Ivo Papousek

Plasmid-Mediated Resistance to Cephalosporins and Fluoroquinolones in Various Escherichia coli Sequence Types Isolated from Rooks Wintering in Europe

ABSTRACT Extended-spectrum-beta-lactamase (ESBL)-producing, AmpC beta-lactamase-producing, and plasmid-mediated quinolone resistance (PMQR) gene-positive strains of Escherichia coli were investigated in wintering rooks (Corvus frugilegus) from eight European countries. Fecal samples (n = 1,073) from rooks wintering in the Czech Republic, France, Germany, Italy, Poland, Serbia, Spain, and Switzerland were examined. Resistant isolates obtained from selective cultivation were screened for ESBL, AmpC, and PMQR genes by PCR and sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were performed to reveal their clonal relatedness. In total, from the 1,073 samples, 152 (14%) cefotaxime-resistant E. coli isolates and 355 (33%) E. coli isolates with reduced susceptibility to ciprofloxacin were found. Eighty-two (54%) of these cefotaxime-resistant E. coli isolates carried the following ESBL genes: bla CTX-M-1 (n = 39 isolates), bla CTX-M-15 (n = 25), bla CTX-M-24 (n = 4), bla TEM-52 (n = 4), bla CTX-M-14 (n = 2), bla CTX-M-55 (n = 2), bla SHV-12 (n = 2), bla CTX-M-8 (n = 1), bla CTX-M-25 (n = 1), bla CTX-M-28 (n = 1), and an unspecified gene (n = 1). Forty-seven (31%) cefotaxime-resistant E. coli isolates carried the bla CMY-2 AmpC beta-lactamase gene. Sixty-two (17%) of the E. coli isolates with reduced susceptibility to ciprofloxacin were positive for the PMQR genes qnrS1 (n = 54), qnrB19 (n = 4), qnrS1 and qnrB19 (n = 2), qnrS2 (n = 1), and aac(6′)-Ib-cr (n = 1). Eleven isolates from the Czech Republic (n = 8) and Serbia (n = 3) were identified to be CTX-M-15-producing E. coli clone B2-O25b-ST131 isolates. Ninety-one different sequence types (STs) among 191 ESBL-producing, AmpC-producing, and PMQR gene-positive E. coli isolates were determined, with ST58 (n = 15), ST10 (n = 14), and ST131 (n = 12) predominating. The widespread occurrence of highly diverse ESBL- and AmpC-producing and PMQR gene-positive E. coli isolates, including the clinically important multiresistant ST69, ST95, ST117, ST131, and ST405 clones, was demonstrated in rooks wintering in various European countries.

Extended spectrum beta‐lactamase and fluoroquinolone resistance genes and plasmids among Escherichia coli isolates from zoo animals, Czech Republic

Commensal Escherichia coli isolates from healthy zoo animals kept in Ostrava Zoological Garden, Czech Republic, were investigated to evaluate the dissemination of extended-spectrum beta-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes. A total of 160 faecal samples of various animal species were inoculated onto MacConkey agar with cefotaxime (2 mg L(-1)) or ciprofloxacin (0.05 mg L(-1)) to obtain ESBL- or PMQR-positive E. coli isolates. Clonality of E. coli isolates was investigated by multilocus sequence typing and pulsed-field gel electrophoresis. Plasmids carrying ESBL or PMQR genes were typed by PCR-based replicon typing, plasmid multilocus sequence typing and restriction fragment length polymorphism. Forty-nine (71%, n = 69) cefotaxime-resistant and 15 (16%, n = 94) ciprofloxacin-resistant E. coli isolates harboured ESBL or PMQR genes. Isolates were assigned to 18 sequence types (ST) and 20 clusters according to their macrorestriction patterns by pulsed-field gel electrophoresis. The genes blaCTX -M-1 and qnrS1 were detected on highly related IncI1 plasmids assigned to clonal complex 3 (ST3, ST38) and on non-related IncN plasmids of ST1 and ST3, respectively. The gene qnrS1 was located on related IncX1 plasmids. Dissemination of antibiotic resistance is associated with spreading of particular E. coli clones and plasmids of specific incompatibility groups among various animal species.

Haemogregarines from western Palaearctic freshwater turtles (genera Emys, Mauremys ) are conspecific with Haemogregarina stepanowi Danilewsky, 1885

The majority of Haemogregarina species have been based on the morphology of their erythrocytic stages and supposed strict host specificity. The quantity of species with a limited number of overlapping diagnostic traits has led to a considerable mess in haemogregarine taxonomy and significant synonymy. We analysed host specificity, intra- and interspecific variability, evolutionary relationships, and the distribution of the type species of the genus Haemogregarina--H. stepanowi. The morphology of blood stages and 18S rDNA sequences of this haemogregarine from four western Palaearctic hard-shelled freshwater turtles (Emys orbicularis, Mauremys caspica, Mauremys leprosa and Mauremys rivulata) were compared with Haemogregarina balli. Additional sequences of 18S rDNA of Haemogregarina-like isolates collected from three species of African hinged terrapins (genus Pelusios) were used to enlarge the dataset for phylogenetic analyses. Thirteen sequences (1085 bp) of Haemogregarina representing all four western Palaearctic turtle species were identical, corresponding to H. stepanowi, which is closely related to the Nearctic species H. balli. In our analyses, Haemogregarina spp. constituted a monophyletic clade sister to the genus Hepatozoon. Haemogregarina stepanowi possesses a wide distribution range from the Maghreb, through Europe, Turkey and the Middle East to Iran. We consider that the genus Haemogregarina has a low host specificity crossing the family level of its vertebrate hosts and that its distribution is likely to be linked to the vector and definitive host--the leech.

Antimicrobial-resistant Enterobacteriaceae from humans and wildlife in Dzanga-Sangha Protected Area, Central African Republic

Antimicrobial resistance is a worldwide concern of public health. Unfortunately, resistant bacteria are spreading to all ecosystems, including the strictly protected ones. We investigated antimicrobial resistance in gastrointestinal Enterobacteriaceae of wild mammals and people living within Dzangha-Sangha Protected Areas, Central African Republic, with an emphasis on extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes. We compare resistance genes found in microbiota of humans, gorillas habituated and unhabituated to humans and other wildlife. In gorillas, we additionally investigate the presence of ESBL resistant isolates after treatment by ceftiofur. We found a considerable prevalence of multiresistant Enterobacteriaceae isolates with ESBL and PMQR genes in humans (10% and 31%, respectively). Among wildlife the most significant findings were CTX-M-15-producing Klebsiella pneumoniae in a habituated gorilla and a multiresistant Escherichia coli isolate with gene qepA in an unhabituated gorilla. Other isolates from wildlife were mostly represented by qnrB-harboring Citrobacter spp. The relatedness of resistant E. coli was investigated in a PFGE-based dendrogram; isolates from gorillas showed less than 80% similarity to each other and less than 80% similarity to human isolates. No ESBL-producing isolates were found in animals treated by ceftiofur. Although we did not detect any bacterial clone common to wildlife and humans, we detected an intersection in the spectrum of resistance genes found in humans and gorillas, represented by blaCTX-M-15 and qepA.

Characteristics of Quinolone Resistance in Escherichia coli Isolates from Humans, Animals, and the Environment in the Czech Republic

Escherichia coli is a common commensal bacterial species of humans and animals that may become a troublesome pathogen causing serious diseases. The aim of this study was to characterize the quinolone resistance phenotypes and genotypes in E. coli isolates of different origin from one area of the Czech Republic. E. coli isolates were obtained from hospitalized patients and outpatients, chicken farms, retailed turkeys, rooks wintering in the area, and wastewaters. Susceptibility of the isolates grown on the MacConkey agar with ciprofloxacin (0.05 mg/L) to 23 antimicrobial agents was determined. The presence of plasmid-mediated quinolone resistance (PMQR) and ESBL genes was tested by PCR and sequencing. Specific mutations in gyrA, gyrB, parC, and parE were also examined. Multilocus sequence typing and pulsed-field gel electrophoresis were performed to assess the clonal relationship. In total, 1050 E. coli isolates were obtained, including 303 isolates from humans, 156 from chickens, 105 from turkeys, 114 from the rooks, and 372 from wastewater samples. PMQR genes were detected in 262 (25%) isolates. The highest occurrence was observed in isolates from retailed turkey (49% of the isolates were positive) and inpatients (32%). The qnrS1 gene was the most common PMQR determinant identified in 146 (56%) followed by aac(6′)-Ib-cr in 77 (29%), qnrB19 in 41 (16%), and qnrB1 in 9 (3%) isolates. All isolates with high level of ciprofloxacin resistance (>32 mg/L) carried double or triple mutations in gyrA combined with single or double mutations in parC. The most frequently identified substitutions were Ser(83)Leu; Asp(87)Asn in GyrA, together with Ser(80)Ile, or Glu(84)Val in ParC. Majority of these isolates showed resistance to beta-lactams and multiresistance phenotype was found in 95% isolates. Forty-eight different sequence types among 144 isolates analyzed were found, including five major clones ST131 (26), ST355 (19), ST48 (13), ST95 (10), and ST10 (5). No isolates sharing 100% relatedness and originating from different areas were identified. In conclusion, our study identified PMQR genes in E. coli isolates in all areas studied, including highly virulent multiresistant clones such as ST131 producing CTX-M-15 beta-lactamases.

Hidden threat of tortoise ticks: high prevalence of Crimean-Congo haemorrhagic fever virus in ticks Hyalomma aegyptium in the Middle East

It is the first time that Crimean-Congo haemorrhagic fever virus (CCHFV), causing potentially lethal disease of humans, has been reported from the Middle East region and from the tortoise tick Hyalomma aegyptium from a tortoise host, whose epidemiological significance may have remained almost completely overlooked so far. We used RT-PCR to screen for 245 ticks collected from 38 Testudo graeca tortoise individuals. Results of our genetic screening provide unambiguous evidence of occurrence of CCHFV in this region and host, suggesting a potentially important role of H. aegyptium in CCHF epidemiology.

Eye trematode infection in small passerines in Peru caused by Philophthalmus lucipetus, an agent with a zoonotic potential spread by an invasive freshwater snail.

Until now, four species of eye trematodes have been found in South America. Of them, Philophthalmus lucipetus (synonymized with Philophthalmus gralli) displays a broad host spectrum, with at least 30 bird species (prevalently large water birds), five mammal species and humans serving as definitive hosts, and with snails Fagotia (Microcolpia) acicularis, Amphimelania holandri, Melanopsis praemorsa and Melanoides tuberculata serving as intermediate hosts. When examining a total of 50 birds of ten species in the wetland of Pantanos de Villa, Lima, Peru in July 2011, eye trematodes were identified visually in the edematous conjunctival sac of 11 (48%) out of 23 resident many-colored rush tyrants Tachuris rubrigastra. Based on morphometric characteristics, the trematodes were identified as P. lucipetus. ITS2 and CO1 gene of the examined specimens combined showed a 99% similarity to an Iranian isolate of Philophthalmus sp. from the intermediate host Melanoides tuberculata, an invasive freshwater snail, suggesting that these two isolates represent the same species with a wide geographical range. Moreover, the prevalence of infection with the philophthalmid cercariae was 31% in 744 Melanoides tuberculata examined in Pantanos de Villa in 2010. It is evident that P. lucipetus occurs throughout the world as well as locally, including Eurasia and South America. Here we report this trematode for the first time in Peru, and we were the first to sequence any of the South American eye trematodes. Low host specificity of P. lucipetus and the invasive character of Melanoides tuberculata as a competent intermediate host suggest that eye trematodosis caused by P. lucipetus may emerge frequently in various parts of the world, especially in the tropics. Increase of the zoonotic potential of the P. lucipetus associated with this invasive snail spreading across the world is predictable and should be of interest for further research.

Plasmid-Mediated Quinolone Resistance Genes in Enterobacteriaceae from American Crows: High Prevalence of Bacteria with Variable qnrB Genes

Though wild birds are not normally exposed to use of antimicrobial agents, they can acquire antibiotic-resistant bacteria through the environment ([1][1]). It was also suggested that rooks may disseminate these bacteria over long distances and pose a risk of contaminating the environment ([2][2]). A

Chewing lice (Phthiraptera: Amblycera and Ischnocera) from wild birds in southern Vietnam, with descriptions of two new species.

A total of 239 individuals of 50 bird species were examined for chewing lice (Insecta: Phthiraptera) in southern Vietnam. Fifty-six birds of 20 species were parasitised by 15 species of lice belonging to 10 genera from two suborders, Amblycera: Menacanthus, Meromenopon, Myrsidea, and Ischnocera: Alcedoecus, Brueelia, Cuculicola, Meropoecus, Penenirmus, Philopteroides and Philopterus. Thirteen louse samples from Passeriformes were identified to genus only because they contain inadequate material. A total of 29 host-louse associations were found, of which nine are new, including: (1) two new species of the genus Brueelia, which are described and named in this paper: Brueelia binhchauensis from Megalaima lineata (Vieillot, 1816) (Piciformes: Megalaimidae), and Brueelia malacocincla from Malacocincla abbotti Blyth, 1845 (Passeriformes: Pellorneidae); (2) first records of lice from Cyornis hainanus (Ogilvie-Grant, 1900); and (3) the first record of Myrsidea claytoni Hellenthal & Price, 2003 from Cymbirhynchus macrorhynchos (Gmelin, 1788) (Passeriformes: Eurylaimidae), here regarded as a case of natural host-switching. A portion of the mitochondrial cytochrome oxidase I (COI) gene for some species of chewing lice was sequenced in order to assess their genetic divergences.

Low rates of antimicrobial-resistant enterobacteriaceae in wildlife in Taï National Park, Côte d’Ivoire, surrounded by villages with high prevalence of multiresistant ESBL-producing Escherichia coli in people and domestic animals

Antimicrobial resistance genes can be found in all ecosystems, including those where antibiotic selective pressure has never been exerted. We investigated resistance genes in a collection of faecal samples of wildlife (non-human primates, mice), people and domestic animals (dogs, cats) in Côte d’Ivoire; in the chimpanzee research area of Taï National Park (TNP) and adjacent villages. Single bacteria isolates were collected from antibiotic-containing agar plates and subjected to molecular analysis to detect Enterobacteriaceae isolates with plasmid-mediated genes of extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR). While the prevalence of ESBL-producing E. coli in the villages was 27% in people (n = 77) and 32% in dogs (n = 38), no ESBL-producer was found in wildlife of TNP (n = 75). PMQR genes, mainly represented by qnrS1, were also present in human- and dog-originating isolates from the villages (36% and 42% in people and dogs, respectively), but no qnrS has been found in the park. In TNP, different variants of qnrB were detected in Citrobacter freundii isolates originating non-human primates and mice. In conclusion, ESBL and PMQR genes frequently found in humans and domestic animals in the villages were rather exceptional in wildlife living in the protected area. Although people enter the park, the strict biosecurity levels they are obliged to follow probably impede transmission of bacteria between them and wildlife.