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.

First report of a Cryptococcus magnus infection in a cat.

This report describes an uncommon case of cryptococcosis in an apparently immunocompetent cat caused by Cryptococcus magnus. An amputation of the complete left foreleg and excision of the ipsilateral cervical lymph node were performed in a young-adult male Domestic Shorthair cat due to suspicion of a tumor. Granulomatous dermatitis, panniculitis, myositis, and lymphadenitis were diagnosed histologically. Intralesional, numerous round-to-ovoid yeast cells showing no capsule were detected within macrophages using special staining methods. The tissue material cultured on Sabouraud's glucose agar at 26°C yielded abundant growth of yeast colonies. Morphological, physiological, and molecular analyses of the yeasts demonstrated that the fungus was C. magnus. Response to treatment with fluconazole was fast and effective, and one year after the end of the therapy no further clinical signs of infection were observed.

Efficacy of pradofloxacin in cats with feline upper respiratory tract disease due to Chlamydophila felis or Mycoplasma infections.

BACKGROUND: Upper respiratory tract disease (URTD) of cats is caused by a number of pathogens, including Chlamydophila felis and Mycoplasma spp. For effective treatment of both infections, doxycycline and enrofloxacin are recommended, but adverse effects limit their use in cats. HYPOTHESIS: That the fluoroquinolone pradofloxacin is effective against C. felis and Mycoplasma infection in cats with URTD or conjunctivitis. ANIMALS: Thirty-nine cats with signs of URTD or conjunctivitis. METHODS: Placebo-controlled, double-blind clinical trial. Cats were randomly entered into 1 of 2 treatment groups: treated PO with either 5 mg/kg pradofloxacin q24h or 5 mg/kg doxycycline q12h for 42 consecutive days. Changes in health status and clinical scores were evaluated. The presence of C. felis and Mycoplasma spp. was determined by quantitative polymerase chain reaction (PCR) and nested PCR of conjunctival swabs, respectively. RESULTS: At the beginning of the study, C. felis and Mycoplasma spp. were detected in 23 and 20 cats, respectively. Cats of both groups responded rapidly with a marked improvement in clinical signs within the 1st week. During treatment with either drug, C. felis DNA copy number declined quickly. Complete elimination of Mycoplasma spp. was achieved in both groups; however, whereas all cats receiving doxycycline eliminated C. felis, 4 cats treated with pradofloxacin remained PCR-positive. CONCLUSION AND CLINICAL IMPORTANCE: This study demonstrates that both pradofloxacin and doxycycline have good efficacy against C. felis and Mycoplasma spp., resulting in a marked improvement of clinical signs. However, C. felis DNA remained in some cats after treatment with pradofloxacin, suggesting that infection might not have been eliminated.

Pharmacokinetics of enrofloxacin and its efficacy in comparison with doxycycline in the treatment of Chlamydophila felis infection in cats with conjunctivitis.

The concentrations of enrofloxacin were measured in the tears, saliva and serum of 14 cats with signs of upper respiratory tract infection and eight with no signs, after daily doses of 5 mg/kg. Enrofloxacin concentrations above the minimum inhibitory concentration of Chlamydophila felis were found in the saliva and tears of the cats with and without signs of upper respiratory tract infection. In a prospective randomised clinical trial, the efficacy of enrofloxacin against C. felis infection in cats with conjunctivitis was compared with the efficacy of doxycycline. Twenty-five cats were randomly assigned to treatment with either enrofloxacin or doxycycline for 14 days; 15 of the cats tested positive for C. felis by an immunofluorescent antibody test on conjunctival swabs. The two treatment groups showed equal improvements in the clinical signs of conjunctivitis and C. felis infection status; in each group three cats were still C. felis antigen-positive after the 14-day course of treatment, indicating a persistent infection. No side effects were observed in the cats treated with enrofloxacin.

Molecular analysis of the macrolide-lincosamide resistance gene region of a novel plasmid from Staphylococcus hyicus.

Resistance to macrolides and lincosamides in Staphylococcus hyicus has been shown to be encoded by a 4.0-kb plasmid designated pSES21. It differed distinctly in its restriction map from all other staphylococcal macrolide resistance plasmids reported so far. Southern blot hybridisation with gene probes specific for staphylococcal erm genes demonstrated that the macrolide resistance gene belonged to hybridisation class C. Analysis of the ermC gene revealed that the deduced amino-acid sequence of the pSES21-encoded ErmC methylase exhibited c. 93% identity with the ErmC methylase encoded by plasmid pE194. The ermC gene of pSES21 was expressed constitutively and sequence analysis of the regulatory region showed multiple base-pair insertions and substitutions in the translational attenuator. As a consequence of these mutations, the reading frame of the small regulatory peptide was destroyed and a novel pair of inverted repeated sequences was generated. Previous studies identified sequence deletions and sequence duplications in the ermC regulatory region as the basis for constitutive ermC gene expression. The multiple point mutations shown in the pSES21-encoded ermC translational attenuator represent a novel kind of structural alteration in this regulatory region and may explain constitutive ermC gene expression by pairing of the newly generated inverted repeated segments in the presence of a functionally deleted reading frame for the small regulatory peptide.

A novel plasmid from Staphylococcus epidermidis specifying resistance to kanamycin, neomycin and tetracycline.

The naturally occurring plasmid pSTS7 from Staphylococcus epidermidis mediated resistance to tetracycline via a tetL gene and to kanamycin and neomycin via an aadD gene. Plasmid pSTS7 showed partial restriction map and sequence homology to the previously described tetracycline resistance plasmid pNS1981 from Bacillus subtilis and to the kanamycin/neomycin/bleomycin resistance plasmid pUB110 from S. aureus. Sequence analysis of the regions flanking the two resistance genes in pSTS7 led to the identification of a novel site for interplasmid recombination which could explain the derivation of pSTS7 from the incompatible pNS1981- and pUB110-like parental plasmids under tetracycline-selective pressure.

Molecular analysis of naturally occuring ermC-encoding plasmids in staphylococci isolated from animals with and without previous contact with macrolide/lincosamide antibiotics.

A total of 16 epidemiologically unrelated macrolide-resistant staphylococcal isolates of various animal origins were investigated for the molecular basis of macrolide resistance with respect to previous contact of their host animals with macrolides and lincosamides. All isolates carried ermC-encoding plasmids of 2.3-4.0 kbp. The eight plasmids of staphylococci from animals which had not received macrolides or lincosamides showed inducible ermC gene expression and did not exhibit alterations in the ermC regulatory region. The remaining eight plasmids expressed the ermC gene constitutively. Six of these plasmids were from staphylococci from animals which had received tylosin or spiramycin as feed additives or lincomycin for therapeutic purposes. All constitutively expressed ermC genes revealed either sequence deletions or sequence duplications in their ermC regulatory region, as detected by a PCR assay and by sequence analysis. These sequence deletions and duplications found in naturally occurring plasmids corresponded closely to the mutations seen in the ermC-encoding plasmids after growth of an inducibly resistant strain in the presence of non-inducing macrolides or lincosamides under in vitro conditions.

Tetracycline resistance in Staphylococcus spp. from domestic animals.

A total of 838 staphylococcal isolates representing 19 different species were obtained from cattle, cats, dogs, ducks, guinea pigs, horses, mink, pigeons, pigs, rabbits, and turkeys. From these 228 (27.2%) isolates were shown to be resistant to tetracycline and to carry one or two of the tetracycline resistance (tet) genes tet (K), tet (L), tet (M), or tet (O) with seven different distribution patterns. Additional resistances to one or more antibiotics were observed in 153 (67.1%) of the tetracycline resistant isolates. The tet (M) gene was found in 94.3% of the resistant S. intermedius isolates while the tet (K) gene predominated in most of the other staphylococcal species irrespective of the host animal. The tet (K) and tet (L) genes were located on plasmids while the tet (M) and tet (O) genes appeared to be associated with the chromosome.

Chloramphenicol resistance in Staphylococcus intermedius from a single veterinary centre: evidence for plasmid and chromosomal location of the resistance genes.

A total of seven Staphylococcus intermedius cultures isolated from cases of canine pyoderma were investigated for the genetic basis of chloramphenicol resistance (Cmr). All of these S. intermedius isolates mediated Cmr via the expression of the Cm-inactivating enzyme chloramphenicol acetyltransferase (CAT); the respective cat genes were found to be located on small multicopy plasmids of 3.1 to 4.1 kb in four of the seven cultures. The four Cmr plasmids, designated pSCS20-23, differed upon restriction endonuclease mapping. Hybridization experiments identified all of them to belong to the pC221-family of staphylococcal Cmr plasmids. The expression of all four plasmid-encoded cat genes was inducible with chloramphenicol. The remaining three S. intermedius isolates also harboured an inducible cat gene of the pC221-type which, however, was found to be located in the chromosomal DNA. These differences in the subcellular localisation and consequently in the number of cat gene copies per S. intermedius cell had no influence on the MIC values of Cm exhibited by the respective S. intermedius isolates.

Dermatophytes in pet Guinea pigs and rabbits.

PROBLEM ADDRESSED: The frequency of dermatophytes in pet Guinea pigs and rabbits. OBJECTIVES: To determine the frequency and types of dermatophytes in pet Guinea pigs and rabbits. METHODS AND APPROACH: First, 2153 samples collected from pet Guinea pigs (n=1132) and rabbits (n=1021) with suspected dermatophytosis and submitted to three different laboratories for fungal culture were analysed. Subsequently, healthy Guinea pigs and rabbits, animals with skin lesions and with noncutaneous diseases were examined prospectively for dermatophytes. RESULTS: Trichophyton (T.) mentagrophytes was the most common fungal species isolated (91.6% and 72.3% of positive cultures from Guinea pigs (n=431) and rabbits (n=83), respectively). Animals with positive fungal culture did not show any gender predisposition, but affected animals were younger than those with negative fungal culture (P<0.0001) or than healthy animals of the prospective part of the study. Dermatophytes were isolated from 14/164 healthy Guinea pigs and 0/140 healthy rabbits. In addition, fungal cultures of Guinea pigs with skin lesions (n=26) and other diseases (n=25) were positive in 7.7% and 8.0% respectively. Samples collected from 17 rabbits with skin lesions and 32 rabbits with noncutaneous disease were all negative in culture. CONCLUSIONS: T. mentagrophytes is the most common dermatophyte in pet Guinea pigs and rabbits, asymptomatic carriers are regularly seen in Guinea pigs, but not in rabbits.

Molecular analysis of the translational attenuator of a constitutively expressed erm(A) gene from Staphylococcus intermedius.

During the course of a study on macrolide, lincosamide and streptogramin B resistance among staphylococci from animal sources, a Staphylococcus intermedius isolate was found to carry a constitutively expressed erm(A) gene on the 70 kb plasmid pSES29. The molecular basis of constitutive erm(A) expression was investigated by cloning and sequence analysis of the erm(A)-associated translational attenuator. Two point mutations in this regulatory region were detected. These mutations cause constitutive erm(A) gene expression by destabilization of mRNA secondary structures required for the inducible type of erm(A) gene expression.

[Resistance to protein biosynthesis inhibitors in Staphylococci: resistance genes and their spread--a review]. / Resistenzen gegenüber Proteinbiosyntheseinhibitoren bei Staphylokokken: Resistenzgene und ihre Ausbreitung--Ubersichtsreferat.

The rapid spread of antibiotic resistances in a wide variety of bacteria is mainly due to the location of antibiotic resistance genes on mobile genetic elements such as plasmids and transposons. Principal ways of transfer of plasmid- and transposon-encoded resistance genes are presented using examples of the predominant genes mediating resistances to protein biosynthesis inhibitors such as tetracyclines, aminoglycosides, macrolide-lincosamide-streptogramin B antibiotics, and chloramphenicol in staphylococci. Transfer between different staphylococcal cells is substantially based on transduction, transformation, conjugation and mobilization while transfer of resistance genes within the same bacterial cell often includes interplasmidic recombination events and chromosomal integration of resistance plasmids or transposons. The abilities of the transferred resistance plasmids or transposons to integrate or to be integrated into DNA molecules, plasmids or chromosomal DNA, of the new host cell are of major importance to circumvent strain-, species- or genusspecific barriers such as restriction/modification systems, plasmid incompatibilities or deficiencies of plasmid replication which may limit efficient resistance gene transfer.

Macrolide-lincosamide-streptogramin B resistance in Staphylococcus lentus results from the integration of part of a transposon into a small plasmid.

The 8.0-kb macrolide-lincosamide-streptogramin B resistance plasmid pSES20 from Staphylococcus lentus harbored part of a Tn917-like transposon including the left terminal repeat, a gene almost identical to ermB, and its regulatory region, as well as the internal direct repeat. Homology between pSES20 and Tn917 ended at a sequence closely related to those of the resolution sites of Tn917 and Tn552 and staphylococcal recombination sites.

Tn5706, a transposon-like element from Pasteurella multocida mediating tetracycline resistance.

The 4,378-bp putative tetracycline resistance transposon Tn5706 of Pasteurella multocida consists of an internal tet(H)-tetR resistance gene region which is flanked by almost identical insertion elements, IS1596 and IS1597. Two reading frames for proteins of 70 and 228 amino acids were detected in each of these insertion sequence elements. The 228-amino-acid protein revealed homology to transposase proteins of gram-positive bacteria.

Identification of a plasmid-borne chloramphenicol-florfenicol resistance gene in Staphylococcus sciuri.

The 16.5-kbp plasmid pSCFS1 from Staphylococcus sciuri mediated combined resistance to chloramphenicol and florfenicol. The gene responsible for this resistance property, cfr, was cloned and sequenced. The amino acid sequence of the Cfr protein revealed no homology to known acetyltransferases or efflux proteins involved in chloramphenicol and/or florfenicol resistance or to other proteins whose functions are known.

Structural alterations in the translational attenuator of constitutively expressed ermC genes.

Sequence deletions of 16, 59, and 111 bp as well as a tandem duplication of 272 bp with respect to the corresponding sequence of pT48 were identified in the regulatory regions of constitutively expressed ermC genes. Constitutive ermC gene expression as a consequence of these structural alterations is based on either the prevention of the formation of mRNA secondary structures in the translational attenuator or the preferential formation of those mRNA secondary structures which do not interfere with the translation of the ermC transcripts. A model for the development of sequence deletions in the ermC translational attenuator by homologous recombination is presented and experimentally tested by in vitro selection of constitutively expressed mutants in staphylococcal strains deficient and proficient in homologous recombination.

Integration of pT181-like tetracycline resistance plasmids into large staphylococcal plasmids involves IS257.

Four large staphylococcal plasmids ranging in size from 31 to 82 kbp have been shown to mediate tetracycline resistance via an integrated copy of the tet(K)-encoding plasmid pT181 which was flanked by copies of the insertion element IS257. In two cases, IS257 elements interrupted the repC reading frame of pT181 and an 8-bp sequence from within the repC gene was duplicated at the interrupted site. In the third plasmid, the IS257 elements interrupted the pT181 DNA immediately upstream of the repC coding sequence with an 8-bp duplication. In the fourth case, the IS257 elements flanked a pT181-like plasmid with one IS257 in the repC coding sequence and the other within the recombinase (pre) coding sequence, so that a section of the pT181 sequence was deleted. All four integration sites detected in this study differ from those previously described for the IS257-mediated integration of pT181-like plasmids into large plasmids or into the chromosomal DNA.

Insertion elements in Staphylococcus intermedius.

Staphylococcus intermedius cultures from dogs, pigeons, horses and mink were investigated for the prevalence of the insertion elements IS256 and IS257 in relation to their antibiotic resistance. Copies of IS256 could not be detected in any of the Staph. intermedius isolates tested whereas single copies of IS257 occurred in the isolates from dogs and horses. The mink strains did not harbour IS257 elements, whereas Staph. intermedius isolates from pigeons carried multiple copies of IS257 as predicted from the hybridization patterns obtained with a gene probe derived from the internal part of the IS257-encoded transposase gene. Independently of the origin of the Staph. intermedius isolates, all IS257 copies were found to be located in the chromosomal DNA. The large number of chromosomal IS257 copies in the pigeon strains might help to explain chromosomal multiresistance in many of those strains.

Antimicrobial resistance in staphylococci from animals with particular reference to bovine Staphylococcus aureus, porcine Staphylococcus hyicus, and canine Staphylococcus intermedius.

Besides their role as commensals on the skin and mucosal surfaces, staphylococci may be involved in a wide variety of diseases in animals. Staphylococcal infections in animals are mainly treated with antimicrobial agents and as a consequence, staphylococci from animal sources have developed and/or acquired resistance to the respective antimicrobial agents. Resistance statistics obtained from national monitoring programmes on staphylococci from cattle and pigs, but also from surveillance studies on staphylococci involved in diseases in dogs are reported and reviewed with regard to their comparability. This review mainly focusses on the genetic basis of antimicrobial resistance in staphylococci of animal origin. Particular attention is paid to resistance to those antimicrobial agents which are most frequently used in veterinary medicine, but also to antimicrobial agents, such as chloramphenicol and mupirocin, which are used in specific cases for the control of staphylococcal infections in pets and companion animals. In addition, plasmids and transposons associated with the respective resistance properties and their ways of spreading between members of the same or different staphylococcal species, but also between staphylococci and other gram-positive bacteria, are described.

Host range of the ermF rRNA methylase gene in bacteria of human and animal origin.

We screened 183 different clinical anaerobic and aerobic bacteria isolated from humans and other animals for the presence of the ermF gene using a polymerase chain reaction (PCR) assay. The ermF gene was detected in 107 (58%) clinical isolates, including 42 (61%) of 69 gram-positive bacteria and 65 (57%) of 114 gram-negative bacteria. Twenty-five ATCC isolates were also tested; 20 (80%) carried the ermF gene. The gene products from the ermF PCR from four isolates were sequenced and showed 95-99% nucleotide homology with the ermF gene and 98-99% amino acid homology with the gene product. Eleven (58%) of the 19 gram-negative donors tested were able to transfer the ermF gene. All nine (100%) of the gram-positive donors tested transferred the ermF gene, using either Enterococcus faecalis or Haemophilus influenzae as the recipients.