Marianne Sunde

Does the Wide Use of Quaternary Ammonium Compounds Enhance the Selection and Spread of Antimicrobial Resistance and Thus Threaten Our Health

Quaternary ammonium compounds (QACs) are widely used biocides that possess antimicrobial effect against a broad range of microorganisms. These compounds are used for numerous industrial purposes, water treatment, antifungal treatment in horticulture, as well as in pharmaceutical and everyday consumer products as preserving agents, foam boosters, and detergents. Resistance toward QACs is widespread among a diverse range of microorganisms and is facilitated by several mechanisms such as modifications in the membrane composition, expression of stress response and repair systems, or expression of efflux pump genes. Development of resistance in both pathogenic and nonpathogenic bacteria has been related to application in human medicine and the food industry. QACs in cosmetic products will inevitably come into intimate contact with the skin or mucosal linings in the mouth and thus are likely to add to the selection pressure toward more QAC-resistant microorganisms among the skin or mouth flora. There is increasing evidence of coresistance and cross-resistance between QACs and a range of other clinically important antibiotics and disinfectants. Use of QACs may have driven the fixation and spread of certain resistance cassette collectors (class 1 integrons), currently responsible for a major part of antimicrobial resistance in gram-negative bacteria. More indiscriminate use of QACs such as in cosmetic products may drive the selection of further new genetic elements that will aid in the persistence and spread of antimicrobial resistance and thus in limiting our treatment options for microbial infections.

Widespread Distribution of Disinfectant Resistance Genes among Staphylococci of Bovine and Caprine Origin in Norway

ABSTRACT We demonstrate here a widespread distribution of genes mediating efflux-based resistance to quaternary ammonium compounds (QACs) in staphylococci from unpasteurized milk from 127 dairy cattle herds and 70 dairy goat herds. QAC resistance genes were identified in 21% of the cattle herds (qacA/B, smr, qacG, and qacJ) and in 10% of the goat herds (qacA/B and smr). Further examination of 42 QAC-resistant bovine and caprine isolates revealed the following genes: qacA/B (12 isolates) was present in four different species of coagulase-negative staphylococci (CoNS), smr (27 isolates) was detected in eight different CoNS species and in Staphylococcus aureus on a previously reported plasmid (pNVH99), qacG (two isolates) was detected on two plasmids (pST94-like) in Staphylococcus cohnii and Staphylococcus warneri, and qacJ (two isolates) was found in Staphylococcus hominis and Staphylococcus delphini on a plasmid (pNVH01) previously found in equine staphylococci. Isolation of indistinguishable pulsed-field gel electrophoresis (PFGE) CoNS types from tank milk and mammary quarter milk samples in a dairy cattle herd suggested that these QAC-resistant staphylococci were of intramammary origin. Indistinguishable or closely related PFGE types of bovine QAC-resistant CoNS were observed in different herds. One particular bovine S. warneri PFGE type was isolated repeatedly from samples collected during a 30-month period in a herd, showing long-term persistence. In conclusion, it seems that the widespread distribution of staphylococci carrying QAC resistance genes in Norwegian dairy cattle and goat herds is the result of both the intra- and interspecies spread of QAC resistance plasmids and the clonal spread of QAC-resistant strains.

Novel Plasmid-Borne Gene qacJ Mediates Resistance to Quaternary Ammonium Compounds in Equine Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus intermedius

ABSTRACT We identified a novel plasmid-borne gene (designated qacJ) encoding resistance to quaternary ammonium compounds (QACs) in three staphylococcal species associated with chronic infections in four horses. qacJ was located on a 2,650-bp plasmid (designated pNVH01), a new member of the pC194 family of rolling-circle replication plasmids. The 107-amino-acid protein, QacJ, showed similarities to known proteins of the small multidrug resistance family: Smr/QacC (72.5%), QacG (82.6%), and QacH (73.4%). The benzalkonium chloride MIC for a qacJ-containing recombinant was higher than those for otherwise isogenic recombinants expressing Smr, QacG, or QacH. Molecular epidemiological analyses by pulsed-field gel electrophoresis suggested both the clonal spread of a qacJ-harboring Staphylococcus aureus strain and the horizontal transfer of pNVH01 within and between different equine staphylococcal species. The presence of pNVH01 of identical nucleotide sequence in different staphylococcal species suggests that recent transfer has occurred. In three of the horses, a skin preparation containing cetyltrimethylammonium bromide had been used extensively for several years; this might explain the selection of staphylococci harboring the novel QAC resistance gene.

Plasmid-Borne smr Gene Causes Resistance to Quaternary Ammonium Compounds in Bovine Staphylococcus aureus

ABSTRACT Resistance to quaternary ammonium compounds (QAC) in staphylococci is common in hospital environments and has been described in the food industry. Little is known about staphylococcal QAC resistance associated with animal disease, although such disinfectants are widely used in veterinary medicine. In order to investigate the occurrence of QAC resistance in staphylococci isolated from QAC-exposed animals, 32 penicillin- and tetracycline-resistant and 23 penicillin- and tetracycline-susceptible Staphylococcus aureus isolates collected from milk from cows with mastitis during a 4-year period were selected for QAC susceptibility studies and genetic characterization. The isolates originated from four different herds that used a common pasture with a joint milking parlor in the summer. During the pasture season, a teat cream containing the QAC cetyltrimethylammonium bromide had been used daily for more than 10 years for mastitis control. Three of the penicillin- and tetracycline-resistant isolates, which were recovered from three different cows during a 20-month period, were resistant to QAC. Plasmid analysis, PCR, and DNA sequencing revealed a novel plasmid of 2,239 bp containing the smr gene. The plasmid, designated pNVH99, has similarities to small,smr-containing staphylococcal plasmids previously found in human and food isolates. pNVH99 is a new member of the pC194 family of rolling-circle replication plasmids. The three QAC-resistant isolates, as well as 28 of the 29 remaining penicillin- and tetracycline-resistant isolates, were indistinguishable by pulsed-field gel electrophoresis. The study indicates that the occurrence and spread of QAC-resistant S. aureus among dairy cows may be a problem that needs further investigation.

Organization of the Antiseptic Resistance Gene qacA and Tn552-Related β-Lactamase Genes in Multidrug- Resistant Staphylococcus haemolyticus Strains of Animal and Human Origins

ABSTRACT A part (12 kb) of a plasmid containing the β-lactamase genes of Tn552, the disinfectant resistance gene qacA, and flanking DNA has been cloned from a Staphylococcus haemolyticus isolate and sequenced. This region was used to map the corresponding regions in six other multiresistant S. haemolyticus isolates of human and animal origin. The organizations of the genetic structures were almost identical in all isolates studied. The β-lactamase and qacA genes from S. haemolyticus have >99.9% identities at the nucleotide level with the same genes from S. aureus, demonstrating that various staphylococcal species able to colonize animal and human hosts can exchange the genetic elements involved in resistance to antibiotics and disinfectants. The use of antibiotics and disinfectants in veterinary practice and animal husbandry may also contribute to the selection and maintenance of resistance factors among the staphylococcal species. Different parts of the 12-kb section analyzed had high degrees of nucleotide identity with regions from several other different Staphylococcus aureus plasmids. This suggests the contribution of interplasmid recombination in the evolutionary makeup of this 12-kb section involving plasmids that can intermingle between various staphylococcal species. The lateral spread of resistance genes between various staphylococcal species is probably facilitated by the generation of large multiresistance plasmids and the subsequent interspecies exchange of them.

Self-Transmissible Multidrug Resistance Plasmids in Escherichia coli of the Normal Intestinal Flora of Healthy Swine

The resistance genes and their surroundings on three self-transmissible plasmids found in Escherichia coli of the enteric normal flora of healthy pigs have been characterized. The resistance elements found are similar to those commonly found in clinical isolates, like the transposon Tn1721 including the Tet A tetracycline resistance determinant, Tn10 with the Tet B determinant, Tn21 including a class 1 integron with the aadA1a cassette inserted, sulII encoding sulfonamide resistance, and the strA-strB genes responsible for streptomycin resistance. The plasmids were able to mobilize into various recipients, including swine pathogens, zoonotic bacteria, and commensals when conjugation experiments were carried out. Transfer of plasmids did not require optimal conditions concerning nutrition and temperature as plasmids were transferred in 0.9% saline at room temperature, suggesting that in vivo transfer might be possible. This study shows that transferable resistance elements appearing in normal flora bacteria from animals are similar to those commonly found in clinical isolates of human origin. The results indicate a probable communication between pathogens and the normal flora with respect to exchange of resistance factors.

Methicillin-resistant Staphylococcus aureus with the novel mecC gene variant isolated from a cat suffering from chronic conjunctivitis

Sir, Methicillin-resistant Staphylococcus aureus (MRSA) is of growing concern in public and animal health and it is important to ensure efficient and reliable methods for MRSA identification. Standard confirmatory tests include PCR for detection of the mecA gene and protein agglutination for identification of the penicillin-binding protein 2a (PBP2a). In 2011, a novel mecA homologue designated mecC (or mecALGA251) was described in S. aureus. This MRSA variant will not be detected with the usual mecA PCR approaches or with the PBP2a agglutination tests, representing a challenge to MRSA confirmation in diagnostic laboratories. Searching for mecC has recently been performed in several countries. Investigated isolates have either been part of historical collections or originated from current diagnostic submissions. The chosen candidates for mecC screening have typically been isolates with a phenotype corresponding to MRSA but lacking mecA, and/or isolates belonging to clonal lineages already associated with mecC carriage. So far the occurrence of the mecC gene has been confirmed in isolates from the typical hosts, humans and/or cattle, in several countries in Northern Europe: the UK, Denmark, Ireland, Germany, France, Sweden and Norway. – 8 Only two studies examining isolates from hosts other than humans and cattle are known; one of these studies investigated a historical isolate collection and mecC was found in S. aureus from a dog, a seal and a chaffinch (all from the UK), from a rabbit and rats from Belgium, and from sheep in Denmark. The other study, recently published by Walther et al., found mecC-positive MRSA from two dogs, seven cats and a guinea pig. The MRSA isolates investigated originated from samples submitted to a laboratory in Germany during the period 2008–11. Based on current knowledge, mecC seems to have a rather wide geographical and host distribution, but occurs at low frequencies. The Norwegian Veterinary Institute has recently detected mecC in an MRSA isolate from a submission to our diagnostic bacteriological service unit in Bergen. The isolate originated from a cat and represents the first finding from current diagnostic activity performed on samples from companion animals. The mecC-positive MRSA (designated 2012-50-2037) was isolated from an eye swab from a 5-year-old house cat with chronic conjunctivitis and stomatitis. The cat had tested negative for all relevant viruses and Chlamydophila felis. Initial treatment with systemic clavulanate-potentiated amoxicillinand topical fusidicacid was ineffective, but improvement was seen after a change of treatment to systemic enrofloxacin and topical dexamethasone, neomycin and polymyxin B. The cat is currently living in a household with two adults, two children and another cat. The MRSA carrier status of the family members and the other cat is not known. The isolate was routinely tested for susceptibility to antimicrobial agents following the disc diffusion method and breakpoints described by EUCAST (www.eucast.org, 7 November 2012, date last accessed). The isolate showed resistance to b-lactams only. MICs were subsequently determined by the use of a broth dilution method (Trek Diagnostics). The following antimicrobial agents were tested: penicillin, cefoxitin, ciprofloxacin, erythromycin, clindamycin, gentamicin, tetracycline, linezolid, fusidic acid, rifampicin, chloramphenicol, trimethoprim, vancomycin, quinupristin/dalfopristin and mupirocin. The isolate exhibited resistance to the b-lactams only, with a cefoxitin MIC of 16 mg/L. The MIC of oxacillin was 2 mg/L, determined by Etest (bioMérieux). The results were interpreted according to EUCAST. S. aureus ATCC 29213 was included as quality control. DNAwas prepared by the boil lysis method and subsequentlysubjected to PCR for detection of mecA, mecC, nuc and 16S rDNA with primers previously described. In addition to a negative control, positive control strains included S. aureus CCUG 29213 (nuc+), S. aureus CCUG 35603 (nuc+, mecA+), Staphylococcus pseudintermedius CCUG 49543 (nuc, mecA) and S. aureus SVA-AB-773 (nuc+, mecC+). The PCR results confirmed MRSA with mecC. The sequence of the mecC amplicon was determined and showed 100% identity with the previously determined mecC sequence. The mecC gene was probably located on a type XI SCCmec element as PCRs with primers for the mecI, mecR, ccrA, ccrB and blaZ genes related to SCCmecXI produced amplicons of correct sizes. The spa typing showed that the isolate belonged to spa type t6902. This spa type has only one other recording in the Ridom spaserver (http://spaserver.ridom.de/, 7 November 2012, date

Methicillin-Resistant Staphylococcus aureus CC398 in Humans and Pigs in Norway: A “One Health” Perspective on Introduction and Transmission

This study provides strong, novel evidence that humans may introduce methicillin-resistant Staphylococcus aureus CC398 into closed pig populations; it also demonstrates that stringent control and eradication measures were effective and prevented dissemination from pig farms to the general human population.

Emergence of AmpC-producing Escherichia coli in the broiler production chain in a country with a low antimicrobial usage profile

The aim of this study was to estimate the prevalence of cephalosporin-resistant Escherichia coli at the different levels of the Norwegian broiler production pyramid and identify the mechanisms responsible for the resistance phenotype. Samples from all levels of the broiler production pyramid and retail chicken meat (fillets) were included (n=649). The occurrence of cephalosporin-resistant E. coli at the different production levels ranged from 8 to 43%. All these isolates had an AmpC-phenotype, and the majority carried the blaCMY-2 gene. In addition, a few isolates with up-regulated chromosomal ampC were identified. The results show that Norway has a relatively high prevalence of cephalosporin-resistant E. coli in the broiler production chain in spite of a very low consumption of antimicrobial agents. Cephalosporins have not been used in the Norwegian broiler production, and it has been hypothesised that import of breeding animals and hatching eggs may be the source of these resistant bacteria. We demonstrate that these bacteria are disseminated in the production pyramid despite the lack of selection pressure from antimicrobial agents.

Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks

ABSTRACT A national survey of Escherichia coli O26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, and E. coli O26 was detected in 17.9% of the flocks. One hundred forty-two E. coli O26 isolates were examined for flagellar antigens (H typing) and four virulence genes, including stx and eae, to identify possible Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC). Most isolates (129 out of 142) were identified as E. coli O26:H11. They possessed eae and may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between the E. coli O26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all the E. coli O26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing on E. coli O26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.