Phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline‐amended and ciprofloxacin‐amended growth media

Aims: The goal of this study was to determine the antimicrobial susceptibility of bacteria isolated from three municipal wastewater treatment plants. Methods and Results: Numerous bacterial strains were isolated from three municipal wastewater treatment facilities on tetracycline‐ (n = 164) and ciprofloxacin‐amended (n = 65) growth media. These bacteria were then characterized with respect to their resistance to as many as 10 different antimicrobials, the presence of 14 common genes that encode resistance to tetracycline, the presence of integrons and/or the ability to transfer resistance via conjugation. All of the characterized strains exhibited some degree of multiple antimicrobial resistance, with nearly 50% demonstrating resistance to every antimicrobial that was tested. Genes encoding resistance to tetracycline were commonly detected among these strains, although intriguingly the frequency of detection was slightly higher for the bacteria isolated on ciprofloxacin‐amended growth media (62%) compared to the bacteria isolated on tetracycline‐amended growth media (53%). Class 1 integrons were also detected in 100% of the queried tetracycline‐resistant bacteria and almost half of the ciprofloxacin‐resistant strains. Conjugation experiments demonstrated that at least one of the tetracycline‐resistant bacteria was capable of lateral gene transfer. Conclusions: Our results demonstrate that multiple antimicrobial resistance is a common trait among tetracycline‐resistant and ciprofloxacin‐resistant bacteria in municipal wastewater. Significance and Impact of the Study: These organisms are potentially important in the proliferation of antimicrobial resistance because they appear to have acquired multiple genetic determinants that confer resistance and because they have the potential to laterally transfer these genetic determinants to strains of clinical importance.     

Genetic transfer of Pseudomonas aeruginosa R factors to plant pathogenic Erwinia species.

The R factors RP1, R68 and R91 were freely transmissible to and from Pseudomonas aeruginosa, Salmonella typhimurium, and various plant pathogenic Erwinia spp. The antibiotic resistance spectrum of R+ Erwinia recipients was similar to those of other bacteria harboring these R factors, but maximum resistance levels differed with each recipient. The sponstaneous elimination of these factors from the Erwinia strains and the ability to transfer multiple antibiotic resistance suggest that these exist as plasmids in these hosts. Several, but not all, RP1-carrying Erwinia strains were sensitive to the RP1 specific phage PRR1. The R factor R18-1 was also transferred from P. aeruginosa to Erwinia spp. R18-1 was unstable in all Erwinia strains. Stable strains were isolated in which R18-1 could not be eliminated by sodium dodecyl sulfate and could not be transferred to other strains.     

Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon

Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in Bacteroides species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene, tetQ, has increased from about 30% to more than 80% of strains. Alleles of tetQ in different Bacteroides species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of tetQ was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF and ermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of tetQ and the erm genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with Bacteroides infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried ermB genes. Two were identical to erm(B)-P from Clostridium perfringens, and the other four had only one to three mismatches. The nine strains with ermG genes had DNA sequences that were more than 99% identical to the ermG of Bacillus sphaericus. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative Bacteroides species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus Bacteroides and among Bacteroides species and gram-positive bacteria.     

The prevalence of tetracycline resistance genes in clinical and commensal strains of Enterococcus faecalis isolated in Gdańsk

The common use of antibiotics is responsible for selecting of drug resistance not only in pathogenic, clinical bacteria but also in commensal, not pathogenic strains which could cause the rapid dissemination of the resistance to these antibacterial agents. However, information regarding the antibiotic resistance of commensal bacteria is very scarce, and the data is based mostly on phenotypical research. Therefore the use of genotyping methods for detection of tetracycline resistance genes, in commensal and medical isolates of bacteria, is essential, for understanding the spread of antibiotic resistance. In this study 24 commensal and 27 clinical isolates of Enterococcus faecalis has been screened by PCR methods for tet(M), tet(S) genes and Tn916 and Tn5397 transpozons. Subsequently, the tet(M) gene amplicones were sequenced and phylogenetic analysis was performed. We have found that the prevalence of tet(S) gene varied significantly between commensal and clinical strains. Moreover, the frequency of transpozons in clinical isolates was much higher comparing to strains isolated from healthy individuals. The phylogenetic analysis did not show significant differences between clinical and commensal strains but it could suggest that the genetic similarity between these two groups could be favourable factor for broad range spread of tet(M) gene.     

Escherichia coli resistance to chlorine and glutathione synthesis in response to oxygenation and starvation

Reduced glutathione (GSH) levels and resistance to chlorine were measured for two isogenic Escherichia coli strains stressed by oxygenation and/or starvation. The E. coli mutant deficient in GSH was not more sensitive to the oxidant than its parent strain when the bacteria were cultured with a low oxygenation rate. Starvation or oxygenation increased the resistance of the parent strain to chlorine, while the resistance of the deficient strain remained unchanged.     

Genetics of resistance to macrolide antibiotics and lincomycin in natural isolates of Streptococcus pyogenes

Of 5 clinically isolated strains of Streptococcus pyogenes, 3 showed high-level resistance to erythromycin and lincomycin that was inducible by subinhibitory concentrations of these drugs (IR strains) while 2 strains exhibited constitutive erythromycin and lincomycin resistance (CR strains) which was expressed without prior exposure to low drug concentrations. The CR strain 15346 showed spontaneous loss of resistance whereas resistance in the other strains was quite stable even under curing conditions. The IR strain 13234 was found to be polylysogenic for at least 4 different phages designated P13234ma, mi, mu, and mo. Phage mo, antigenically distinct from the other three, was shown to mediate the transfer of the resistance determinant ERL1 of strain 13234. ERL1 if borne by appropriate strains was also transducible by the virulent phage A25. ERL1 behaved as a discrete genetic unit in transduction experiments, was not linked to either of two chromosomal regions governing resistance to antibiotics that affect the ribosome, could be transferred to recombination deficient hosts, represented a relatively large UV inactivation target, and showed no stimulation of transduction by low UV doses. These findings suggest that resistance to erythromycin and lincomycin in certain natural isolates of S. pyogenes is specified by, or under the control of, a plasmid.     

Escherichia coli Resistance to Chlorine and Glutathione Synthesis in Response to Oxygenation and Starvation

Reduced glutathione (GSH) levels and resistance to chlorine were measured for two isogenic Escherichia coli strains stressed by oxygenation and/or starvation. The E. coli mutant deficient in GSH was not more sensitive to the oxidant than its parent strain when the bacteria were cultured with a low oxygenation rate. Starvation or oxygenation increased the resistance of the parent strain to chlorine, while the resistance of the deficient strain remained unchanged.     

Acceptance and transfer of plasmid Rts1 by bacteria of the genus Erwinia]

The ability of 13 Erwinia strains to accept, to inherit and to transmit the Rts1 factor by conjugation was studied. 11 strains accepted the Rts1 factor from Escherichia coli K-12 CSH-2 with the frequency of about 10(-7)--10(-3). The Rts1 factor was genetically stable in the Erwinia cells and was not eliminated by acriflavine and under the temperature of 37 and 42 degrees C. All the R+ exconjugants were characterized with more high degree of the resistance of kanamycin than E. coli cells harbouring the same R factor. Erwinia strains harbouring the Rts1 plasmid transferred it by conjugation into homologic (Erwinia) and heterologic (E. coli) bacteria. The study of kinetics of the transfer of the Rts1 factor in different mating systems showed that the transfer of this plasmid from R+ Erwinia into R- Erwinia and R- E. coli--in the liquid medium. It is concluded that Erwinia can be the host and the donor of the Rts1 factor.     

Cefamandole resistance transfer in bacterial strains from two newborn units

Transfer of Cefamandole resistance was demonstrated from strains of Citrobacter freundii as well as from individual strains of Enterobacter cloacae, Acinetobacter anitratus and Klebsiella pneumoniae isolated from patients in two newborn units. In Citrobacter freundii, Cefamandole resistance was transferred always with Cephalotin resistance as well as with a TEM-like beta lactamase (conferring resistance to Ampicillin, Carbenicillin and Azlocillin). Citrobacter freundii strains from Hospital I were completely susceptible to gentamicin, while strains of other species, resistant to Cefamandole plus Cephalotin, were resistant to Gentamicin as well, and transferred this resistance, too. In one Enterobacter cloacae strain from Hospital I, Cefamandole resistance could be separated from resistance to Cephalotin, but only in clones selected with gentamicin and not with any of the cephalosporins. Acinetobacter anitratus strain was also resistant to Cefotaxime, but did not transfer this resistance. It might be concluded that special nosocomial bacteria may carry plasmids conferring a transferable type of resistance to Cefamandole together with resistance to classical cephalosporines. Second cycle of transfers, i.e. between two variants of E. coli K-12 strains confirmed the contransferability of Cefamandole and Cephalotin resistance.     

Occurrence of Chloramphenicol-acetylating Enzymes in Various Gram-negative Bacilli

The occurrence of a chloramphenicol-acetylating enzyme, similar to that found in Escherichia coli, carrying an R factor was investigated in various gram-negative bacilli. The acetylated products of chloramphenicol were identified by chromatography and quantitatively assayed after benzene extraction. The investigated strains were of the Salmonella-Arizona group, the Klebsiella-Aerobacter group, Serratia marcescens, the Proteus group, and Pseudomonas aeruginosa, most of which were isolated from 1947 to 1957. Both chloramphenicol-sensitive and -resistant strains were included, but none of them was able to transfer chloramphenicol resistance by conjugation. In the Proteus group, a significant level of a chloramphenicol-acetylating enzyme was found in most strains, whether they were sensitive or resistant to chloramphenicol; the resistant strains showed higher levels of the enzyme. Some chloramphenicol-sensitive strains lacked this enzyme. Only the sensitive strains containing the enzyme could easily produce chloramphenicol-resistant mutants with higher enzyme activity. Thus, the chloramphenicol resistance of this group can be reasonably explained on the basis of the chloramphenicol-acetylating enzyme. All of the Pseudomonas aeruginosa strains were resistant to chloramphenicol, and most strains showed low levels of the enzyme (which, however, did not appear sufficient to explain their resistance). All of the strains of the other groups (except one strain of Enterobacter cloacae) lacked the enzyme, although most strains of the Klebsiella-Aerobacter group and of S. marcescens were resistant to chloramphenicol. With respect to the origin of the resistance gene of the R factor, it is noteworthy that the strains of Proteus mirabilis isolated in 1947 possessed this enzyme before the discovery of chloramphenicol.     

The complete genome sequences of four new IncN plasmids from wastewater treatment plant effluent provide new insights into IncN plasmid diversity and evolution

The dissemination of antibiotic resistance genes among bacteria often occurs by means of plasmids. Wastewater treatment plants (WWTP) were previously recognized as hot spots for the horizontal transfer of genetic material. One of the plasmid groups that is often associated with drug resistance is the incompatibility group IncN. The aim of this study was to gain insights into the diversity and evolutionary history of IncN plasmids by determining and comparing the complete genome sequences of the four novel multi-drug resistance plasmids pRSB201, pRSB203, pRSB205 and pRSB206 that were exogenously isolated from the final effluent of a municipal WWTP. Their sizes range between 42,875 bp and 56,488 bp and they share a common set of backbone modules that encode plasmid replication initiation, conjugative transfer, and plasmid maintenance and control. All plasmids are transferable at high rates between Escherichia coli strains, but did not show a broad host range. Different genes conferring resistances to ampicillin, streptomycin, spectinomycin, sulfonamides, tetracycline and trimethoprim were identified in accessory modules inserted in these plasmids. Comparative analysis of the four WWTP IncN plasmids and IncN plasmids deposited in the NCBI database enabled the definition of a core set of backbone genes for this group. Moreover, this approach revealed a close phylogenetic relationship between the IncN plasmids isolated from environmental and clinical samples. Phylogenetic analysis also suggests the existence of host-specific IncN plasmid subgroups. In conclusion, IncN plasmids likely contribute to the dissemination of resistance determinants between environmental bacteria and clinical strains. This is of particular importance since multi-drug resistance IncN plasmids have been previously identified in members of the Enterobacteriaceae that cause severe infections in humans.     

Fertility factors in Pseudomonas putida: selection and properties of high-frequency transfer and chromosome donors.

The octane plasmid (OCT) in Pseudomonas putida strains has been shown to be transferred at low frequency. However, bacteria which had newly received this plasmid showed a transient increase in donor ability. Using Octane+ P. putida as the donor, the transfer of most chromosomal markers was shown to be independent of OCT transfer, whereas the mobilization of the octanoate catabolism genes (octanoic and acetate) was dependent on OCT plasmid transfer. The presence of a fertility factor termed FPo has been postulated to explain these results. Strains carrying only this fertility factor have been obtained from strains carrying both OCT and FPo plasmids. Strains in which the OCT plasmid was transferred at high frequencies have also been isolated, and chromosome mobilization by OCT and FPo has been compared. A different gradient of transmission by OCT and FPo has been observed. It has also been shown that chromosome transfer by OCT was dependent on the bacterial recombination system, whereas the chromosome transfer by FPo was unaffected by the presence of a rec mutation in the donor strain.     

Evidence for Extensive Resistance Gene Transfer among Bacteroides spp. and among Bacteroides and Other Genera in the Human Colon

Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in Bacteroides species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene, tetQ, has increased from about 30% to more than 80% of strains. Alleles of tetQ in different Bacteroides species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of tetQ was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF and ermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of tetQ and the erm genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with Bacteroides infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried ermB genes. Two were identical to erm(B)-P from Clostridium perfringens, and the other four had only one to three mismatches. The nine strains with ermG genes had DNA sequences that were more than 99% identical to the ermG of Bacillus sphaericus. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative Bacteroides species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus Bacteroides and among Bacteroides species and gram-positive bacteria.     

Specific Distribution of R Factors in Serratia marcescens Strains Isolated from Hospital Infections

Serratia marcescens strains from three hospitals in the city of New York were tested for antibiotic susceptibility patterns and the presence of transmissible antibiotic resistance factors. There appears to be a pattern characteristic for each hospital with regard to the sensitivity to nalidixic acid, tetracycline, chloramphenicol, and sulfonamides, whereas the resistance to ampicillin, cephalothin, and streptomycin is similar in the strains isolated from all three hospitals. In one hospital, a single type of R factor was found which transfers resistance to streptomycin, kanamycin, ampicillin, and sulfonamides, whereas strains isolated from a second hospital transfer only ampicillin resistance. No R factors could be detected in multiply resistant Serratia strains isolated in a third hospital. The presence of a single type of R factor probably reflects the relative ecological isolation of S. marcescens and could be useful for epidemiological studies of hospital infections with Serratia.     

Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia

AIMS: To carry out a preliminary assessment of the occurrence of resistance to antimicrobials in bacteria that has been isolated from a variety of aquaculture species and environments in Australia. METHOD AND RESULTS: A total of 100 Gram-negative (Vibrio spp. and Aeromonas spp. predominantly) and four Gram-positive bacteria isolated from farmed fish, crustaceans and water from crab larval rearing tanks were obtained from diagnostic laboratories from different parts of Australia. All the isolates were tested for sensitivity to 19 antibiotics and Minimal Inhibitory Concentrations were determined by the agar dilution method. Plasmid DNA was isolated by the alkali lysis method. Resistance to ampicillin, amoxycillin, cephalexin and erythromycin was widespread; resistance to oxytetracycline, tetracycline, nalidixic acid and sulfonamides was common but resistance to chloramphenicol, florfenicol, ceftiofur, cephalothin, cefoperazone, oxolinic acid, gentamicin, kanamycin and trimethoprim was less common. All strains were susceptible to ciprofloxacin. Multiple resistance was also observed and 74.4% of resistant isolates had between one and ten plasmids with sizes ranging 2-51 kbp. CONCLUSIONS: No antibiotics are registered for use in aquaculture in Australia but these results suggest that there has been significant off-label use. SIGNIFICANCE AND IMPACT OF STUDY: Transfer of antibiotic resistant bacteria to humans via the food chain is a significant health concern. In comparison with studies on terrestrial food producing animals, there are fewer studies on antibiotic resistance in bacteria from aquaculture enterprises and this study provides further support to the view that there is the risk of transfer of resistant bacteria to humans from consumption of aquaculture products. From the Australian perspective, although there are no products registered for use in aquaculture, antimicrobial resistance is present in isolates from aquaculture and aquaculture environments.     

SdrI of Staphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site

The transferability of a large plasmid that harbors a tetracycline resistance gene tet (S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients ( Lactococcus garvieae and Listeria monocytogenes ), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L . garvieae . A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes , even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.     

Mupirocin resistance in staphylococci: development and transfer of isoleucyl-tRNA synthetase-mediated resistance in vitro

Mupirocin resistance could be transferred from highly resistant clinical isolates of Staphylococcus aureus to highly sensitive recipients of Staph. aureus, Staph. epidermidis and Staph. haemolyticus. Transconjugants of the latter two organisms could transfer this resistance into mupirocin-sensitive Staph. aureus. Moderately resistant strains did not transfer this resistance to sensitive recipients, nor did strains with high-level mupirocin resistance developed by serial transfer or habituation. The inhibitory effects of mupirocin on crude isoleucyl-tRNA synthetases (IRS) isolated from mupirocin-sensitive and -resistant strains of Staph. aureus have been determined. Drug concentrations needed to produce 50% inhibition, I50 values, were very low against IRS from a highly sensitive strain, somewhat higher against IRS from moderately resistant strains, much higher against enzyme from strains trained in vitro to high-level resistance, and considerably higher still against IRS extracted from clinical isolates possessing high-level mupirocin resistance and from the transconjugates of such strains resulting from crosses with mupirocin-sensitive strains. It is concluded that high-level resistance in clinical isolates is plasmid-mediated involving a second, mupirocin-resistant IRS whereas in moderately resistant strains, and in strains trained in vitro to high-level resistance, chromosomal mutations are likely to be responsible for decreasing IRS sensitivity.     

Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.     

Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.     

Mesophilic strains of Aeromonas spp. can acquire the multidrug resistance plasmid pRAS1 in horizontal transfer experiments at low temperatures

Both biotic and abiotic characteristics of an ecosystem play an important role in the horizontal transfer of DNA in nature. The abiotic factor temperature has a great impact on such transfers as it controls the metabolic activity of mesophilic microorganisms. Moreover, psychrophilic bacteria, which are not affected by low temperatures, are considered to be potential donors of DNA to mesophilic bacteria under temperature stress conditions. In our study, mesophilic Aeromonas spp. strains isolated from fresh fish were genotypically identified and used as recipients in in vitro conjugal transfer experiments using plasmid pRAS1 from psychrophilic strain Aeromonas salmonicida 718 at three different temperatures (8, 15 and 20 °C). The transfer of the plasmid was confirmed by identifying the elements of the integron in pRAS1. A low temperatures did not prevent the transfer of the pRAS1 plasmid to Aeromonas veronii, A. media, A. hydrophila and A. caviae strains, which showed detectable conjugation frequencies of 10–8 at 8 °C. In other strains of the same species, transconjugants were not detected, which indicated that the genetic background of each strain directly affected the ability to be a recipient of this plasmid at the temperatures tested. Our results demonstrate that mesophilic Aeromonas spp. strains are potential reservoirs of extrachromosomal genetic material. Implications of this plasmid transfer at low temperatures and its possible consequences for human health are discussed.