Rolf Bauerfeind

Prevalence of Enteropathogens in Suckling and Weaned Piglets with Diarrhoea in Southern Germany

Faecal samples from suckling (n=205) and weaned piglets (n=82) with diarrhoea from 24 farms in Southern Germany were examined for shedding of important metazoic parasitic, viral and bacterial pathogens using culture, microscopic and electronmicroscopic methods. Escherichia coli isolates were tested further for the enterotoxin genes est‐Ia and elt‐I by colony blot hybridization. Isospora suis was diagnosed in 26.9 % and Cryptosporidium parvum in 1.4 % of the piglets investigated. The proportion of coronavirus‐positive animals was 13.4 % and 4 % were positive for rotavirus. It was found that 17.6 % of the animals were infected with enterotoxigenic E. coli (ETEC; 10.1 % ETEC‐ST‐Ia and 8.6 % ETEC‐LT‐I, respectively). The occurrence of the pathogens was significantly associated with the age of the animals examined (P < 0.001). Isospora suis was predominantly isolated from suckling piglets (in the second and third week of life), while in weaned piglets (fourth week of life) rotavirus and ETEC were most prevalent. On 22 of the 24 piglet production farms examined at least one of the investigated pathogens was detected. Coronavirus was diagnosed in 66.7 %, I. suis in 62.5 %, rotavirus in 20.8 % and C. parvum in 8.3 % of the farms. These results underline the fact that despite the hygienic, technical and immune preventive efforts during the last years, enteropathogens are still common in German piglet production units.

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: An approach to quantify the distribution of ESBL types between different reservoirs

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

Multiresistant extended-spectrum β-lactamase- producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany

BackgroundMultiresistant Gram-negative bacteria producing extended-spectrum β-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of β-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses.ResultsIn this study 153 (83.6%) of the human isolates (n = 183) and 163 (91.6%) of the animal isolates (n = 178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac(‘6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates.ConclusionsIsolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations.

Characterization of Shiga-like Toxin Producing Escherichia coli (SLTEC) Isolated from Calves with and without Diarrhoea

To determine if shiga-like toxin producing Escherichia coli (SLTEC) are involved in neonatal calf diarrhoea, isolated E. coli strains from diarrhoeic and non-diarrhoeic calves were characterized for shiga-like toxin (SLT) by colony blot hybridization and cytotoxicity assays. None of 150 E. coli strains isolated from diarrhoeic calves in 1985-1988 was positive for SLT, while 7/232 (3.0%) isolated in 1989 were positive for SLT. In contrast, samples collected during 1989 and 1990 from diarrhoeic calves were 21.9% SLTEC positive, and samples from non-diarrhoeic calves were 12.9% SLTEC positive. SLT I positive E. coli strains were isolated more often from diseased (17.8%) than from healthy animals (5.0%), while SLT II positive E. coli were more often detected in non-diarrhoeic (8.9%) than in diarrhoeic calves (4.1%). The mean percentage of SLT I positive E. coli in the whole E. coli flora of the samples was significantly higher in diarrhoeic than in healthy animals, implying a pathogenic role of SLT I producing E. coli in neonatal calf diarrhoea. Enterohemolysin was produced by 70.8% of the SLT I producing E. coli strains examined. Determination of O- and K-antigens of SLT positive E. coli revealed a highly diverse spectrum of SLTEC O-groups in calves. While no E. coli isolate belonged to serotype O157:H7, classical human enteropathogenic E. coli O-groups (O26, O111, O128) were detected. These results support the theory that cattle serve as a reservoir for human SLTEC infection.

Compensation of preliminary blood phagocyte immaturity in the newborn calf

To estimate the functional maturity of the phagocytic defence in neonatal calves, we analyzed the characteristics of blood phagocytes from calves (n = 10) 1 h post partum (p.p.) and 4 h p.p. At 1 h p.p., all calves were colostrum-deprived, while 5 calves had received colostrum before the 4 h p.p. sampling. The results were compared to those obtained from 3-9-week-old calves (n = 10). Phagocytic and oxidative burst activity of polymorphonuclear leukocytes (PMNL) and monocytes were determined in whole blood and separately analyzed by flow cytometry. In neonates prior to colostrum ingestion (1 h p.p.), phagocytic activity of PMNL against non-preopsonized E. coli was lower when compared to PMNL of 3-9-week-old calves. Opsonization of bacteria with pooled plasma from adult animals only partially restituted this lower PMNL phagocytic activity, indicating that humoral as well as cellular aspects of PMNL phagocytosis are altered in neonatal calves. In contrast to PMNL, monocytes of neonates exhibited an enhanced phagocytic activity. The oxidative burst activity of PMNL, as well as of monocytes was higher in newborn calves. During the first 4 h of life, the activities of blood phagocytes changed. Colostrum ingestion was accompanied by an increase in the percentage of phagocytizing PMNL and monocytes. This increase was absent in colostrum-deprived calves. In contrast, the oxidative burst activity of phagocytes decreased with age. In monocytes, the decrease of oxidative burst activity was only observed in colostrum-fed calves. In conclusion, some blood phagocyte functions in calves were found to be immature at birth, but these functions are presumably compensated by high absolute PMNL numbers and by other the more active mechanisms.

Neutralizing antibodies against Shiga-like toxins from Escherichia coli in colostra and sera of cattle

Previous or present infection with Shiga-like toxin producing E. coli (SLTEC) was detected by an indirect neutralization assay of antibody titer. Bovine colostra and sera blocked the cytotoxic effects of Shiga-like toxin on Vero cell monolayers. SLT neutralizing antibodies were present in 84.0% (189/225) of the colostrum samples from randomly chosen cows in Bavaria, Germany. While all of the colostra with neutralizing activity reacted with SLT-I, only 14.7% neutralized both SLT-I and -II. Approximately 93.0% (37/40) of sera from heifers had SLT neutralizing activity. To quantify the neutralizing antibodies, colostra were tested in the Vero cell assay for their capability to reduce the 50% cytotoxic dose (CD50) of SLT standards, where the neutralizing units/ml (nu/ml) equal the log10 of CD50 reduction. Almost half of reactive colostra (48.7%) reduced the CD50 of the SLT-I standard by 10(4) to 10(5) (4-5 nu/ml). Higher reactivity (5-7 nu/ml) was found in 46.5% of positive colostra. The remaining colostra samples had over 7 nu/ml. To determine if the colostra were blocking receptors for SLT on Vero cells, cells were preincubated with colostra, and SLT was later added. No neutralizing activity was detected, indicating the reactivity of colostra was directed against SLT. When the colostra were subjected to ammonium sulphate precipitation and DEAE anion exchange chromatography, high levels of neutralizing activity were found in the IgG1 containing fractions. Colostrum fractions were tested for SLT-I binding antibodies in a capture ELISA, based on the binding of SLT-I to the toxin receptor analogue P1-glycoprotein. Only fractions from colostra with over 5 nu/ml were reactive in this assay, indicating the ELISA was less sensitive than the Vero cell assay. The results support the theory that SLTEC exposure of cows in Germany is more widespread than expected from epidemiological studies based on bacterial isolation. This possibly indicates a higher risk of human SLTEC infection via beef and milk products.

Promiscuous Shiga toxin 2e and its intimate relationship to Forssman

Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Stx2e-producing strains from the intestine of slaughtered pigs (n = 3), feces of piglets with postweaning diarrhea or edema disease (n = 12) and feces of humans with asymptomatic infections or mild diarrhea (n = 13) were comparatively analyzed for the binding specificities of Stx2e to glycosphingolipids (GSLs) of the globo-series. Besides equivalent binding towards globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), we could demonstrate specific interaction of Stx2e preparations from human and porcine STEC isolates with Forssman GSL. Notably, Forssman GSL was recognized neither by structurally closely related Stx2 nor by Stx1 derived from human STEC isolates conferring Stx2e a unique recognition feature. Noteworthy, 7 (54%) of the 13 human and 8 (53%) of the 15 pig Stx2e samples exhibited cytotoxic action towards human brain microvascular endothelial cells. Our findings provide a basis for further exploring the functional role of the promiscuous receptor repertoire of Stx2e and the exact nature of the mechanisms that underlie different pathological outcomes of Stx2e-producing STEC in humans and pigs.

Detection of colostrum-derived alloantibodies in calves with bovine neonatal pancytopenia.

Bovine neonatal pancytopenia (BNP) is an emerging calf disease of unknown cause characterized by a pronounced susceptibility to bleeding as a result of a pancytopenia and bone marrow depletion. In this study we investigated whether this phenomenon is related to colostrum-derived alloantibodies directed against neonatal leukocytes. In a first experiment and using a flow cytometric approach sera from 6 BNP-dams (had given birth to BNP-calves; vaccinated against bovine viral diarrhea virus [BVDV]) and 6 control-dams (no herd history of BNP; no BVDV vaccination) were analyzed for the presences of alloantibodies (IgG) able to bind to the surface of leukocytes isolated from 7 calves from a herd with no history of BNP (no BVDV vaccination). In a second experiment, 4 neonates from 3 BNP-dams were fed colostrum from their corresponding mothers and sampled on a regular basis from birth up to day 21 of life under clinically controlled conditions. Sample analysis of the 4 neonates included hematology (white blood cell count and platelets), bone marrow cytology and histopathology as well as the flow cytometric detection of the percentage of IgG+-lymphocytes/monocytes in the peripheral blood. Experiment #1 showed that all BNP-dam sera harbored significantly higher alloantibody titers than the control dam sera (p<0.001). In the peripheral blood of the two neonates (Experiment #2), the percentage of IgG+-cells increased dramatically within 12h post colostrum intake (p.c.i.), remaining at over 95% for up to 3 days. Both calves developed BNP-associated clinical symptoms, one died. Both twin calves showed no clinical symptoms accompanied by a minor increase of IgG+ cells for up to 12h. Thus, the level of IgG+-cells and the duration of the detection thereof correlated with the severity of BNP developed by these animals. The results show that BNP-dams harbor alloantibodies against surface antigens of neonatal leukocytes in their sera that are readily transferred to the offspring via colostrum. These alloantibodies probably play a crucial role in the pathogenesis of BNP.

Circulation of clonal populations of fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST410 in humans and animals in Germany

Multidrug-resistant Escherichia coli encoding CTX-M-type extended-spectrum β-lactamases (ESBLs) are isolated in increasing numbers from humans, companion animals and livestock, raising concern regarding the exchange and spread of isolates in these populations. In this study, whole-genome sequencing of CTX-M-15-producing E. coli isolates recently sampled from humans, companion animals, livestock and farm environments was performed. In total, 26 different sequence types (STs) were detected, of which ST410 was the most frequent and was the only ST present in all populations studied. Five clades (designated A-E) were detected within the ST410 isolates. In particular, isolates of clade B were present in all four populations and had core genomes that differed by less than 70 single nucleotide polymorphisms (SNPs). Isolates of clades B and C were also clonally marked, exhibiting identical chromosomal insertions of blaCTX-M-15 at distinct loci. These data provide strong evidence for the clonal dissemination of specific clades of CTX-M-15-producing E. coli ST410 in human and animal populations.

First report of an Escherichia coli strain from swine carrying an OXA-181 carbapenemase and the colistin resistance determinant MCR-1

Plasmid-mediated resistance to carbapenems and colistin in Enterobacteriaceae represents an emerging public health threat. Although animals have been identified as a relevant source of multidrug-resistant (MDR) bacteria, there are only a few reports on the presence of carbapenemases in animal isolates. In this study, 7850 faecal Escherichia coli isolates obtained from 2160 pigs were screened for carbapenem non-susceptibility using Mueller-Hinton agar supplemented with meropenem. Eleven isolates showed growth on meropenem-containing agar but only two proved positive by PCR for a carbapenemase gene, namely blaOXA-48-like. The two isolates were obtained from different pigs housed at the same farm in Italy and were not genetically related by multilocus sequence typing (MLST), comprising ST359 and ST641. Whole-genome sequencing revealed the presence of blaOXA-181 in both isolates; in addition, the colistin resistance gene mcr-1 and aminoglycoside resistance gene armA were found in one isolate. The blaOXA-181 resistance gene was located on a 51.5-kb non-conjugative plasmid of replicon type IncX3 and the mcr-1 gene on a 33.3-kb transferable IncX4 plasmid. The high nucleotide similarity (>99%) of plasmids pEcIHIT31346-OXA-181 and pEcIHIT31346-MCR-1 to published plasmids from various human and animal sources suggests that specific antibiotic resistance plasmids are circulating among E. coli strains worldwide and across vertebrate species barriers. Although carbapenems are not licensed for use in livestock and the overall prevalence of carbapenemases in porcine E. coli appears to be low, the current findings indicate that even pigs can host MDR strains with accumulated plasmid-mediated resistance against several last-line antibiotics.