Vincent Perreten

Antibiotic resistance spread in food

Nutritive and therapeutic treatment of farm animals with antibiotics, amounting to half of the worlds antibiotic output, has selected for resistant bacteria that may contaminate the food produced. Antibiotic-resistant enterococci and staphylococci from animals are found in food when they survive the production processes, as in raw cured sausages and raw milk cheeses. The broad host ranges of some plasmids and the action of transposons in many bacteria allow antibiotic-resistance genes to be communicated by conjugation between different species and genera,. A multi-antibiotic resistance plasmid from a lactococcus found in cheese provides a historical record of such events.

Microarray-Based Detection of 90 Antibiotic Resistance Genes of Gram-Positive Bacteria

ABSTRACT A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

A New Sulfonamide Resistance Gene (sul3) in Escherichia coli Is Widespread in the Pig Population of Switzerland

ABSTRACT A new gene, sul3, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized. Expression of the cloned sul3 gene conferred resistance to sulfamethoxazole on E. coli. Two copies of the insertion element IS15Δ/26 flanked the region containing sul3. The sul3 gene was detected in one-third of the sulfonamide-resistant pathogenic E. coli isolates from pigs in Switzerland.

Characterization of New Staphylococcal Cassette Chromosome mec (SCCmec) and Topoisomerase Genes in Fluoroquinolone- and Methicillin-Resistant Staphylococcus pseudintermedius

ABSTRACT Fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius isolates harbor two new staphylococcal cassette chromosome mec (SCCmec) elements that belong to class A, allotype 3 (SCCmec II-III), and to the new allotype 5 (SCCmec VII). Analysis of the complete nucleotide sequences of the topoisomerase loci gyrB/gyrA and grlB/grlA revealed mutations involved in fluoroquinolone resistance.

Presence of New mecA and mph(C) Variants Conferring Antibiotic Resistance in Staphylococcus spp. Isolated from the Skin of Horses before and after Clinic Admission

ABSTRACT Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genes mecA and mph(C), respectively. After 3 days, the percentage of Staphylococcus isolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment. Staphylococcus species displaying multiple resistance were found to harbor mainly genes conferring resistance to β-lactams (mecA and blaZ), aminoglycosides [str and aac(6′)-Ie-aph(2′)-Ia], and trimethoprim [dfr(A) and dfr(D)]. Additional genes conferring resistance to macrolides [mph(C), erm(C), and erm(B)], tetracycline [tet(K) and tet(M)], chloramphenicol [cat(pC221) and cat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.

Extended-spectrum cephalosporin-resistant gram-negative organisms in livestock: An emerging problem for human health?

Escherichia coli, Salmonella spp. and Acinetobacter spp. are important human pathogens. Serious infections due to these organisms are usually treated with extended-spectrum cephalosporins (ESCs). However, in the past two decades we have faced a rapid increasing of infections and colonization caused by ESC-resistant (ESC-R) isolates due to production of extended-spectrum-β-lactamases (ESBLs), plasmid-mediated AmpCs (pAmpCs) and/or carbapenemase enzymes. This situation limits drastically our therapeutic armamentarium and puts under peril the human health. Animals are considered as potential reservoirs of multidrug-resistant (MDR) Gram-negative organisms. The massive and indiscriminate use of antibiotics in veterinary medicine has contributed to the selection of ESC-R E. coli, ESC-R Salmonella spp. and, to less extent, MDR Acinetobacter spp. among animals, food, and environment. This complex scenario is responsible for the expansion of these MDR organisms which may have life-threatening clinical significance. Nowadays, the prevalence of food-producing animals carrying ESC-R E. coli and ESC-R Salmonella (especially those producing CTX-M-type ESBLs and the CMY-2 pAmpC) has reached worryingly high values. More recently, the appearance of carbapenem-resistant isolates (i.e., VIM-1-producing Enterobacteriaceae and NDM-1 or OXA-23-producing Acinetobacter spp.) in livestock has even drawn greater concerns. In this review, we describe the aspects related to the spread of the above MDR organisms among pigs, cattle, and poultry, focusing on epidemiology, molecular mechanisms of resistance, impact of antibiotic use, and strategies to contain the overall problem. The link and the impact of ESC-R organisms of livestock origin for the human scenario are also discussed.

Human infection associated with methicillin-resistant Staphylococcus pseudintermedius ST71.

Sir, Staphylococcus pseudintermedius is one of the most common pathogens isolated from skin and post-operative infections in dogs and cats and can also occasionally cause infections in humans. People working or living with animals are more likely to be colonized with S. pseudintermedius in their nasal cavities and human infection acquired from dogs has been recently reported. In recent years, there have been increasing numbers of infections in dogs and cats caused by methicillin-resistant S. pseudintermedius (MRSP) and a predominant MRSP clone is disseminating in dogs and cats throughout Europe. This MRSP clone belongs to the multilocus sequence type (MLST) ST71, spa type t02 and SmaI PFGE group J (ST71-t02-J) and contains the staphylococcal cassette chromosome element SCCmec II–III. It displays resistance to many classes of antibiotics and represents a challenge for therapy. Here, we report, to our knowledge, the first case of a human infection caused by MRSP ST71 emphasizing its zoonotic potential and therapeutic challenge. An adult patient presented to the doctor in 2009 with headache, watery eyes and a small oedema over the right eye. The patient had a history of recurrent rhinosinusitis and three surgical interventions had been performed over the previous 6 years, the last one in 2005. CT illustrated an obliteration of the right sinus frontalis with an expansive mucocele. Yet another surgical intervention was absolutely essential and post-operative treatment for 3 weeks with 500 mg of ciprofloxacin twice per day and 300 mg of clindamycin three times per day was given. Five weeks post-operatively, a purulent infection appeared. After 10 days of treatment with 875 mg of amoxicillin/125 mg of clavulanic acid twice daily and 300 mg of clindamycin three times daily and no improvement, a sample was sent for bacteriological analysis. The laboratory identified a multidrug-resistant Staphylococcus belonging to the Staphylococcus intermedius group (SIG) using the ID 32 STAPH system (bioMerieux, Marcy l’Etoile, France) and disc diffusion susceptibility testing (Oxoid Ltd, Basingstoke, UK). Based on these results, a local treatment with fusidic acid gauze and application of 2% mupirocin ointment four times a day for 8 days followed by a topical glucocorticoid/antibiotic combination therapy (0.5 mg/g fluocinonide, 2.5 mg/g neomycin, 0.25 mg/g gramicidin and 100000 IU/g nystatin) packing for 4 days improved the local condition. Six weeks after the purulent infection, the wound had healed with scarring and no bacterium was detectable in a repeat smear. Local follow-up treatment with steroids and routine check-ups confirmed the absence of residual infection. The multidrug-resistant Staphylococcus isolate was further identified as MRSP ST71-t02-J containing SCCmec II–III using MLST typing, spa typing, PFGE and SCCmec typing methods described previously. MICs of antibiotics were determined in Mueller–Hinton broth using custom Sensititre susceptibility NLV73 plates (Trek Diagnostics System, East Grinstead, UK) except for rifampicin, trimethoprim, kanamycin and fusidic acid, which were tested using home-made microbroth dilution plates. Antibiotic resistance genes were detected using a microarray. Mutations in topoisomerase genes gyrA and grlA were identified by sequencing of PCR products (Table 1). The isolate was susceptible to the antibiotics amikacin, chloramphenicol, fusidic acid, linezolid, nitrofurantoin, the combination quinupristin/dalfopristin, rifampicin and vancomycin (Table 1). The patient owned a male dog that needed home care because of various clinical problems such as diabetes, recurrent warts and abdominal tumour. The dog had consultations in various clinics and underwent several antibiotic treatments (details of substances could not be ascertained). The dog was euthanized before samples could be taken to determine whether it was a carrier of the strain. The patient also owned horses and cats; all of them were in good health without clinical signs. Even if the ultimate source of the MRSP infection could not be identified, the patient was infected with the same MRSP clone (ST71-t02-J with SCCmec II–III) that has been disseminating in dogs over Europe in recent years. A case similar to the one reported here has been described in the USA, where a woman developed sinusitis caused by a methicillinresistant SIG whose origin could be attributed to her pet dog. In that case, vancomycin and linezolid were used for treatment. The increasing number of dogs carrying MRSP constitutes a risk for pet owners to become colonized with MRSP, and MRSP infections in humans may also increase in the near future. It is therefore important to recognize MRSP as a zoonotic pathogen and have antibiotics available for the treatment of MRSP infections in humans. Therefore, antibiotics such as mupirocin, linezolid, quinupristin/dalfopristin, rifampicin and vancomycin that are used for decolonization or as ‘last resort antibiotics’ against methicillin-resistant staphylococci in humans should not be used for treatment in animals.

Guidelines for Reporting Novel mecA Gene Homologues

Methicillin-resistant staphylococci are disseminated all over the world and are frequent causes of health care- and community-associated infections. Methicillin-resistant strains typically carry the acquired mecA gene that encodes a low-affinity penicillin-binding protein (PBP), designated PBP2a or

Genetic Diversity and Ecological Success of Staphylococcus aureus Strains Colonizing Humans

ABSTRACT The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Antibiotic Resistance Genes in Coagulase-negative Staphylococci Isolated from Food

Coagulase-negative staphylococci were isolated from different raw milk cheeses and raw meat products and screened for their antibiotic resistances. They were identified as Staphylococcus xylosus, S. lentus, S. caprae, S. epidemidis and S. haemolyticus. The most frequent resistances found were those to chloramphenicol, tetracycline, erythromycin and lincomycin. They have been characterized on the molecular level. The chloramphenicol resistance genes were localized in several S. xylosus and S. caprae on plasmids with sizes ranging from 3.8-kb to 4.3-kb and were identified as chloramphenicol acetyltransferase (cat). All the tetracycline resistant strains were identified as S. xylosus and harboured a 4.4-kb plasmid carrying the tetracycline efflux resistance gene (tetK). The two erythromycin/lincomycin resistant S. caprae and S. epidermidis strains did not hybridize with the MLSB resistance genes ermAM, ermA, ermB and ermC. Three erythromycin resistant Staphylococcus sp. strains harboured an erythromycin efflux resistance gene (msr) localized twice on a 18-kb plasmid and once on the chromosome. A S. haemolyticus strain showing resistance to both lincomycin and clindamycin harboured a linA gene-carrying 2.2-kb plasmid. Further resistances to gentamicin, penicillin and kanamycin were less frequently observed and yet not characterized on a molecular level.