Plasmid Content Research Articles

ESBL-plasmid carriage in E. coli enhances in vitro bacterial competition fitness and serum resistance in some strains of pandemic sequence types without overall fitness cost

BackgroundExtended spectrum beta lactamase (ESBL)-producing extraintestinal pathogenic Escherichia coli infections are of global interest because of their clinical and economic impact. The ESBL resistance genes disseminate through plasmids, and are found in successful global lineages such as ST131 and ST648. The carriage of plasmids has been suggested to result in a fitness burden, but recently it was shown that ESBL-plasmids enhanced virulence in pandemic ST131 and ST648 lineages without affecting their fitness. Herein, we investigated the influence of ESBL-plasmids on bacterial competition and serum resistance, both of which are essential characteristics of ExPEC during infections.MethodsTriplets of ESBL-plasmid-carrying wildtype (WT), plasmid-cured variant (PCV) and transformant (T) of five ExPEC strains of ST131 and ST648 were used for bacterial competition experiments with colicin-producing commensal E. coli, competitive adhesion experiments and serum survival. In addition, resilience after SDS, acid, osmotic challenges and RNA sequence data were analyzed.ResultsIn all five strains tested, ESBL-plasmid carriage did not negatively influence E. coli fitness in direct bacterial competition with commensal E. coli in vitro. That is, WTs did not show any disadvantages when compared to their isogenic plasmid-free PCV. For one strain we even found the opposite as PCV17433 was out-competed by a commensal strain, which suggests an even protective role of the ESBL-plasmid carried by the WT17433. Similarly, in the serum-resistance experiments, the PCVs of two strains (PCV17433 and PCV17887) were more sensitive to serum, unlike WTs and Ts. The observed inter-strain differences could be explained by the different genetic content of plasmids carried in those strains.ConclusionsOverall, we found no compelling evidence for an increased burden resulting from the carriage of ESBL-plasmids in the absence of antimicrobial selection pressure in the strains of pandemic ST131 and ST648; rather, the possession of certain ESBL-plasmids was beneficial for some strains in regarding competition fitness and serum survival.

Is blaCTX-M-1 Riding the Same Plasmid Among Horses in Sweden and France?

A predominance of the blaCTX-M-1/IncHI1 plasmid combination in horses has been reported in Czech-Republic, Denmark, and The Netherlands. To clarify a possible specific plasmid epidemiology of blaCTX-M-1 in horses in a European perspective, a collection of 74 extended-spectrum beta-lactamase-producing Escherichia coli recovered from diseased horses in France and Sweden during the period 2009-2014 was investigated in respect of their genetic relatedness, plasmid content, and molecular features. Overall, 80% of E. coli isolates from diseased horses harbored blaCTX-M-1 on large IncHI1 plasmids with plasmid sequence type (pST) 2 and pST9 more prevalent in Sweden and France, respectively. In French isolates, IncI1/pST3 plasmids were also identified. The CTX-M-1-producing E. coli belonged principally to the clonal complex 10. ST641, together with its single locus variant, and ST1730 constituted two other major groups. The rep-PCR clustering highlighted a clonal dissemination of E. coli CTX-M-1 producers in different regions of the same country and during several years. The STs found in our isolates were also reported in The Netherlands, suggesting a common source of contamination in Europe, although only further cooperative investigation will clarify this issue. On the other hand, the spread of IncI1/pST3 plasmids among horses constitutes another worrisome issue considering their successful spread in other animal hosts such as chicken or bovines. Monitoring evolution and propagation of antimicrobial resistance in equine environment is a priority to avoid further propagation of antimicrobial resistance mechanisms threatening human and animal health.

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens

BackgroundClostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome.ResultsDistinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis.ConclusionsThe high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.

INFLUENCE OF YERSINIA PESTIS PLASMID CONTENT ON BIOFILM FORMATION IN FLEAS WITH DIFFERENT VECTOR ACTIVITY

Aim. Influence of the plague agent plasmid content on biofilm formation in vivo and death rate of fleas-vectors with different vector activity in experiment were analyzed. Materials and methods. Three Yersinia pestis strains: virulent I-3230 (pYT, pYV, pYP) and I-2638 (pYT, pYV, pYP, pTP 33), and its selected avirulent isogenic clone I-3480 lacking two plasmids (pYV, pYP) were used. Three species of fleas were artificially infected: 477 individuals of Xenopsylla cheopis (a highly active vector), 441 - Citellophilus tesquorum (an active vector), 519 - Frontopsylla lucu-lenta (a low-active vector). The peculiarities of Y. pestis biofilm formation in fleas were estimated by a portion of individuals with bacterial «conglomerates» and «blocks» for a feeding. Death rate of the insects was defined by the percent of the dead fleas at each feeding. Results. All three flea species infected by Y. pestis strains carrying an additional plasmid pTP33 (I-2638 and I-3480) demonstrated the increase of the individual number with various biofilm forms in comparison with the three-plasmid strain I-3230. In X. cheopis it occurred due to the blocked insects, in C. tesquo-rum - mainly due to the fleas containing «conglomerates», in F. luculenta it was completely connected with ectoparasites with «conglomerates». A share of X. cheopis and C. tesquorum died at a feeding was higher in ectoparasites infected with I-3230 strain and F. luculenta - infected by I-2638. Conclusion. Y. pestis strains possessing an additional replicon pTP33 formed a biofilm in the infected insects more often and larger size than a classical three-plasmid variant. Influence of the strain plasmid content on death rate of the infected fleas depended on a vector species.

High genetic diversity of extended-spectrum β-lactamases producing Escherichia coli in feces of horses

Extended-spectrum beta-lactamases (ESBLs), especially those of the CTX-M type, represent a major public health problem throughout the world. Although the carriage of ESBL-producing Enterobacteriaceae (EPE) in feces of horses is now well recognized, little is known about the diversity of EPE after treatment of horses with antibiotics. We undertook this study to assess and follow the diversity of EP Escherichia coli isolated from horses after antibiotic treatment for an infection. Fecal samples from two horses treated and two that were untreated were tested for the presence of EPE on different days. All isolated E. coli strains were evaluated for antimicrobial resistance (AMR) and by whole-genome sequencing. Multi locus sequence typing, phylogrouping, resistance genes and plasmid content were extracted from genomic data. A phylogenetic analysis based on single nucleotide polymorphism (SNP) divergence was also performed on the core genome. We isolated 35 strains belonging to the A, B1 and C phylo-groups. All but one expressed SHV-12 enzymes and one expressed CTX-M-1. Intra- and inter-horse genetic diversity of E. coli strains was identified in the genome analysis and 10 AMR profiles. Two distinct EP E. coli-resistant populations (phylo-group B1: ST4164-AMR3 and ST155-AMR2) were found in one horse, and five other resistant populations were found in the second horse (phylo-group A: ST1250-AMR1; phylo-group B1: ST1250-AMR1, ST6981-AMR1 and phylo-group C: ST10-AMR4). Some persistent EP E. coli strains were detected at least 1 month after treatment. These results indicate that EP E. coli strains isolated from horse feces show intra- and inter-host genetic diversity, even in a region with low ESBL prevalence and in horses that are rarely treated with third-generation cephalosporins. These results also suggest that horizontal gene transfer and/or selection of resistance genes probably occurs in vivo within the horse gut microbiome. Follow-up of EP E. coli resistance profiles for at least 1 month after treatment is warranted to prevent persistence of EP E. coli.

Correlation between bacterial G+C content, genome size and the G+C content of associated plasmids and bacteriophages.

Based on complete bacterial genome sequence data, we demonstrate a correlation between bacterial chromosome length and the G+C content of the genome, with longer genomes having higher G+C contents. The correlation value decreases at shorter genome sizes, where there is a wider spread of G+C values. However, although significant (P<0.001), the correlation value (Pearson R=0.58) suggests that other factors also have a significant influence. A similar pattern was seen for plasmids; longer plasmids had higher G+C values, although the large number of shorter plasmids had a wide spread of G+C values. There was also a significant (P<0.0001) correlation between the G+C content of plasmids and the G+C content of their bacterial host. Conversely, the G+C content of bacteriophages tended to reduce with larger genome sizes, and although there was a correlation between host genome G+C content and that of the bacteriophage, it was not as strong as that seen between plasmids and their hosts.

Pseudomonas furukawaii sp. nov., a polychlorinated biphenyl-degrading bacterium isolated from biphenyl-contaminated soil in Japan.

Strain KF707T was isolated from a biphenyl-contaminated site in Kitakyushu, Japan. Analysis of 16S rRNA gene sequences, retrieved from the whole-genome sequence, revealed that the isolate was closely related to members of the genus Pseudomonas, sharing the highest sequence similarities with Pseudomonas balearica strain SP1402T (DSM 6083) (97.8 %). The DNA G+C chromosome and plasmid content of strain KF707T were 65.5 and 60.5 mol%. The major cellular fatty acids were iso-C15 : 0 and C16 : 1ω7c/C16 : 1ω6c. Polyphasic analysis indicated that strain KF707T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas furukawaii sp. nov. is proposed. The type strain is KF707T (=DSM 10086T=NBRC 110670T).

Azithromycin Resistance in Shigella spp. in Southeast Asia.

ABSTRACTInfection by Shigella spp. is a common cause of dysentery in Southeast Asia. Antimicrobials are thought to be beneficial for treatment; however, antimicrobial resistance in Shigella spp. is becoming widespread. We aimed to assess the frequency and mechanisms associated with decreased susceptibility to azithromycin in Southeast Asian Shigella isolates and use these data to assess appropriate susceptibility breakpoints. Shigella isolates recovered in Vietnam and Laos were screened for susceptibility to azithromycin (15 μg) by disc diffusion and MIC. Phenotypic resistance was confirmed by PCR amplification of macrolide resistance loci. We compared the genetic relationships and plasmid contents of azithromycin-resistant Shigella sonnei isolates using whole-genome sequences. From 475 available Shigella spp. isolated in Vietnam and Laos between 1994 and 2012, 6/181 S. flexneri isolates (3.3%, MIC ≥ 16 g/liter) and 16/294 S. sonnei isolates (5.4%, MIC ≥ 32 g/liter) were phenotypically resistant to azithromycin. PCR amplification confirmed a resistance mechanism in 22/475 (4.6%) isolates (mphA in 19 isolates and ermB in 3 isolates). The susceptibility data demonstrated the acceptability of the S. flexneri (MIC ≥ 16 g/liter, zone diameter ≤ 15 mm) and S. sonnei (MIC ≥ 32 g/liter, zone diameter ≤ 11 mm) breakpoints with a <3% discrepancy. Phylogenetic analysis demonstrated that decreased susceptibility has arisen sporadically in Vietnamese S. sonnei isolates on at least seven occasions between 2000 and 2009 but failed to become established. While the proposed susceptibility breakpoints may allow better recognition of resistant isolates, additional studies are required to assess the impact on the clinical outcome. The potential emergence of azithromycin resistance highlights the need for alternative options for management of Shigella infections in countries where Shigella is endemic.

In Vivo Transfer and Microevolution of Avian Native IncA/C2blaNDM-1-Carrying Plasmid pRH-1238 during a Broiler Chicken Infection Study.

ABSTRACTThe emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) in wildlife and livestock animals pose an important safety concern for public health. With our in vivo broiler chicken infection study, we investigated the transfer and experimental microevolution of the blaNDM-1-carrying IncA/C2 plasmid (pRH-1238) introduced by avian native Salmonella enterica subsp. enterica serovar Corvallis without inducing antibiotic selection pressure. We evaluated the dependency of the time point of inoculation on donor (S. Corvallis [12-SA01738]) and plasmid-free Salmonella recipient [d-tartrate-fermenting (d-Ta+) S. Paratyphi B (13-SA01617), referred to here as S. Paratyphi B (d-Ta+)] excretion by quantifying their excretion dynamics. Using plasmid profiling by S1 nuclease-restricted pulsed-field gel electrophoresis, we gained insight into the variability of the native plasmid content among S. Corvallis reisolates as well as plasmid acquisition in S. Paratyphi B (d-Ta+) and the enterobacterial gut microflora. Whole-genome sequencing enabled us to gain an in-depth insight into the microevolution of plasmid pRH-1238 in S. Corvallis and enterobacterial recipient isolates. Our study revealed that the fecal excretion of avian native carbapenemase-producing S. Corvallis is significantly higher than that of S. Paratyphi (d-Ta+) and is not hampered by S. Paratyphi (d-Ta+). Acquisition of pRH-1238 in other Enterobacteriaceae and several events of plasmid pRH-1238 transfer to different Escherichia coli sequence types and Klebsiella pneumoniae demonstrated an interspecies broad host range. Regardless of the microevolutionary structural deletions in pRH-1238, the single carbapenem resistance marker blaNDM-1 was maintained on pRH-1238 throughout the trial. Furthermore, we showed the importance of the gut E. coli population as a vector of pRH-1238. In a potential scenario of the introduction of NDM-1-producing S. Corvallis into a broiler flock, the pRH-1238 plasmid could persist and spread to a broad host range even in the absence of antibiotic pressure.

Light emitting diode irradiation induced shape conversion of DNA-capped silver nanoparticles and their antioxidant and antibacterial activities

Due to size- and shape-dependent properties, a shape-controlled synthesis of silver nanoparticles (AgNPs) is one of the research challenging topics for their production and potential applications. This work reported the simple eco-friendly syntheses of different shaped AgNPs controlled by the plasmid DNA content and light emitting diodes (LEDs) irradiation. The synthesized AgNPs appeared as yellow, orange and green colloidal AgNPs, which transmission electron microscope (TEM) images revealed their different shapes; spherical AgNPs, a mixture of spherical and hexagonal AgNPs, and a mixture of spherical, hexagonal and corner-truncated triangle AgNPs, respectively. The average sizes of spherical, hexagonal and corner-truncated triangle AgNPs in the green colloidal solution were 12.32 ± 2.22, 23.03 ± 6.62 and 15.84 ± 4.31 nm, respectively. The analyses of X-ray diffraction, selected area electron diffraction and high-resolution TEM indicated the crystalline nature of the synthesized particles as the face-centred cubic silver. All synthesized AgNPs exhibited antioxidant activities similarly, whereas the yellow colloidal AgNPs exhibited the strongest antibacterial activity against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus as compared with the green and orange colloidal AgNPs.

Genomic analyses of the Chlamydia trachomatis core genome show an association between chromosomal genome, plasmid type and disease

BackgroundChlamydia trachomatis (Ct) plasmid has been shown to encode genes essential for infection. We evaluated the population structure of Ct using whole-genome sequence data (WGS). In particular, the relationship between the Ct genome, plasmid and disease was investigated.ResultsWGS data from 157 Ct isolates deposited in the Chlamydiales pubMLST database (http://pubMLST.org/chlamydiales/) were annotated with 902 genes including the core and accessory genome. Plasmid associated genes were annotated and a plasmid MLST scheme was defined allowing plasmid sequence types to be determined. Plasmid allelic variation was investigated. Phylogenetic relationships were examined using the Genome Comparator tool available in pubMLST. Phylogenetic analyses identified four distinct Ct core genome clusters and six plasmid clusters, with a strong association between the chromosomal genotype and plasmid. This in turn was linked to ompA genovars and disease phenotype. Horizontal genetic transfer of plasmids was observed for three urogenital-associated isolates, which possessed plasmids more commonly found in isolates resulting from ocular infections. The pgp3 gene was identified as the most polymorphic plasmid gene and pgp4 was the most conserved.ConclusionA strong association between chromosomal genome, plasmid type and disease was observed, consistent with previous studies. This suggests co-evolution of the Ct chromosome and their plasmids, but we confirmed that plasmid transfer can occur between isolates. These data provide a better understanding of the genetic diversity occurring across the Ct genome in association with the plasmid content.

Genomic Diversity in the Endosymbiotic Bacterium Rhizobium leguminosarum.

Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

Salmonella enterica serovars Typhimurium and Enteritidis causing mixed infections in febrile children in Mozambique.

Background and purposeInvasive nontyphoidal salmonellosis, mostly caused by serovars Typhimurium and Enteritidis of Salmonella enterica, has emerged as a major public health problem in sub-Saharan Africa. The aim of this study was the clinical and microbiological characterization of nontyphoidal salmonellosis episodes affecting febrile children in Mozambique.Patients and methodsThe clinical records of the patients were evaluated, and S. enterica isolates were characterized with regard to serovar, phage type, antimicrobial resistance (phenotype/responsible genes), plasmid content, pulsed-field gel electrophoresis, and multilocus sequence typing.ResultsFifteen S. Typhimurium and 21 S. Enteritidis isolates were recovered from blood samples of 25 children, the majority with underlying risk factors. With regard to phage typing, most isolates were either untypeable or reacted but did not conform, revealing that a number of previously unrecognized patterns are circulating in Mozambique. Most isolates were multidrug-resistant, with nearly all of the responsible genes located on derivatives of serovar-specific virulence plasmids. ST313 and ST11 were the predominant sequence types associated with S. Typhimurium and S. Enteritidis, respectively, and the uncommon ST1479 was also detected in S. Enteritidis. A distinct XbaI fragment of ~350 kb was associated with pulsed-field gel electrophoresis patterns of multidrug-resistant isolates of S. Enteritidis. Nearly half of the children were coinfected with both serovars, a fact expected to aggravate the disease and hamper the treatment. However, particularly poor outcomes were not observed for the coinfected patients.ConclusionMixed Salmonella infections could frequently occur in febrile children in Mozambique. Additional studies are required to determine their actual impact and consequences, not only in this country, but also in other African countries.

A blaOXA-181-harbouring multi-resistant ST147 Klebsiella pneumoniae isolate from Pakistan that represent an intermediate stage towards pan-drug resistance.

Carbapenem resistant Klebsiella pneumoniae (CR-KP) infections are an ever-increasing global issue, especially in the Indian subcontinent. Here we report genetic insight into a blaOXA-181 harbouring Klebsiella pneumoniae, belonging to the pandemic lineage ST147, that represents an intermediate stage towards pan-drug resistance. The CR-KP isolate DA48896 was isolated from a patient from Pakistan and was susceptible only to tigecycline and colistin. It harboured blaOXA-181 and was assigned to sequence type ST147. Analysis from whole genome sequencing revealed a very high sequence similarity to the previously sequenced pan-resistant K. pneumoniae isolate MS6671 from the United Arab Emirates. The two isolates are very closely related with only 46 chromosomal nucleotide differences, 14 indels and differences in plasmid content. Both carry a substantial number of plasmid-borne and chromosomally encoded resistance determinants. Interestingly, the two differences in susceptibility between the isolates could be attributed to DA48896 lacking an insertion of blaOXA-181 into the mgrB gene that results in colistin resistance in MS6671 and SNPs affecting AcrAB efflux pump expression likely to result in tigecycline resistance. These differences between the otherwise very similar isolates indicate that strong selection has occurred for resistance towards these last-resort drugs and illustrates the trajectory of resistance evolution of OXA-181-producing versions of the ST147 international risk clone.

Whole genome sequencing for the molecular characterization of carbapenem-resistant Klebsiella pneumoniae strains isolated at the Italian ASST Fatebenefratelli Sacco Hospital, 2012\u20132014

BackgroundThe emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment.MethodsSixty-eight carbapenemase-producing K. pneumoniae strains isolated at Luigi Sacco University Hospital-ASST Fatebenefratelli Sacco (Milan, Italy) between 2012 and 2014 were characterised microbiologically and molecularly. They were tested for drug susceptibility and carbapenemase phenotypes, investigated by means of repetitive extra-genic palindromic polymerase chain reaction (REP-PCR), and fully sequenced by means of next-generation sequencing for the in silico analysis of multi-locus sequence typing (MLST), their resistome, virulome and plasmid content, and their core single nucleotide polymorphism (SNP) genotypes.ResultsAll of the samples were resistant to carbapenems, other β-lactams and ciprofloxacin; many were resistant to aminoglycosides and tigecycline; and seven were resistant to colistin. Resistome analysis revealed the presence of blaKPC genes and, less frequently blaSHV, blaTEM, blaCTX-M and blaOXA, which are related to resistance to carbapenem and other β-lactams. Other genes conferring resistance to aminoglycoside, fluoroquinolone, phenicol, sulphonamide, tetracycline, trimethoprim and macrolide-lincosamide-streptogramin were also detected. Genes related to AcrAB-TolC efflux pump-dependent and pump-independent tigecycline resistance mechanisms were investigated, but it was not possible to clearly correlate the genomic features with tigecycline resistance because of the presence of a common mutation in susceptible, intermediate and resistant strains. Concerning colistin resistance, the mgrB gene was disrupted by an IS5-like element, and the mobile mcr-1 and mcr-2 genes were not detected in two cases. The virulome profile revealed type-3 fimbriae and iron uptake system genes, which are important during the colonisation stage in the mammalian host environment. The in silico detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX1, IncX3, IncFII(K), IncN, IncL/M(pMU407) and IncFIA(HI1). REP-PCR showed five major clusters, and MLST revealed six different sequence types: 512, 258, 307, 1519, 745 and 101. Core SNP genotyping, which led to four clusters, correlated with the MLST data. Isolates of the same sequencing type often had common genetic traits, but the SNP analysis allowed greater strain tracking and discrimination than either the REP-PCR or MLST analysis.ConclusionOur findings support the importance of implementing bacterial genomics in clinical medicine in order to complement traditional methods and overcome their limited resolution.

Horizontal Dissemination of Antimicrobial Resistance Determinants in Multiple Salmonella Serotypes following Isolation from the Commercial Swine Operation Environment after Manure Application.

The aim of this study was to characterize the plasmids carrying antimicrobial resistance (AMR) determinants in multiple Salmonella serotypes recovered from the commercial swine farm environment after manure application on land. Manure and soil samples were collected on day 0 before and after manure application on six farms in North Carolina, and sequential soil samples were recollected on days 7, 14, and 21 from the same plots. All environmental samples were processed for Salmonella, and their plasmid contents were further characterized. A total of 14 isolates including Salmonella enterica serotypes Johannesburg (n = 2), Ohio (n = 2), Rissen (n = 1), Typhimurium var5- (n = 5), Worthington (n = 3), and 4,12:i:- (n = 1), representing different farms, were selected for plasmid analysis. Antimicrobial susceptibility testing was done by broth microdilution against a panel of 14 antimicrobials on the 14 confirmed transconjugants after conjugation assays. The plasmids were isolated by modified alkaline lysis, and PCRs were performed on purified plasmid DNA to identify the AMR determinants and the plasmid replicon types. The plasmids were sequenced for further analysis and to compare profiles and create phylogenetic trees. A class 1 integron with an ANT(2″)-Ia-aadA2 cassette was detected in the 50-kb IncN plasmids identified in S Worthington isolates. We identified 100-kb and 90-kb IncI1 plasmids in S Johannesburg and S Rissen isolates carrying the blaCMY-2 and tet(A) genes, respectively. An identical 95-kb IncF plasmid was widely disseminated among the different serotypes and across different farms. Our study provides evidence on the importance of horizontal dissemination of resistance determinants through plasmids of multiple Salmonella serotypes distributed across commercial swine farms after manure application.IMPORTANCE The horizontal gene transfer of antimicrobial resistance (AMR) determinants located on plasmids is considered to be the main reason for the rapid proliferation and spread of drug resistance. The deposition of manure generated in swine production systems into the environment is identified as a potential source of AMR dissemination. In this study, AMR gene-carrying plasmids were detected in multiple Salmonella serotypes across different commercial swine farms in North Carolina. The plasmid profiles were characterized based on Salmonella serotype donors and incompatibility (Inc) groups. We found that different Inc plasmids showed evidence of AMR gene transfer in multiple Salmonella serotypes. We detected an identical 95-kb plasmid that was widely distributed across swine farms in North Carolina. These conjugable resistance plasmids were able to persist on land after swine manure application. Our study provides strong evidence of AMR determinant dissemination present in plasmids of multiple Salmonella serotypes in the environment after manure application.

Curing of plasmid contents of Proteus spp. isolated from urinary tract infections in AL- Diwaniyah city

This study included isolation and identification of Proteus spp . from urinary tract infections of Gynecology and padiatrics Hospital teaching in Diwaniyah cityand investigated the virulence factors connected with pathogenicity of this bacteria. It aims also at curing of plasmid contact by means of using some chemical materials. The bacteria Proteus spp. Isolates formed 16% which belong P. mirabilis and P. vulgaris from bacterial causes of urinary tract infection in children. The isolates of this bacteria has some physiological characteristics which represent virulence factors related to the ability of bacteria to make infection . It seems that 80% from these isolates were capable to Haemagglutination of RBCs . These isolates produce also Haemolysine enzyme. It was found that percentage of production was 60% . As for Blactamase enzyme production 40% of these bacteria isolates showed ability to produce his enzyme. Moreover, only 20% from Proteus isolates showed ability to produce bacteriocin production. All these isolates were not able to produce capsule production. When testing the resistance of Proteus isolates toward 8 different commonly used antibiotics in treatment of urinary tract infection, results showed that the isolates have different resistance depending on the type of antibiotic and the strain of the isolates. All these isolates were completely resistance Nitrofurantion in 100% while the least resistance toward Nalidixic acid and Gentamicin reached 20% for both. The results showed that there were DNA plasmid in 3 bacteria isolates but there was no relationship between the plasmid bands and resistance toward antibiotics. The P. mirabilis isolate which contained 4 plasmid bands was treated with SDS and Ethidiun bromide in an attempt to curing plasmid bands. The result showed that this isolate lost the ability to produce the majority of virulence factors that caused this disease and lost its resistance for som antibiotics especially Nalidix acid , Ampicillin and Choramphenicol. The ethidium bromide was the most effective in curing process since the resultant strain aest all plasmid bands.

Characterization of a Large Antibiotic Resistance Plasmid Found in Enteropathogenic Escherichia coli Strain B171 and Its Relatedness to Plasmids of Diverse E. coli and Shigella Strains.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline (tetA), sulfonamides (sulI), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor (csi). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 (csi and traI) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli.

First description in Europe of the emergence of Enterococcus faecium ST117 carrying both vanA and vanB genes, isolated in Greece.

An Enterococcus faecium isolate (Efa-125) carrying both the vanA and vanB genes was recovered from a patient with bacteraemia treated in a Greek hospital. Since this is the first description in Europe of E. faecium carrying both vanA and vanB genes, the isolate was further studied. Susceptibility to several antibiotics was determined using the VITEK®2 automated system. The isolate was typed by multilocus sequence typing (MLST). To define the genetic units of the vanA and vanB genes, the plasmid content of Efa-125 was analysed by pulsed-field gel electrophoresis (PFGE) of total DNA digested with S1 nuclease followed by hybridisation with digoxigenin-labelled vanA and vanB probes. In addition, plasmids and chromosomes were sequenced using the Illumina MiSeq platform. E. faecium Efa-125 belonged to ST117 and expressed resistance both to vancomycin and teicoplanin, with minimum inhibitory concentrations (MICs) for both of 256mg/L. The vanA gene was carried on a 29 320-bp plasmid exhibiting high similarity to pA6981 previously characterised from Enterococcus gallinarum A6981, whereas vanB was part of a Tn1549-like transposon integrated into the chromosome. Expression of the VanA phenotype was correlated with the presence of intact vanZ and vanS genes. This is the first detection in Greece of vanA-vanB genotype/VanA phenotype E. faecium and indicates an evolving epidemiology of vancomycin-resistant enterococci.

Prevalence of a New Variant OXA-204 and OXA-48 Carbapenemases Plasmids Encoded inKlebsiella pneumoniaeClinical Isolates in Tunisia

Carbapenemase-producing Klebsiella pneumoniae strains have emerged as a major problem for healthcare systems. The aim of this study was to determine the role and diversity of plasmids harboring carbapenemases encoding genes from a collection of K. pneumoniae isolates recovered between July 2011 and January 2012, with decreased susceptibility to carbapenems. Imipenem (IPM), ertapenem (ETP), meropenem (MEM), and doripenem (DOR) minimum inhibitory concentrations (MICs) were determined by E-test. Carbapenemase production was detected with the modified Hodge test. β-Lactamases encoding genes were amplified by PCR and sequenced. Plasmid incompatibility groups harbored by carbapenemases producers were investigated using the PCR-based replicon typing method and the clonal relationship of the isolates was investigated by pulse filed electrophoresis. IMP, ertapenem, meropenem, and doripenem MICs ranged between 0.25 and 16 mg/L. Carbapenemase activity was detected in 14 isolates. Two carbapenemases were identified: OXA-48 in 13 isolates and a new variant OXA-204 in 1 isolate, in combination with extended-spectrum β-lactamases, CTX-M-1, CTX-M-9, CTX-M-14, CTX-M-15, and VEB-8. One isolate produced CMY-2. OXA-48 and the new variant OXA-204 were confirmed as transferable plasmid encoded. The carbapenemase-producing K. pneumoniae harbored plasmids of the A/C, LVPK, and L/M replicon types. Thirteen different pulso types were observed. Three pairs of isolates showed a clonal relatedness. This diversity in β-lactamases, in pulso types and in plasmid content, shows the ability of OXA-type carbapenemase to disseminate. This is worrying for the control of the increase in antibiotic resistance frequency and necessitates that continuous investigations in the clinical setting remain a high priority to clarify the contribution of antimicrobial use into multiresistance bacterial dissemination.