Partial nucleotide sequencing of the mecA genes of Staphylococcus aureus isolates from cats and dogs.
ABSTRACT: Methicillin-resistant staphylococci (MRS) pose a challenge to clinicians and health administrators in human medicine, but MRS infections in cats and dogs are not perceived as a problem in veterinary medicine. Ten methicillin-resistant staphylococcal isolates obtained from healthy and diseased cats and dogs were subjected to partial DNA sequencing of the mecA gene. Sequence analysis shows that MRS isolates from both healthy and diseased cats and dogs can harbor the mecA gene. The mecA genes of animal isolates were identical to that found in human MRS strains, and therefore the possibility of zoonotic transfer must be considered.
Project description:The aim of this study was to detect the prevalence of methicillin-resistant Staphylococcus sp. (MRS) in populations of companion animals that either have previously been exposed or have not been exposed to antibiotic therapy or veterinary facilities, and if owners' healthcare profession had an influence on colonization with MRS. In addition, the antimicrobial resistance pheno- and genotype were investigated and risks for colonization with MRS were assessed. During this study, 347 nasal swabs (dogs n = 152; cats n = 107; rabbits n = 88) were investigated for the presence of methicillin-resistant Staphylococcus aureus (MRSA). In addition, 131 nasal swabs (dogs n = 79; cats n = 47; rabbits = 3; guinea pigs = 2) were examined for the presence of MRSA but also other MRS. In total, 23 MRS isolates belonged to nine staphylococcal species: Staphylococcus epidermidis (n = 11), Staphylococcus warneri (n = 3), Staphylococcus hominis (n = 2), Staphylococcus pseudintermedius (n = 2), and singletons Staphylococcus cohnii, Staphylococcus sciuri, Staphylococcus fleurettii, Staphylococcus lentus, and Staphylococcus haemolyticus. Twenty isolates displayed a multidrug-resistant phenotype. Various resistance and biocide resistance genes were detected among the examined staphylococci. Risk assessment for MRS colonization was conducted using a number of factors, including animal species, breed, age, gender, recent veterinary health care hospitalization, and antibiotic prescription, resulting in recent veterinary health care hospitalization being a significant risk factor. The detection of multidrug-resistant MRS in healthy animals is of importance due to their zoonotic potential.
Project description:BACKGROUND:Mupirocin is one of the few antimicrobials active against methicillin-resistant Staphylococcus aureus (MRSA), and is frequently used for the eradication of MRSA nasal colonisation in humans. Initially, mupirocin resistance was recognised in human S. aureus, including MRSA isolates, then also among coagulase-negative staphylococci (CoNS). Nowadays, mupirocin resistance is occasionally observed in canine staphylococci, along with Staphylococcus pseudintermedius (MRSP) strains, as well as CoNS, which usually show methicillin resistance. In the current study, high-level mupirocin resistance in methicillin-resistant staphylococci isolated from diseased dogs and cats was investigated. RESULTS:Among 140 methicillin-resistant staphylococci isolates from dogs and cats, three showed high-level mupirocin resistance in a screening test using the agar disk diffusion method. One was recognised as methicillin-resistant S. aureus, one as methicillin-resistant S. pseudintermedius, and one as methicillin-resistant Staphylococcus haemolyticus. S. pseudintermedius and S. aureus were isolated from dogs, S. haemolyticus was obtained from a cat. All isolates showed high-level mupirocin resistance, confirmed by minimum inhibitory concentration (MIC) values of above 1024 μg/ml and the presence of the plasmid-located gene ileS2. This is the first report on the detection of high-level mupirocin resistance (HLMR) in S. haemolyticus of feline origin. CONCLUSIONS:This study revealed the occurrence of HLMR in three Staphylococcus isolates obtained from companion animals in Poland. The results of this study indicate that the monitoring of mupirocin resistance in staphylococci of animal origin, especially in methicillin-resistant isolates, is strongly recommended.
Project description:mecA, the gene that mediates methicillin resistance, and its accompanying mec locus DNA, insert near the gyrA gene in Staphylococcus aureus. To investigate whether there is a similar relationship between mecA and gyrA in coagulase-negative staphylococci (CNS), mecA- and gyrA-specific DNA fragments were used to probe methicillin-resistant isolates of Staphylococcus epidermidis (MRSE) (n = 11) and Staphylococcus haemolyticus (MRSH) (n = 11). The gyrA probe hybridized to the same SmaI DNA fragment as the mecA probe in all strains tested. However, since the size of the SmaI fragments containing mecA and gyrA varied from 73 to 600 kb, the distance between the two genes was determined more precisely. Cloned mecA or gyrA fragments plus vector sequences each containing a SmaI site were introduced into the chromosome of three isolates each of MRSE and methicillin-resistant S. aureus (MRSA), and the sizes of the generated SmaI fragments were determined by pulsed-field gel electrophoresis. The distance between gyrA and mecA was found to be between 38 and 42 kb in both MRSE and MRSA, and the two genes were in the same relative orientation in all strains. Restriction fragment length polymorphism (RFLP) patterns around the gyrA gene in CNS were identical, but species specific, for all 10 MRSE and 10 MRSH isolates examined. In contrast, 8 of 11 methicillin-susceptible S. epidermidis isolates and 7 of 7 methicillin-susceptible S. haemolyticus isolates had different gyrA RFLP patterns. These data show that mecA is site and orientation specific, relative to gyrA, in both MRSE and MRSA. In addition, the local environment around gyrA in methicillin-resistant CNS, in contrast to methicillin-susceptible isolates, is similar, suggesting clonality or the requirement for specific DNA sequences with which the mec complex must interact for chromosomal integration to occur.
Project description:Objectives:To evaluate temporal trends in antimicrobial resistance, over 16 years, in bacteria isolated from dogs and cats with urinary tract infection (UTI) and the clonal lineages of bacteria harbouring critical antimicrobial resistance mechanisms. Methods:Antimicrobial susceptibility testing was conducted for 948 bacteria isolated from dogs and cats with UTI (1999-2014). Resistance mechanisms were detected by PCR, namely ESBL/AmpC in third-generation cephalosporin (3GC)-resistant Escherichia coli and Proteus mirabilis, mecA in methicillin-resistant staphylococci, and aac(6')-Ieaph(2?)-Ia and aph(2?)-1d in high-level gentamicin-resistant (HLGR) enterococci. Resistant bacteria were typed by MLST, and temporal trends in E. coli and Enterobacteriaceae antimicrobial resistance were determined by logistic regression. Results:Enterobacteriaceae had a significant temporal increase in resistance to amoxicillin/clavulanate, 3GCs, trimethoprim/sulfamethoxazole, fluoroquinolones, gentamicin and tetracycline (P?<?0.001). An increase in MDR was also detected (P?<?0.0001). 3GC resistance was mainly caused by the presence of blaCTX-M-15 and blaCMY-2 in E. coli and the presence of blaCMY-2 in P. mirabilis. Two major 3GC-resistant E. coli clonal lineages were detected: O25b:H4-B2-ST131 and ST648. The mecA gene was detected in 9.2% (n?=?11/119) of Staphylococcus spp., including MRSA clonal complex (CC) 5 (n?=?2) and methicillin-resistant Staphylococcus epidermidis CC5 (n?=?4). A temporal increase in MDR methicillin-resistant Staphylococcus pseudintermedius was detected (P?=?0.0069). Some ampicillin-resistant and/or HLGR Enterococcus spp. were found to belong to hospital-adapted CCs, namely Enterococcus faecalis ST6-CC6 (n?=?1) and Enterococcus faecium CC17 (n?=?8). Conclusions:The temporal increase in antimicrobial resistance and in MDR bacteria causing UTI in dogs and cats creates important therapeutic limitations in veterinary medicine. Furthermore, the detection of MDR high-risk clonal lineages raises public health concerns since companion animals with UTI may contribute to the spread of such bacteria.
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important causes of hospital infections worldwide. High-level resistance to methicillin is caused by the mecA gene, which encodes an alternative penicillin-binding protein, PBP 2a. To determine the clonal relationships between methicillin-susceptible S. aureus (MSSA) and MRSA, we typed 1,069 S. aureus isolates (493 MSSA isolates and 576 MRSA isolates), collected mainly in North American and European hospitals between the 1960s and the year 2000, using pulsed-field gel electrophoresis and ribotyping. Of 10 widespread S. aureus lineages recognized, 8 had corresponding mecA-positive strains. Multiresistant MRSA strains are found in hospitals worldwide, while unrelated and more susceptible strains represent less than 1% of the MRSA population. This supports the hypothesis that horizontal transfer plays an important role in the dissemination of the mecA gene in the S. aureus population.
Project description:The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA+ and 135 were MecA-. For the coagulase-negative staphylococci, 150 were MecA+ and 111 were MecA-. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA+ but oxacillin sensitive and 1 isolate was MecA- but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA+ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.
Project description:The staphylococcal methicillin resistance determinant, mecA, resides on a mobile genetic element, staphylococcus chromosomal cassette mec (SCCmec). The distribution of SCCmec in nature is limited to relatively few clonal complexes of related methicillin-resistant Staphylococcus aureus (MRSA). We have previously reported that some genetic backgrounds are restrictive of mecA and penicillin-binding protein 2a expression, which could account for the restricted clonal distribution of SCCmec in nature. In this study, we investigate the potential role of the host chromosome in the transformability and expression of mecA in 103 naturally occurring methicillin-susceptible S. aureus clinical isolates. The isolates, which had been genotyped previously by multilocus sequence typing, were classified into one of two mutually exclusive categories based on whether the isolates belonged to "major" MRSA lineages or to "other" lineages that are never or occasionally MRSA. We introduced mecA expressed on the low-copy-number plasmid pYK20 into each MSSA strain and assayed the phenotype of resistance to nafcillin by population analysis to assess the relationship between the stability of mecA expression and genetic background. Strains from the major MRSA lineages were more transformable with pYK20 and better able to maintain the plasmid and express resistance in comparison to strains from other lineages. These data support the hypothesis that the presence of mecA within relatively few clonal complexes is partly due to genetic factors that are permissive of mecA and its gene product.
Project description:Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to ?-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (?SCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ?SCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ?SCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ?SCCmec57395 element amplified by long-range PCR revealed the presence of ?SCCmec57395 in the 33 additional isolates of MRSP CC45. The ?SCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.
Project description:BACKGROUND:Staphylococcus aureus is responsible for large numbers of hospital-related and community-acquired infections. In this study, we investigated the presence of S. aureus and methicillin-resistant S. aureus (MRSA) in 100 samples from animals (55 cattle, 36 dogs, and 9 cats) and 150 samples from hospitalized human patients. The samples were collected from healthy and diseased animals and from diseased humans and included milk, wound swab, pus, exudates, nasal swab and diabetic ulcer. Initially, S. aureus was isolated and identified by colony morphology, Gram staining, and biochemical tests (catalase and coagulase tests). The S. aureus-positive samples were examined by polymerase chain reaction (PCR) to determine their MRSA status. RESULTS:Of the 100 animal samples, 29 were positive for S. aureus. Four samples (13.8%) from dogs were MRSA-positive, but samples from cattle and cats were MRSA-negative. Of the 150 human samples we collected, 64 were S. aureus-positive and, of these, 34 (53.1%) were MRSA-positive. Most (28%) of the MRSA samples were isolated from surgical wound swabs, followed by the pus from skin infections (11%), exudates from diabetic ulcers (6%), exudates from burns (4%), and aural swabs (3%). By contrast, a low MRSA detection rate (n?=?4) was seen in the non-human isolates, where all MRSA bacteria were isolated from nasal swabs from dogs. The antimicrobials susceptibility testing results showed that S. aureus isolates with mecA genes showed resistance to penicillin (100%), oxacillin (100%), erythromycin (73.5%), ciprofloxacin (70.6%), and gentamicin (67.7%). The lowest resistance was found against ceftazidime, and no vancomycin-resistant isolates were obtained. CONCLUSIONS:We detected S. aureus and MRSA in both human and canine specimens. Isolates were found to be resistant to some of the antimicrobials available locally. MRSA carriage in humans and animals appears to be a great threat to effective antimicrobials treatment. The prudent use of antimicrobials will reduce the antimicrobial resistance. Our findings will help to find the most appropriate treatment and to reduce antimicrobial resistance in the future by implementing prudent use of antimicrobials. Further studies are required to better understand the epidemiology of MRSA human-animal inter-species transmission in Bangladesh.
Project description:INTRODUCTION: The purpose of this study is to develop a nuc and mecA gene specific Loop-mediated isothermal Amplification (LAMP) assay for rapid identification and detection of methicillin resistant Staphylococcus aureus among clinical isolates. MATERIALS AND METHODS: A total of 100 (70 from pus and 30 from blood), clinical isolates of Staphylococcus spp were screened for the nuc gene to differentiate between S.aureus and Coagulase negative Staphylococci (CONS) by a nuc gene specific LAMP assay. The isolates were also screened for the presence of the mec Agene by the mecA specific LAMP assay. The results were compared with the phenotypic identification and methicillin resistance by Vitek-2 system (bioMérieux, Marcy l'Etoile, France) and conventional PCR. RESULTS: Among 100 Staphylococcus isolates, there were 82 (82%) Staphylococcus aureus isolates and 18 (18%) coagulase negative Staphylococcus as detected by the Vitek 2, conventional PCR and the LAMP assay using the nuc gene. The mecA gene was detected by the LAMP assay in 56(56%) isolates (44 Methicillin resistant Staphylococcus aureus (MRSA) and 12 Methicillin resistant coagulase negative Staphylococcus (MRCONS), which were also identified by the Vitek 2 and conventional PCR as methicillin resistant. The results of the LAMP assay were available within 90min as compared to the Vitek 2 results (18- 24hours) and conventional PCR (3-4 hours). CONCLUSION: The present study proved that LAMP assay can be used for the simultaneous differentiation of Staphylococcal spp and detection of methicillin resistance.