Sebastian Guenther

Extended-spectrum β-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective

The possible zoonotic spread of antimicrobial-resistant bacteria is controversial. This review discusses global molecular epidemiological data combining both analyses of the chromosomal background, using multilocus sequence typing (MLST), and analyses of plasmid (episomal) extended-spectrum β-lactamase (ESBL)/AmpC genes in Escherichia coli present in humans and animals. For consideration of major epidemiological differences, animals were separated into livestock and companion animals. MLST revealed the existence of ESBL-producing isolates thoughout the E. coli population, with no obvious association with any ancestral EcoR group. A similar distribution of major ESBL/AmpC types was apparent only in human isolates, regardless of their geographical origin from Europe, Asia, or the Americas, whereas in animals this varied extensively between animal groups and across different geographical areas. In contrast to the diversity of episomal ESBL/AmpC types, isolates from human and animals mainly shared identical sequence types (STs), suggesting transmission or parallel micro-evolution. In conclusion, the opinion that animal ESBL-producing E. coli is a major source of human infections is oversimplified, and neglects a highly complex scenario.

Emergence of human pandemic O25:H4-ST131 CTX-M-15 extended-spectrum-β-lactamase-producing Escherichia coli among companion animals

OBJECTIVES In view of the intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15 extended-spectrum beta-lactamase (ESBL) in human clinical settings it would be of great interest to explore its existence in animals to unravel a possible reservoir function and the origin and transmission of this group of multiresistant strains. METHODS A total of 177 clinical phenotypically ESBL-producing E. coli isolates, mainly obtained from companion animals with urinary tract infections, wound infections and diarrhoea, were collected in a veterinary diagnostic laboratory covering a European-wide service area. They were screened for molecular subtype O25b and multilocus sequence type 131. O25b-ST131 isolates were subsequently tested for ESBL types, and phenotypic and genotypic resistance determinants. Further characterization of the strains was performed by PFGE and virulence gene typing. RESULTS Ten (5.6%) of 177 phenotypically ESBL-producing E. coli isolates, nine strains from dogs and one strain from a horse, were allocated to the B2-O25b-ST131 lineage. Nine of these isolates harboured a CTX-M-15-type beta-lactamase enzyme while one strain possessed an SHV-12-type ESBL. Macrorestriction analysis revealed a cluster formation of six of the animal CTX-M-15-type ESBL-producing strains from five different European countries together with a human control strain constituting a group of clonally related strains at a similarity value of 87.0%. CONCLUSIONS Our findings demonstrate that the group of clonally related human B2-O25:H4-ST131 CTX-M-15-type ESBL-producing E. coli strains is present in companion animals from various European countries. This highlights the possibility of inter-species transmission of these multiresistant strains from human to animal and vice versa.

Extended-spectrum beta-lactamases producing E. coli in wildlife, yet another form of environmental pollution?

Wildlife is normally not exposed to clinically used antimicrobial agents but can acquire antimicrobial resistant bacteria through contact with humans, domesticated animals and the environment, where water polluted with feces seems to be the most important vector. Escherichia coli, an ubiquitous commensal bacterial species colonizing the intestinal tract of mammals and birds, is also found in the environment. Extended-spectrum beta-lactamases producing E. coli (ESBL-E. coli) represent a major problem in human and veterinary medicine, particular in nosocomial infections. Additionally an onset of community-acquired ESBL-E. coli infections and an emergence in livestock farming has been observed in recent years, suggesting a successful transmission as well as persistence of ESBL-E. coli strains outside clinical settings. Another parallel worldwide phenomenon is the spread of ESBL-E. coli into the environment beyond human and domesticated animal populations, and this seems to be directly influenced by antibiotic practice. This might be a collateral consequence of the community-onset of ESBL-E. coli infections but can result (a) in a subsequent colonization of wild animal populations which can turn into an infectious source or even a reservoir of ESBL-E. coli, (b) in a contribution of wildlife to the spread and transmission of ESBL-E. coli into fragile environmental niches, (c) in new putative infection cycles between wildlife, domesticated animals and humans, and (d) in problems in the medical treatment of wildlife. This review aims to summarize the current knowledge on ESBL-E. coli in wildlife, in turn underlining the need for more large scale investigations, in particular sentinel studies to monitor the impact of multiresistant bacteria on wildlife.

Methicillin-resistant staphylococci (MRS) and extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae in companion animals: Nosocomial infections as one reason for the rising prevalence of these potential zoonotic pathogens in clinical samples

The ongoing change in the relationship between humans and companion animals is hallmarked by the increasing intensive care provided to companion animals in veterinary medicine, resulting in growing numbers of high-risk animal patients. The emergence of nosocomial infections in small animal clinics is one of the major drawbacks of this development, especially in terms of multidrug-resistance and potentially zoonotic pathogens. This mini-review therefore addresses recent findings regarding the increasing prevalence of multi-resistant bacterial pathogens like methicillin-resistant staphylococci (MRS), including Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) as well as extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae in companion animals. Along with the steady increase of nosocomial infection rates in veterinary clinics, particular attention has recently been drawn to the genetic background of multi-resistant strains, resulting in the identification of certain genetic lineages which frequently appear in both, human and animal samples. These sequence types (ST), included ST254, ST8 and ST22 in terms of MRSA and ST131, ST405 and ST648 for ESBL-producing E. coli. The interspecies distribution of these STs resulted in the assumption that certain extended-host spectrum genotypes (EHSG) might exist both for MRS and ESBL-producing E. coli. These initial findings underline the necessity to investigate the major molecular or functional driving forces facilitating interspecies transferability of such EHSG strains. Due to the zoonotic potential of these multi-resistant bacteria, another aspect of the changing social role of companion animals needs to be addressed: the close contact of pets with their owners, resulting in presumptive new transmission and infection routes. We therefore envision retaliatory actions like initial surveillance and monitoring programs not only in livestock, but also particularly in companion animals. Interdisciplinary approaches including human and veterinary experts should be implemented to develop reliable investigation procedures with respect to the current reality of animal owners and their pets. Additionally, consequent basic hygienic measures, prudent use of antimicrobials in companion animals and efforts regarding implementation of antibiotic stewardships should be fostered.

Emergence of OXA-48 carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in dogs

OBJECTIVES To evaluate the possible occurrence of carbapenemase-producing Escherichia coli and Klebsiella spp. strains in domestic animals. METHODS Veterinary clinical E. coli (n = 1175) and Klebsiella spp. (n = 136) isolates consecutively collected from livestock and companion animals in Germany from June 2012 to October 2012 were screened for their susceptibility to carbapenems using the agar disc diffusion test. Carbapenemase genes were characterized by PCR and sequencing; conjugation assays were performed. Carbapenemase-positive isolates were assigned to phylogenetic lineages by multilocus sequence typing and the clonal relatedness was determined using macrorestriction analysis and subsequent PFGE. RESULTS Carbapenem non-susceptible isolates of Klebsiella pneumoniae (n = 5) and E. coli (n = 3) were obtained from six dogs hospitalized in a single veterinary clinic in Hessia, Germany, partly at the same time and consecutively over the study period. All isolates harboured carbapenemase gene blaOXA-48 located within Tn1999.2 transposons on conjugative ~60 kb plasmids. The K. pneumoniae isolates belonged to sequence type ST15, pulsotype 1, and coexpressed CTX-M-15, SHV-28, OXA-1 and TEM-1. Two E. coli isolates were assigned to ST1196 and pulsotype 2 and coproduced CMY-2, SHV-12 and TEM-1, while the third E. coli isolate was of ST1431 (pulsotype 3), and possessed blaCTX-M-1, blaOXA-2 and blaTEM-1. CONCLUSIONS This is the first known report of OXA-48-producing bacteria from companion animals. The clonal nature of the K. pneumoniae and two E. coli isolates suggests a nosocomial dissemination rather than repeated introduction by individual patients into the clinic.

CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence?

OBJECTIVES To discern the relevance of ST648 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli as a putative new group of multiresistant and extraintestinal pathogenic strains in animals, its frequency, ESBL types, antimicrobial resistance patterns and virulence gene (VG) profiles should be determined and compared with ST131 strains from the same collection of strains. METHODS ESBL-producing E. coli isolates (n = 1152), consecutively sampled from predominantly dogs, cats and horses between 2008 and 2011, were assigned to a phylogenetic group by PCR. Partial multilocus sequence typing was performed for group D and B2 strains and strains presumed to be D-ST648 and B2-ST131 were fully typed. ESBL genes and extraintestinal pathogenic E. coli (ExPEC)-like VGs were characterized by PCR and sequence analysis and antimicrobial resistance was determined by broth dilution. Clonal analysis was done by PFGE. RESULTS Forty (3.5%) ESBL-producing E. coli were determined as D-ST648, whereas B2-ST131 isolates occurred less frequently (2.8%). Although the predominant ESBL type in both groups was CTX-M-15 (72.5% versus 46.9%), ST648 strains from companion animals and horses displayed a lower variety of ESBL types (CTX-M-1, -3, -14, -15 and -61 versus CTX-M-1,-2,-14,-15,-27 and -55 and SHV-12). In contrast to ST131 strains, a higher proportion of ST648 strains showed resistance to most non-β-lactam antibiotics. Overall, VGs were less abundant in ST648 strains, although some strains had VG profiles comparable to those of ST131 strains. ExPEC-associated serotype O1:H6 was predominant (46.8%) among the ST648 strains. Some PFGE clusters comprised ST648 isolates from pets, horses and wild birds and humans included from previous studies. CONCLUSIONS Our findings demonstrate that certain subgroups of E. coli D-ST648-CTX-M may represent a novel genotype that combines multiresistance, extraintestinal virulence and zoonotic potential.

CTX-M-15-producing Escherichia coli clone B2-O25b-ST131 and Klebsiella spp. isolates in municipal wastewater treatment plant effluents

OBJECTIVES The global occurrence of antibiotic resistance genes in bacteria in water environments is an increasing concern. Treated wastewater was sampled daily over a 45 day period from the outflow of a municipal wastewater treatment plant in Brno, Czech Republic, and examined for extended-spectrum β-lactamase (ESBL)-producing bacteria. METHODS Water samples were cultivated on MacConkey agar with cefotaxime (2 mg/L) and individual colonies were examined for ESBL production. Phenotypic ESBL-positive bacteria identified as Escherichia coli or Klebsiella spp. were tested for the presence of antibiotic resistance genes, the virulence gene afa/dra and the bla(CTX-M) upstream region. Genetic relatedness was analysed by PFGE, multilocus sequence typing and plasmid analysis. RESULTS A total of 68 ESBL-producing Enterobacteriaceae isolates were detected in 34 out of 45 wastewater samples. ESBL-producing isolates included 26 E. coli isolates, 4 Klebsiella pneumoniae isolates and 1 Klebsiella oxytoca isolate. The pandemic and multiresistant B2-O25b-ST131 clone was predominant, being detected among 19 E. coli isolates, and 17 of the B2-O25b-ST131 isolates were positive for the FIA replicon and the afa/dra operon and had an IS26 element flanking bla(CTX-M-15). Seventeen of the B2-O25b-ST131 isolates showed closely related PFGE profiles (defined by 84% band similarity) and belonged to identical clonal groups. CONCLUSIONS The results highlight the inadequacy of the treatment process in removing multiresistant bacteria from municipal wastewater and point to a risk of transmission of clinically important multiresistant strains, such as the pandemic ST131 clone, to the environment. This is the first study demonstrating the pandemic ST131 clone in wastewater.

Antimicrobial resistance profiles of Escherichia coli from common European wild bird species.

The emergence and spread of multiresistant bacteria in natural environments constitute a serious impact on animal and human health. To gain more insight into the role of wild birds as carriers and reservoir of multiresistant Escherichia coli we tested a broad spectrum of common European bird species for the occurrence of E. coli strains and their antimicrobial resistance by minimal inhibitory concentration testing and PCR analysis of several resistance genes. Nine of the 187 E. coli isolates (4.8%) exhibited multiresistant phenotypes including resistances against beta-lactams, aminoglycosides, fluoroquinolones, tetracyclines and sulfonamides. By comparing avian E. coli resistance frequencies with frequencies known for E. coli isolated from livestock and companion animals analogous profiles were identified. Multiresistant E. coli strains were isolated from synanthropic avian species as well as from birds of prey, waterfowl and passerines. By that, all these avian hosts are suggested to represent a considerable reservoir of resistant E. coli strains. Consequently wild birds might constitute a potential hazard to human and animal health by transmitting multiresistant strains to waterways and other environmental sources via their faecal deposits.

The broader context of antibiotic resistance: Zinc feed supplementation of piglets increases the proportion of multi-resistant Escherichia coli in vivo

Following the Europe-wide ban of antimicrobial growth promoters, feed supplementation with zinc has increased in livestock breeding. In addition to possible beneficial effects on animal health, feed supplementation with heavy metals is known to influence the gut microbiota and might promote the spread of antimicrobial resistance via co-selection or other mechanisms. As Escherichia coli is among the most important pathogens in pig production and often displays multi-resistant phenotypes, we set out to investigate the influence of zinc feed additives on the composition of the E. coli populations in vivo focusing on phylogenetic diversity and antimicrobial resistance. In a piglet feeding trial, E. coli were isolated from ileum and colon digesta of high dose zinc-supplemented (2500ppm) and background dose (50ppm) piglets (control group). The E. coli population was characterized via pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) for the determination of the phylogenetic background. Phenotypic resistance screening via agar disk diffusion and minimum inhibitory concentration testing was followed by detection of resistance genes for selected clones. We observed a higher diversity of E. coli clones in animals supplemented with zinc compared to the background control group. The proportion of multi-resistant E. coli was significantly increased in the zinc group compared to the control group (18.6% vs. 0%). For several subclones present both in the feeding and the control group we detected up to three additional phenotypic and genotypic resistances in the subclones from the zinc feeding group. Characterization of these subclones suggests an increase in antimicrobial resistance due to influences on plasmid uptake by zinc supplementation, questioning the reasonability of zinc feed additives as a result of the ban of antimicrobial growth promoters.

Rat hepatitis E virus: geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus).

Zoonotic hepatitis E virus (HEV) infection in industrialised countries is thought to be caused by transmission from wild boar, domestic pig and deer as reservoir hosts. The detection of HEV-specific antibodies in rats and other rodents has suggested that these animals may represent an additional source for HEV transmission to human. Recently, a novel HEV (ratHEV) was detected in Norway rats from Hamburg, Germany, showing the typical genome organisation but a high nucleotide and amino acid sequence divergence to other mammalian and to avian HEV strains. Here we describe the multiple detection of ratHEV RNA and HEV-specific antibodies in Norway rats from additional cities in north-east and south-west Germany. The complete genome analysis of two novel strains from Berlin and Stuttgart confirmed the association of ratHEV to Norway rats. The present data indicated a continuing existence of this virus in the rat populations from Berlin and Hamburg. The phylogenetic analysis of a short segment of the open reading frame 1 confirmed a geographical clustering of the corresponding sequences. Serological investigations using recombinant ratHEV and genotype 3 capsid protein derivatives demonstrated antigenic differences which might be caused by the high amino acid sequence divergence in the immunodominant region. The high amount of animals showing exclusively ratHEV RNA or anti-ratHEV antibodies suggested a non-persistent infection in the Norway rat. Future studies have to prove the transmission routes of the virus in rat populations and its zoonotic potential. The recombinant ratHEV antigen generated here will allow future seroepidemiological studies to differentiate ratHEV and genotype 3 infections in humans and animals.