Oxygen concentration affects frequency and range of transconjugants for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1

ABSTRACT The frequency of transconjugants were compared for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1 under aerobic and anaerobic conditions. Filter mating assays were performed with one donor strain and one recipient strain using different donors of Pseudomonas and recipient strains, including Pseudomonas, Pantoea, and Buttiauxella. Under anaerobic condition, frequencies of transconjugants for both plasmids were 101-103-fold lower than those under aerobic condition regardless of whether aerobically or anaerobically grown donors and recipients were used. To compare the transconjugant ranges under aerobic and anaerobic conditions, conjugation was performed between the donor of pBP136 and recipient bacteria extracted from environmental samples. Several transconjugants were uniquely obtained from each aerobic or anaerobic condition. Our findings indicate that a plasmid can differently spread among bacteria depending on the oxygen concentrations of the environment.

Linezolid-resistant (Tn6246::fexB-poxtA) Enterococcus faecium strains colonizing humans and bovines on different continents: similarity without epidemiological link

Objectives poxtA is the most recently described gene conferring acquired resistance to linezolid, a relevant antibiotic for treating enterococcal infections. We retrospectively screened for poxtA in diverse enterococci and aimed to characterize its genetic/genomic contexts. Methods poxtA was screened by PCR in 812 enterococci from 458 samples (hospitals/healthy humans/wastewater/animals/retail food) obtained in Portugal/Angola/Tunisia (1996–2019). Antimicrobial susceptibility testing was performed for 13 antibiotics (EUCAST/CLSI). poxtA stability (∼500 generations), transfer (filter mating), clonality (SmaI-PFGE) and location (S1-PFGE/hybridization) were tested. WGS (Illumina-HiSeq) was performed for clonal representatives. Results poxtA was detected in Enterococcus faecium from six samples (1.3%): a healthy human (rectal swab) in Porto, Portugal (ST32/2001); four farm cows (milk) in Mateur, Tunisia (ST1058/2015); and a hospitalized patient (faeces) in Matosinhos, Portugal (ST1058/2015). All expressed resistance to linezolid (MIC = 8 mg/L), chloramphenicol, tetracycline and erythromycin, with variable resistance to ciprofloxacin and streptomycin. ST1058-poxtA-carrying isolates from Tunisia and Portugal differed by two SNPs and had similar plasmid content. poxtA, located in an IS1216-flanked Tn6246-like element, co-hybridized with fexB on one or more plasmids per isolate (one to three plasmids of 30–100 kb), was stable after several generations and transferred only from ST1058. ST1058 strains carried resistance/virulence genes (Efmqnr/acm) possibly induced under selective quinolone treatment. Conclusions poxtA has been circulating in Portugal since at least 2001, corresponding to the oldest description worldwide to date. We also extend the reservoir of poxtA to bovines. The similar linezolid-resistant poxtA-carrying strains colonizing humans and livestock on different continents, and without a noticeable relationship, suggests a recent transmission event or convergent evolution of E. faecium populations in different hosts and geographic regions.

From farm to fork: identical clones and Tn6674-like elements in linezolid-resistant Enterococcus faecalis from food-producing animals and retail meat

Objectives Increasing numbers of linezolid-resistant Enterococcus carrying optrA are being reported across different niches worldwide. We aimed to characterize the first optrA-carrying Enterococcus faecalis obtained from food-producing animals and retail meat samples in Tunisia. Methods Seven optrA-carrying E. faecalis obtained from chicken faeces (n=3, August 2017) and retail chicken meat (n=4, August 2017) in Tunisia were analysed. Antimicrobial susceptibility was determined by disc diffusion, broth microdilution and Etest against 13 antibiotics, linezolid and tedizolid, respectively (EUCAST/CLSI). optrA stability (∼600 bacterial generations), transfer (filter mating) and location (S1-PFGE/hybridization) were characterized. WGS (Illumina-HiSeq) was done for four representatives that were analysed through in silico and genomic mapping tools. Results Four MDR clones carrying different virulence genes were identified in chicken faeces (ST476) and retail meat (the same ST476 clone plus ST21 and ST859) samples. MICs of linezolid and tedizolid were stably maintained at 8 and 1–2 mg/L, respectively. optrA was located in the same transferable chromosomal Tn6674-like element in ST476 and ST21 clones, similar to isolates from pigs in Malaysia and humans in China. ST859 carried a non-conjugative plasmid of ∼40 kb with an impB-fexA-optrA segment, similar to plasmids from pigs and humans in China. Conclusions The same chromosomal and transferable Tn6674-like element was identified in different E. faecalis clones from humans and animals. The finding of retail meat contaminated with the same linezolid-resistant E. faecalis strain obtained from a food-producing animal highlights the potential role of the food chain in the worrisome dissemination of optrA that can be stably maintained without selective pressure over generations.

SalmonellaGenomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance ofSalmonella enterica

ABSTRACTSalmonellagenomic island 3 (SGI3) was first described as a chromosomal island inSalmonella4,[5],12:i:–, a monophasic variant ofSalmonella entericasubsp.entericaserovar Typhimurium. The SGI3 DNA sequence detected fromSalmonella4,[5],12:i:– isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using theS. entericaserovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genespheVorpheR. The length of the target site was 52 or 55 bp, and a 55-bpattIsequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4compared to the recipient strains under anaerobic conditions. Tolerance was defined by thecusgene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance ofS. enterica.

Draft Genome Sequences of a Ceftazidime-Resistant Acinetobacter baumannii Donor and a Conjugal Escherichia coli Recipient with Acquired Resistance

A ceftazidime-resistant Acinetobacter baumannii strain was isolated from hospital wastewater and used as the donor in a filter mating experiment with an Escherichia coli strain as the recipient. Recipient, donor, and transconjugant were sequenced, and both donor and transconjugant were found to harbor highly similar plasmid sequences, suggesting that plasmid transfer had occurred.

Salmonellagenomic island 3 is an integrative and conjugative element and contributes to copper and arsenic resistance ofSalmonella enterica

ABSTRACTSalmonellagenomic island 3 (SGI3) was first described as a chromosomal island inSalmonella4,[5],12:i:-, a monophasic variant ofSalmonella entericasubsp.entericaserovar Typhimurium. The SGI3 DNA sequence detected fromSalmonella4,[5],12:i:-isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic resistance operon in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter mating experiments using theS. entericaserovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genespheVorpheR. The length of the target site was 52 or 55 bp, and a 55-bpattIsequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4compared with the recipient strains under anaerobic conditions. Resistance was defined by thecusgene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4compared with recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic resistance ofS. enterica.

Conjugative Selectivity of Plasmids Is Affected by Coexisting Recipient Candidates

ABSTRACTUnderstanding the mechanisms underlying plasmid behavior under conditions of various environments is important to predict the fate of plasmids in nature. Most previous studies on plasmid transfer employed two strains: one as a donor and the other as a recipient. However, in natural environments, there are usually different recipient cells available to which plasmid can be transferred. In this study, to reveal the underlying mechanisms, we assessed the transferability of plasmids from one donor strain to either of two recipient candidates as the most simplified model. We usedPseudomonas putidaKT2440 andPseudomonas resinovoransCA10dm4 as model hosts and pCAR1 (IncP-7), NAH7 (IncP-9), pB10 (IncP-1β), and R388 (IncW) as model plasmids. As expected, in most cases these plasmids were generally transferred more frequently to a recipient of the same species than to a recipient of a different one under conditions of liquid and filter mating, although NAH7 was transferred fromP. resinovoransmore frequently toP. putidathan toP. resinovoransduring filter mating. With the exception of pCAR1, which was less affected, the coexistence of other recipients enhanced the preferences of conjugative transfer to the same species. In particular, preferences corresponding to transfer fromP. putidato a different recipient (P. resinovorans) were reduced by the presence of a coexisting same recipient (P. putida) during transfer of NAH7 in liquid and transfer of R388 in filter mating. We determined that large cell aggregates and substances secreted into culture supernatant were not responsible for this phenomenon. Overall, the results of this study suggest the existence of unknown factors determining optimal plasmid transfer to native recipients.IMPORTANCEMost previous studies on plasmid conjugal transfer employed experimental setups with two strains: one as a donor and the other as a recipient. However, the results obtained sometimes failed to agree with observations obtained under natural environmental conditions or in a model microcosm using natural soil and water samples. Therefore, we consider that there is a “gap” in our understanding of plasmid behavior in the context of bacterial consortia that exist under the actual environmental conditions. In this study, we clearly showed that the conjugation selectivity of a plasmid can be affected by the recipient candidates existing around the donor strain by the use of a simplified experimental setup with one strain as the donor and two strains as recipients. These phenomena could not be explained by factors known to affect plasmid transfer as suggested by previous studies. Therefore, we suggest the presence of novel elements regulating plasmid transfer within consortia.

Different impacts of naturally occurring variants of the globally disseminated carbapenemase-encoding plasmid pKpQIL on the biology of host strains

SynopsisKlebsiella-associated plasmid pKpQIL and its variant have been isolated globally. Our study aimed to determine whether a naturally occurring variant has altered host range and impacts on the fitness of different bacterial host strains. The plasmids pKpQIL-UK and pKpQIL-D2 were transferred from the original clinical isolate host strains of Klebsiella pneumoniae into Escherichia coli, Salmonella Typhimurium, Enterobacter cloacae and Serratia marcescens strains by filter-mating and conjugation frequencies determined and compared. The fitness of the resulting transconjugants was assessed by determining growth kinetics, ability to form a biofilm and persistence of the plasmids in each host was also measured. Transfer of either plasmid into Salmonella and S. marcescens was similar. However, pKpQIL-UK transferred into E. coli at a higher rate than did pKpQIL-D2; the reverse was found for E. cloacae. Both plasmids were rapidly lost from the E. coli population. Plasmid pKpQIL-UK, but not -D2, was able to persist in Salmonella. Although pKpQIL-UK imposed a greater fitness cost (inferred from an increased generation time) than -D2 on E. cloacae, it was able to persist as well as pKpQIL-D2 in this host. The pKpQIL-D2 plasmid did not confer any fitness benefit on any of the hosts under the conditions tested. Variants of the globally important pKpQIL plasmid have arisen in patients due to recombination. The impacts of the pKpQIL-UK plasmid and the -D2 variant in various Enterobacteriaceae are host-dependent. Continuing evolution of pKpQIL may alter its host range in the future.