Astrid Bethe

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 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.

The chicken as a natural model for extraintestinal infections caused by avian pathogenic Escherichia coli (APEC)

E. coli infections in avian species have become an economic threat to the poultry industry worldwide. Several factors have been associated with the virulence of E. coli in avian hosts, but no specific virulence gene has been identified as being entirely responsible for the pathogenicity of avian pathogenic E. coli (APEC). Needless to say, the chicken would serve as the best model organism for unravelling the pathogenic mechanisms of APEC, an extraintestinal pathogen. Five-week-old white leghorn SPF chickens were infected intra-tracheally with a well characterized APEC field strain IMT5155 (O2:K1:H5) using different doses corresponding to the respective models of infection established, that is, the lung colonization model allowing re-isolation of bacteria only from the lung but not from other internal organs, and the systemic infection model. These two models represent the crucial steps in the pathogenesis of APEC infections, including the colonization of the lung epithelium and the spread of bacteria throughout the bloodstream. The read-out system includes a clinical score, pathomorphological changes and bacterial load determination. The lung colonization model has been established and described for the first time in this study, in addition to a comprehensive account of a systemic infection model which enables the study of severe extraintestinal pathogenic E. coli (ExPEC) infections. These in vivo models enable the application of various molecular approaches to study host-pathogen interactions more closely. The most important application of such genetic manipulation techniques is the identification of genes required for extraintestinal virulence, as well as host genes involved in immunity in vivo. The knowledge obtained from these studies serves the dual purpose of shedding light on the nature of virulence itself, as well as providing a route for rational attenuation of the pathogen for vaccine construction, a measure by which extraintestinal infections, including those caused by APEC, could eventually be controlled and prevented in the field.

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.

No evidence of the Shiga toxin-producing E. coli O104:H4 outbreak strain or enteroaggregative E. coli (EAEC) found in cattle faeces in northern Germany, the hotspot of the 2011 HUS outbreak area

BackgroundRuminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli (STEC), but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST) 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC). Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain (stx2, terD, rfbO104, fliCH4), STEC (stx1, stx2, escV), EAEC (pAA, aggR, astA), and ESBL-production (blaCTX-M, blaTEM, blaSHV).ResultsThe faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28%) shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli.ConclusionsWhile we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.

CTX-M-15-type extended-spectrum beta-lactamases-producing Escherichia coli from wild birds in Germany.

The isolation of Escherichia coli from wild birds in Germany revealed the occurrence of four CTX-M-15-producing strains from four different birds (2.3% of 172 isolates). CTX-M producers were recovered from two Eurasian Blackbirds, one Rock Pigeon and a Greater White-fronted Goose. All CTX-M-producing E. coli revealed a clonal relationship as determined by pulsed-field gel electrophoresis (PFGE) and were assigned to multilocus sequence type (ST) 648. Our findings suggest the emergence of a new clone with epidemiological importance and strengthen the role of wild bird species other than waterfowl as possible reservoirs of ESBL-producing Enterobacteriaceae.

Clonal spread of highly successful ST15-CTX-M-15 Klebsiella pneumoniae in companion animals and horses

OBJECTIVES To investigate the clinical relevance and molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Klebsiella species in animals. METHODS Antimicrobial susceptibilities and presence of ESBLs were examined among Klebsiella spp. (n = 1519) from clinical samples (>1200 senders from Germany and other European countries) mainly from companion animals and horses from October 2008 to March 2010. Multilocus sequence typing (MLST) and PFGE were performed including human isolates for comparative purposes. RESULTS The overall ESBL rate was 8% for Klebsiella pneumoniae subsp. pneumoniae. Most K. pneumoniae subsp. pneumoniae ESBL producers were isolated from soft tissue infections (29.3%) and urinary tract infections (14.9%). The major ESBL type was CTX-M-15 (85.4%), located on different plasmid scaffolds (HI2, I1, FIA, FIB, FII, A/C, R and N). Other ESBL genes, such as bla(CTX-M-1) (5.6%), bla(CTX-M-3), bla(CTX-M-9), bla(SHV-2) and bla(SHV-12) (1.1% each), were also detected. Additional resistances, e.g. to fluoroquinolones (89.9%), were frequently present. ST15-CTX-M-15, a clonal group that recently emerged in humans, accounted for 75.8% of the strains analysed by MLST and there was evidence for nosocomial events in five veterinary clinics. Human ST15-CTX-M-15 strains shared PFGE clusters with animal isolates, suggesting the dissemination of this clonal group between both populations. CONCLUSIONS Our data indicate a wide spread of ST15-CTX-M-15 K. pneumoniae subsp. pneumoniae, which should be considered as a zoonotic agent of high clinical relevance for humans and animals. Further research should be undertaken to unravel both microevolutionary and biological aspects probably contributing to this global success.

Frequent combination of antimicrobial multiresistance and extraintestinal pathogenicity in Escherichia coli isolates from urban rats (Rattus norvegicus) in Berlin, Germany.

Urban rats present a global public health concern as they are considered a reservoir and vector of zoonotic pathogens, including Escherichia coli. In view of the increasing emergence of antimicrobial resistant E. coli strains and the on-going discussion about environmental reservoirs, we intended to analyse whether urban rats might be a potential source of putatively zoonotic E. coli combining resistance and virulence. For that, we took fecal samples from 87 brown rats (Rattus norvegicus) and tested at least three E. coli colonies from each animal. Thirty two of these E. coli strains were pre-selected from a total of 211 non-duplicate isolates based on their phenotypic resistance to at least three antimicrobial classes, thus fulfilling the definition of multiresistance. As determined by multilocus sequence typing (MLST), these 32 strains belonged to 24 different sequence types (STs), indicating a high phylogenetic diversity. We identified STs, which frequently occur among extraintestinal pathogenic E. coli (ExPEC), such as STs 95, 131, 70, 428, and 127. Also, the detection of a number of typical virulence genes confirmed that the rats tested carried ExPEC-like strains. In particular, the finding of an Extended-spectrum beta-lactamase (ESBL)-producing strain which belongs to a highly virulent, so far mainly human- and avian-restricted ExPEC lineage (ST95), which expresses a serogroup linked with invasive strains (O18:NM:K1), and finally, which produces an ESBL-type frequently identified among human strains (CTX-M-9), pointed towards the important role, urban rats might play in the transmission of multiresistant and virulent E. coli strains. Indeed, using a chicken infection model, this strain showed a high in vivo pathogenicity. Imagining the high numbers of urban rats living worldwide, the way to the transmission of putatively zoonotic, multiresistant, and virulent strains might not be far ahead. The unforeseeable consequences of such an emerging public health threat need careful consideration in the future.

Feeding the Probiotic Enterococcus faecium Strain NCIMB 10415 to Piglets Specifically Reduces the Number of Escherichia coli Pathotypes That Adhere to the Gut Mucosa

ABSTRACT Feed supplementation with the probiotic Enterococcus faecium for piglets has been found to reduce pathogenic gut microorganisms. Since Escherichia coli is among the most important pathogens in pig production, we performed comprehensive analyses to gain further insight into the influence of E. faecium NCIMB 10415 on porcine intestinal E. coli. A total of 1,436 E. coli strains were isolated from three intestinal habitats (mucosa, digesta, and feces) of probiotic-supplemented and nonsupplemented (control) piglets. E. coli bacteria were characterized via pulsed-field gel electrophoresis (PFGE) for clonal analysis. The high diversity of E. coli was reflected by 168 clones. Multilocus sequence typing (MLST) was used to determine the phylogenetic backgrounds, revealing 79 sequence types (STs). Pathotypes of E. coli were further defined using multiplex PCR for virulence-associated genes. While these analyses discerned only a few significant differences in the E. coli population between the feeding groups, analyses distinguishing clones that were uniquely isolated in either the probiotic group only, the control group only, or both groups (shared group) revealed clear effects at the habitat level. Interestingly, extraintestinal pathogenic E. coli (ExPEC)-typical clones adhering to the mucosa were significantly reduced in the probiotic group. Our data show a minor influence of E. faecium on the overall population of E. coli in healthy piglets. In contrast, this probiotic has a profound effect on mucosa-adherent E. coli. This finding further substantiates a specific effect of E. faecium strain NCIMB 10415 in piglets against pathogenic E. coli in the intestine. In addition, these data question the relevance of data based on sampling fecal E. coli only.