Janine Beutlich

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.

Virulotyping and antimicrobial resistance typing of Salmonella enterica serovars relevant to human health in Europe

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

The global establishment of a highly-fluoroquinolone resistant Salmonella enterica serotype Kentucky ST198 strain

While the spread of Salmonella enterica serotype Kentucky resistant to ciprofloxacin across Africa and the Middle-East has been described recently, the presence of this strain in humans, food, various animal species (livestock, pets, and wildlife) and in environment is suspected in other countries of different continents. Here, we report results of an in-depth molecular epidemiological study on a global human and non-human collection of S. Kentucky (n = 70). We performed XbaI-pulsed field gel electrophoresis and multilocus sequence typing, assessed mutations in the quinolone resistance-determining regions, detected β-lactam resistance mechanisms, and screened the presence of the Salmonella genomic island 1 (SGI1). In this study, we highlight the rapid and extensive worldwide dissemination of the ciprofloxacin-resistant S. Kentucky ST198-X1-SGI1 strain since the mid-2000s in an increasingly large number of contaminated sources, including the environment. This strain has accumulated an increasing number of chromosomal and plasmid resistance determinants and has been identified in the Indian subcontinent, Southeast Asia and Europe since 2010. The second substitution at position 87 in GyrA (replacing the amino acid Asp) appeared helpful for epidemiological studies to track the origin of contamination. This global study provides evidence leading to the conclusion that high-level resistance to ciprofloxacin in S. Kentucky is a simple microbiological trait that facilitates the identification of the epidemic clone of interest, ST198-X1-SGI1. Taking this into account is essential in order to detect and monitor it easily and to take rapid measures in livestock to ensure control of this infection.

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.

Antimicrobial Resistance and Virulence Determinants in European Salmonella Genomic Island 1-Positive Salmonella enterica Isolates from Different Origins

ABSTRACT Salmonella genomic island 1 (SGI1) contains a multidrug resistance region conferring the ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance phenotype encoded by bla PSE-1, floR, aadA2, sul1, and tet(G). Its increasing spread via interbacterial transfer and the emergence of new variants are important public health concerns. We investigated the molecular properties of SGI1-carrying Salmonella enterica serovars selected from a European strain collection. A total of 38 strains belonging to S. enterica serovar Agona, S. enterica serovar Albany, S. enterica serovar Derby, S. enterica serovar Kentucky, S. enterica serovar Newport, S. enterica serovar Paratyphi B dT+, and S. enterica serovar Typhimurium, isolated between 2002 and 2006 in eight European countries from humans, animals, and food, were subjected to antimicrobial susceptibility testing, molecular typing methods (XbaI pulsed-field gel electrophoresis [PFGE], plasmid analysis, and multilocus variable-number tandem-repeat analysis [MLVA]), as well as detection of resistance and virulence determinants (PCR/sequencing and DNA microarray analysis). Typing experiments revealed wide heterogeneity inside the strain collection and even within serovars. PFGE analysis distinguished a total of 26 different patterns. In contrast, the characterization of the phenotypic and genotypic antimicrobial resistance revealed serovar-specific features. Apart from the classical SGI1 organization found in 61% of the strains, seven different variants were identified with antimicrobial resistance properties associated with SGI1-A (S. Derby), SGI1-C (S. Derby), SGI1-F (S. Albany), SGI1-L (S. Newport), SGI1-K (S. Kentucky), SGI1-M (S. Typhimurium), and, eventually, a novel variant similar to SGI1-C with additional gentamicin resistance encoded by aadB. Only minor serovar-specific differences among virulence patterns were detected. In conclusion, the SGI1 carriers exhibited pathogenetic backgrounds comparable to the ones published for susceptible isolates. However, because of their multidrug resistance, they may be more relevant in clinical settings.

Characterization of illegal food items and identification of foodborne pathogens brought into the European Union via two major German airports

Foods of animal origin brought illegally from third party countries into the European Community pose a risk for the introduction of diseases. This can lead to animal disease outbreaks with significant economic and social costs and subsequent severe trade restrictions. Further, disease outbreaks in humans due to illegally imported foods of animal origin have been described, yet, there are very few studies examining the potential human health impact. Passenger baggage is the most likely route by which illegal products enter a country. Therefore, the volume and geographic origin of foods of animal origin introduced illegally into Germany via the Frankfurt International Airport and Berlin-Schönefeld Airport by passenger luggage were characterized. Further, the occurrence of foodborne zoonotic bacteria such as Salmonella spp., Listeria spp., Campylobacter spp., Yersinia spp., Verocytotoxin-producing Escherichia coli (VTEC) and Brucella spp. and the microbial quality of the foods were analysed by total bacterial count. Between 2012 and 2013, a total of 663 food items were seized from 296 passengers arriving in Germany from 35 different departure countries. The majority of confiscates (51%) originated from Turkey and Russia. A selection of 474 samples was subjected to microbiological analyses. Twenty-three food products tested positive for at least one of the pathogens analysed. The majority of the contaminated foods were meat (33%) or meat products (42%), and milk products (21%). Considering that only a small fraction of arriving passengers is subjected to airport custom controls and only a small number of confiscated foods could be analysed during this study, further investigations are needed to understand the public health risks posed by illegally introduced food items.

Diversity of Plasmids Encoding Virulence and Resistance Functions in Salmonella enterica subsp. enterica Serovar Typhimurium Monophasic Variant 4,[5],12:i:- Strains Circulating in Europe

Plasmids encoding resistance and virulence properties in multidrug resistant (MDR) Salmonella enterica (S.) serovar Typhimurium monophasic variant 4,[5],12:i:- isolates recovered from pigs and humans (2006-2008) in Europe were characterised. The isolates were selected based on the detection by PCR-amplification of S. Typhimurium virulence plasmid pSLT genes and were analysed by multi-locus sequence typing (MLST). The resistance genes present in the isolates and the association of these genes with integrons, transposons and insertion sequences were characterised by PCR-sequencing, and their plasmid location was determined by alkaline lysis and by S1-nuclease pulsed-field gel electrophoresis (PFGE) Southern-blot hybridisation. Plasmids were further analysed by replicon typing, plasmid MLST and conjugation experiments. The 10 S. 4,[5],12,i:- selected isolates belonged to ST19. Each isolate carried a large plasmid in which MDR with pSLT-associated virulence genes were located. After analysis, eight different plasmids of three incompatibility groups (IncA/C, IncR and IncF) were detected. Two IncA/C plasmids represented novel variants within the plasmid family of the S. 4,[5],12:i:- Spanish clone, and carried an empty class 1 integron with a conventional qacEΔ1-sul1 3′ conserved segment or an In-sul3 type III with estX-psp-aadA2-cmlA1-aadA1-qacH variable region linked to tnpA440-sul3, part of Tn2, Tn21 and Tn1721 transposons, and ISCR2. Four newly described IncR plasmids contained the resistance genes within In-sul3 type I (dfrA12-orfF-aadA2-cmlA1-aadA1-qacH/tnpA440-sul3) and part of Tn10 [tet(B)]. Two pSLT-derivatives with FIIs-ST1+FIB-ST17 replicons carried cmlA1-[aadA1-aadA2]-sul3-dfrA12 and bla TEM-1 genes linked to an In-sul3 type I integron and to Tn2, respectively. In conclusion, three emerging European clones of S. 4,[5],12:i:- harboured MDR plasmids encoding additional virulence functions that could contribute significantly to their evolutionary success.

A Predominant Multidrug-Resistant Salmonella enterica Serovar Saintpaul Clonal Line in German Turkey and Related Food Products

ABSTRACT Recently, Salmonella enterica subsp. enterica serovar Saintpaul has increasingly been observed in several countries, including Germany. However, the pathogenic potential and epidemiology of this serovar are not very well known. This study describes biological attributes of S. Saintpaul isolates obtained from turkeys in Germany based on characterization of their pheno- and genotypic properties. Fifty-five S. Saintpaul isolates from German turkeys and turkey-derived food products isolated from 2000 to 2007 were analyzed by using antimicrobial agent, organic solvent, and disinfectant susceptibility tests, isoelectric focusing, detection of resistance determinants, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and hybridization experiments. These isolates were compared to an outgroup consisting of 24 S. Saintpaul isolates obtained from humans and chickens in Germany and from poultry and poultry products (including turkeys) in Netherlands. A common core resistance pattern was detected for 27 German turkey and turkey product isolates. This pattern included resistance (full or intermediate) to ampicillin, amoxicillin-clavulanic acid, gentamicin, kanamycin, nalidixic acid, streptomycin, spectinomycin, and sulfamethoxazole and intermediate resistance or decreased susceptibility to ciprofloxacin (MIC, 2 or 1 μg/ml, respectively) and several third-generation cephalosporins (including ceftiofur and cefoxitin [MIC, 4 to 2 and 16 to 2 μg/ml, respectively]). These isolates had the same core resistance genotype, with blaTEM-1, aadB, aadA2, sul1, a Ser83→Glu83 mutation in the gyrA gene, and a chromosomal class 1 integron carrying the aadB-aadA2 gene cassette. Their XbaI, BlnI, and combined XbaI-BlnI PFGE patterns revealed levels of genetic similarity of 93, 75, and 90%, respectively. This study revealed that a multiresistant S. Saintpaul clonal line is widespread in turkeys and turkey products in Germany and was also detected among German human fecal and Dutch poultry isolates.

Identification and molecular characterization of pathogenic bacteria in foods confiscated from non-EU flights passengers at one Spanish airport

Two hundred food samples of animal origin confiscated from passengers arriving on flights from non-European countries at the International Airport of Bilbao (Spain) were tested for the presence of four main bacterial foodborne pathogens (Campylobacter spp., Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp.) during 2012 and 2013. Overall, 20 samples were positive for L. monocytogenes (10%) and 11 for Salmonella spp. (5.5%), whereas Campylobacter spp. and E. coli O157:H7 were not detected in any sample. The positive isolates were widely clustered: 14 and 7 different pulsotypes for L. monocytogenes and Salmonella spp. isolates, respectively. Nine sequence types (ST) were detected for L. monocytogenes: ST2 (45%), ST9 (15% isolates), ST8 and ST87 (10%), and ST308, ST37, ST155 and ST378 (5%). The Salmonella spp. isolates belonged to seven serovars: monophasic serovar 4,12:d:- (3; 27.3%), Rauform (2; 18.2%), Anatum (2; 18.2%), Oranienburg, Enteritidis, Newport and Typhimurium (1; 9.1% each). Antibiotic resistance among L. monocytogenes isolates was high, especially for clindamycin and daptomycin (more than 95% of the isolates). These results indicate that food samples imported by travelers in their personal luggage may harbor the most prevalent L. monocytogenes genotypes and Salmonella spp. serovars responsible for foodborne outbreaks worldwide. Consequently, international travel can play an important role in the prevalence and dissemination of successful clones of foodborne pathogenic bacteria, and continuous monitoring of international movements is of importance to better understand clonal evolution and emergence and dissemination of successful lineages.

Virulence and antimicrobial resistance determinants of verotoxigenic Escherichia coli (VTEC) and of multidrug-resistant E. coli from foods of animal origin illegally imported to the EU by flight passengers.

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx(1) (variants stx1(a) or stx(1c)) or st(x2) (variants stx(2a), stx(2b), stx(2a/d) or stx(2c/d)) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe.