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.

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.

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

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 beta-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.

Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple-antibiotic-resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

Active surveillance of antibiotic resistance prevalence in urinary tract and skin infections in the outpatient setting

The aim of the study was to evaluate the need for active surveillance of antibiotic resistance in ambulatory infections. We measured the prevalence of antibiotic resistance in urinary tract infections (UTIs) (n = 1018) and skin infections (n = 213) diagnosed in outpatients between September 2008 and February 2009 in the Canton of Bern, Switzerland. Samples were stratified into 'solicited' (diagnostic work-up for study purpose only) and 'routine' (diagnostic work-up as part of standard care). Susceptibility patterns were compared for 463 Escherichia coli isolates from UTIs (231 solicited; 232 routine) and 87 Staphylococcus aureus isolates from skin infections (35 solicited; 52 routine). Overall, E. coli showed higher susceptibility to ampicillin, amoxicillin-clavulanic acid and norfloxacin in solicited than in routine samples. Among 15-45-year-old patients, susceptibility rates were comparable between solicited and routine samples for all antibiotics except for amoxicillin-clavulanic acid. However, among patients >45 years old, isolates from routine samples showed lower susceptibility to all β-lactams tested and quinolones than those from solicited samples. Extended-spectrum β-lactamase (ESBL)-producing E. coli isolates were rare (solicited, 0.4%; routine, 1.7%; p 0.4). Susceptibility patterns of S. aureus were comparable between solicited and routine samples. Therefore, in the outpatient setting, susceptibility rates for E. coli isolates differ by indication for urinary culture and age. Surveillance based on samples taken during standard care may underestimate susceptibility rates for uncomplicated infections, especially among the elderly. Reports of resistance data should include age stratification.

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

AIMS: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. METHODS AND RESULTS: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which - in combination with MLST - increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37.5, 33.1 and 8.1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone-sensitive human isolates showed overlapping MLST-flaB types with those of chicken origin, resistant strains showed only 39% of matching types. CONCLUSION: Mainly quinolone-sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.

Two novel antibiotic resistance genes, tet(44) and ant(6)-Ib, are located within a transferable pathogenicity island in Campylobacter fetus subsp. fetus

New tetracycline and streptomycin resistance genes, tet(44) and ant(6)-Ib, were identified in Campylobacter fetus subsp. fetus within a transferable pathogenicity island that is typically unique to Campylobacter fetus subsp. venerealis. The 640-amino-acid tetracycline resistance determinant, Tet 44, belongs to a class of proteins that confers resistance to tetracycline and minocycline by ribosomal protection. The 286-amino-acid streptomycin resistance determinant, ANT(6)-Ib, belongs to a family of aminoglycoside nucleotidyltransferases. The resistance phenotypes were demonstrated by gene inactivation and expression.

Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks

To obtain genetic information about Campylobacter jejuni and Campylobacter coli from broilers and carcasses at slaughterhouses, we analyzed and compared 340 isolates that were collected in 2008 from the cecum right after slaughter or from the neck skin after processing. We performed rpoB sequence-based identification, multilocus sequence typing (MLST), and flaB sequence-based typing; we additionally analyzed mutations within the 23S rRNA and gyrA genes that confer resistance to macrolide and quinolone antibiotics, respectively. The rpoB-based identification resulted in a distribution of 72.0% C. jejuni and 28.0% C. coli. The MLST analysis revealed that there were 59 known sequence types (STs) and 6 newly defined STs. Most of the STs were grouped into 4 clonal complexes (CC) that are typical for poultry (CC21, CC45, CC257, and CC828), and these represented 61.8% of all of the investigated isolates. The analysis of 95 isolates from the cecum and from the corresponding carcass neck skin covered 44 different STs, and 54.7% of the pairs had matching genotypes. The data indicate that cross-contamination from various sources during slaughter may occur, although the majority of Campylobacter contamination on carcasses appeared to originate from the slaughtered flock itself. Mutations in the 23S rRNA gene were found in 3.1% of C. coli isolates, although no mutations were found in C. jejuni isolates. Mutations in the gyrA gene were observed in 18.9% of C. jejuni and 26.8% of C. coli isolates, which included two C. coli strains that carried mutations conferring resistance to both classes of antibiotics. A relationship between specific genotypes and antibiotic resistance/susceptibility was observed.

Antibiotic resistance profile of Staphylococcus rostri, a new species isolated from healthy pigs

Staphylococcus rostri is a newly described Staphylococcus species that is present in the nasal cavity of healthy pigs. Out of the 225 pigs tested at slaughterhouse, 46.7% carried the new species alone and 22% in combination with Staphylococcus aureus. An antibiotic resistance profile was determined for S. rostri and compared to that of S. aureus isolated from the same pig. Resistance to tetracycline specified by tet(M), tet(K) and tet(L), streptomycin (str(pS194)), penicillin (blaZ), trimethoprim (dfr(G)), and erythromycin and clindamycin (erm genes), were found in both species; however, with the exception of streptomycin and trimethoprim, resistance was higher in S. aureus. S. rostri isolates display very low genetic diversity as demonstrated by pulsed-field gel electrophoresis, which generated two major clusters. Several clonal complexes (CC1, CC5, CC9, CC30 and CC398) were identified in S. aureus with CC 9 and CC 398 being the most frequent. Our study gives the first overview of the distribution, genetic relatedness, and resistance profile of one coagulase-negative Staphylococcus species that is commonly present in the nares of healthy pigs in Switzerland, and shows that S. rostri may harbor resistance genes associated with transferable elements like Tn916.

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.

[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.