50-kb Plasmid Research Articles

Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid.

The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of bla NDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including bla KPC, bla IMP, bla VIM, bla OXA-48 and bla NDM-1 were screened and sequenced. Ninety isolates were identified as harboring the bla KPC-2 genes, and five bla NDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three bla NDM-1-positive K. pneumoniae isolates belonged to two different clones. S1-PFGE and southern blot suggested that the bla NDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4) and 104.5 to 138.9 kb (n=1), respectively, all of which could easily transfer to Escherichia coli by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around bla NDM-1 (bla NDM-1-trpF- dsbC-cutA1-groEL-ΔInsE,) was detected. PCR mapping and sequencing demonstrated that four smaller bla NDM-1 plasmids contained a common gene environment around bla NDM-1 (IS5-bla NDM-1-trpF- dsbC-cutA1-groEL). We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the bla NDM-1 gene among the CRE.

Molecular epidemiology of carbapenemase-producing Escherichia coli and the prevalence of ST131 subclone H30 in Shanghai, China.

The molecular characteristics and epidemiology of carbapenemase-producing Escherichia coli (CPEC) isolates from Shanghai, China, were investigated using 21 imipenem-resistant E. coli isolates obtained from a Shanghai teaching hospital from 2011 to 2014. The presence of bla KPC, bla IMP, bla VIM, bla OXA-48, and bla NDM was assessed by polymerase chain reaction (PCR) amplification and sequencing. CPEC isolates were characterized by the Etest®, multilocus sequence typing (MLST), and pulse-field gel electrophoresis (PFGE). Plasmids carrying resistance genes were analyzed by conjugation experiments, replicon typing, plasmid MLST (pMLST), S1 nuclease PFGE (S1-PFGE), and Southern hybridization. The genetic environment of the resistance genes was determined by PCR and sequencing. Among the 21 E. coli isolates, 16 produced carbapenemases; of these, ten isolates transferred carbapenemase-encoding plasmids to recipient bacteria. Nine of the 16 isolates were clonally related, and their PFGE patterns were designated type A. ST131 was the predominant sequence type (11 isolates, 68.8 %); the H30 subclone comprised 81.8 % of the ST131 strains. In all three isolates, bla IMP-4 was located on 50-kb IncN plasmids. All but two bla KPC-2 genes were carried on IncF plasmids of various sizes. Hence, both clone-spread and horizontal transfer mediated the dissemination of carbapenemase-producing genes in the Shanghai isolates.

Dissemination of blaOXA-23 in Acinetobacter spp. in China: main roles of conjugative plasmid pAZJ221 and transposon Tn2009.

Production of the OXA-23 carbapenemase is the most common reason for the increasing carbapenem resistance in Acinetobacter spp. This study was conducted to reveal the genetic basis of blaOXA-23 dissemination in Acinetobacter spp. in China. A total of 63 carbapenem-resistant OXA-23-producing Acinetobacter sp. isolates, representing different backgrounds, were selected from 28 hospitals in 18 provinces for this study. Generally, two patterns of plasmids carrying blaOXA-23 were detected according to S1-nuclease pulsed-field gel electrophoresis and Southern blot hybridization. A ca. 78-kb plasmid, designated pAZJ221, was found in 23 Acinetobacter baumannii and three Acinetobacter nosocomialis isolates, while a novel ca. 50-kb plasmid was carried by only two other A. baumannii isolates. Three of these isolates had an additional copy of blaOXA-23 on the chromosome. Transformation of the two plasmids succeeded, but only pAZJ221 was conjugative. Plasmid pAZJ221 was sequenced completely and found to carry no previously known resistance genes except blaOXA-23. The blaOXA-23 gene of the remaining 35 isolates was chromosome borne. The blaOXA-23 genetic environments were correlated with Tn2009 in 57 isolates, Tn2008 in 5 isolates, and Tn2006 in 1 isolate. The MIC values for the carbapenems with these isolates were not significantly associated with the genomic locations or the copy numbers of blaOXA-23. Overall, these observations suggest that the plasmid pAZJ221 and Tn2009 have effectively contributed to the wide dissemination of blaOXA-23 in Acinetobacter spp. in China and that horizontal gene transfer may play an important role in dissemination of the blaOXA-23 gene.

Plasmid Profile Analysis and Antibiotic Resistance Patterns of Shigella Isolates from Kwale County, Kenya

Shigellosis caused by multidrug-resistant (MDR) Shigella strains is an important cause of morbidity and mortality in developing countries. Shigella strains usually harbour heterogeneous population of plasmids which can confer resistance to different antibiotics. A hospital based survey was conducted to determine the pattern of antimi- crobial resistance and plasmid profiles in Shigella species from diarrheal patients in Kwale county. Twenty nine (8.1%) Shigella spp were obtained from 360 stool samples collected and each strain screened for antimicrobial resistance to common antimicrobial agents and presence of plasmids. High antibiotic resistance was found to Sulfamethoxazole-tri- methoprim (79%), Ampicillin (75.9%) and Streptomycin (75.9%). All isolates were sensitive to ciprofloxacin, ceph- alosporins and Aztreonam antibiotics. Ninety seven percent of the isolates screened contained one or more plasmids ranging from 1-6. Majority were small plasmids. Species specific plasmids of 3.2kb, 9.0kb, and 3.8kb were found in Shigella flexneri, Shigella dysenteriae and Shigella sonnei respectively, while 34.5% and 79.3% MDR strains harbored large (>100kb) and middle range self-transmissible plasmids (10-100kb). In all species, no association was found be- tween specific plasmid profile and antibiotic resistance patterns. Significant association was found between carriage of 80kb plasmid in S. dysenteriae (P<0.001), combined 48kb and 80kb plasmids in S. flexneri (P=0.002) and S. sonnei (P=0.041) and MDR phenotype. The middle range plasmids specified resistance to Sulfamethoxazole-trimethoprim, Ampicillin, Streptomycin and Tetracycline. This study reports the usefulness of plasmid profiling in detection and discrimination of Shigella strains in Kenya. The study also shows that middle range self-transferable R-plasmids are widespread among the circulating MDR Shigella strains indicating the important role of these genetic elements in the development and dissemination of multidrug resistance.

Characteristics of KPC-2–producing Klebsiella pneumoniae (ST258) clinical isolates from outbreaks in 2 Mexican medical centers

The KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) is an important pathogen widely spread in nosocomial infections. In this study, we identified the KPC-2–producing K. pneumoniae clinical isolates of 2 unrelated outbreaks that corresponded to pandemic strain ST258. The isolates showed high resistance to cephalosporins, carbapenems, quinolones, and colistin. The KPC-2–producing K. pneumoniae isolates were compared to the previously studied KPC-3–producing K. pneumoniae isolates from an outbreak in Mexico; they showed an unrelated pulsed-field gel electrophoresis fingerprinting pattern and a different plasmid profile. The KPC-2 carbapenemase gene was identified in two 230- and 270-kb non-conjugative plasmids; however, 1 isolate transferred the KPC-2 gene onto an 80-kb plasmid. These findings endorse the need of carrying out a continuous molecular epidemiological surveillance of carbapenem-resistant isolates in hospitals in Mexico.

IS1R-mediated plasticity of IncL/M plasmids leads to the insertion of bla OXA-48 into the Escherichia coli Chromosome.

The OXA-48 carbapenemase is mainly encoded by ∼ 62-kb IncL/M plasmids. However, chromosome-mediated genes have been observed in Escherichia coli isolates. In this work, we investigated the genetic environment of OXA-48 in members of the family Enterobacteriaceae (n = 22) to understand how the OXA-48-encoding gene is transferred into the E. coli chromosome. The OXA-48-encoding gene was located within intact Tn1999.2 transposons in the ∼ 62-kb plasmids or within a truncated variant of Tn1999.2 for the OXA-48-encoding genes located in the chromosomes of E. coli bacteria. The analysis of the Tn1999.2 genetic environment revealed an inverted orientation of the transposon in five ∼ 62-kb plasmids (5/14 [35%]) and in all chromosome inserts (n = 8). The sequencing of pRA35 plasmid showed that this orientation of Tn1999.2 and the acquisition of an IS1R insertion sequence generated a 21.9-kb IS1R-based composite transposon encoding OXA-48 and designated Tn6237. The sequencing of a chromosomal insert encoding OXA-48 also revealed this new transposon in the E. coli chromosome. PCR mapping showed the presence of this element in all strains harboring an OXA-48-encoding chromosomal insert. However, different insertion sites of this transposon were observed in the E. coli chromosome. Overall, these findings indicate a plasticity of the OXA-48 genetic environment mediated by IS1R insertion sequences. The insertion sequences can induce the transfer of the OXA-encoding gene into E. coli chromosomes and thereby promote its persistence and expression at low levels.

OXA-253, a Variant of the Carbapenem-Hydrolyzing Class D β - Lactamase OXA-143 in Acinetobacter baumannii

The carbapenem-hydrolyzing class D β-lactamase OXA-253 was identified in an Acinetobacter baumannii clinical isolate belonging to sequence type 113 (ST113) in Brazil. OXA-253 shares 93.8% amino acid identity with OXA-143. The blaOXA-253 gene is located on a ca. 20-kb plasmid. The genetic environment of the blaOXA-253 gene shares the highest identity with ubiquitous GR2 group plasmids usually carrying blaOXA-24/-40 genes.

Evaluation of Virulence and Antimicrobial Resistance inSalmonella entericaSerovar Enteritidis Isolates from Humans and Chicken- and Egg-Associated Sources

Salmonella enterica serovar Enteritidis is a leading cause of salmonellosis throughout the world and is most commonly associated with the consumption of contaminated poultry and egg products. Salmonella Enteritidis has enhanced ability to colonize and persist in extraintestinal sites within chickens. In this study, 54 Salmonella Enteritidis isolates from human patients (n=28), retail chicken (n=9), broiler farms (n=9), and egg production facilities (n=8) were characterized by antimicrobial susceptibility testing, plasmid analysis, genetic relatedness using XbaI and AvrII pulsed-field gel electrophoresis (PFGE), and the presence of putative virulence genes. Nine isolates were evaluated for their abilities to invade and survive in intestinal epithelial and macrophage cell lines. Overall, 56% (n=30) of isolates were resistant to at least one antimicrobial agent tested, yet no isolates showed resistance to more than three antimicrobials. All isolates carried a common ∼55-kb plasmid, with some strains containing additional plasmids ranging from 3 to 50 kb. PFGE analysis revealed five XbaI and AvrII clusters. There were significant overlaps in the PFGE patterns of the isolates from human, chicken, and egg houses. All isolates tested PCR positive for iacP, purR, ttrB, spi4H, rmbA, sopE, invA, sopB, spvB, pagC, msgA, spaN, orgA, tolC, and sifA, and negative for iss, virB4, and sipB. Of the isolates selected for virulence testing, those containing the iron acquisition genes, iutA, sitA, and iucA, and ∼50-kb plasmids demonstrated among the highest levels of macrophage and epithelial cell invasion, which may indicate their importance in pathogenesis.

The First Case of Septicemia Caused by Imipenem-Susceptible, Meropenem-Resistant Klebsiella pneumoniae

The First Case of Septicemia Caused by Imipenem-Susceptible, Meropenem-Resistant <i>Klebsiella pneumoniae</i>

In planta induced changes in the native plasmid profile of Pseudomonas syringae pathover phaseolicola strain 1302A

Pseudomonas syringae pv. phaseolicola (Pph) strain 1302A, a causative agent of halo blight in the common bean Phaseolus vulgaris, contains four native plasmids designated pAV505 (150kb), pAV506 (50kb), pAV507 (47kb) and pAV508 (42kb). Pph 1302A also contains a 106kb genomic island PPHGI-1 which shares features with integrative and conjugative elements (ICElands) and carries the effector gene avrPphB (hopAR1) which triggers a defensive response in bean cultivars carrying the matching R3 resistance gene. It has been shown that when Pph 1302A is sequentially inoculated (passaged) through resistant bean cultivar Tendergreen (TG) in which the hypersensitive response (HR) is generated, the three largest plasmids are lost and an extra ∼100kb plasmid is gained, which tests confirmed to be the 106kb circular form of PPHGI-1. The aim of the current study was to determine if upon further passaging though bean plants, the plasmid profile of Pph 1302A would alter again and if the missing plasmids had been integrated into the chromosome. Pph 1302A-P6, the strain with the altered plasmid profile was passaged twice through TG and of the four re-isolated strains examined all displayed the plasmid profile associated with wildtype Pph 1302A, that is, all four native plasmids had reappeared and the PPHGI-1 plasmid was absent. This demonstrated that the plasmid composition of Pph 1302A-P6 could indeed change on further exposure to the plant environment and also that the seemingly missing native plasmids were still present within the genome, lending evidence to the theory that they had integrated into the chromosome. Furthermore two of these re-isolated strains had lost PPHGI-1 entirely, meaning they no longer triggered a HR on TG and instead generated a disease response. This study clearly demonstrates the plasticity of the bacterial genome and the extent it can be influenced by the plant environment and conditions generated during the HR.

Outbreak Caused by an Ertapenem-Resistant, CTX-M-15-Producing Klebsiella pneumoniae Sequence Type 101 Clone Carrying an OmpK36 Porin Variant

Although numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae (CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum β-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining β-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the blaCTX-M-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniae isolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak.

Characterization of TEM-1 β-Lactamase-Producing Kingella kingae Clinical Isolates

Kingella kingae is a human pathogen that causes pediatric osteoarticular infections and infective endocarditis in children and adults. The bacterium is usually susceptible to β-lactam antibiotics, although β-lactam resistance has been reported in rare isolates. This study was conducted to identify β-lactam-resistant strains and to characterize the resistance mechanism. Screening of a set of 90 K. kingae clinical isolates obtained from different geographic locations revealed high-level resistance to penicillins among 25% of the strains isolated from Minnesota and Iceland. These strains produced TEM-1 β-lactamase and were shown to contain additional ≥50-kb plasmids. Ion Torrent sequencing of extrachromosomal DNA from a β-lactamase-producing strain confirmed the plasmid location of the blaTEM gene. An identical plasmid pattern was demonstrated by multiplex PCR in all β-lactamase producers. The porin gene's fragments were analyzed to investigate the relatedness of bacterial strains. Phylogenetic analysis revealed 27 single-nucleotide polymorphisms (SNPs) in the por gene fragment, resulting in two major clusters with 11 allele types forming bacterial-strain subclusters. β-Lactamase producers were grouped together based on por genotyping. Our results suggest that the β-lactamase-producing strains likely originate from a single plasmid-bearing K. kingae isolate that traveled from Europe to the United States, or vice versa. This study highlights the prevalence of penicillin resistance among K. kingae strains in some regions and emphasizes the importance of surveillance for antibiotic resistance of the pathogen.

First Report of the Multiresistance Gene cfr in Streptococcus suis

The multiresistance gene cfr was identified for the first time in streptococci, namely, in porcine Streptococcus suis isolate S10. The cfr gene was detected on the ~100-kb plasmid pStrcfr, where it was bracketed by two copies of the novel insertion sequence ISEnfa5, located in the same orientation. The detection of a cfr- and ISEnfa5-containing amplicon by inverse PCR suggests that ISEnfa5 may play a role in the dissemination of cfr.

A Plasmid-Encoded Phosphatase Regulates Bacillus subtilis Biofilm Architecture, Sporulation, and Genetic Competence

Bacillus subtilis biofilm formation is tightly regulated by elaborate signaling pathways. In contrast to domesticated lab strains of B. subtilis which form smooth, essentially featureless colonies, undomesticated strains such as NCIB 3610 form architecturally complex biofilms. NCIB 3610 also contains an 80-kb plasmid absent from laboratory strains, and mutations in a plasmid-encoded homolog of a Rap protein, RapP, caused a hyperrugose biofilm phenotype. Here we explored the role of rapP phrP in biofilm formation. We found that RapP is a phosphatase that dephosphorylates the intermediate response regulator Spo0F. RapP appears to employ a catalytic glutamate to dephosphorylate the Spo0F aspartyl phosphate, and the implications of the RapP catalytic glutamate are discussed. In addition to regulating B. subtilis biofilm formation, we found that RapP regulates sporulation and genetic competence as a result of its ability to dephosphorylate Spo0F. Interestingly, while rap phr gene cassettes routinely form regulatory pairs; i.e., the mature phr gene product inhibits the activity of the rap gene product, the phrP gene product did not inhibit RapP activity in our assays. RapP activity was, however, inhibited by PhrH in vivo but not in vitro. Additional genetic analysis suggests that RapP is directly inhibited by peptide binding. We speculate that PhrH could be subject to posttranslational modification in vivo and directly inhibit RapP activity or, more likely, PhrH upregulates the expression of a peptide that, in turn, directly binds to RapP and inhibits its Spo0F phosphatase activity.

Commensal Enterobacteriaceae as reservoirs of extended-spectrum beta-lactamases, integrons, and sul genes in Portugal.

Bacteria colonizing the human intestine have a relevant role in the spread of antimicrobial resistance. We investigated the faecal carriage of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in healthy humans from Portugal and analyzed the distribution of sul genes and class 1 and 2 integrons. Faecal samples (n = 113) were recovered from healthy persons (North/Centre of Portugal, 2001–2004) and plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). Isolates representing different morphotypes/plate and antibiotic susceptibility patterns (n = 201) were selected. Isolates resistant to sulfonamides and/or streptomycin, gentamicin, and trimethoprim were screened (PCR and sequencing) for sul genes (sul1, sul2, sul3) and class 1 and 2 integrons. Presence of ESBLs was inferred using the double disk synergy test (DDST) and further confirmed by PCR and sequencing. ESBL producers were selected for clonal analysis, plasmid characterization and conjugation assays by standard methods. ESBL-producing isolates were found in 1.8% (2/113) of samples, corresponding to Escherichia coli of phylogroups A (n = 1) and B1 (n = 1) carrying transferable blaCTX-M-14 and the new blaTEM-153, respectively. A 80kb IncK plasmid bearing blaCTX-M-14 was found, being highly related to that widely spread among CTX-M-14 producers of humans and animals from Portugal and other European countries. sul genes were found in 88% (22/25; sul2-60%, sul1-48%, sul3-4%) of the sulfonamide resistant isolates. Class 1 integrons were more frequently found than class 2 (7%, 14/201 vs. 3%, 6/201). Interestingly, gene cassette arrangements within these platforms were identical to those commonly observed among Enterobacteriaceae from Portuguese food-producing animals, although aadA13 is here firstly described in Morganella morganii. These results reinforce the relevance of human commensal flora as reservoir of clinically relevant antibiotic resistance genes including blaESBLs, and highly transferable genetic platforms as IncK epidemic plasmids.

New Delhi Metallo-β-Lactamase 4–producingEscherichia coliin Cameroon

New Delhi Metallo-β-Lactamase 4–producing<i>Escherichia coli</i>in Cameroon

Imipenem Resistance inKlebsiella pneumoniaeIs Associated to the Combination of Plasmid-Mediated CMY-4 AmpC β-Lactamase and Loss of an Outer Membrane Protein

This study was conducted to identify the molecular mechanisms of imipenem resistance in a Klebsiella pneumoniae (Kp16137) isolate recovered in August 2008 at the University Hospital Sahloul, Sousse, Tunisia. The strain was identified with the API 20E system; antibiotic-containing disks were used for detection of antibiotic susceptibility by a disk diffusion assay. We investigated the presence of β-lactamases by PCR, using specific primers for bla(TEM), bla(SHV), bla(CTX-M), bla(OXA), bla(CMY), bla(ACC), bla(FOX), bla(IMP), bla(KPC), bla(VIM), and by sequencing. Extraction of plasmid DNA from Kp16137 and the transconjugant was performed by the method of Kado. Southern transfer was performed on nylon. The membrane was hybridized with a specific probe for the bla(CMY-2) gene. Outer membrane proteins were isolated and were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% polyacrylamide gel. K. pneumoniae Kp16137 was resistant to all available β-lactams, including third generation cephalosporins and carbapenems. The screening of β-lactamases showed the presence of three β-lactamases: TEM-1, SHV-61, and CMY-4. The CMY-4 β-lactamase was located on an 80-kb plasmid. An analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of 42 kDa. The loss of this outer membrane protein band correlated with imipenem resistance in this strain. In K. pneumoniae 16137, synthesis of a plasmid-mediated β-lactamase: AmpC CMY-4, together with alteration in permeability led to resistance to all available β-lactams and carbapenems.

Complete Genome Sequence of the Highly Hemolytic Strain Bacillus cereus F837/76

Highly hemolytic strain Bacillus cereus F837/76 was isolated in 1976 from a contaminated prostate wound. The complete nucleotide sequence of this strain reported here counts nearly 36,500 single-nucleotide differences from the closest sequenced strain, Bacillus thuringiensis Al Hakam. F827/76 also contains a 10-kb plasmid that was not detected in the Al Hakam strain.

Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

BackgroundMost Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1). F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.MethodsPhage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.ResultsSeven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.ConclusionsOur high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.

Human and Swine Hosts Share Vancomycin-Resistant Enterococcus faecium CC17 and CC5 and Enterococcus faecalis CC2 Clonal Clusters Harboring Tn 1546 on Indistinguishable Plasmids

VRE isolates from pigs (n = 29) and healthy persons (n = 12) recovered during wide surveillance studies performed in Portugal, Denmark, Spain, Switzerland, and the United States (1995 to 2008) were compared with outbreak/prevalent VRE clinical strains (n = 190; 23 countries; 1986 to 2009). Thirty clonally related Enterococcus faecium clonal complex 5 (CC5) isolates (17 sequence type 6 [ST6], 6 ST5, 5 ST185, 1 ST147, and 1 ST493) were obtained from feces of swine and healthy humans. This collection included isolates widespread among pigs of European Union (EU) countries since the mid-1990s. Each ST comprised isolates showing similar pulsed-field gel electrophoresis (PFGE) patterns (≤6 bands difference; >82% similarity). Some CC5 PFGE subtype strains from swine were indistinguishable from hospital vancomycin-resistant enterococci (VRE) causing infections. A truncated variant of Tn1546 (encoding resistance to vancomycin) and tcrB (coding for resistance to copper) were consistently located on 150- to 190-kb plasmids (rep(pLG1)). E. faecium CC17 (ST132) isolates from pig manure and two clinical samples showed identical PFGE profiles and contained a 60-kb mosaic plasmid (rep(Inc18) plus rep(pRUM)) carrying diverse Tn1546-IS1216 variants. The only Enterococcus faecalis isolate obtained from pigs (CC2-ST6) corresponded to a multidrug-resistant clone widely disseminated in hospitals in Italy, Portugal, and Spain, and both animal and human isolates harbored an indistinguishable 100-kb mosaic plasmid (rep(pRE25) plus rep(pCF10)) containing the whole Tn1546 backbone. The results indicate a current intra- and international spread of E. faecium and E. faecalis clones and their plasmids among swine and humans.