Annemieke Smet

Diversity of Extended-Spectrum β-Lactamases and Class C β-Lactamases among Cloacal Escherichia coli Isolates in Belgian Broiler Farms

ABSTRACT A total of 295 ceftiofur-resistant Escherichia coli isolates were obtained from 489 cloacal samples collected at five different Belgian broiler farms with the aim to evaluate the diversity of this resistance at the farm level. Strains were examined for resistance against β-lactam antibiotics and other antimicrobial agents by using disk diffusion tests. Three different β-lactam resistance phenotypes suggested the presence of an extended-spectrum β-lactamase (ESBL), a class C β-lactamase, or the combination of an ESBL with a class C β-lactamase. Seventy-six percent of these isolates also showed acquired resistance to other antimicrobial agents. After genotyping by repetitive extragenic palindromic-PCR, 51 unrelated E. coli strains were selected for further analyses. Isoelectric focusing and sequencing of the amplicons obtained in PCRs for the detection of genes encoding broad-spectrum β-lactamase enzymes revealed the following ESBLs: TEM-52 (13.2%), TEM-106 (2%), CTX-M-1 (27.4%), CTX-M-2 (7.8%), CTX-M-14 (5.9%), and CTX-M-15 (2%). The only plasmidic AmpC β-lactamase found in this study was the CMY-2 enzyme (49%). Mutations in the promoter and attenuator regions of the chromosomal ampC gene were found only in association with blaCMY-2 genes and ESBL genes. The combination of an ESBL (CTX-M-1) with a plasmidic AmpC β-lactamase (CMY-2) was found in 7.8% of the isolates. These data show that ceftiofur-resistant E. coli strains are often present in cloacal samples of broilers at the farm level in Belgium. The diversity of broad-spectrum β-lactamases among these isolates is high, and they may act as a reservoir of ESBL and ampC genes.

Broad-spectrum β-lactamases among Enterobacteriaceae of animal origin: molecular aspects, mobility and impact on public health.

Broad-spectrum β-lactamase genes (coding for extended-spectrum β-lactamases and AmpC β-lactamases) have been frequently demonstrated in the microbiota of food-producing animals. This may pose a human health hazard as these genes may be present in zoonotic bacteria, which would cause a direct problem. They can also be present in commensals, which may act as a reservoir of resistance genes for pathogens causing disease both in humans and in animals. Broad-spectrum β-lactamase genes are frequently located on mobile genetic elements, such as plasmids, transposons and integrons, which often also carry additional resistance genes. This could limit treatment options for infections caused by broad-spectrum β-lactam-resistant microorganisms. This review addresses the growing burden of broad-spectrum β-lactam resistance among Enterobacteriaceae isolated from food, companion and wild animals worldwide. To explore the human health hazard, the diversity of broad-spectrum β-lactamases among Enterobacteriaceae derived from animals is compared with respect to their presence in human bacteria. Furthermore, the possibilities of the exchange of genes encoding broad-spectrum β-lactamases - including the exchange of the transposons and plasmids that serve as vehicles for these genes - between different ecosystems (human and animal) are discussed.

Characterization of Extended-Spectrum β-Lactamases Produced by Escherichia coli Isolated from Hospitalized and Nonhospitalized Patients: Emergence of CTX-M-15-Producing Strains Causing Urinary Tract Infections

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates were obtained from hospitalized and nonhospitalized patients in Belgium between August 2006 and November 2007. The antimicrobial susceptibility of these isolates was determined and their ESBL genes were characterized. Clonal relationships between the CTX-M-producing E. coli isolates causing urinary tract infections were also studied. A total of 90 hospital- and 45 community-acquired cephalosporin-resistant E. coli isolates were obtained. Tetracycline, enrofloxacine, gentamicin, and trimethoprim-sulfamethaxozole resistance rates were significantly different between the community-onset and hospital-acquired isolates. A high diversity of different ESBLs was observed among the hospital-acquired E. coli isolates, whereas CTX-M-15 was dominating among the community-acquired E. coli isolates (n = 28). Thirteen different pulsed-field gel electrophoresis profiles were observed in the community-acquired CTX-M-15-producing E. coli, indicating that multiple clones have acquired the bla(CTX-M-15) gene. All community-acquired CTX-M-15-producing E. coli isolates of phylogroups B2 and D were assigned to the sequence type ST131. The hospital-acquired CTX-M-15-producing E. coli isolates of phylogroups B2, B1, A, and D corresponded to ST131, ST617, ST48, and ST405, respectively. In conclusion, CTX-M-type ESBLs have emerged as the predominant class of ESBLs produced by E. coli isolates in the hospital and community in Belgium. Of particular concern is the predominant presence of the CTX-M-15 enzyme in ST131 community-acquired E. coli.

Genome sequence of Helicobacter suis supports its role in gastric pathology

Helicobacter (H.) suis has been associated with chronic gastritis and ulcers of the pars oesophagea in pigs, and with gastritis, peptic ulcer disease and gastric mucosa-associated lymphoid tissue lymphoma in humans. In order to obtain better insight into the genes involved in pathogenicity and in the specific adaptation to the gastric environment of H. suis, a genome analysis was performed of two H. suis strains isolated from the gastric mucosa of swine. Homologs of the vast majority of genes shown to be important for gastric colonization of the human pathogen H. pylori were detected in the H. suis genome. H. suis encodes several putative outer membrane proteins, of which two similar to the H. pylori adhesins HpaA and HorB. H. suis harbours an almost complete comB type IV secretion system and members of the type IV secretion system 3, but lacks most of the genes present in the cag pathogenicity island of H. pylori. Homologs of genes encoding the H. pylori neutrophil-activating protein and γ-glutamyl transpeptidase were identified in H. suis. H. suis also possesses several other presumptive virulence-associated genes, including homologs for mviN, the H. pylori flavodoxin gene, and a homolog of the H. pylori vacuolating cytotoxin A gene. It was concluded that although genes coding for some important virulence factors in H. pylori, such as the cytotoxin-associated protein (CagA), are not detected in the H. suis genome, homologs of other genes associated with colonization and virulence of H. pylori and other bacteria are present.

Helicobacter suis causes severe gastric pathology in mouse and mongolian gerbil models of human gastric disease.

Background “Helicobacter (H.) heilmannii” type 1 is the most prevalent gastric non-H. pylori Helicobacter species in humans suffering from gastric disease. It has been shown to be identical to H. suis, a bacterium which is mainly associated with pigs. To obtain better insights into the long-term pathogenesis of infections with this micro-organism, experimental infections were carried out in different rodent models. Methodology/Principal Findings Mongolian gerbils and mice of two strains (BALB/c and C57BL/6) were infected with H. suis and sacrificed at 3 weeks, 9 weeks and 8 months after infection. Gastric tissue samples were collected for PCR analysis, histological and ultrastructural examination. In gerbils, bacteria mainly colonized the antrum and a narrow zone in the fundus near the forestomach/stomach transition zone. In both mice strains, bacteria colonized the entire glandular stomach. Colonization with H. suis was associated with necrosis of parietal cells in all three animal strains. From 9 weeks after infection onwards, an increased proliferation rate of mucosal epithelial cells was detected in the stomach regions colonized with H. suis. Most gerbils showed a marked lymphocytic infiltration in the antrum and in the forestomach/stomach transition zone, becoming more pronounced in the course of time. At 8 months post infection, severe destruction of the normal antral architecture at the inflamed sites and development of mucosa-associated lymphoid tissue (MALT) lymphoma-like lesions were observed in some gerbils. In mice, the inflammatory response was less pronounced than in gerbils, consisting mainly of mononuclear cell infiltration and being most severe in the fundus. Conclusions/Significance H. suis causes death of parietal cells, epithelial cell hyperproliferation and severe inflammation in mice and Mongolian gerbil models of human gastric disease. Moreover, MALT lymphoma-like lesions were induced in H. suis-infected Mongolian gerbils. Therefore, the possible involvement of this micro-organism in human gastric disease should not be neglected.

Non-Helicobacter pylori Helicobacter Species in the Human Gastric Mucosa: A Proposal to Introduce the Terms H. heilmannii Sensu Lato and Sensu Stricto

Dear Editor, Helicobacter (H.) pylori is by far the most prevalent Helicobacter species in the human stomach. However, already in the early days of H. pylori research, pathologists examining stomach biopsies have reported the presence of morphologically distinct, typically long spiral shaped bacteria. These gastric non-H. pylori helicobacters were originally referred to as Gastrospirillum hominis and later as H. heilmannii which was further subdivided in two taxa, types 1 and 2 [1]. A general characteristic of these bacteria is that these are very fastidious microorganisms which, as far as we know, have been cultured from the gastric mucosa of only two human patients [2,3]. Although the name H. heilmannii has for many years been used to refer to the long spiral shaped bacteria in the human stomach, it was not formally recognized as a valid species name until recently. Gastric non-H. pylori helicobacters actually comprise several Helicobacter species, all of them known to colonize the gastric mucosa of animals. Microorganisms previously referred to as H. heilmannii type 1 are identical to H. suis, a species colonizing the stomachs of pigs. The former H. heilmannii type 2 does not represent a single species, but rather a group of species all of them known to colonize the gastric mucosa of dogs and cats: H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus, H. baculiformis and a bacterium which was in 2004 given the provisional name ‘‘Candidatus H. heilmannii’’ because, at that time, it could not be cultured in vitro [1,4]. Only recently have in vitro cultures been obtained from the latter microorganism, resulting in the formal and valid description of H. heilmannii as a novel Helicobacter species [5]. Unfortunately, this will further add to the already existing confusion on the nomenclature of this complex and expanding group of gastric microorganisms. Even for specialists in the field, it is not always easy or even impossible to figure out which bacterial species some of the papers on gastric non-H. pylori helicobacters exactly deal with. We expect that clinicians and clinical bacteriologists will continue to use the name H. heilmannii to refer to the group of long spiral shaped microorganisms although according to taxonomic rules it represents only one member of this group. To avoid further confusion, we propose to introduce the terms H. heilmannii sensu lato (H. heilmannii s.l.) and H. heilmannii sensu stricto (H. heilmannii s.s.). H. heilmannii s.l. may then be used to refer to the whole group of non-H. pylori helicobacters detected in the human or animal stomach, for instance when only results of histopathology, electron microscopy or crude taxonomic data are available. The name H. heilmannii s.s. or the other species names should be used whenever bacteria are really identified to the species level. Although differences in morphology between different non-H. pylori helicobacters have been described, it is not an accurate method for species identification since variation in morphology within a species occurs and different species may be morphologically very similar. Sequencing of the 16S or 23S rRNA-encoding genes allows differentiation of H. suis from the other gastric non-H. pylori helicobacters mentioned above, but it cannot distinguish between H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus, H. baculiformis and H. heilmannii s.s. [1]. Although for differentiation between these species, sequencing of the hsp60 or gyrB gene is useful, sequencing of the urease A and B genes seems currently to be the most suitable method since sequences of these genes are available for all H. heilmannii s.l. species [1,3,4]. Whole cell protein profiling may also be useful but it is only applicable if pure in vitro cultures are available, which is a serious drawback for these fastidious microorganisms [1]. H. bizzozeronii is the only H. heilmannii s.l. species isolated from the human gastric mucosa thus far [2,3], although PCR-based screening studies revealed that human patients are more often colonized with other species, including H. suis [1]. For describing new species of the genus Helicobacter, a polyphasic approach is necessary and the recommendations by Dewhirst et al. [6] for required phenotypic and molecular data should be followed.

Prevalence and Persistence of Antimicrobial Resistance in Broiler Indicator Bacteria

This study explored the prevalence and persistence of acquired antimicrobial resistance in Escherichia coli and Enterococcus faecium from healthy broilers. In 32 broiler farms, cloacal samples were taken during two production rounds, with one production round in between. For 10 of the sampled flocks, samples from the carcasses at the slaughterhouse were also collected. For E. coli, high levels of resistance were found for ampicillin, nalidixic acid, streptomycin, tetracycline, and the combination of trimethoprim and sulfonamide. Over 58% of all the isolates showed resistance to four or more antimicrobial agents. Only 4.8% were fully susceptible for all 14 drugs tested. A remarkably high resistance rate (up to 41%) to ceftiofur was found. The enterococci were frequently resistant to macrolides, tetracycline, and the combination quinopristin/dalfopristin. Over 80% displayed acquired resistance to four or more antimicrobial agents, and 3.9% were fully susceptible for the eight agents tested. Resistance was found to persist over consecutive production rounds. There was a good correlation between results obtained with cloacal samples of the live animals and caecal content samples collected in the slaughterhouse for both E. coli and E. faecium. For E. coli but not for E. faecium, the resistance profile of neck skin isolates was different from that of cloacal isolates.

Gastric epithelial cell death caused by Helicobacter suis and Helicobacter pylori γ‐glutamyl transpeptidase is mainly glutathione degradation‐dependent

Helicobacter (H.) suis is the most prevalent non‐H. pylori Helicobacter species colonizing the stomach of humans suffering from gastric disease. In the present study, we aimed to unravel the mechanism used by H. suis to induce gastric epithelial cell damage. H. suis lysate induced mainly apoptotic death of human gastric epithelial cells. Inhibition of γ‐glutamyl transpeptidase (GGT) activity present in H. suis lysate and incubation of AGS cells with purified native and recombinant H. suis GGT showed that this enzyme was partly responsible for the observed apoptosis. Supplementation of H. suis or H. pylori GGT‐treated cells with glutathione strongly enhanced the harmful effect of both enzymes and resulted in the induction of oncosis/necrosis, demonstrating that H. suis and H. pylori GGT‐mediated degradation of glutathione and the resulting formation of glutathione degradation products play a direct and active role in the induction of gastric epithelial cell death. This was preceded by an increase of extracellular H2O2 concentrations, generated in a cell‐independent manner and causing lipid peroxidation. In conclusion, H. suis and H. pylori GGT‐mediated generation of pro‐oxidant glutathione degradation products brings on cell damage and causes apoptosis or necrosis, dependent on the amount of extracellular glutathione available as a GGT substrate.

The local immune response of mice after Helicobacter suis infection: strain differences and distinction with Helicobacter pylori

Helicobacter (H.) suis colonizes the stomach of pigs and is the most prevalent gastric non-H. pylori Helicobacter species in humans. Limited information is available on host immune responses after infection with this agent and it is unknown if variation in virulence exists between different H. suis strains. Therefore, BALB/c and C57BL/6 mice were used to compare colonization ability and gene expression of various inflammatory cytokines, as determined by real-time PCR, after experimental infection with 9 different H. suis strains. All strains were able to persist in the stomach of mice, but the number of colonizing bacteria at 59 days post inoculation was higher in stomachs of C57BL/6 mice compared to BALB/c mice. All H. suis strains caused an upregulation of interleukin (IL)-17, which was more pronounced in BALB/c mice. This upregulation was inversely correlated with the number of colonizing bacteria. Most strains also caused an upregulation of regulatory IL-10, positively correlating with colonization in BALB/c mice. Only in C57BL/6 mice, upregulation of IL-1β was observed. Increased levels of IFN-γ mRNA were never detected, whereas most H. suis strains caused an upregulation of the Th2 signature cytokine IL-4, mainly in BALB/c mice. In conclusion, the genetic background of the murine strain has a clear impact on the colonization ability of different H. suis strains and the immune response they evoke. A predominant Th17 response was observed, accompanied by a mild Th2 response, which is different from the Th17/Th1 response evoked by H. pylori infection.

OXA-23-producing Acinetobacter species from horses: a public health hazard?

Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; Department of Bacteriology and Immunology, CODA-CERVA-VAR, Groeselenberg 99, 1180 Brussels, Belgium; Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; Laboratory of Bacterial Genetics, National Institute of Public Health, Srobarova 48, 100 42 Prague, Czech Republic; Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine & Health Sciences, Ghent University, De Pintenlaan 185, Ghent, Belgium