Clonal and plasmid-mediated flow of ESBL/AmpC genes in Escherichia coli in a commercial laying hen farm.

Resistance to third- and fourth-generation cephalosporins in Escherichia coli is mainly due to extended-spectrum beta-lactamases (ESBL) and AmpC cephalosporinases, which have been increasingly reported, mainly in isolates from humans and poultry. The aim of this study was to address the flow of antimicrobial resistance determinants in the full laying hen production cycle (four batches followed from day-old chicks to 83/84-week-old layers), using cephalosporin-resistant E. coli as a model and their characterization using whole genome sequencing (WGS). Fifteen out of 22 samples analysed yielded growth on MacConkey agar with cefotaxime (1 mg/L). Of these, 141 isolates were identified as E. coli and 47 were characterized by WGS. Genes detected were three ESBL (blaCTX-M-1 (n = 19); blaCTX-M-14 (n = 1); and blaSHV-12 (n = 9)) and one AmpC (blaCMY-2 (n = 13)). Some isolates only harboured blaTEM-1B (n = 2) or blaTEM-1D (n = 1). IncI1 plasmids were the main platform for ESBL/AmpC genes. In addition, five clones were identified harbouring blaCTX-M-1 (two), blaSHV-12 (one) and blaCMY-2 (two), drawing a clone-plasmid mixed flow model. Gene blaCTX-M-1 was found in the chromosomal DNA of clone 1 over 14 months, and in IncI1/ST3 plasmids over six months; over six months blaSHV-12 was found harboured by clone 3 (IncI1/ST26 plasmids), and 15 months later in a non-replicon detected plasmid. Finally, blaCMY-2 spread for at least 16 months, mainly by IncK2 (including clone 4) and IncI1/ST12 (clone 5) plasmids. Proper use of antimicrobials should be combined with other farm management strategies for the effective control of cephalosporin-resistant E. coli isolates in commercial layer farms.

Opportunistic feeding behaviour and Leishmania infantum detection in Phlebotomus perniciosus females collected in the human leishmaniasis focus of Madrid, Spain (2012-2018).

BACKGROUND: An outbreak of human leishmaniasis due to Leishmania infantum has been registered in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys carried out in the municipalities of Fuenlabrada, Leganés, Getafe and Humanes de Madrid showed that Phlebotomus perniciosus is the only potential vector. In this work, an intensive molecular surveillance was performed in P. perniciosus females captured in the region between 2012 and 2018. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1805 P. perniciosus females were analyzed for Leishmania infection, and 1189 of them also for bloodmeal identification. Eleven different species of vertebrate were detected by amplification and subsequent sequencing of the 359 bp cytb fragment. The most prevalent blood source identified was hare (n = 553, 46.51%), followed by rabbit (n = 262, 21.95%). Less frequent were cat (n = 45, 3.80%), human (n = 34, 2.90%), pig (n = 14, 1.20%), horse (n = 11, 0.93%), sheep (n = 3, 0.25%), rhea (n = 3, 0.25%), partridge (n = 1, 0.09%) and chicken (n = 1, 0.09%). The distribution of the blood meal sources varied between the different locations. Regarding L. infantum detection, PCR amplification of a fragment of kDNA, cpb gene and ITS1 region showed 162 positive specimens (8.97%). The highest infection rate was found in the municipality of Leganés (15.17%). CONCLUSIONS: The results of this molecular survey in P. perniciosus, the only leishmaniasis vector in the outbreak occurred in southwestern Madrid region, showed its opportunistic blood-feeding behaviour, high infection rates and the differences between the different points. This study was an essential part of the intensive surveillance plan in the area and the results obtained have supported the implementation of control measures in the outbreak.

Leishmania sp. detection and blood-feeding behaviour of Sergentomyia minuta collected in the human leishmaniasis focus of southwestern Madrid, Spain (2012-2017).

Phlebotomine sand flies are the only known vectors of Leishmania spp. protozoan which causes leishmaniasis in 98 countries. In Spain, 11 sand fly species are described, but only Phlebotomus perniciosus and Phlebotomus ariasi are proven vectors of the disease. On the other hand, Sergentomyia minuta is one of the most abundant and ubiquitous sand flies in this territory, although scarce information is available about this species. Sand flies from this genus are known for their preference to feed on cold-blooded animals and are traditionally involved in the transmission of reptile Leishmania. However, studies have suggested that Sergentomyia spp. could be implicated in the transmission of human pathogenic Leishmania. This study analyses blood meal preferences and Leishmania sp. infection of S. minuta sand flies from the largest human leishmaniasis outbreak in Europe. Sand flies were collected during entomological surveillance carries out from 2012 to 2017 in the active season of these dipterans, from May to October. Molecular detection of Leishmania spp. showed 68 positive specimens of S. minuta out of 377 (18%). The analysis of blood meal preferences by amplification of 359 bp fragment of cytochrome b gene revealed that blood preference of S. minuta is not only limited to reptiles, but they also feed on mammals, including humans. Results suggest the presence of a Leishmania sp., related to Leishmania tarentolae, cycle in S. minuta from the studied area. Although there is no evidence about its incrimination in the L. infantum transmission more investigation is needed to elucidate the intravectorial cycle of Leishmania spp. in S. minuta sand flies, their feeding behaviour and their potential contribution in Leishmania spp. epidemiology in the country.