Proteomic characterization of van A-containing Enterococcu s recovered from Seagulls at the Berlengas Natural Reserve, W Portugal

Background

The human immunodeficiency virus type 1 (HIV-1) pandemic has continued unabated for nearly 30 years. To better understand the influence of virus on host cells, we performed the differential proteome research of peripheral blood mononuclear cells (PBMCs) from HIV-positive patients and healthy controls.

Results

26 protein spots with more than 1.5-fold difference were detected in two dimensional electrophoresis (2DE) gels. 12 unique up-regulated and one down-regulated proteins were identified in HIV-positive patients compared with healthy donors. The mRNA expression of 10 genes was analyzed by real time RT-PCR. It shows that the mRNA expression of talin-1, vinculin and coronin-1C were up-regulated in HIV positive patients and consistent with protein expression. Western blotting analysis confirmed the induction of fragments of vinculin, talin-1 and filamin-A in pooled and most part of individual HIV-positive clinical samples. Bioinformatic analysis showed that a wide host protein network was disrupted in HIV-positive patients.

Conclusions

Together, this work provided useful information to facilitate further investigation of the underlying mechanism of HIV and host cell protein interactions, and discovered novel potential biomarkers such as fragment of vinculin, filamin-A and talin-1 for anti-HIV research.     

Structure and dynamics of CTX-M enzymes reveal insights into substrate accommodation by extended-spectrum beta-lactamases

Oxyimino-cephalosporin antibiotics, such as ceftazidime, escape the hydrolytic activity of most bacterial β-lactamases. Their widespread use prompted the emergence of the extended-spectrum β-lactamases CTX-Ms, which have become highly prevalent. The C7 β-amino thiazol-oxyimino-amide side chain of ceftazidime has a protective effect against most CTX-M β-lactamases. However, Asp240Gly CTX-M derivatives demonstrate enhanced hydrolytic activity against this compound. In this work, we present the crystallographic structures of Asp240Gly-harboring enzyme CTX-M-16 in complex with ceftazidime-like glycylboronic acid (resolution 1.80 Å) and molecular dynamics simulations of the corresponding acyl-enzyme complex. These experiments revealed breathing motions of CTX-M enzymes and the role of the substitution Asp240Gly in the accommodation of ceftazidime. The substitution Asp240Gly resulted in insertion of the C7β side chain of ceftazidime deep in the catalytic pocket and orchestrated motions of the active serine Ser70, the β3 strand and the omega loop, which favored the key interactions of the residues 237 and 235 with ceftazidime.     

High presence of extended-spectrum beta-lactamases and resistance to quinolones in clinical isolates of Escherichia coli

A study was conducted to detect the presence of extended-spectrum β-lactamases (ESBLs) in 706 isolates of Escherichia coli, largely from outpatients (75.2%). The Clinical and Laboratory Standards Institute (formerly NCCLS)-recommended disk diffusion procedure was used to detect ESBL presence; the VITEK 2 system (bioMérieux, Marcy L’Etoile, France) was used to determine the susceptibility to antibiotics of clinical interest, and the ESBLs were characterized by biochemical study, determining the isoelectric point, and by molecular study with PCR. Clonal distribution was studied in eight hospital isolates. There were 115 ESBL-producing isolates (16.3%), with a predominance of CTX-M9 type (58.3%). We draw attention to the high resistance to quinolones (>70%) in CTX-M9 and SHV enzyme producing isolates and the lower aminoglycoside activity in the latter.     

Contribution of extended-spectrum AmpC (ESAC) beta-lactamases to carbapenem resistance in Escherichia coli

ESAC beta-lactamases have increased catalytic efficiencies toward extended-spectrum cephalosporins and to a lesser extent toward imipenem as compared with the wild-type cephalosporinases. We show here that ESAC expression associated with the loss of both OmpC and OmpF porins conferred in Escherichia coli a high level of resistance to ertapenem and reduced the susceptibility to imipenem. On the contrary, ESAC expressed in the OmpC- or OmpF-deficient E. coli strains or narrow-spectrum cephalosporinase expressed in the OmpC-and OmpF-deficient strain do not confer reduced susceptibility to any of the carbapenems. The production of ESAC beta-lactamase in favorable E. coli background may represent an additional mechanism of resistance to ertapenem.     

Investigation of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea

AIMS: Isolates obtained from various regions in Korea in 2002 were identified and their susceptibility to extended-spectrum cephalosporins, monobactams and/or cephamycins was studied along with any production of extended-spectrum beta-lactamases (ESBLs). METHODS AND RESULTS: Bacteria identified by the conventional techniques and Vitek GNI card were Klebsiella pneumoniae and Escherichia coli. Using disk diffusion and double-disk synergy tests, we found that 39.2% of strains produced ESBLs. About 52% of isolates transferred resistance to ceftazidime by conjugation. Banding patterns of PCR amplification with the designed primers showed that 837- and 259-bp fragments specific to bla(TEM) genes were amplified in 63.3% of strains. 929- and 231-bp fragments (bla(SHV)), 847- and 520-bp fragments (bla(CMY)), 597- and 858-bp fragments (bla(CTX-M)) were amplified in 61.5, 17.3 and 7.7% of strains respectively. About 51.9% of strains contained more than two types of beta-lactamase genes. Especially, one strain contained bla(TEM), bla(CMY) and bla(CTX-M) genes. SIGNIFICANCE: Resistance mechanisms to beta-lactams, comprising mostly ESBL production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also make therapeutic failure and lack of eradiation of these strains by extended-spectrum cephalosporins or cephamycins.     

Antibiotic-resistant Salmonella and Escherichia coli isolates with integrons and extended-spectrum beta-lactamases in surface water and sympatric black-headed gulls

Aim: To examine surface water from a pond in the northeastern part of the Czech Republic and young black‐headed gulls (Larus ridibundus) nesting on the same pond for the presence of antibiotic‐resistant Salmonella and Escherichia coli. Methods and Results: A total of 16% (n = 87) of water and 24% (n = 216) of gull samples yielded Salmonella. Salmonella Enteritidis PT8 and PT4 were the most prevalent. Antibiotic resistance was found in 12% (n = 14) of water and 28% (n = 51) of gull salmonellae. Escherichia coli were found in 83 (95%) and 213 (99%) of pond water and gull samples, respectively. Totals of 18% (n = 83) of water and 28% (n = 213) of gull E. coli isolates were resistant to antimicrobial agents tested. Class 1 integrons were found in 21% (n = 14) of water and 15% (n = 60) of gull antibiotic‐resistant E. coli isolates. Class 2 integrons and extended‐spectrum beta‐lactamase‐producing E. coli isolates (with bla_CTX‐M‐1, bla_CTX‐M‐15‐like, bla_SHV‐2 and bla_SHV‐12) were found in 13% (eight positive, n = 60 gull‐resistant E. coli isolates) and 3% (seven positive, n = 216 gull E. coli isolates) of gull isolates, respectively. Antibiotic‐resistant E. coli isolates with identical pulsed field gel electrophoresis (PFGE) patterns were found in either gulls or water, but not both. Salmonellae of the same serotype and PFGE profile were found in both gulls and water. Conclusion: A high prevalence of antibiotic‐resistant salmonellae and E. coli were found in both pond water and in sympatric black‐headed gulls. Significance and Impact of the Study: Intensive contamination of pond surface water by antibiotic‐resistant E. coli and salmonellae was documented. Black‐headed gulls were identified as important reservoirs of antibiotic‐resistant salmonellae and E. coli, including extended‐spectrum beta‐lactamase‐producing isolates.     

A preliminary survey of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Japan

We conducted a survey of extended-spectrum beta-lactamases (ESBLs) among 16805 Escherichia coli and 9794 Klebsiella pneumoniae clinical isolates recovered from 196 separate medical institutions during the period January 1997 to January 1998. Using the criteria for minimal inhibitory concentrations (MICs) of oxyimino-cephalosporins of >/=8 microg ml(-1) and confirmation by double-disk test, we detected 15 E. coli and 34 K. pneumoniae isolates producing ESBLs. Genotypes of ESBLs determined by PCR with type-specific primers included one TEM-derived and 24 SHV-derived ESBLs, in addition to 24 Toho-1-type ESBLs, one of the major types of ESBLs reported in Japan. Nucleotide sequence analysis of SHV-specific PCR products revealed that SHV-12 was the dominant type of SHV-derived ESBL. In addition, we also identified TEM-26 and SHV-2. This is the first report characterizing TEM- and SHV-derived ESBLs in Japan.     

Active sites of beta-lactamases. The chromosomal beta-lactamases of Pseudomonas aeruginosa and Escherichia coli.

An acyl-enzyme was isolated from certain chromosomal beta-lactamases and a penicillin. The penicillin was cloxacillin which, although it is a substrate for these enzymes, has such a low kcat. that it functions as an inhibitor. The enzymes were from the mutant of Pseudomonas aeruginosa 18 S that produces the beta-lactamase constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol., in the press] and from Escherichia coli K-12 (the ampC beta-lactamase) [Boman, Nordström & Normak (1974) Ann. N.Y. Acad. Sci. 235, 569-586]. The acyl-enzymes have been degraded to determine the residue labelled, and the sequence around it. The residue labelled is serine. The sequences around the labelled serine in these two beta-lactamases are exceedingly similar. However, the sequences are quite different from those around the active site serine in the beta-lactamases previously studied. There is thus more than one class of serine beta-lactamases.     

Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences

A strategy has been designed for the construction of recombinant bacterial strains which eventually may become useful as live vaccines and which may also be relevant for the preparation of conventional vaccines. The approach used is the fusion of small antigenic peptide sequences into specific segments of a protein whose location on the bacterial surface ensures that the recombinant organism is able to present the inserted antigen to the host (animal or human) infected by the bacterium. The chosen surface protein is a naturally occurring polymer of Escherichia coli, viz., type 1 fimbriae. The results obtained show that fusion of such foreign sequences into selected points of the structural protein of the fimbriae results in the production of functionally normal type 1 fimbriae. Furthermore, hybrid fimbriae carrying such small epitope sequences can be recognized by antibodies directed against the foreign parent protein. This observation is an important prerequisite for the eventual design of useful vaccines. The analysis of the fimbrial protein and its potential as a carrier of foreign peptides from hepatitis B surface antigen, foot-and-mouth disease virus and poliovirus indicated that there may be several positions in the protein which may turn out to be relevant for this purpose and be important fusion sites.     

Detection of the expression of extended-spectrum beta-lactamases (ESBL) in clinical strains of Klebsiella pneumoniae and Escherichia coli: comparison of E-test and two-disc methods

The aim of this study was to compare the effectiveness of E-test and two disc methods applied for the detection of extended spectrum beta-lactamases. All strains were tested by E-test, by double-disc synergy test (DDST) according to Jarlier (cefotaxim, ceftazidim, aztreonam and clavulanic acid) and also by disc test according to Appleton (cefpodoxime and cefpodoxime with clavulanic acid, CPD and CD01 disc). We tested 148 clinical strains of E. coli and 78 strains of K. Pneumoniae. In case of K. pneumoniae, the activity of the ESBLs was detected among 30 strains--both in E-test, Jarlier test and Appleton test. Among E. coli, four strains were found ESBL-positive in the test according to Jarlier but only three strain of these when E-test and Appleton test was used. The results of investigations performed suggest, that E-test and disc methods according both Jarlier and Appleton have the same effectiveness in detection ESBLs among K. pneumoniae strains. However, in case of E. coli, interpretation of results may present a problem.     

Variable within- and between-herd diversity of CTX-M cephalosporinase-bearing Escherichia coli isolates from dairy cattle

bla(CTX-M) beta-lactamases confer resistance to critically important cephalosporin drugs. Recovered from both hospital- and community-acquired infections, bla(CTX-M) was first reported in U.S. livestock in 2010. It has been hypothesized that veterinary use of cephalosporins in livestock populations may lead to the dissemination of beta-lactamase-encoding genes. Therefore, our objectives were to estimate the frequency and distribution of coliform bacteria harboring bla(CTX-M) in the fecal flora of Ohio dairy cattle populations. In addition, we characterized the CTX-M alleles carried by the isolates, their plasmidic contexts, and the genetic diversity of the bacterial isolates themselves. We also evaluated the association between ceftiofur use and the likelihood of recovering cephalosporinase-producing bacteria. Thirty fresh fecal samples and owner-reported ceftiofur use data were collected from each of 25 Ohio dairy farms. Fecal samples (n = 747) yielded 70 bla(CTX-M)-positive Escherichia coli isolates from 5/25 herds, 715 bla(CMY-2) E. coli isolates from 25/25 herds, and 274 Salmonella spp. from 20/25 herds. The within-herd prevalence among bla(CTX-M)-positive herds ranged from 3.3 to 100% of samples. Multiple pulsed-field gel electrophoresis (PFGE) patterns, plasmid replicon types, and CTX-M genes were detected. Plasmids with CTX-M-1, -15, and -14 alleles were clonal by restriction fragment length polymorphism (RFLP) within herds, and specific plasmid incompatibility group markers were consistently associated with each bla(CTX-M) allele. PFGE of total bacterial DNA showed similar within-herd clustering, with the exception of one herd, which revealed at least 6 different PFGE signatures. We were unable to detect an association between owner-reported ceftiofur use and the probability of recovering E. coli carrying bla(CTX-M) or bla(CMY-2).     

Emergence of uropathogenic extended-spectrum beta lactamases-producing Escherichia coli strains in the community

The aim of this study was to determine the virulence characteristics and resistance pattern of the extended-spectrum/lactamases (ESBLs)-producing Escherichia coli strains isolated from urine of outpatients in the Zagreb region during a five-month period, and to compare them with the non ESBLs-producing E. coli strains isolated in the same period. Out of 2451 E. coli strains isolated from urine of nonhospitalized patients with significant bacteriuria, a total of 39 ESBLs-producing strains (1.59%) were detected by a double-disk diffusion technique and by the broth-dilution minimal inhibitory concentration reduction method. The 45 non ESBLs-producing strains were randomly chosen, and phenotype of the two groups of strains was characterized and compared. Serogroup O4, hemolysin production, expression of P- and type 1 fimbriae as well as resistance to gentamicin and amikacin were significantly more prevalent characteristics among the ESBLs-producing strains than among non ESBLs-producing strains (p < 0.01), while higher prevalence of trimethoprim-sulfamethoxazole resistance among ESBLs-producing strains was not statistically significant (p > 0.05). Chromosomal DNA analysis by pulsed-field gel electrophoresis exhibited a great genomic similarity among ESBLs-producing strains and revealed that those highly virulent and resistant E. coli strains isolated from urine of outpatients in the Zagreb region had a clonal propagation.     

Application of bacteriophages in simultaneously controlling Escherichia coli O157:H7 and extended-spectrum beta-lactamase producing Escherichia coli

Applied Microbiology and Biotechnology - Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of...     

Identification of fecal Escherichia coli from humans and animals by ribotyping

Fecal pollution of water resources is an environmental problem of increasing importance. Identification of individual host sources of fecal Escherichia coli, such as humans, pets, production animals, and wild animals, is prerequisite to formulation of remediation plans. Ribotyping has been used to distinguish fecal E. coli of human origin from pooled fecal E. coli isolates of nonhuman origin. We have extended application of this technique to distinguishing fecal E. coli ribotype patterns from human and seven individual nonhuman hosts. Classification accuracy was best when the analysis was limited to three host sources. Application of this technique to identification of host sources of fecal coliforms in water could assist in formulation of pollution reduction plans.     

Comparison of culturable fecal coliforms and Escherichia coli enumeration in freshwaters

Fecal coliforms (FC) counts were compared with Escherichia coli counts in differently contaminated freshwater samples (n = 166). FC were enumerated by plate count on triphenyl 2,3,5-tetrazolium chloride Tergitol medium. Escherichia coli were enumerated by the most probable number microplate method based on the detection of glucuronidase activity. FC and E. coli counts were highly correlated; an average E. coli/FC ratio equal to 0.77 was found, meaning that on average, 77% of FC were E. coli. Knowing the E. coli/FC ratio allows us to convert the historical microbiological quality data expressed in FC counts into E. coli abundance and thus to compare with present and future monitoring data that are (or will be) based on E. coli enumeration.     

Escherichia coli harboring a natural IncF conjugative F plasmid develops complex mature biofilms by stimulating synthesis of colanic acid and Curli

It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F⁺ cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms.     

Structure and urovirulence characteristics of the fecal Escherichia coli population among healthy women

The fecal Escherichia coli population structure may influence the occurrence and etiology of extraintestinal infection, but is poorly understood. Accordingly, fecal E. coli from 39 healthy women (30 putative colonies per subject) were characterized for clonal identity, urinary tract infection-associated virulence traits, and phylogenetic background. The 120 unique E. coli clones (mean, three per sample) were distributed by phylogenetic group as follows: A (33%), D (31%), B1 (19%), and B2 (17%). However, 36% of women carried ≥1 clone from group B2, and 87% had clones from groups B2 and/or D. Of the B2 clones, 90% were from pauciclonal fecal samples (≤4 clones), compared with 47% and 52% of A and B1 clones (P = .001 and P = .007, respectively). Group B2 and D clones more often were dominant within the source sample than group A and B1 clones (60% vs. 41%: P = .05). Dominant clones exhibited higher virulence scores than non-dominant clones (mean 4.4 vs. 3.1: P = .015). In multilevel regression models, pauciclonal sample, B2, and clonal prevalence significantly predicted virulence score. In conclusion, within the intestinal E. coli population, virulence-associated traits, clonal prevalence, and low fecal clonal diversity are related. Virulence-associated traits of group B2/D E. coli may enhance fitness within the gut, thereby increasing strains’ likelihood of causing extraintestinal infection.     

Dissemination of Escherichia coli with CTX-M Type ESBL between Humans and Yellow-Legged Gulls in the South of France

Extended Spectrum β-Lactamase (ESBL) producing Enterobacteriaceae started to appear in the 1980s, and have since emerged as some of the most significant hospital-acquired infections with Escherichia coli and Klebsiella being main players. More than 100 different ESBL types have been described, the most widespread being the CTX-M β-lactamase enzymes (bla _CTX-M genes). This study focuses on the zoonotic dissemination of ESBL bacteria, mainly CTX-M type, in the southern coastal region of France. We found that the level of general antibiotic resistance in single randomly selected E. coli isolates from wild Yellow-legged Gulls in France was high. Nearly half the isolates (47,1%) carried resistance to one or more antibiotics (in a panel of six antibiotics), and resistance to tetracycline, ampicillin and streptomycin was most widespread. In an ESBL selective screen, 9,4% of the gulls carried ESBL producing bacteria and notably, 6% of the gulls carried bacteria harboring CTX-M-1 group of ESBL enzymes, a recently introduced and yet the most common clinical CTX-M group in France. Multi locus sequence type and phylogenetic group designations were established for the ESBL isolates, revealing that birds and humans share E. coli populations. Several ESBL producing E. coli isolated from birds were identical to or clustered with isolates with human origin. Hence, wild birds pick up E. coli of human origin, and with human resistance traits, and may accordingly also act as an environmental reservoir and melting pot of bacterial resistance with a potential to re-infect human populations.