Luca Guardabassi

Population Genetic Structure of the Staphylococcus intermedius Group: Insights into agr Diversification and the Emergence of Methicillin-Resistant Strains

The population genetic structure of the animal pathogen Staphylococcus intermedius is poorly understood. We carried out a multilocus sequence phylogenetic analysis of isolates from broad host and geographic origins to investigate inter- and intraspecies diversity. We found that isolates phenotypically identified as S. intermedius are differentiated into three closely related species, S. intermedius, Staphylococcus pseudintermedius, and Staphylococcus delphini. S. pseudintermedius, not S. intermedius, is the common cause of canine pyoderma and occasionally causes zoonotic infections of humans. Over 60 extant STs were identified among the S. pseudintermedius isolates examined, including several that were distributed on different continents. As the agr quorum-sensing system of staphylococci is thought to have evolved along lines of speciation within the genus, we examined the allelic variation of agrD, which encodes the autoinducing peptide (AIP). Four AIP variants were encoded by S. pseudintermedius isolates, and identical AIP variants were shared among the three species, suggesting that a common quorum-sensing capacity has been conserved in spite of species differentiation in largely distinct ecological niches. A lack of clonal association of agr alleles suggests that assortive recombination may have contributed to the distribution of agr diversity. Finally, we discovered that the recent emergence of methicillin-resistant strains was due to multiple acquisitions of the mecA gene by different S. pseudintermedius clones found on different continents. Taken together, these data have resolved the population genetic structure of the S. intermedius group, resulting in new insights into its ancient and recent evolution.

spa type distribution in Staphylococcus aureus originating from pigs, cattle and poultry

Methicillin-resistant S. aureus (MRSA) of clonal complex 398 (CC398) is emerging globally among production animals such as cattle, pigs and poultry as well as among humans. However, little is known about the prevalence of CC398 among methicillin sensitive S. aureus (MSSA) or the relative clonal distribution of S. aureus isolated from these three animal reservoirs. To study this, we have analyzed a random sample of S. aureus consisting of 296 epidemiologically unrelated isolates from infections and colonisation of pigs, cattle and poultry. These were examined and compared by spa and multi-locus sequence typing (MLST) and the result was compared to the most common spa types found among human blood isolates. Little overlap in spa types was seen between isolates from the three animal reservoirs or between animals and humans. Most of the porcine isolates had the spa types t034 (CC398), t1333 (CC30) and t337 (CC9), while the bovine isolates mainly had spa types t518 (CC50), t524 (CC97) and t529 (CC151). None of these spa types are common among human blood isolates in Denmark. Surprisingly, almost all of the poultry isolates (96%) belonged to CC5 (spa types t002 and t306), which is also known to be commonly found among human blood isolates and subsequent pulsed-field gel electrophoresis (PFGE) analysis identified indistinguishable PFGE patterns among a poultry isolate and selected human isolates. In conclusion, strains of MSSA CC398 were commonly present in pigs but not present at all in the other reservoirs tested.

The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria

The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria were investigated in two large-scale municipal treatment plants during a period of six months. Total and relative numbers of resistant bacteria were determined in raw sewage, treated sewage and anaerobically digested sludge by bacteriological counts on media selective for coliforms (MacConkey agar) and Acinetobacter spp. (Baumann agar). In addition, the level of antimicrobial susceptibility was determined by the disc-diffusion method in 442 Acinetobacter isolates identified by colony hybridisation with a genus-specific DNA probe. Independent of the different antibiotics and media used, the total numbers of resistant bacteria in treated sewage were 10-1000 times lower than in raw sewage. Based on linear regression analysis of data on bacteriological counts, the prevalences of antimicrobial-resistant presumptive coliforms and Acinetobacter spp. in treated sewage and digested sludge were not significantly higher compared with raw sewage. On the contrary at one plant, statistically significant decreases were observed in the prevalence of ampicillin-resistant presumptive Acinetobacter spp. (p = 0.0188) following sewage treatment, and in the prevalence of either ampicillin-resistant presumptive Acinetobacter spp. (p = 0.0013) or ampicillin- and gentamicin-resistant presumptive coliforms (p = 0.0273 and p = 0.0186) following sludge treatment. The results obtained by bacteriological counts were confirmed by antimicrobial susceptibility testing of Acinetobacter isolates. Based on logistic regression analysis, isolates from treated sewage and digested sludge were generally not significantly more resistant compared with isolates from raw sewage. Based on these evidences, it was concluded that tertiary wastewater treatment did not result in a selection of antimicrobial resistant bacteria.

Antimicrobial Use Guidelines for Treatment of Urinary Tract Disease in Dogs and Cats: Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases

Urinary tract disease is a common reason for use (and likely misuse, improper use, and overuse) of antimicrobials in dogs and cats. There is a lack of comprehensive treatment guidelines such as those that are available for human medicine. Accordingly, guidelines for diagnosis and management of urinary tract infections were created by a Working Group of the International Society for Companion Animal Infectious Diseases. While objective data are currently limited, these guidelines provide information to assist in the diagnosis and management of upper and lower urinary tract infections in dogs and cats.

Transmission of IncN plasmids carrying blaCTX-M-1 between commensal Escherichia coli in pigs and farm workers.

ABSTRACT CTX-M-1-producing Escherichia coli were isolated from 56 pigs, three farm personnel, two manure samples, and two air samples from two Danish pig farms where an association between prophylactic ceftiofur use and the occurrence of cephalosporin resistance was previously demonstrated. Human, animal, and environmental strains displayed high genetic diversity but harbored indistinguishable or closely related IncN plasmids carrying blaCTX-M-1, indicating that IncN plasmids mediating cephalosporin resistance were transmitted between pigs and farm workers across multiple E. coli lineages.

Carriage of Methicillin-Resistant Staphylococcus pseudintermedius in Small Animal Veterinarians: Indirect Evidence of Zoonotic Transmission

Methicillin‐resistant Staphylococcus pseudintermedius (MRSP) is increasingly reported in small animals and cases of human infections have already been described despite its recent emergence in veterinary practice. We investigated the prevalence of MRSP and methicillin‐resistant Staphylococcus aureus (MRSA) among small animal dermatologists attending a national veterinary conference in Italy. Nasal swabs were obtained from 128 veterinarians, seven of which harboured MRSP (n = 5; 3.9%) or MRSA (n = 2; 1.6%). A follow‐up study of two carriers revealed that MRSP persisted for at least 1 month in the nasal cavity. Methicillin‐susceptible S. aureus (MSSA) was isolated from 32 (25%) conference participants, whereas methicillin‐susceptible S. pseudintermedius (MSSP) was not detected, suggesting that MRSP may have a particular ability to colonize humans compared to MSSP. All isolates were characterized by spa typing. Methicillin‐resistant isolates were further typed by antimicrobial susceptibility testing, SCCmec and multi‐locus sequence typing. Two lineages previously associated with pets were identified among the five MRSP isolates; the European epidemic clone ST71‐SCCmec II‐III and ST106‐SCCmec IV. One of the two MRSA isolates displayed a genotype (ST22‐ SCCmecIV) frequently reported in dogs and cats. MRSP isolates were resistant to more antimicrobial agents compared with MRSA isolates and displayed the typical multidrug resistance patterns of MRSP in pets. The 32 MSSA isolates belonged to 20 spa types and the most frequent types (t12, t15 and t166) were associated with common S. aureus lineages in humans (CC30 and CC45). Although low, the 3.9% MRSP carriage rate found among small animal dermatologists was surprising in consideration of the rare occurrence of S. pseudintermedius in humans, the lack of MSSP detection and the recent appearance of MRSP in Europe. As cases of human MRSP infection have been linked with pets, veterinarians should be aware of this zoonotic risk and proper preventative measures should be taken to avoid MRSP transmission from animal patients.

Tandem repeat sequence analysis of staphylococcal protein A (spa) gene in methicillin-resistant Staphylococcus pseudintermedius.

A putative staphylococcal protein A (spa) gene was discovered in the genome of Staphylococcus pseudintermedius and used for developing a species-specific spa typing protocol. Thirty-one clinical methicillin-resistant S. pseudintermedius (MRSP) isolates from dogs and cats in four countries were characterized by spa typing, pulsed-field gel electrophoresis (PFGE) and staphylococcal cassette chromosome (SCCmec) typing. The results indicated the occurrence of two MRSP clones that acquired distinct SCCmec elements in Europe (t02, PFGE type A, SCCmec type III,) and California (t06, PFGE type B, SCCmec type V). Sequence analysis of mecA revealed the occurrence of four alleles (mecA1 to mecA4), which correlated with the geographical origin of the isolates and enabled discrimination of two distinct subtypes within the European clone. The newly developed spa typing method appeared to be a promising tool for easy and rapid typing of MRSP, either alone or in combination with SCCmec and mecA typing for fine-structure epidemiological analysis.

Guide to Antimicrobial Use in Animals

Foreword: David Lloyd (Royal Veterinary College). Preface. Table of Contents. List of contributors. 1. Principles of Prudent and Rational Antimicrobial Use in Animals: Luca Guardabassi (University of Copenhagen) and Hilde Kruse (National Veterinary Institute Norway). 2. Human Health Risks Associated with Antimicrobial Use in Animals: Lars B. Jensen (Technical University of Denmark), Frederick J. Angulo (Centers for Disease Control and Prevention, USA), Kare Molbak (Statens Serum Institut) and Henrik C. Wegener (Technical University of Denmark). 3. Antimicrobial Resistance Risk Assessment: Emma Snary (Veterinary Laboratories Agency, UK) and Scott McEwen (University of Guelph). 4. Clinical Importance of Antimicrobial Drugs in Human Medicine: Peter Collignon (Australian National University), Patrice Courvalin (Institut Pasteur) and Awa Aidara-Kane (World Health Organization). 5. Geographical Differences in Market Availability, Regulation and Use of Antimicrobial Products: Angelo A. Valois (Australian Government Department of Agriculture Fisheries and Forestry), Yuuko S. Endoh (Ministry of Agriculture, Forestry and Fisheries (MAFF), Tokyo, Japan), Kornelia Grein (European Medicines Agency) and Linda Tollefson (US Food and Drug Administration). 6. Strategies to Minimize the Impact of Antimicrobial Treatment on the Selection of Resistant Bacteria: Peter Lees (Royal Veterinary College), Ove Svendsen (University of Copenhagen) and Camilla Wiuff (Health Protection Scotland). 7. Guidelines for Antimicrobial Use in Swine: David G. S. Burch (Octagon Services Ltd), C. Oliver Duran (Moss Veterinary Partners) and Frank M. Aarestrup (Technical University of Denmark). 8. Guidelines for Antimicrobial Use in Poultry: Ulrich Loehren (Lohmann & Co.), Antonia Ricci (Istituto Zooprofilattico Sperimentale delle Venezie) and Timothy S. Cummings (Mississippi State University). 9. Guidelines for Antimicrobial Use in Ruminants: Peter D. Constable (Purdue University), Satu Pyorala (University of Helsinki) and Geoffrey W. Smith (North Carolina State University). 10. Guidelines for Antimicrobial Use in Horses: J. Scott Weese (University of Guelph), Keith Edward Baptiste (University of Copenhagen), Viveca Baverud (National Veterinary Institute Sweden) and Pierre-Louis Toutain (Ecole Nationale Veterinaire). 11. Guidelines for Antimicrobial Use in Dogs and Cats: Luca Guardabassi (University of Copenhagen), G. Houser (University of Copenhagen), L. A. Frank (University of Tennessee) and M. G. Papich (North Carolina State University). 12. Guidelines for Antimicrobial Use in Aquaculture: Peter R. Smith (National University of Ireland, Galway), Alain Le Breton (Fish Consultant), Tor Einar Horsberg (Norwegian School of Veterinary Science) and Flavio Corsin (Ministry of Fisheries, Vietnam). Acknowledgements

Rapid PCR detection of Staphylococcus aureus clonal complex 398 by targeting the restriction-modification system carrying sau1-hsdS1.

ABSTRACT A PCR targeting sau1-hsdS1 was developed for rapid detection of Staphylococcus aureus clonal complex 398 (CC398). High sensitivity (100%) and specificity (100%) were shown by evaluating the test on a large strain collection (n = 1,307). We recommend this test for accurate, rapid, and inexpensive diagnosis of methicillin-resistant S. aureus (MRSA) CC398 in hospitals and on farms.

Occurrence of Campylobacter jejuni in Pets Living with Human Patients Infected with C. jejuni

ABSTRACT Campylobacter jejuni was recovered from four dogs (11%) and four cats (33%) living with Danish human patients infected with C. jejuni. Pulsed-field gel electrophoresis (PFGE) analysis revealed the occurrence of the same quinolone-resistant strain in a girl and her dog. C. jejuni isolates with closely related (>95% similarity) PFGE profiles occurred in humans and pets from different Danish counties.