spiked with doxycycline. This effect was unexpectedly lower in the rhizosphere than in bulk soil, suggesting a faster dissipation of the antibiotic and a more resilient prokaryotic community in the rhizosphere. Interestingly, the tetracycline resistance gene tetA(P) was highly enriched in manure-treated bulk soil and rhizosphere, with highest values observed in doxycycline-treated bulk soil, concurring with an enrichment of Clostridia. Thus, the gene tetA(P) might be a suitable marker of soil contamination by ARB, ARGs, and antibiotics of manure origin. These findings illustrate that the effects of manure and doxycycline on ARGs and MGEs differ between rhizosphere and bulk soil, which needs to be considered when assessing risks for human health connected to the spread of ARGs in the environment.

High antibiotic releases from manufacturing facilities have been identified as a risk factor for antibiotic resistance development in bacterial pathogens. However, the role of antibiotic pollution in selection and transferability of antibiotic resistance genes (ARGs) is still limited. In this study, we analyzed effluents from azithromycin-synthesis and veterinary-drug formulation facilities as well as sediments from receiving river and creek taken at the effluent discharge sites, upstream and downstream of discharge. Culturing showed that the effluent discharge significantly increased the proportion of antibiotic resistant bacteria in exposed sediments compared to the upstream ones. Quantitative real-time PCR revealed that effluents from both industries contained high and similar relative abundances of resistance genes [sul1, sul2, qacE/qacEΔ1, tet(A)], class 1 integrons (intI1) and IncP-1 plasmids (korB). Consequently, these genes significantly increased in relative abundances in receiving sediments, with more pronounced effects being observed for river than for creek sediments due to lower background levels of the investigated genes in the river. In addition, effluent discharge considerably increased transfer frequencies of captured ARGs from exposed sediments into Escherichia coli CV601 recipient as shown by biparental mating experiments. Most plasmids exogenously captured from effluent and polluted sediments belonged to the broad host range IncP-1ε plasmid group, conferred multiple antibiotic resistance and harbored class 1 integrons. Discharge of pharmaceutical waste from antibiotic manufacturing sites thus poses a risk for development and dissemination of multi-resistant bacteria, including pathogens.