Angela H.A.M. van Hoek

Acquired Antibiotic Resistance Genes: An Overview

In this review an overview is given on antibiotic resistance (AR) mechanisms with special attentions to the AR genes described so far preceded by a short introduction on the discovery and mode of action of the different classes of antibiotics. As this review is only dealing with acquired resistance, attention is also paid to mobile genetic elements such as plasmids, transposons, and integrons, which are associated with AR genes, and involved in the dispersal of antimicrobial determinants between different bacteria.

Molecular Characterization of Intrinsic and Acquired Antibiotic Resistance in Lactic Acid Bacteria and Bifidobacteria

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.

Variability of Escherichia coli O157 Strain Survival in Manure-Amended Soil in Relation to Strain Origin, Virulence Profile, and Carbon Nutrition Profile

ABSTRACT The variation in manure-amended soil survival capability among 18 Escherichia coli O157 strains (8 animal, 1 food, and 9 human isolates) was studied using a single sandy soil sample and a single sample of cattle manure as the inoculum carrier. The virulence profiles of E. coli O157 strains were characterized by detection of virulence determinants (73 genes, 122 probes in duplicate) by using the Identibac E. coli genotyping DNA miniaturized microarray. Metabolic profiling was done by subjecting all strains to the Biolog phenotypic carbon microarray. Survival times (calculated as days needed to reach the detection limit using the Weibull model) ranged from 47 to 266 days (median, 120 days). Survival time was significantly higher for the group of human isolates (median, 211 days; minimum [min.], 71; maximum [max.], 266) compared to the group of animal isolates (median, 70 days; min., 47; max., 249) (P = 0.025). Although clustering of human versus animal strains was observed based on pulsed-field gel electrophoresis (PFGE) patterns, no relation between survival time and the presence of virulence genes was observed. Principal component analysis on the metabolic profiling data revealed distinct clustering of short- and long-surviving strains. The oxidization rate of propionic acid, α-ketobutyric acid, and α-hydroxybutyric acid was significantly higher for the long-surviving strains than for the short-surviving strains. The oxidative capacity of E. coli O157 strains may be regarded as a phenotypic marker for enhanced survival in manure-amended soil. The large variation observed in survival is of importance for risk assessment models.

Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli on Flies at Poultry Farms

ABSTRACT In the Netherlands, extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli bacteria are highly prevalent in poultry, and chicken meat has been implicated as a source of ESBL-producing E. coli present in the human population. The current study describes the isolation of ESBL-producing E. coli from house flies and blow flies caught at two poultry farms, offering a potential alternative route of transmission of ESBL-producing E. coli from poultry to humans. Overall, 87 flies were analyzed in 19 pools. ESBL-producing E. coli bacteria were detected in two fly pools (10.5%): a pool of three blow flies from a broiler farm and a pool of eight house flies from a laying-hen farm. From each positive fly pool, six isolates were characterized and compared with isolates obtained from manure (n = 53) sampled at both farms and rinse water (n = 10) from the broiler farm. Among six fly isolates from the broiler farm, four different types were detected with respect to phylogenetic group, sequence type (ST), and ESBL genotype: A0/ST3519/SHV-12, A1/ST10/SHV-12, A1/ST58/SHV-12, and B1/ST448/CTX-M-1. These types, as well as six additional types, were also present in manure and/or rinse water at the same farm. At the laying-hen farm, all fly and manure isolates were identical, carrying bla TEM-52 in an A1/ST48 genetic background. The data imply that flies acquire ESBL-producing E. coli at poultry farms, warranting further evaluation of the contribution of flies to dissemination of ESBL-producing E. coli in the community.

Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet(L) and Chromosomally Located Transposon-Associated tet(M), Coexist in Lactobacillus sakei Rits 9

ABSTRACT Lactobacillus sakei is extensively used as functional starter culture in fermented meat products. One of the safety criteria of a starter culture is the absence of potentially transferable antibiotic resistance determinants. However, tetracycline-resistant L. sakei strains have already been observed. In this paper, we show that tetracycline resistance in L. sakei Rits 9, a strain isolated from Italian Sola cheese made from raw milk, is mediated by a transposon-associated tet(M) gene coding for a ribosomal protection protein and a plasmid-carried tet(L) gene coding for a tetracycline efflux pump. pLS55, the 5-kb plasmid carrying the tet(L) gene, is highly similar to the pMA67 plasmid recently described for Paenibacillus larvae, a species pathogenic to honeybees. pLS55 could be transferred by electroporation into the laboratory strain L. sakei 23K. While the L. sakei 23K transformant containing pLS55 displayed an intermediate tetracycline resistance level (MIC, <32 μg/ml), L. sakei Rits 9, containing both tetracycline-resistant determinants, had a MIC of <256 μg/ml, suggesting that Tet L and Tet M confer different levels of resistance in L. sakei. Remarkably, in the absence of tetracycline, a basal expression of both genes was detected for L. sakei Rits 9. In addition, subinhibitory concentrations of tetracycline affected the expression patterns of tet(M) and tet(L) in different ways: the expression of tet(M) was induced only at high tetracycline concentrations, whereas the expression of tet(L) was up-regulated at lower concentrations. This is the first time that two different mechanisms conferring resistance to tetracycline are characterized for the same strain of a lactic acid bacterium.

Mosaic Tetracycline Resistance Genes and Their Flanking Regions in Bifidobacterium thermophilum and Lactobacillus johnsonii

ABSTRACT For the first time, mosaic tetracycline resistance genes were identified in Lactobacillus johnsonii and in Bifidobacterium thermophilum strains. The L. johnsonii strain investigated contains a complex hybrid gene, tet(O/W/32/O/W/O), whereas the five bifidobacterial strains possess two different mosaic tet genes: i.e., tet(W/32/O) and tet(O/W). As reported by others, the crossover points of the mosaic tet gene segments were found at similar positions within the genes, suggesting a hot spot for recombination. Analysis of the sequences flanking these genes revealed that the upstream part corresponds to the 5′ end of the mosaic open reading frame. In contrast, the downstream region was shown to be more variable. Surprisingly, in one of the B. thermophilum strains a third tet determinant was identified, coding for the efflux pump Tet(L).

Prevalence and characterization of ESBL- and AmpC-producing Enterobacteriaceae on retail vegetables

In total 1216 vegetables obtained from Dutch stores during 2012 and 2013 were analysed to determine the prevalence of 3rd-generation cephalosporin (3GC) resistant bacteria on soil-grown fresh produce possibly consumed raw. Vegetables grown conventionally and organically, from Dutch as well as foreign origin were compared. Included were the following vegetable types; blanched celery (n=192), bunched carrots (n=190), butterhead lettuce (n=137), chicory (n=96), endive (n=188), iceberg lettuce (n=193) and radish (n=120). Overall, 3GC-resistant Enterobacteriaceae were detected on 5.2% of vegetables. Based on primary habitat and mechanism of 3GC-resistance, these bacteria could be divided into four groups: ESBL-producing faecal species (Escherichia coli, Enterobacter spp.), AmpC-producing faecal species (Citrobacter freundii, Enterobacter spp.), ESBL-producing environmental species (Pantoea spp., Rahnella aquatilis, Serratia fonticola), and AmpC-producing environmental species (Cedecca spp., Hafnia alvei, Pantoea spp., Serratia plymuthica), which were detected on 0.8%, 1.2%, 2.6% and 0.4% of the vegetables analysed, respectively. Contamination with faecal 3GC-resistant bacteria was most frequently observed in root and bulb vegetables (average prevalence 4.4%), and less frequently in stem vegetables (prevalence 1.6%) and leafy greens (average prevalence 0.6%). In Dutch stores, only four of the included vegetable types (blanched celery, bunched carrots, endive, iceberg lettuce) were available in all four possible variants: Dutch/conventional, Dutch/organic, foreign/conventional, foreign/organic. With respect to these vegetable types, no statistically significant difference was observed in prevalence of 3GC-resistant Enterobacteriaceae between country of origin or cultivation type (5.2%, 5.7%, 5.7% and 3.3%, respectively). Vegetables consumed raw may be a source of dissemination of 3GC-resistant Enterobacteriaceae and their resistance genes to humans. The magnitude of the associated public health risk presumably depends on the types of bacteria that are ingested, i.e., faecal or environmental species, and may therefore be higher for root and bulb vegetables compared to leafy greens.

Prevalence and characteristics of ESBL-producing E. coli in Dutch recreational waters influenced by wastewater treatment plants

Outside health care settings, people may acquire ESBL-producing bacteria through different exposure routes, including contact with human or animal carriers or consumption of contaminated food. However, contact with faecally contaminated surface water may also represent a possible exposure route. The current study investigated the prevalence and characteristics of ESBL-producing Escherichia coli in four Dutch recreational waters and the possible role of nearby waste water treatment plants (WWTP) as contamination source. Isolates from recreational waters were compared with isolates from WWTP effluents, from surface water upstream of the WWTPs, at WWTP discharge points, and in connecting water bodies not influenced by the studied WWTPs. ESBL-producing E. coli were detected in all four recreational waters, with an average concentration of 1.3 colony forming units/100ml, and in 62% of all samples. In surface waters not influenced by the studied WWTPs, ESBL-producing E. coli were detected in similar concentrations, indicating the existence of additional ESBL-E. coli contamination sources. Isolates with identical ESBL-genes, phylogenetic background, antibiotic resistance profiles, and sequence type, were obtained from effluent and different surface water sites in the same watershed, on the same day; occasionally this included isolates from recreational waters. Recreational waters were identified as a potential exposure source of ESBL-producing E. coli. WWTPs were shown to contribute to the presence of these bacteria in surface waters, but other (yet unidentified) sources likely co-contribute.

Genetic Basis of Tetracycline Resistance in Bifidobacterium animalis subsp lactis

ABSTRACT All strains of Bifidobacterium animalis subsp. lactis described to date show medium level resistance to tetracycline. Screening of 26 strains from a variety of sources revealed the presence of tet(W) in all isolates. A transposase gene upstream of tet(W) was found in all strains, and both genes were cotranscribed in strain IPLAIC4. Mutants with increased tetracycline resistance as well as tetracycline-sensitive mutants of IPLAIC4 were isolated and genetically characterized. The native tet(W) gene was able to restore the resistance phenotype to a mutant with an alteration in tet(W) by functional complementation, indicating that tet(W) is necessary and sufficient for the tetracycline resistance seen in B. animalis subsp. lactis.

Molecular Characteristics of Extended-Spectrum Cephalosporin-Resistant Enterobacteriaceae from Humans in the Community

Objective To investigate the molecular characteristics of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae collected during a cross-sectional study examining the prevalence and risk factors for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in humans living in areas with high or low broiler density. Methods ESC-resistant Enterobacteriaceae were identified by combination disc-diffusion test. ESBL/AmpC/carbapenemase genes were analysed using PCR and sequencing. For E. coli, phylogenetic groups and MLST were determined. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing. Results 175 ESC-resistant Enterobacteriaceae were cultured from 165/1,033 individuals. The isolates were Escherichia coli(n=65), Citrobacter freundii (n=52), Enterobacter cloacae (n=38), Morganella morganii (n=5), Enterobacter aerogenes (n=4), Klebsiella pneumoniae (n=3), Hafnia alvei (n=2), Shigella spp. (n=2), Citrobacter amalonaticus (n=1), Escherichia hermannii (n=1), Kluyvera cryocrescens (n=1), and Pantoea agglomerans (n=1). The following ESBL genes were recovered in 55 isolates originating from 49 of 1,033 (4.7 %) persons: bla CTX-M-1 (n=17), bla CTX-M-15 (n=16), bla CTX-M-14 (n=9), bla CTX-M-2 (n=3), bla CTX-M-3 (n=2), bla CTX-M-24 (n=2), bla CTX-M-27 (n=1), bla CTX-M-32 (n=1), bla SHV-12 (n=2), bla SHV-65 (n=1) and bla TEM-52 (n=1). Plasmidic AmpC (pAmpC) genes were discovered in 6 out of 1,033 (0.6 %) persons. One person carried two different E. coli isolates, one with bla CTX-M-1 and the other with bla CMY-2 and therefore the prevalence of persons carrying Enterobacteriaceae harboring ESBL and/or pAmpC genes was 5.2 %. In eight E. coli isolates the AmpC phenotype was caused by mutations in the AmpC promoter region. No carbapenemase genes were identified. A large variety of E. coli genotypes was found, ST131 and ST10 being most common. Conclusions ESBL/pAmpC genes resembled those from patients in Dutch hospitals, indicating that healthy humans form a reservoir for transmission of these determinants to vulnerable people. The role of poultry in the transmission to humans in the community remains to be elucidated.