Comparison of antibiotic resistance of udder pathogens in dairy cows kept on organic and on conventional farms

There has been a rapid rise in the emergence of multi-drug-resistant pathogens in the past 10 to 15 yr and some bacteria are now resistant to most antimicrobial agents. Antibiotic use is very restricted on Swiss organic dairy farms, and a purely prophylactic use, such as for dry cow mastitis prevention, is forbidden. A low prevalence of antibiotic resistance in organic farms can be expected compared with conventional farms because the bacteria are infrequently or not exposed to antibiotics. The occurrence of antibiotic resistance was compared between mastitis pathogens (Staphylococcus aureus, nonaureus staphylococci, Streptococcus dysgalactiae, Streptococcus uberis) from farms with organic and conventional dairy production. Clear differences in the percentage of antibiotic resistance were mainly species-related, but did not differ significantly between isolates from cows kept on organic and conventional farms, except for Streptococcus uberis, which exhibited significantly more single resistances (compared with no resistance) when isolated from cows kept on organic farms (6/10 isolates) than on conventional farms (0/5 isolates). Different percentages were found (albeit not statistically significant) in resistance to ceftiofur, erythromycin, clindamycin, enrofloxacin, chloramphenicol, penicillin, oxacillin, gentamicin, tetracycline, and quinupristin-dalfopristin, but, importantly, none of the strains was resistant to amoxicillin-clavulanic acid or vancomycin. Multidrug resistance was rarely encountered. The frequency of antibiotic resistance in organic farms, in which the use of antibiotics must be very restricted, was not different from conventional farms, and was contrary to expectation. The antibiotic resistance status needs to be monitored in organic farms as well as conventional farms and production factors related to the absence of reduced antibiotic resistance in organic farms need to be evaluated.

Role for tandem duplication and lon protease in AcrAB-TolC- dependent multiple antibiotic resistance (Mar) in an Escherichia coli mutant without mutations in marRAB or acrRAB

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.

[Antibiotic resistance: infections of the upper respiratory tract and bronchi. When are antibiotics necessary?]

Antimicrobial resistance among respiratory tract pathogens has become an increasing problem worldwide during the last 10-20 years. The wide use of antimicrobial agents in ambulatory practice has contributed to the emergence and spread of antibiotic-resistant bacteria in the community, namely Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. The pneumococcus has developed resistance to most antibiotics used for its treatment. Classes with important resistance problems include the beta-lactams, the macrolides, the lincosamides, trimethoprim-sulfamethoxazole, and the tetracyclines. Unfortunately, resistance to more than one class of antibiotics is common. In Haemophilus influenzae and Moraxella catarrhalis, resistance to beta-lactam antibiotics is the main concern currently. It is important to know the local resistance pattern of the most common respiratory tract pathogens in order to make reasonable recommendations for an empirical therapy for respiratory tract infection, when antibiotic therapy is indeed indicated.

Antibiotic resistance and genetic diversity in Staphylococcus aureus from slaughter pigs in Switzerland

Nasal carriage of Staphylococcus aureus was evaluated in pigs at slaughterhouse. The nasal cavities of 304 pigs from 54 herds were screened. Eighty-nine percent of the farms harbored pigs that were colonized with S. aureus. Among them, no MRSA were found, indicating a low prevalence. However, pigs were found to harbor S. aureus, which displayed resistance to penicillin (blaZ) (62.5%), tetracycline [tet(M)] (33.3%), streptomycin (strpS194) (27%), clindamycin [erm(B)] (4.1%), erythromycin [erm(B)] (4.1%), kanamycin (4.1%), chloramphenicol (catpC194) (2%) and gentamicin [aac(6')-Ie-aph(2')-Ia] (2%). The S. aureus isolates mainly belong to Ridom spa type t034 (31.3%), t208 (14.6%) and t899 (12.5%). These pig-associated spa types have not yet been detected in hospitalized human patients in Switzerland. Surveillance programs are now necessary at both inland and import levels to rapidly detect and suppress the emergence of MRSA in pigs in Switzerland.

Genotypes and antibiotic resistance of Campylobacter coli in fattening pigs

Campylobacter coli is a food-borne zoonotic pathogen causing human gastroenteritis worldwide. The organism is a commensal in the intestine of many food production animals including fattening pigs. The role of the pig as a potential reservoir for C. coli affecting human either directly or via poultry has hardly been investigated and genetic characterization of porcine strains is needed to address this question. For this aim multilocus sequence typing (MLST) and flaB typing was applied to 256 C. coli isolates from faeces of fattening pig collected during 2009 at different slaughterhouses in Switzerland. In addition genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. Of the 67 sequence types (STs) obtained by MLST, 37 were found for the first time. flaB typing revealed 46 different types with 14 of them being novel and was useful to further differentiate strains with an identical ST. Quinolone resistance was detected in 33.6% and macrolide resistance was found in 10.6% of isolates. Comparison with 99 C. coli pig isolates from 2001 revealed a significant decrease in antibiotic resistance towards both groups of antibiotics and there was high overlap between genotypes of 2001 and 2009. Little overlap of porcine genotypes was found with 97 C. coli isolates from poultry collected 2008, however, macrolide resistance was significantly higher in pig isolates. In conclusion, C. coli from Swiss pig are heterogeneous containing many novel STs, findings that could reflect the partitioned Swiss pig production with almost no international breed exchange. The antibiotic resistance echoes the use of corresponding drugs in the Swiss livestock production and indicates the efficacy of restrictive application of antibiotics in order to reduce resistances.

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

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.

Genotypes and antibiotic resistance of canine Campylobacter jejuni isolates

Campylobacter jejuni is the most important cause of bacterial gastroenteritis in humans. It is a commensal in many wild and domestic animals, including dogs. Whereas genotypes of human and chicken C. jejuni isolates have been described in some detail, only little information on canine C. jejuni genotypes is available. To gain more information on genotypes of canine C. jejuni and their zoonotic potential, isolates from routine diagnostics of diarrheic dogs as well as isolates of a prevalence study in non-diarrheic dogs were analyzed. Prevalence of thermophilic Campylobacter among non-diarrheic dogs was 6.3% for C. jejuni, 5.9% for Campylobacter upsaliensis and 0.7% for Campylobacter coli. The C. jejuni isolates were genotyped by multi locus sequence typing (MLST) and flaB typing. Resistance to macrolides and quinolones was genetically determined in parallel. Within the 134 genotyped C. jejuni isolates 57 different sequence types (ST) were found. Five STs were previously unrecognized. The most common STs were ST-48 (11.2%), ST-45 (10.5%) and ST-21 (6.0%). Whereas no macrolide resistance was found, 28 isolates (20.9%) were resistant to quinolones. ST-45 was significantly more prevalent in diarrheic than in non-diarrheic dogs. Within the common time frame of isolation 94% of the canine isolates had a ST that was also found in human clinical isolates. In conclusion, prevalence of C. jejuni in Swiss dogs is low but there is a large genetic overlap between dog and human isolates. Given the close contact between human and dogs, the latter should not be ignored as a potential source of human campylobacteriosis.

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Analysis of clonality and antibiotic resistance among early clinical isolates of Enterococcus faecium in the United States.

BACKGROUND: The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments. METHODS: Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994, we determined the multilocus sequence type; the presence of 16 putative virulence genes (hyl(Efm), esp(Efm), and fms genes); resistance to ampicillin (AMP) and vancomycin (VAN); and high-level resistance to gentamicin and streptomycin. RESULTS: Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the United States. The earliest CC17 isolates were part of an outbreak that occurred in 1982 in Richmond, Virginia. The characteristics of CC17 isolates included increases in resistance to AMP, the presence of hyl(Efm) and esp(Efm), emergence of resistance to VAN, and the presence of at least 13 of 14 fms genes. Eight of 41 of the early isolates with resistance to AMP, however, were not in CC17. CONCLUSIONS: Although not all early US AMP isolates were clonally related, E. faecium CC17 isolates have been circulating in the United States since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment.

Cotransfer of antibiotic resistance genes and a hylEfm-containing virulence plasmid in Enterococcus faecium.

The hyl(Efm) gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hyl(Efm)-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hyl(Efm) gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hyl(Efm), whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hyl(Efm)-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hyl(Efm) plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

Eluding antibiotic resistance: capitalizing on antimicrobial peptides interaction with the lipid bilayer

It is widely accepted that the emergence of drug-resistant pathogens is the result of the overuse and misuse of antibiotics. Infectious Disease Society of America, Center for Disease Control and World Health Organization continue to view, with concern, the lack of antibiotics in development, especially those against Gram-negative bacteria. Antimicrobial peptides (AMPs) have been proposed as an alternative to antibiotics due to their selective activity against microbes and minor ability to induce resistance. For example, the Food and Drug Administration approved Daptomycin (DAP) in 2003 for treatment of severe skin infections caused by susceptible Gram-positive organisms. Currently, there are 12 to 15 examples of modified natural and synthetic AMPs in clinical development. But most of these agents are against Gram-positive bacteria. Therefore, there is unmet medical need for antimicrobials used to treat infections caused by Gram-negative bacteria. In this study, we show that a pro-apoptotic peptide predominantly used in cancer therapy, (KLAKLAK)2, is an effective antimicrobial against Gram-negative laboratory strains and clinical isolates. Despite the therapeutic promise, AMPs development is hindered by their susceptibility to proteolysis. Here, we demonstrate that an all-D enantiomer of (KLAKLAK)2, resistant to proteolysis, retains its activity against Gram-negative pathogens. In addition, we have elucidated the specific site and mechanism of action of D(KLAKLAK)2 through a repertoire of whole-cell and membrane-model assays. Although it is considered that development of resistance does not represent an obstacle for AMPs clinical development, strains with decreased susceptibility to these compounds have been reported. Staphylococci resistance to DAP was observed soon after its approval for use and has been linked to alterations of the cell wall (CW) and cellular membrane (CM) properties. Immediately following staphylococcal resistance, Enterococci resistance to DAP was seen, yet the mechanism of resistance in enterococci remains unknown. Our findings demonstrate that, similar to S. aureus, development of DAP-resistance in a vancomycin-resistant E. faecalis isolate is associated with alterations of the CW and properties of the CM. However, the genes linked to these changes in enterococci appear to be different from those described in S. aureus.

Antibiotic resistance in Lactococcus species from bovine milk: presence of a mutated multidrug transporter mdt(A) gene in susceptible Lactococcus garvieae strains

A total of 72 Lactococcus strains (41 Lactococcus lactis and 31 Lactococcus garvieae) isolated from bovine milk were tested for susceptibility to 17 antibiotics and screened for the presence of antibiotic resistance genes using a microarray. Resistance to tetracycline, clindamycin, erythromycin, streptomycin, nitrofurantoin were found. The tetracycline-resistant L. garvieae and L. lactis harbored tet(M) and tet(S). L. lactis that were resistant to clindamycin were also resistant to erythromycin and possessed the erm(B) gene. The multidrug transporter mdt(A), originally described in L. lactis, was detected for the first time in L. garvieae and does not confer decreased susceptibility to erythromycin nor tetracycline in this species. Mdt(A) of L. garvieae contains one mutation in each antiporter motif C, which is known to play an essential role in drug efflux antiporters. This suggests that the mutations found in the C-motifs of Mdt(A) from L. garvieae may be responsible for susceptibility. The study revealed the presence of antibiotic resistance genes in non-pathogenic and pathogenic lactococci from bovine milk, including a mutated multidrug transporter in L. garvieae.

Mdt(A), a new efflux protein conferring multiple antibiotic resistance in Lactococcus lactis and Escherichia coli

The mdt(A) gene, previously designated mef214, from Lactococcus lactis subsp. lactis plasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lincosamides, streptogramins, and tetracyclines in L. lactis and Escherichia coli, but not in Enterococcus faecalis or in Staphylococcus aureus. Glucose-dependent efflux of erythromycin and tetracycline was demonstrated in L. lactis and in E. coli.

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.

Antibiotic resistance spread in food

Nutritive and therapeutic treatment of farm animals with antibiotics, amounting to half of the world's 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 cheeses1. 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 genera2,3. A multi-antibiotic resistance plasmid from a lactococcus found in cheese provides a historical record of such events.