DNA fingerprinting is a technique for comparing DNA patterns that has applications in a wide variety of contexts. Several commercial and freely-available tools can be used to analyze DNA fingerprint ...gel images; however, commercial tools are expensive and usually difficult to use; and, free tools support the basic functionality for DNA fingerprint analysis, but lack some instrumental features to obtain accurate results.
In this paper, we present GelJ, a feather-weight, user-friendly, platform-independent, open-source and free tool for analyzing DNA fingerprint gel images. Some of the outstanding features of GelJ are mechanisms for accurate lane- and band-detection, several options for computing migration models, a number of band- and curve-based similarity methods, different techniques for generating dendrograms, comparison of banding patterns from different experiments, and database support.
GelJ is an easy to use tool for analyzing DNA fingerprint gel images. It combines the best characteristics of both free and commercial tools: GelJ is light and simple to use (as free programs), but it also includes the necessary features to obtain precise results (as commercial programs). In addition, GelJ incorporates new functionality that is not supported by any other tool.
Sixty-eight owners and 66 pets, from 43 unrelated pet-owning households were screened for methicillin-resistant coagulase negative staphylococci (MRCoNS), potential cases of MRCoNS interspecies ...transmission (IT), and persistence. MRCoNS isolates were identified by microbiological and molecular tests. MLST-based phylogenetic analysis was performed in
isolates. Antimicrobial susceptibility was evaluated using phenotypic and molecular methods. SCC
type and the presence of biofilm-related
locus was PCR-tested. Isolates suspected for MRCoNS IT cases were subjected to
-PFGE analysis and individuals from positive households were followed-up for 1 year for carriage dynamics (every 3 months, T0-T4). Nineteen MRCoNS isolates from owners (27.9%) and 12 from pets (16.7%) were detected, coming from 20 households (46.5%).
was predominant (90 and 67% of human and animal strains, respectively), showing high phylogenetic diversity (16 STs among 24 strains). Methicillin-resistant
(MRSE) strains belonged to CC5 (75%), CC11 (12.5%), singleton S556 (8.3%), and S560 (4.17%). Significant host-associated differences were observed for resistance to aminoglycosides, co-trimoxazole, chloramphenicol (higher in animal isolates) and tetracycline (higher among human strains). Multidrug resistance (MDR) was common (68.4%) and associated with human strains. Great diversity of
and
complexes were detected, most strains being non-typeable, followed by SCC
IV and V. Over one third of isolates (most from owners), carried the
locus, all MRSE CC5. Two sporadic IT cases (T0) were identified in owners and dogs from two households (4.7%), with diverse interspecies-exchanged clones detected along the sampling year, especially in dogs. A comparative analysis of all MRCoNS, with all nasal coagulase positive staphylococci (CoPS) recovered from the same individuals at T0, revealed that CoPS alone was predominant in owners and pets, followed by co-carriage of CoPS and MRCoNS in owners but single MRCoNS in pets. Statistical analyses revealed that owners are more prone to co-carriage and that co-existence of IT cases and co-carriage are positively interrelated. MRCoNS from healthy owners and their pets are genetically heterogeneous MDR strains that are spread in the community. Therefore, pets also contribute to the dissemination of successful human clones. Owner-pet inhabitancy increases the risk for staphylococcal temporal concomitance with its subsequent risk for bacterial infection and genetic exchange.
Objectives
To characterize the location and genetic environments of qnr, aac(6′)-Ib-cr and qepA genes related to quinolone resistance in 19 Escherichia coli, Klebsiella pneumoniae and Klebsiella ...oxytoca strains.
Methods
Genetic environments of the indicated genes were studied by cloning, PCR mapping and sequencing. The location of these genes was analysed by S1-PFGE and PFGE-I-CeuI and hybridization with specific probes. Associated antibiotic resistance mechanisms and molecular typing of strains were also investigated.
Results
The studied strains carried the aac(6′)-Ib-cr, qepA, qnrS1, qnrB6, qnrB4 and oqxAB genes, with aac(6′)-Ib-cr being the most prevalent. E. coli strains belonged to sequence types (STs) ST648, ST131, ST224 and ST205, and K. pneumoniae strains to ST433, ST341, ST152, ST15 and ST431. Different genetic environments of quinolone resistance genes were observed, and some of them had not been previously detected and registered in GenBank. The aac(6′)-Ib-cr gene was mainly located in class 1 integrons or associated with the Tn1721 transposon in E. coli and associated with the aac(3)-II gene in Klebsiella. All these structures contained mechanisms of gene acquisition and/or dissemination, such as IS26. The studied quinolone resistance genes were mostly detected in IncF and IncN plasmids in E. coli and in IncR plasmids in Klebsiella, but in some strains the chromosomal location of the aac(6′)-Ib-cr gene was detected for the first time. The bla
CTX-M-15, bla
OXA-1, tet(A), aac(3)-II and aph(3′)-Ia genes and class 1 integrons were found in most strains.
Conclusions
The aac(6′)-Ib-cr gene was detected for the first time in the chromosome, although a plasmidic location was the most frequently found, with differentiation of plasmids types in E. coli versus Klebsiella.
Staphylococcus pseudintermedius (SP) and Staphylococcus aureus (SA) are common colonizers of companion animals, but they are also considered opportunistic pathogens, causing diseases of diverse ...severity. This study focused on the identification and characterization of 33 coagulase-positive staphylococci isolated from diseased pets (28 dogs and five cats) during 2009-2011 in a veterinary hospital in Spain in order to stablish the circulating lineages and their antimicrobial resistance profile.
Twenty-eight isolates were identified as SP and five as SA. Nine methicillin-resistant (MR) isolates (27%) carrying the mecA gene were detected (eight MRSP and one MRSA). The 55% of SP and SA isolates were multidrug-resistant (MDR). MRSP strains were typed as ST71-agrIII-SCCmecII/III-(PFGE) A (n=5), ST68-agrIV-SCCmecV-B1/B2 (n=2), and ST258-agrII-SCCmecIV-C (n=1). SP isolates showed resistance to the following antimicrobials percentage of resistant isolates/resistance genes: penicillin 82/blaZ, oxacillin 29/mecA erythromycin/clindamycin 43/erm(B), aminoglycosides 18-46/aacA-aphD, aphA3, aadE, tetracycline 71/tet(M), tet(K), ciprofloxacin 29, chloramphenicol 29/cat
, and trimethoprim-sulfamethoxazole 50/dfrG, dfrK. The dfrK gene was revealed as part of the radC-integrated Tn559 in two SP isolates. Virulence genes detected among SP isolates were as follow percentage of isolates: siet 100, se-int 100, lukS/F-I 100, sec
7, and expB 7. The single MRSA-mecA detected was typed as t011-ST398/CC398-agrI-SCCmecV and was MDR. The methicillin-susceptible SA isolates were typed as t045-ST5/CC5 (n=2), t10576-ST1660 (n=1), and t005-ST22/CC22 (n=1); the t005-ST22 feline isolate was PVL-positive and the two t045-ST45 isolates were ascribed to Immune Evasion Cluster (IEC) type F. Moreover, the t10576-ST1660 isolate, of potential equine origin, harbored the lukPQ and scneq genes. According to animal clinical history and data records, several strains seem to have been acquired from different sources of the hospital environment, while some SA strains appeared to have a human origin.
The frequent detection of MR and MDR isolates among clinical SP and SA strains with noticeable virulence traits is of veterinary concern, implying limited treatment options available. This is the first description of MRSA-ST398 and MRSP-ST68 in pets in Spain, as well the first report of the dfrK-carrying Tn559 in SP. This evidences that current transmissible lineages with mobilizable resistomes have been circulating as causative agents of infections among pets for years.
Nasal carriage of coagulase-positive staphylococci (CoPS) in healthy dogs could indicate increased risks of colonization for in-contact people or vice versa. This study determined the nasal carriage ...rate of CoPS among healthy dogs and in-contact people, their genotypic characteristics and phylogenetic relatedness.
Nasal samples were collected from 27 households (34 dogs and 41 humans) in Spain. Staphylococci were identified by MALDI-TOF-MS, their antimicrobial resistance (AMR) genes and
-types were tested by PCR/sequencing. The relatedness of CoPS from the same households was assessed by core genome single nucleotide polymorphisms (SNPs) analyses.
carriage was found in 34.1% of humans (including one methicillin-resistant
MRSA-CC5-t2220-SCC
type-IV2B) and 5.9% of dogs;
in 2.4% of humans and 32.4% of dogs; while
was only detected in dogs (5.4%). Remarkably, one human co-carried
/
, while a dog co-carried the three CoPS species. Household density was significantly associated with
carriage in households with > than 1 dog and >than 1 human (OR = 18.10, 95% CI: 1.24-260.93,
= 0.034). Closely related (<15 SNPs)
or
were found in humans or dogs in three households. About 56.3%
carriers (dog or human) harboured diverse within-host
-types or AMR genotypes. Ten clonal complexes (CCs) were detected among the
, of which methicillin-susceptible
-CC398-IEC-type C (t1451 and t571) was the most frequent, but exclusive to humans.
and
isolates harboured resistance genes or mutations associated to 9 classes of antimicrobials including linezolid (G2261A & T1584A point mutations in 23S rDNA). The
isolates were susceptible to all antimicrobials. Most of the
carried
and
genes, and all
were negative for
-PV
and
genes.
Clonally related human-to-human MSSA and dog-to-human MSSP were found. The detection of the MSSA-CC398 clade highlights the need for its continuous surveillance from One Health perspective.
The presence of methicillin-resistant or -susceptible S. aureus in pig nostrils has been known for a long time, but the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli has ...hardly been investigated. Here, we collected 25 E. coli recovered from nasal samples of 40 pigs/10 farmers of four farms. Nine ESBL-producing isolates belonging to ST48, ST117, ST847, ST5440, ST14914 and ST10 were retrieved from seven pigs. All blaESBL genes (blaCTX-M-32,blaCTX-M-14,blaCTX-M-1,blaCTX-M-65, and blaSHV-12) were horizontally transferable by conjugation through plasmids belonging to IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1) types. IncI1-plasmids displayed different genetic environments: i) IS26-blaSHV-12-deoR-IS26, ii) wbuC-blaCTX-M-32-ISKpn26 (IS5), and iii) IS930-blaCTX-M-14-IS26. The IncHI2-plasmid contained the genetic environment IS903-blaCTX-M-65-fipA with multiple resistance genes associated either to: a) Tn21-like transposon harbouring genes conferring aminoglycosides/beta-lactams/chloramphenicol/macrolides resistance located on two atypical class 1 integrons with an embedded ΔTn5393; or b) Tn1721-derived transposon displaying an atypical class 1 integron harbouring aadA2-arr3-cmlA5-blaOXA-10-aadA24-dfrA14, preceding the genetic platform IS26-blaTEM-95-tet(A)-lysR-floR-virD2-ISVsa3-IS3075-IS26-qnrS1, as well as the tellurite resistance module. Other plasmids harbouring clinically relevant genes were detected, such as a ColE-type plasmid carrying the mcr-4.5 gene. Chromosomally encoded genes (fosA7) or integrons (intI1-dfrA1-aadA1-qacE-sul1/intI1-IS15-dfrA1-aadA2) were also identified. Finally, an IncY plasmid harbouring a class 2 integron (intI2-dfrA1-sat2-aadA1-qacL-IS406-sul3) was detected but not associated with a blaESBL gene. Our results evidence that pig nostrils might favour the spread of ESBL-E. coli and mcr-mediated colistin-resistance. Therefore, enhanced monitoring should be considered, especially in a sector where close contact between animals in intensive farming increases the risk of spreading antimicrobial resistance.
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•ESBL-producing E. coli were detected in seven pig nostrils (7/40, 17.5%).•SHV-12, CTX-M-1, CTX-M-14, CTX-M-32 and CTX-M-65 enzymes were identified.•ESBL-E. coli belonged to ST10, ST101, ST117, ST93, ST5440 and the newly ST14914.•Plasmids involved in ESBL-transfer were IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1).•Tn21-like/Tn1721-like, tellurite-resistance, new integron arrangements were shown.
Bacteriocins are antimicrobial peptides with relevance in the modulation of human and animal microbiota that have gained interest in biomedical and biotechnological applications. In this study, the ...production of bacteriocin-like inhibitory substances (BLIS) was tested among a collection of 890 staphylococci of different origins (humans, animals, food, and the environment) and species, both coagulase-positive (CoPS, 238 isolates of 3 species) and coagulase-negative staphylococci (CoNS, 652 isolates of 26 species). Of the 890 staphylococci, 60 (6.7%) showed antimicrobial activity by the
method against at least one of the 25 indicator bacteria tested. BLIS-producer (BLIS
) isolates were detected in 8.8% of CoPS and 6.0% of CoNS. The staphylococcal species with the highest percentages of BLIS
isolates were
(38.5%),
(26.7%), and
(23.1%). The production of BLIS was more frequently detected among isolates of pets, wild animals, and food. Moreover, 13 BLIS
isolates showed wide antimicrobial activiy spectrum, and 7 of these isolates (of species
,
,
, and
) demonstrated antimicrobial activity against more than 70% of the indicator bacteria tested. The genetic characterization (by PCR and sequencing) of the 60 BLIS
isolates revealed the detection of (a) 11 CoNS and CoPS isolates carrying putative lantibiotic-like genes; (b) 3
isolates harboring the genes of BacSp222 bacteriocin; and (c) 2
isolates that presented the gene of a putative cyclic bacteriocin (uberolysin-like), being the first report in this CoNS species. Antimicrobial susceptibility testing was performed in BLIS
isolates and one-third of the CoNS isolates showed susceptibility to all antibiotics tested, which also lacked the virulence genes studied. These BLIS
CoNS are good candidates for further characterization studies.
This study investigated the diversity and carriage rate of nasal Staphylococcus spp., and within-host variability of antimicrobial resistance (AMR), virulence determinants, immune evasion cluster ...(IEC) types and genetic lineages of S. aureus isolates. Also, the co-carriage rate of CoNS with S. aureus in the same nasal niche of healthy pigs and pig-farmers were studied in four pig-farms (A-D) in Aragon (Spain). Nasal samples of 40 pigs (10 pigs/farm) and 10 pig-farmers (2–3/farm) were collected for staphylococci recovery and isolates (up to 9 per sample) were identified by MALDI-TOF-MS. The virulence and AMR genes and spa-types of S. aureus isolates were investigated by PCR/sequencing. Of the 243 staphylococci identified (10 different species), 142 were S. aureus and 51 distinct isolates were selected for further characterization (that corresponded to one S. aureus/sample or more than one if they showed different AMR phenotypes). The highest carriage rate in pigs was S. aureus (65%) and S. chromogenes (22.5%), whereas in the pig-farmers, S. aureus (80%) and S. epidermidis (40%). Methicillin-resistant S. aureus (MRSA) were detected in 60% of pigs and 70% of pig-farmers. Only six S. aureus isolates were methicillin-susceptible (MSSA), all from farm-C. A multidrug resistance (MDR) phenotype was detected in all MRSA and in 83.3% of the MSSA isolates. All MRSA isolates were CC398 with spa-type t011 being the predominant (92.7%), while t034, t1451 (only in pig-farmers) and t4571 (in pigs) were also found. MSSA-CC9 isolates (t191, t1430) were detected in farm-C. All S. aureus isolates were negative for luk-S/F-PV, tst, and scn genes, except one MSSA-CC45-t065-IEC-type C isolate from a pig-farmer. About 34.6% and 75.0% of the pigs and pig-farmers S. aureus carriers, respectively, harboured within-host varied spa-types or resistomes. Moreover, 40% of pigs and pig-farmers with MRSA-CC398 had no CoNS nasal co-carriage, and 23.3% had ≥2 CoNS carriage. Conversely, only 16.7% of MSSA carriers had no CoNS co-carriage, whereas 50% had ≥2 CoNS carriages. The very high MRSA level and within-host resistome diversities highlight the need for multiple samplings to account for the dynamics of AMR crisis and control of inter-host transmission of S. aureus in pig-farms using “One Health” approach.
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