Applications of optical DNA mapping in microbiology.

Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches. In addition, designing lab-on-a-chip systems based on OM is now feasible and will allow the integrated and automated microbiological analysis of biological fluids. Here, we review the basic technology of OM, detail the current state of the art of the field, and look ahead to possible future developments in OM technology for microbiological applications.

Antimicrobial resistance and virulence genes in Escherichia coli and enterococci from red foxes (Vulpes vulpes).

The aims of the study were to analyse the prevalence of antimicrobial resistance and the mechanisms implicated, as well as the virulence factors, in faecal Escherichia coli and Enterococcus spp. from red foxes. From 52 faecal samples, 22 E. coli (42.3%) and 50 enterococci (96.2%) isolates were recovered (one/sample). A high percentage of E. coli isolates exhibited resistance to streptomycin, tetracycline, trimethoprim-sulfamethoxazole or ampicillin (54-27%), and they harboured the aadA, tet(A) and/or tet(B), sul1 and blaTEM resistance genes, respectively. The E. coli isolates were ascribed to the 4 major phylogroups, D (41% of isolates), A (31.8%), B1 (18.2%) and B2 (9.1%), and carried the fimA (63.3%) or aer (13.6%) virulence genes. Among enterococcal isolates, Enterococcus faecium was the most prevalent species (50%). A high percentage of enterococcal isolates showed tetracycline resistance (88%) harbouring different combinations of tet(M) and tet(L) genes. The erm(B) or the aph(3')-IIIa gene were identified in most of our erythromycin- or kanamycin-resistant enterococci, respectively. This report suggests the role of red foxes from rural areas in the cycle of transmission and spread of antimicrobial-resistant E. coli and enterococci into the environment, representing a reservoir of these antimicrobial-resistant microorganisms.

Detection of antibiotic resistant enterococci and Escherichia coli in free range Iberian Lynx (Lynx pardinus).

Thirty fecal samples from wild specimens of Iberian lynx were collected and analyzed for Enterococcus spp. (27 isolates) and Escherichia coli (18 isolates) recovery. The 45 isolates obtained were tested for antimicrobial resistance, molecular mechanisms of resistance, and presence of virulence genes. Among the enterococci, Enterococcus faecium and Enterococcus hirae were the most prevalent species (11 isolates each), followed by Enterococcus faecalis (5 isolates). High percentages of resistance to tetracycline and erythromycin (33% and 30%, respectively) were detected among enterococcal isolates. The tet(M) and/or tet(L), erm(B), aac(6')-Ie-aph(2″)-Ia, ant(6)-Ia, or aph(3')-IIIa genes were detected among resistant enterococci. Virulence genes were detected in one E. faecalis isolate (cpd, cylB, and cylL) and one E. hirae isolate (cylL). High percentages of resistance were detected in E. coli isolates to tetracycline (33%), streptomycin (28%), nalidixic acid (28%), and sulfamethoxazole-trimethoprim (SXT, 22%). Additionally, the blaTEM, tet(A), aadA, cmlA, and different combinations of sul genes were detected among most ampicillin, tetracycline, streptomycin, chloramphenicol and SXT-resistant isolates, respectively. Two isolates contained a class 1 integron with the gene cassette arrays dfrA1 + aadA1 and dfrA12 + aadA2. The E. coli isolates were ascribed to phylo-groups A (n=5); B1 (n=4); B2 (n=6), and D (n=3), with the virulence gene fimA present in all E. coli isolates. This study found resistance genes in wild specimens of Iberian lynx. Thus, it is important to notice that multiresistant bacteria have reached species as rare and completely non-synanthropic as the Iberian lynx. Furthermore, the susceptibility of this endangered species to bacterial infection may be affected by the presence of these virulence and resistance genes.

High prevalence of ESBL-producing Escherichia coli isolates among hemodialysis patients in Portugal: appearance of ST410 with the bla(CTX-M-14) gene.

Ten extended-spectrum ß-lactamase-producing Escherichia coli isolates were detected among 121 fecal samples (8.3%) recovered from hemodialysis patients in Portugal. The isolates harbored the bla(CTX-M-15), bla(CTX-M-14a), and/or bla(CTX-M-1) genes. A new sequence type, ST2229, was detected, and this study also reports, for the first time, ST410 CTX-M-14-producing isolates.

Molecular characterization of vanA-containing Enterococcus from migratory birds: song thrush (Turdus philomelos)

Vancomycin-resistant enterococci (VRE) were detected in two faecal samples (1.3%) of song thrush in Portugal. vanA isolates showed high level vancomycin/teicoplanin resistance, as well as resistance to ciprofloxacin, quinupristin-dalfopristin and cloranfenicol. Thrush can be a reservoir of VRE and transmit these resistant bacteria to other animals including humans.

Wild birds as biological indicators of environmental pollution: antimicrobial resistance patterns of Escherichia coli and enterococci isolated from common buzzards (Buteo buteo).

A total of 36 Escherichia coli and 31 enterococci isolates were recovered from 42 common buzzard faecal samples. The E. coli isolates showed high levels of resistance to streptomycin and tetracycline. The following resistance genes were detected: bla(TEM) (20 of 22 ampicillin-resistant isolates), tet(A) and/or tet(B) (16 of 27 tetracycline-resistant isolates), aadA1 (eight of 27 streptomycin-resistant isolates), cmlA (three of 15 chloramphenicol-resistant isolates), aac(3)-II with/without aac(3)-IV (all seven gentamicin-resistant isolates) and sul1 and/or sul2 and/or sul3 [all eight sulfamethoxazole/trimethoprim-resistant (SXT) isolates]. intI1 and intI2 genes were detected in four SXT-resistant isolates. The virulence-associated genes fimA (type 1 fimbriae), papC (P fimbriae) and aer (aerobactin) were detected in 61.1, 13.8 and 11.1% of the isolates, respectively. The isolates belonged to phylogroups A (47.2%), B1 (8.3%), B2 (13.9%) and D (30.5%). For the enterococci isolates, Enterococcus faecium was the most prevalent species (48.4%). High levels of tetracycline and erythromycin resistance were found among our isolates (87 and 81%, respectively). Most of the tetracycline-resistant strains carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in 80% of erythromycin-resistant isolates. The vat(D) and/or vat(E) genes were found in nine of the 17 quinupristin-dalfopristin-resistant isolates. The enterococcal isolates showing high-level resistance for kanamycin, gentamicin and streptomycin contained the aph(3')-IIIa, aac(6')-aph(2″) and ant(6)-Ia genes, respectively. This report reveals that common buzzards seem to represent an important reservoir, or at least a source, of multi-resistant E. coli and enterococci isolates, and consequently may represent a considerable hazard to human and animal health by transmission of these isolates to waterways and other environmental sources via their faecal deposits.

Molecular characterization of vanA-containing Enterococcus from migratory birds: song thrush (Turdus philomelos).

Vancomycin-resistant enterococci (VRE) were detected in two faecal samples (1.3%) of song thrush in Portugal. vanA isolates showed high level vancomycin/teicoplanin resistance, as well as resistance to ciprofloxacin, quinupristin-dalfopristin and cloranfenicol. Thrush can be a reservoir of VRE and transmit these resistant bacteria to other animals including humans.

Iberian wolf as a reservoir of extended-spectrum ß-lactamase-producing Escherichia coli of the TEM, SHV, and CTX-M groups.

The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment. Moreover, the presence of resistant genes in integrons and the existence of virulence determinants were shown. The association between antibiotic resistance and virulence determinants should be monitored, as it constitutes a serious public health problem.

Molecular characterization of vancomycin-resistant enterococci and extended-spectrum ß-lactamase-containing Escherichia coli isolates in wild birds from the Azores Archipelago.

To study the prevalence of vancomycin-resistant enterococci (VRE) and extended-spectrum ß-lactamase (ESBL)-containing Escherichia coli isolates, and the mechanisms of resistance implicated, 220 faecal samples from wild birds were collected between 2006 and 2010 in the Azores Archipelago. Samples were spread on Slanetz-Bartley agar plates supplemented with 4 mg/l vancomycin and on Levine agar plates supplemented with 2 mg/l cefotaxime for VRE and ESBL-containing E. coli isolation, respectively. vanA-containing enterococcal isolates (four Enterococcus faecium and two Enterococcus durans) and vanC-1 Enterococcus gallinarum isolates were detected in six and seven faecal samples, respectively. VRE isolates showed ampicillin (n=11), ciprofloxacin (n=9), tetracycline (n=6), erythromycin (n=5), quinupristin/dalfopristin (n=3) and high-level kanamycin resistance (n=1). The tet(L) and/or tet(M) gene was found in all tetracycline-resistant isolates and the erm(B) gene in all erythromycin-resistant isolates. Three vanA-containing E. faecium and two E. gallinarum presented specific sequences of the Tn5397 transposon. Four VRE isolates harboured the ace virulence gene. One faecal sample revealed one ESBL-containing E. coli isolate that belongs to the A phylogenetic group, showed a phenotype of resistance to ß-lactams and tetracycline, and harboured the bla (CTX-M-14), bla (SHV-12) and the tet(A) genes. To our knowledge, this is the first study to focus on defining the prevalence of VRE and/or ESBL-containing E. coli strains in wild birds from the Azores. The data recovered are essential to improve knowledge about the dissemination of resistant strains through wild ecosystems and their possible implications by transferring these resistances to other animals or to humans.