Survey of antimicrobial susceptibility of bacterial pathogens isolated from dogs and cats with respiratory tract infections in Europe: ComPath results.

AIMS: To present antimicrobial susceptibilities for bacteria from dogs and cats with respiratory tract infection (RTI) across Europe in 2013-2014 and compare with data from 2008-2010. METHODS AND RESULTS: Minimal inhibitory concentrations were determined for 464 isolates following Clinical and Laboratory Standards Institute standards using antibiotics approved for RTI treatment. Where possible, susceptibility was calculated using predominantly human-derived breakpoints whilst some antibiotics had no breakpoints. The main pathogen from dogs was Staphylococcus pseudintermedius which was > 90% susceptible to fluoroquinolones and oxacillin (92·5%; six isolates confirmed mecA-positive) and 53·8, 80·0 and 88·8% susceptible to tetracycline, penicillin and trimethoprim/sulfamethoxazole. Streptococci, Escherichia coli, Bordetella bronchiseptica, Staphylococcus aureus and Pseudomonas aeruginosa were also present in dog RTI. Streptococci were fully susceptible to penicillin, ampicillin and pradofloxacin. None were enrofloxacin-resistant but 31·4% had intermediate susceptibility. The least active agent against streptococci was tetracycline (51·4% susceptible). For E. coli, 90·9% were amoxicillin/clavulanic acid-susceptible; susceptibility to other compounds ranged from 63·6 to 81·8%. There are no breakpoints for B. bronchiseptica and Ps. aeruginosa. For Staph. aureus, penicillin susceptibility was low (34·8%); for other compounds 87·0-100%. The main RTI pathogen from cats was Pasteurella multocida, where only pradofloxacin has breakpoints (100% susceptible). Susceptibility of coagulase-negative staphylococci ranged from 66·7% (penicillin) to 97·2% (pradofloxacin). Streptococci from cats were 100% susceptible to all antibiotics except enrofloxacin and tetracycline (both 65·2% susceptible). CONCLUSIONS: Overall, antimicrobial resistance was low to medium in RTI in dogs and cats, although susceptibility varied widely among pathogens studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Responsible use of antibiotics is crucial to maintain susceptibility and continued resistance monitoring is important to support this goal. These findings support the need for the setting of RTI-specific breakpoints for pathogens of dogs and cats.

Antimicrobial susceptibility monitoring of dermatological bacterial pathogens isolated from diseased dogs and cats across Europe (ComPath results).

AIMS: The ComPath project is a pan-European programme dedicated to the monitoring of antimicrobial susceptibility of pathogens from diseased dogs and cats using standardized methods and centralized minimum inhibitory concentration (MIC) determination. Here, the susceptibility of major pathogens is reported from antimicrobial nontreated animals with acute clinical signs of skin, wound or ear infections in 2008-2010. METHODS AND RESULTS: MICs were determined by agar dilution for commonly used antibiotics and interpreted using CLSI breakpoints, if available. Of the 1408 strains recovered, the main canine species was Staphylococcus pseudintermedius, followed by Pseudomonas and Streptococcus. In cats, Pasteurella multocida and Staph. pseudintermedius were most prevalent. For Staph. pseudintermedius, resistance was 18·4-25·2% for penicillin, clindamycin and chloramphenicol, but below 11% for ampicillin, amoxi/clav and fluoroquinolones. For Staphylococcus aureus, beta-lactam resistance was high (26·7-62·1%) but low (0·0-4·4%) for other antibiotics. 6·3% of Staph. pseudintermedius and 5·4% of Staph. aureus were confirmed mecA-positive. Gentamicin and fluoroquinolones exhibited moderate activity against Pseudomonas aeruginosa. For streptococci, resistance was absent/very low for penicillin, ampicillin, chloramphenicol and fluoroquinolones. For Escherichia coli, resistance was low to fluoroquinolones, chloramphenicol and gentamicin. No resistance was observed in Past. multocida. CONCLUSIONS: Overall, antimicrobial resistance was low in skin and soft tissue infections in dogs and cats. The results show the need for ongoing monitoring. SIGNIFICANCE AND IMPACT OF THE STUDY: The results are a reference baseline for future surveillance. The paucity of clinical breakpoints underlines the need to set breakpoints for relevant antibiotics.

Pharmacokinetic/pharmacodynamic evaluation of marbofloxacin in the treatment of Haemophilus parasuis and Actinobacillus pleuropneumoniae infections in nursery and fattener pigs using Monte Carlo simulations.

This study evaluated the theoretical clinical outcome of three marbofloxacin posology regimens in two groups of pigs (weaners and fatteners) for the treatment of Actinobacillus pleuropneumoniae (App) and Haemophilus parasuis (Hp) infection and the appearance of resistant bacteria due to the antibiotic treatment. The probability of target attainment (PTA) for pharmacokinetic/pharmacodynamics (PK/PD) ratios associated with clinical efficacy and with the appearance of antimicrobial resistance for fluoroquinolones at each minimum inhibitory concentration (MIC) or mutant prevention concentration (MPC) were calculated, respectively. The cumulative fraction of response (CFR) was calculated for the three posology regimens against App and they ranged from 91.12% to 96.37% in weaners and from 93% to 97.43% in fatteners, respectively. In the case of Hp, they ranged from 80.52% to 85.14% in weaners and from 82.01% to 88.49% in fatteners, respectively. Regarding the PTA of the PK/PD threshold associated with the appearance of antimicrobial resistance, results showed that marbofloxacin would prevent resistances in most of the animals up to the MPC value of 1 µg/mL.

StaphVar-DNA microarray analysis of accessory genome elements of community-acquired methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Approximately 75% of the genome of Staphylococcus aureus (the 'core' genome) is highly conserved between strains, whereas the remaining 25% (the 'accessory' genome) is composed of mobile genetic elements (MGEs), containing virulence and resistance genes. We developed a composite microarray focused on resistance and virulence genes located on the accessory or core-variable genome to characterize a collection of Belgian community-acquired methicillin-resistant S. aureus (CA-MRSA) strains. METHODS: Oligonucleotide probes targeting 403 genes encoding antimicrobial resistance (35%), virulence (28%) and adhesion (31%) factors were designed among eight S. aureus sequenced genomes. The StaphVar Array was validated by testing five of the strains used for the design and utilized to characterize 13 CA-MRSA strains representative of the multilocus sequence typing (MLST) sequence types circulating in Belgium. RESULTS: Analysis of the gene content of the five reference strains by the StaphVar Array matched 90% to 97% of the theoretical results. Analysis of CA-MRSA strains showed that 54.4% of the genes tested were strain-dependent. Strains presented specific exotoxin, enterotoxin, cytolysin and adhesin gene profiles by MLST lineage. One exception to these 'lineage-specific' profiles was the variable presence of the arginine catabolic mobile element (characteristic of the USA300 clone) within ST8 strains. CONCLUSIONS: The StaphVar Array enables the characterization of approximately 400 variable resistance and virulence determinants in S. aureus. CA-MRSA strains displayed extensive diversity in virulence and resistance profiles. The presence of the USA300 clone in Belgium was confirmed. Although mainly located on MGEs, associations of virulence genes were highly conserved within strains of the same MLST lineage.

Survey of susceptibility to marbofloxacin in bacteria isolated from diseased pigs in Europe.

A monitoring programme of marbofloxacin susceptibility of bacteria from Europe causing respiratory tract infection and meningitis in pigs has been active since 1994 and 2002, respectively. Monitoring digestive, metritis and urinary tract infection (UTI) in pigs has been active since 2005 and susceptibility results until 2013 are presented. Minimum inhibitory concentration (MIC) was determined by broth microdilution. For MIC interpretation, Vétoquinol-evaluated breakpoints were applied. For digestive pathogens, Escherichia coli and Salmonella species (1717 and 300 isolates, respectively) exhibited 7.5 per cent resistance in E coli and no resistance in Salmonella species. Similarly, E coli from metritis (369 isolates) had 7.0 per cent resistance to marbofloxacin. However, E coli from UTI (633 isolates) had higher resistance (10.4 per cent). For Streptococcus suis causing meningitis (585 isolates), marbofloxacin susceptibility was very high with only 0.5 per cent resistance and 0.4 per cent resistance was observed with S suis causing respiratory disease (729 isolates). Other respiratory pathogens were also highly susceptible to marbofloxacin with no resistance in Actinobacillus pleuropneumoniae (647 isolates) or Bordetella bronchiseptica (504 isolates), 0.1 per cent resistance in Pasteurella multocida (1373 isolates) and 1.4 per cent resistance in Haemophilus parasuis (145 isolates). There was no apparent change in marbofloxacin MIC over time for any bacterial pathogen based on MIC50/90 These data confirm previously published MIC results from porcine and other animal infections.

mcr-1 is borne by highly diverse Escherichia coli isolates since 2004 in food-producing animals in Europe.

OBJECTIVES: In November 2015, a plasmid-mediated colistin resistance, MCR-1, was described in animals, food and humans in China, and it was considered as a potential emerging threat to public health. Therefore, we screened for the mcr-1 gene a European collection of colistin-resistant Escherichia coli (n=218) and Salmonella spp. (n=74) isolated from diseased food-producing animals between 2004 and 2014 and characterized the mcr-1-positive clones. METHODS: Screening for mcr-1 gene was performed by PCR on isolates for which inhibition diameter was <15 mm around a 50 µg disk of colistin. Positive E. coli isolates were then characterized by phylogrouping, multilocus sequence typing and pulsed-field gel electrophoresis typing. Antibiotic susceptibility was determined by disk diffusion testing or by broth microdilution. RESULTS: Among the collection, 42 E. coli and three Salmonella spp. were positive for mcr-1, with continuous detection since 2004 mainly from bovine and swine digestive infections. Most of the mcr-1-positive strains were resistant to amoxicillin and cotrimoxazole but remained susceptible to cephalosporins, carbapenems and piperacillin/tazobactam. All but one isolate were resistant to colistin, with a minimum inhibitory concentration of >2 mg/L. Most of the mcr-1-positive E. coli belonged to the phylogroup A with two prevalent clonal complexes, CC10 and CC165, in which sequence type 10 and sequence type 100 were overrepresented and pulsed-field gel electrophoresis typing revealed a high diversity of pulsotypes. CONCLUSIONS: MCR-1 was detected yearly in European food-producing animal since 2004 with a high diversity of pulsotypes supporting the dissemination of mcr-1 via plasmids.

EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis.

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase.

A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.

Diversity of accessory genome of human and livestock-associated ST398 methicillin resistant Staphylococcus aureus strains.

BACKGROUND: Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) has documented the diversity of the genetic background of strains associated with healthcare (HA-MRSA), community (CA-MRSA) and livestock (LA-MRSA). The accessory and core-variable genomes of those strains however remain largely unknown. OBJECTIVE: To compare the genetic background and accessory and core variable gene content of ST398 LA-MRSA strains with those of HA-and CA-MRSA strains from the same region. METHODS: Representative strains of HA- (n=21), CA- (n=13) and ST398 LA-MRSA (n=18) were selected from Belgian National Reference Laboratory collections. The accessory and core-variable genomes of these strains were characterized by a DNA-microarray composed of oligonucleotide probes targeting ~400 resistance, adhesion and virulence associated genes. RESULTS: ST398 strains displayed very homogenous hybridization profiles irrespective of their host origin. This ST398 genomic profile was moderately related to that of certain human HA- or CA-lineages but distinctively lacked several virulence- and colonization-associated genes implicated in carriage in humans, such as proteases and adhesins. No enterotoxin gene was found among ST398 strains. Differences were observed in the mobile resistance gene content of ST398 strains, including antibiotic resistance determinants. CONCLUSION: LA-MRSA strains represent a homogenous lineage distinct from co-local HA- and CA-MRSA strains, especially in its accessory genome content characterized by a lack of human-associated virulence and adhesion determinants. The absence of detectable enterotoxin gene among ST398 LA-MRSA strains from a wide host range is reassuring regarding their foodborne pathogenic potential.

[Mechanism of adaptive resistance to aminoglycosides of Pseudomonas aeruginosa]. / Mécanisme de la résistance adaptative de Pseudomonas aeruginosa aux aminosides.

Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, << adaptive >> resistance to these antibiotics. In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in P. aeruginosa. Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1. Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration). In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance. Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.

[Two efflux systems expressed simultaneously in clinical Pseudomonas aeruginosa]. / Deux systèmes d'efflux exprimés simultanément chez des souches cliniques de Pseudomonas aeruginosa.

Active efflux systems MexAB-OprM and MexXY were found to be overexpressed simultaneously in 12 multiresistant clinical isolates of Pseudomonas aeruginosa. Nine of these strains (agrZ mutants) harbored mutations in gene mexZ, the product of which down-regulates expression of operon mexXY. Eight of the 12 strains exhibited mutations in genes known to control transcription of operon mexAB-oprM, such as mexR (four nalB mutants) or PA3721 (three nalC mutants). One strain was a nalB/nalC double mutant. For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the over-expression levels of genes mexA or mexX, and (iii) the resistance levels to effluxed antibiotics. Finally, three and four isolates overproduced MexXY (agrW mutants) or MexAB-OprM (nalD mutants), respectively, without any mutation in the known regulator genes. These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY.