Giorgia Dotto

High Prevalence of oqxAB in Escherichia coli Isolates from Domestic and Wild Lagomorphs in Italy

This study aimed to identify and characterize class 1 and 2 integrons and plasmid-mediated quinolones resistance (PMQR) genes in a collection of 113 multidrug resistance (MDR) Escherichia coli isolated from farm and wild lagomorphs between 2006 and 2008 in Northern Italy. Strains were examined for antimicrobial susceptibility by agar disk diffusion method and E-test for colistin (COL); integrons and gene cassettes content by real-time polymerase chain reaction (PCR) and DNA sequencing; PMQR genes by PCR and DNA sequencing; clonal relatedness by multilocus sequence typing; and plasmids by PCR-based replicon typing. Class 1 integrons were detected in 69 isolates (47 farm rabbits, 14 wild rabbits, and 8 wild hares). No class 2 integrons were found. Five different gene cassettes arrays were identified (aadA1, dfrA1-aadA1, orf in682-dhfrA5, orf in682-dfrA5-orfD ins21, and dfrA17-aadA5). Fifteen percent (17/113) of isolates carried oqxAB, no other PMQR determinants. All but one oqxAB-positive E. coli strains were recovered from farm rabbits. Seven out of 17 strains were associated with the predominant ST238 and carried from three to six different plasmid types, such as IncF, IncHI1, IncI1, IncN, IncP, IncX1, IncY, and ColE. COL resistance was identified in 6/113 strains (5.3%). This study provides new insights on the resistance phenotypes and the prevalence and dissemination of oqxAB in E. coli from farm and wild lagomorphs, suggesting that these animals may be reservoir of these genetic determinants in Italy and thus a potential source of PMQR E. coli for humans. PMQR mediated by oqxAB has not been detected in farm and wild lagomorphs before.

International trades, local spread and viral evolution: The case of porcine circovirus type 2 (PCV2) strains heterogeneity in Italy

Porcine circovirus type 2 is one of the most widespread and economically relevant infections of swine. Four genotypes have been recognized, but currently, only three (PCV2a, PCV2b and PCV2d) are effectively circulating. The widespread livestock trade and rapid viral evolution have contributed to determining the high heterogeneity of PCV2 and the dispersal of potentially more virulent strains. Italian swine farming and the related processing industry are relevant in the national economy. Despite the noteworthy losses associated with direct and control measure costs, no data are currently available on the molecular epidemiology of PCV2 in Italy. Our study, which was intended to fill this gap, considered 75 completed genome PCV2 sequences, which were obtained from samples collected from the highly densely populated area of Northern Italy between 2007 and 2014. Phylogenetic analysis and comparison with reference sequences demonstrated the co-circulation, with different prevalences, of PCV2a, PCV2b and PCV2d within the national borders, with PCV2b being the most prevalent. Recombination between different genotypes was also proven to be frequent. Phylogeographic analysis demonstrated that the marked variability of Italian PCV2 strains can be attributable to multiple introduction events. The comparison of the phylogenetic analysis results, the location of different haplotypes and the international commercial routs of live pigs allow the speculation of several links as well as the role of Italy as both an importer and exporter of PCV2 haplotypes, mainly from and to European and Asian countries. A similarly intricate contact network was demonstrated within national borders, with different haplotypes being detected in the same province and different provinces harbouring the same haplotype. Overall, this paper represents the first description of PCV2 in Italy and demonstrates that the high variability of circulating Italian strains is due to multiple introduction events, wide circulation within national boundaries and rapid viral evolution.

Class 1 and class 2 integrons in avian pathogenic Escherichia coli from poultry in Italy

The aim of this study was to investigate the occurrence of class 1 and 2 integrons in avian pathogenic Escherichia coli (APEC) from poultry in northern Italy. Strains were tested for phenotypic resistance to aminoglycosides and sulphonamides, and the association between the presence of integrons and the resistance to these antimicrobials was evaluated. A total of 299 isolates (158 from turkeys, 110 from broilers, and 31 from layer hens) were collected from 200 industrial farms. Antimicrobial susceptibility test by the disk diffusion method was performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. All strains were screened for the presence of class 1 and 2 integrons by PCR and sequencing. About 55% of APEC contained integrons (class 1, 49.8%; class 2, 10.4%). Different variants of the aadA (5 variants) and the dfrA (4 variants) genes, encoding for streptomycin and trimethoprim resistance respectively, were detected in integron-positive isolates. Less common gene cassettes, such as sat, estX, and orfF, were also identified. Fifteen and 4 gene cassette arrays were found among class 1 and 2 integrons, respectively. High levels of resistance were observed for triple sulphonamides (79.3%), streptomycin (67.2%), and sulfamethoxazole combined with trimethoprim (62.2%), whereas resistance against gentamycin (16.7%), kanamycin (14.7%), and apramycin 3.0%) was low. Integron positivity was significantly higher in isolates phenotypically resistant to aminoglycosides (63.6% vs. 37.8%, P<0.001) and sulfonamides (64.1% vs. 21.1%, P<0.001) than in susceptible ones. Integron-borne aminoglycoside and sulfonamide resistance in APEC represents a concern for the poultry industry in Italy, since they are among the most commonly used antimicrobials in poultry therapy.

Absence of class 1 and class 2 integrons among Campylobacter jejuni and Campylobacter coli isolated from poultry in Italy

in the GenBank non-redundant DNA database was examined. Two additional cases of ISAba1 associated with distinct ampC alleles were found in accession numbers EU604835 8 and AY325306. 3 In both of them, the IS was again in the same orientation and separated from the ATG initiation codon of ampC by 9 bp. The number of single nucleotide differences between the various ampC alleles is shown in Table 1. Two ceftazidime and cefotaxime resistant isolates in our collection that did not belong to GC1 or GC2 were found to carry ISAba1 upstream of ampC, and these were also sequenced. D46 isolated in 2010 in Sydney, Australia was ST110 (Oxford scheme) and RBH2 isolated in 1999 in Brisbane, Australia was ST125 (Oxford scheme). Each contained a distinct ampC allele (Table 1) and ISAba1 was again appropriately oriented and 9 bp away from the ampC initiation codon (GenBank accession numbers KF030679 and KF030678). The simplest explanation for the finding that ISAba1 was found in the same position and orientation relative to six different ampC alleles is that ISAba1 has repeatedly inserted at exactly the same position. Additional support for this conclusion comes from an examination of the sequences of ISAba1. A total of 10 single nucleotide polymorphisms, most of them near the ends of the IS, were found in various combinations when the six ISAba1 sequences were compared, and this suggests that different IS variants were inserted. The currently unexplained site specificity could contribute to the importance of this mechanism of resistance to third-generation cephalosporins. A detailed examination of the location of ISAba1 upstream of the intrinsic oxa-Ab gene, which it also activates, may shed further light on this issue. 5 Nigro SJ, Post V, Hall RM. Aminoglycoside resistance in multiply antibiotic-resistant Acinetobacter baumannii belonging to global clone 2 from Australian hospitals. 6 Mak JK, Kim MJ, Pham J et al. Antibiotic resistance determinants in nosocomial strains of multidrug-resistant Acinetobacter baumannii. 7 Hamidian M, Hall RM. AbaR4 replaces AbaR3 in a carbapenem resistant Acinetobacter baumannii isolate belonging to global clone 1 from an Australian hospital. 8 Lee HY, Chen CL, Wang SB et al. Imipenem heteroresistance induced by imipenem in multidrug-resistant Acinetobacter baumannii: mechanism and clinical implications. 9 Hamidian M, Hancock DP, Hall RM. Horizontal transfer of an ISAba125-activated ampC gene between Acinetobacter baumannii strains leading to cephalosporin resistance. Sir, Since their discovery in the late 1980s, integrons have been revealed to play …

Post-mortem investigations on a leatherback turtle Dermochelys coriacea stranded along the Northern Adriatic coastline

Leatherback sea turtles Dermochelys coriacea are regularly reported in the Mediterranean Sea but rarely reach the northern Adriatic Sea. In the summer of 2009, a well-preserved carcass of an adult female of this species was found dead along the coast of Lido di Venezia. A complete necropsy was carried out, along with evaluation of levels of tissue trace elements. The the post-mortem revealed acute severe bacterial gastroenteritis caused by Photobacterium damselae ssp. piscicida, an opportunistic agent that infected an apparently debilitated animal weakened by ingested plastic debris. High levels of heavy metals (Hg, Pb, Cd and As) found in the liver and kidneys might have contributed to the animals demise. These findings support previous indications that marine debris is one of the major threats to marine animals, particularly for critically endangered species such as the leatherback turtle.

Lethal septic shock after dental scaling in a healthy dog due to Ochrobactrum anthropi-contaminated propofol

A 10-year-old, 6-kg male Yorkshire terrier dog was scheduled for routine dental cleaning. No significant problem was observed either during anaesthesia, which was induced with propofol, or during recovery. However, 2 hours after discharge, the dogs owner returned to the clinic, complaining that the animal was lethargic and had had bloody diarrhoea. On physical examination the dog was depressed, dyspnoeic, tachycardic and hypoglycaemic. Despite supportive treatment, the dog deteriorated and died within a few hours.A presumed diagnosis of sepsis was confirmed by laboratory testing. Bacteriological and molecular examinations of both premortem blood samples and the anaesthetic, highlighted the presence of Ochrobactrum anthropi, an opportunistic pathogen usually associated with immunocompromised hosts with indwelling medical devices. To the authors’ knowledge, this is the first case of sepsis in a healthy dog due to contamination of an anaesthetic solution by O. anthropi, suggesting a potential role of this microorganism as an emerging pathogen.

Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy

This study is aimed at determining the antimicrobial resistance (AMR) and the presence of class 1 and 2 integrons in 48 avian pathogenic Escherichia coli (APEC) strains isolated from meat turkeys during three sequential production cycles. Thirty avian faecal E. coli (AFEC) strains from the first cycle were also analysed. Strains were tested for AMR against 25 antimicrobials by disk diffusion test and were screened for the presence of integrons and associated gene cassettes by polymerase chain reaction followed by sequencing. Genetic relatedness of isolates was established by pulsed-field gel electrophoresis. High levels of resistance were detected to tetracyclines, penicillins and sulphonamides in APEC and AFEC. Resistance to aminoglycosides, fluoroquinolones, cephalosporins and phenicols was variable, based on the antimicrobial drug and the isolate (APEC vs. AFEC). Full susceptibility to colistin was detected. Multidrug resistance of up to seven antimicrobial classes was exhibited by APEC (93.8%) and AFEC (100%). Nearly 44% of strains tested positive for class 1 and/or class 2 integrons containing the dfrA, aadA and sat2 genes, alone or in combination, coding for streptomycin/spectinomycin, trimethoprim and streptothricin resistance, respectively. The estX and orfF genes of unknown function were also detected. A significant association was found between the presence of integrons and the resistance to aminoglycosides and potentiated sulphonamides. The results of this study showed that AMR, multidrug resistance and class 1 and 2 integrons are widespread among pathogenic and commensal E. coli from Italian turkeys. More attention should be addressed to limit the use of antimicrobials in turkeys and the AMR of turkey E. coli.

Multilocus Sequence Typing of Commensal and Enteropathogenic Escherichia Coli from Domestic and Wild Lagomorphs in Italy

The aim of the study was to determine the multilocus sequence types of Escherichia coli from diseased farm rabbits and apparently healthy wild lagomorphs, and the genetic relatedness among them. Fifty-five enteropathogenic E. coli from reared rabbits and 32 from wild rabbits and hares were characterised by multilocus sequence typing (MLST) according to the Michigan State University Ec MLST scheme. Isolates were differentiated into 37 sequence types (STs), which were grouped into 8 clonal complexes (CCs). The most common ST was ST140 (CC31), followed by ST238 and ST119 (CC17). MLST analysis revealed 22 novel STs. Phylogenetic analyses showed a heterogeneous distribution of STs into 3 clusters of genetically related strains. The genetic relationship among STs of different origin and the detection of new, as well as previously described STs as human pathogens, indicate a widespread distribution and adaptability of particular lineages to different hosts. These findings highlight the need for further research to improve the knowledge about E. coli populations colonising the gut of lagomorphs and their zoonotic potential.