Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10⁶ to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10⁶ cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

Detection of methicillin-resistant Staphylococcus pseudintermedius with commercially available selective media

Aims: Commercially available selective media for methicillin‐resistant Staphylococcus aureus (MRSA) were tested for the detection and isolation of methicillin‐resistant Staphylococcus pseudintermedius (MRSP). Methods and Results: Five different screening agars [mannitol salt agar with oxacillin and BD BBL? Chromagar? MRSA (BD Diagnostics); chromID? MRSA agar (bioMérieux); Oxacillin resistance screening agar base (ORSAB); and Brilliance MRSA agar (Oxoid)] were analysed for the detection of MRSP. Bacteria that may be isolated together with MRSP and may grow on the screening agars were included in the study to determine possible interference with the growth of MRSP. MRSP grew well on all selective media except on BD BBL? Chromagar? MRSA (BD Diagnostics) and chromID? MRSA agar (bioMérieux), on which a low to moderate growth rate was noted. Conclusions: ORSAB (Oxoid) and Brilliance MRSA agar (Oxoid) are most suitable for the detection and isolation of MRSP from clinical material. Significance and Impact of the Study: The importance of MRSP in veterinary medicine is increasing. Diagnostic systems are needed to detect MRSP carrier as soon as possible. This study provides information about selected MRSA screening agars for the detection of MRSP to the clinical microbiologists.     

Longitudinal study on methicillin-resistant Staphylococcus pseudintermedius in households

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an emerging pathogen in dogs and has been found in Europe, Asia and North America. To date most studies are one-point prevalence studies and therefore little is known about the dynamics of MRSP in dogs and their surrounding. In this longitudinal study MRSP colonization in dogs and the transmission of MRSP to humans, contact animals and the environment was investigated. Sixteen dogs with a recent clinical MRSP infection were included. The index dogs, contact animals, owners and environments were sampled once a month for six months. Samples taken from the nose, perineum and infection site (if present) of the index cases and contact animals, and the nares of the owners were cultured using pre-enrichment. Index cases were found positive for prolonged periods of time, in two cases during all six samplings. In five of the 12 households that were sampled during six months, the index case was intermittently found MRSP-positive. Contact animals and the environment were also found MRSP-positive, most often in combination with a MRSP-positive index dog. In four households positive environmental samples were found while no animals or humans were MRSP-positive, indicating survival of MRSP in the environment for prolonged periods of time. Genotyping revealed that generally similar or indistinguishable MRSP isolates were found in patients, contact animals and environmental samples within the same household. Within two households, however, genetically distinct MRSP isolates were found. These results show that veterinarians should stay alert with (former) MRSP patients, even after repeated MRSP-negative cultures or after the disappearance of the clinical infection. There is a considerable risk of transmission of MRSP to animals in close contact with MRSP patients. Humans were rarely MRSP-positive and never tested MRSP-positive more than once suggesting occasional contamination or rapid elimination of colonization of the owners.     

Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

Methicillin-resistant Staphylococcus pseudintermedius isolated from canine pyoderma in North China

Aims: To determine the prevalence of carriage of methicillin‐resistant Staphylococcus pseudintermedius (MRSP) among dogs with pyoderma from two small animal hospitals in North China during a 21‐month period and to characterize these isolates. Methods and Results: Swabs were taken from 260 dogs with pyoderma, and the staphylococcal species isolated and methicillin resistance were confirmed phenotypically and genotypically. The identified MRSP isolates were characterized by multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome (SCC) mec typing, testing for susceptibility to nine antimicrobial agents and SmaI‐digested pulsed‐field gel electrophoresis. Thirty‐three (12·7%) dogs were positive for MRSP. The most prevalent genotypes detected among MRSP were ST71(MLST)‐t06(spa)‐II‐III(SCCmec) (n = 22, 66·7%), followed by ST5‐t19 (n = 8, 24·2%), ST126‐III(n = 2, 6·1%) and ST6‐t02‐V (3·0%). All MRSP isolates showed extended resistance to tested antimicrobial agents. Eight different SmaI patterns were observed in 21 typeable MRSP isolates. Conclusions: Clinical isolates of MSRP isolated from dogs in North China belonged to two major clonal lineages ST71 and ST5. Significance and Impact of the Study: This study is the first report on MRSP from canine pyoderma in China. Further surveillance study is needed to gain more detailed data concerning this major clinical challenge in veterinary medicine.     

A novel microfluidic wound model for testing antimicrobial agents against Staphylococcus pseudintermedius biofilms

Background

Current methods for testing treatments for veterinary surgical site infections can successfully emulate elements of a chronic wound, but these are time consuming and costly, requiring specialized laboratory equipment and considerable space to house study animals. Microfluidic devices however, can be coated with collagen and maintained at basal body temperature, providing a more cost-effective and space-saving model of a chronic wound. Our study assesses the applicability of a new microfluidic model by testing the activity of DispersinB against biofilms of methicillin-resistant Staphylococcus pseudintermedius (MRSP); DispersinB has been shown to prevent biofilm growth of Staphylococcus epidermidis, another prominent wound colonizer.

Results

We successfully developed a microfluidic model to examine the effects of antimicrobial therapy on biofilms formed by organisms associated with wound infections in companion animals (e.g. MRSP). Although, we were unable to recapitulate previous findings that DispersinB-Gentamycin is highly effective against Staphylococcal biofilms using this model, we were able to confirm its effect in a microtitre plate. Differences in the experimental conditions likely account for this result (e.g. strains tested, flow conditions, treatment time, etc.). In the microtitre plate assay, DispersinB inhibited biofilm growth after a 24 hour period; there was an inverse relationship between the concentration of DispersinB-Gentamycin and the amount of biofilm remaining following treatment. Collagen-coated microtitre plates showed a similar result, but this did not correlate as well; collagen, the most abundant protein in the body may help to retain the biomass of treated biofilms.

Conclusions

Our model may be useful in examining the effect of treatment on wound infections, although we acknowledge that in this model the test organisms may be more recalcitrant to antimicrobials than in other published systems. We contend that this may in fact better represent the conditions in vivo, where organisms associated with chronic wound infections are highly resistant to antimicrobials.     

PrimerCE: Designing Primers for Cloning and Gene Expression

A number of primer design programs have been developed for diverse applications. However, none of these programs can be used to design primers for gene cloning aimed at expressing protein. Here we report the design of PrimerCE, which can be used to cover the whole process of gene cloning and expression. The main features of PrimerCE include inspection of restriction enzyme recognition sequence, open reading frame verification, stop codon inspection, base adjustment, primer optimization, sequence assembly and protein analysis. In addition to this, the program can be modified based on the different needs of users, e.g. new vector sequence and restriction enzyme recognition sequences can be integrated. With the use of PrimerCE, a pair of primers can be designed within minutes. The program has been proven to be efficient in designing primers in our high throughput cloning and gene expression experiments. The software is freely available at .     

Prevalence of staphylococcal enterotoxins in Staphylococcus pseudintermedius isolates from dogs with pyoderma and healthy dogs

To investigate the role of staphylococcal enterotoxins (SEs) produced by Staphylococcus pseudintermedius in the pathogenesis of pyoderma, isolates from dogs with pyoderma and healthy dogs were analyzed. According to reverse passive latex agglutination, 14/184 isolates (7.6%) from dogs with pyoderma and 9/87 (10.3%) from healthy dogs produced SEs (SEA, SEC or SED). According to multiplex PCR, 99 isolates (53.7%) from dogs with pyoderma and 97 (90.8%) from healthy dogs possessed one or more se genes. There was no significant difference regarding ses between dogs with pyoderma and healthy dogs. Therefore, SEs may not be a direct virulence factor in pyoderma.     

Occurrence and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus pseudintermedius in an Academic Veterinary Hospital

Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in human hospitals. The prevalence of hospital-acquired MRSA (HA-MRSA) infection among inpatients in intensive care units (ICUs) continues to increase steadily in Japan. Recently, cases of community-acquired MRSA (CA-MRSA) have been documented in persons without an established risk factor for HA-MRSA infection (14, 32, 36, 49).There has also been an increase in the number of reports of the isolation of MRSA from veterinarians and companion animals (5, 21, 23-26, 28, 31, 34, 38, 44, 50, 51, 53). Values reported for the prevalence of MRSA among veterinary staff include 17.9% in the United Kingdom (21), 10% in Japan (38), 3.9% in Scotland (13), and 3.0% in Denmark (28). Loeffler et al. reported that the prevalence of MRSA among dog patients and healthy dogs owned by veterinary staff members was 8.9% (21). In Japan, an MRSA isolate was detected in only one inpatient dog (3.8%) and could not be detected in any of 31 outpatient dogs (38). In the United States, MRSA isolates were detected in both dog (0.1%) and cat (0.1%) patients (31). The prevalence of MRSA among healthy dogs has been reported to be 0.7% (5). Hanselman et al. suggested that MRSA colonization may be an occupational risk for large-animal veterinarians (12). Recently, Burstiner et al. reported that the frequency of MRSA colonization among companion-animal veterinary personnel was equal to the frequency among large-animal veterinary personnel (6).In addition, other methicillin-resistant coagulase-positive staphylococci (MRCPS), such as methicillin-resistant Staphylococcus pseudintermedius (MRSP) and methicillin-resistant Staphylococcus schleiferi (MRSS), isolated from dogs, cats, and a veterinarian have been reported (11, 31, 38, 40, 52). MRSP isolates have also been detected among inpatient dogs (46.2%) and outpatient dogs (19.4%) in a Japanese veterinary teaching hospital (38). In Canada, however, MRSP and MRSS isolates were detected in only 2.1% and 0.5% of dog patients, respectively (11).Methicillin-resistant staphylococci produce penicillin-binding protein 2′, which reduces their affinity for β-lactam antibiotics. This protein is encoded by the mecA gene (48), which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is a mobile genetic element characterized by the combination of the mec and ccr complexes (16), and it is classified into subtypes according to differences in the junkyard regions (43). SCCmec typing can be used as a molecular tool (22, 27, 30, 33, 36, 55) for examining the molecular epidemiology of methicillin-resistant staphylococci.In this study, we investigated the occurrence and characteristics of MRCPS isolates in a veterinary hospital in order to establish the transmission route of MRCPS in a veterinary hospital and with a view to preventing the spread of MRCPS infection. In addition, we evaluated the factors associated with MRCPS. Further, as Heller et al. have reported the distribution of MRSA within veterinary hospital environments and suggested the necessity to review cleaning protocols of hospital environments (13), we also attempted to isolate MRCPS from environmental samples collected in a veterinary hospital for an evaluation of MRSA transmission cycle though environmental surfaces in the veterinary hospital.     

Detection and Identification of Gastrointestinal Lactobacillus Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.     

Linkage Mapping of DNA Markers Generated with Specific and Non-Specific Gene Primers in Soybean

Polymerase chain reaction (PCR) has been used extensively in the construction of linkage maps for many cultivated crops including soybean, [Glycine max (L.) Merr]. In this study, four sets of oligonucleotide primer pairs of known genes (pearl millet Adh 1, nodule specific proline-rich protein, Drosophila homeobox, heat shock protein), several different combinations from kits A, D, E, and J of arbitrary primers and five primer pairs of soybean simple sequence repeats of varying length (Satt 9, Satt 20, Satt 42, Satt 64, and Satt 30) were utilized in PCR to identify molecular markers which were then used to construct a genetic linkage map. DNA for the PCR reactions was isolated from 65 recombinant inbred soybean lines resulting from crossing PI 290,136 and BARC-2 (Rj ₄ ), followed by self-pollination for seven generations without selection. Mapmaker 3.0, a computer package, was used for construction of the linkage map. A total of 43 polymorphic markers were identified; 30 markers were linked and distributed among 5 linkage groups while 13 markers were unlinked. Arbitrary primers revealed more polymorphisms than specific primers. A combination of arbitrary primers A5 and A18 revealed the maximum number of polymorphic bands. Five observed linkage groups can be expanded in future soybean research by using additional markers.     

Universal Primers for Amplification of Mitochondrial Small Subunit Ribosomal RNA-Encoding Gene in Scleractinian Corals

We describe the construction of polymerase chain reaction primers designed to amplify a portion of the mitochondrial (mt) small subunit ribosomal (SSU) RNA-encoding genes in scleractinian corals. Combinations of cloning and sequencing show that the amplified fragments are between 694 and 896 bp in length. Alignment of the amplified DNA sequences to the published mt SSU rRNA genes of Metridium senile and Sarcophyton glaucum indicates several conserved regions among actiniarian, corallimorpharian, octocorallian, and scleractinians, suggesting this primer set can successfully amplify over 80% of the mt SSU rDNA region of scleractinian corals. Surveys of sequence variation and estimation of the rate of evolution show an extremely slow divergence of the SSU rRNA gene in the family Acroporidae. Received June 11, 1999; accepted October 4, 1999.     

Antimicrobial Resistance, Virulence Genes, and Genetic Lineages of Staphylococcus pseudintermedius in Healthy Dogs in Tunisia

Nasal swabs of 100 healthy dogs were obtained in 2011 in Tunisia and tested for Staphylococcus pseudintermedius recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST) and SmaI-pulsed-field gel electrophoresis (PFGE) were investigated. S. pseudintermedius was recovered in 55 of the 100 tested samples (55 %), and one isolate per sample was further studied. All 55 S. pseudintermedius isolates were susceptible to methicillin (MSSP) but showed resistance to the following antimicrobials (% resistant isolates/resistance gene): penicillin (56.4/blaZ), tetracycline (40/tetM), trimethoprim-sulfamethoxazole (23.7), fusidic acid (9), kanamycin (3.7/aph(3´)-Ia), erythromycin-clindamycin (1.8/erm(B)), streptomycin (1.8/ant(6)-Ia), chloramphenicol (1.8) and ciprofloxacin (1.8). The following toxin genes were identified (% of isolates): lukS/F-I (98.2), expA (5.5), se-int (98.2), sec _canine (1.8), siet (100), sea (5.5), seb (3.6), sec (10.9), sed (54.5), sei (5.5), sej (29.1), sek (3.6), ser (9.1), and hlg _v (38.2). Ten different sequence-types were detected among 11 representative MSSP isolates: ST20, ST44, ST69, ST70, ST78, ST100, ST108, ST160, ST161, and ST162, the last three ones revealing novel alleles or allele combinations. Eleven different PFGE-patterns were identified in these isolates. The nares of healthy dogs could be a reservoir of antimicrobial resistant and virulent MSSP, highlighting the presence of the recently described exfoliating gene expA and several enterotoxin genes.     

Clonal Dynamics of Nasal Staphylococcus aureus and Staphylococcus pseudintermedius in Dog-Owning Household Members. Detection of MSSA ST398

The objective of this study was to investigate the dynamics of nasal carriage by Staphylococcus aureus (SA) and Staphylococcus pseudintermedius (SP) among healthy dog-owning household members involved in 7 previous index cases of suspected anthropozoonotic (n = 4) and zoonotic (n = 3) interspecies transmission [4 direct cases, identical SA (n = 3) or SP (n = 1) in owner and dog; three indirect, SP in owner (n = 2) or SA in dog (n = 1)]. Co-carriage with methicillin-resistant coagulase-negative staphylococci (MRCoNS) was also evaluated. Sixteen owners and 10 dogs were sampled once every three months for one year. In total, 50 SA and 31 SP were analysed by MLST, and SA also by spa typing. All isolates were subjected to ApaI/SmaI-PFGE and antimicrobial resistance and virulence profiles were determined. All index owners were persistent SA carriers in all direct-anthropozoonotic transmission cases, while only one dog was persistent SA carrier. Owner and dog exhibited a persistent SP carriage status in the direct-zoonotic transmission case. SP was maintained in the index human over time in one indirect-zoonotic transmission case. Only one SP was methicillin-resistant. SA belonged to genetic backgrounds of MRSA pandemic clones: CC45, CC121, CC30, CC5 and CC398. Three individuals carried a MSSA t1451-ST398 clone with the erm(T)-cadD/cadX resistance genes. SA or SP were persistently detected in the nasal cavity of 7 (43.8%) and 2 (12.5%) owners, and in one and 2 dogs, respectively. SA was recovered as the single species in 10 owners and in one dog; SP in 3 owners and 4 dogs; and both bacterial species in one owner and 4 dogs. Co-carriage of SA or SP with MRCoNS isolates was common (30.7%). This is the first study on the dynamics of nasal carriage of SA and SP in healthy pet-owning household members. Dog-contact may play a role in the staphylococcal species distribution of in-contact individuals.