Motile aeromonads from farmed and wild freshwater fish in northern Italy: an evaluation of antimicrobial activity and multidrug resistance during 2013 and 2016

Background Antimicrobial resistant bacteria are emerging biological contaminants of the environment. In aquatic ecosystems, they originate mainly from hospitals, livestock manure and private households sewage water, which could contain antimicrobial agents and resistant microorganisms. Aeromonas spp. occur ubiquitously in aquatic environments and they cause disease in fish. Motile aeromonads are also associated with human gastrointestinal and wound infections and fish can act as a transmission route of antimicrobial resistance (AMR) aeromonads to humans. The environmental ubiquity, the natural susceptibility to antimicrobials and the zoonotic potential of Aeromonas spp. make them optimal candidates for studying the AMR in aquatic ecosystems. Results The AMR patterns of 95 motile aeromonads isolated from freshwater fish during 2013 and 2016 were analyzed. All samples from fish came from farms and natural water bodies located in northern Italy, which is an area characterized by high anthropic impact on the environment. The isolates were biochemically identified as Aeromonas hydrophila, Aeromonas sobria or Aeromonas caviae and AMR was determined by the standard disk diffusion method. All isolates were resistant to cloxacillin, spiramycin and tilmicosin. High AMR frequencies (>\u200995%) were detected for tylosin, penicillin and sulfadiazine. AMR to danofloxacin, enrofloxacin, flumequine, ceftiofur, aminosidine, colistin, doxycycline, gentamicin, marbocyl and florfenicol was observed at low levels (<\u200910%). No AMR to cefquinome was found. Logistic regression showed several differences in antimicrobial activity between complexes. According to the source of aeromonads, only few differences in AMR between isolates from farmed and wild fish were observed. Conclusions Our data revealed an increasing trend of AMR to neomycin and apramycin among Aeromonas isolates during the study period, while resistance to erythromycin, tetracycline and thiamphenicol decreased. All isolates were multidrug resistance (MDR), but A. caviae showed the highest number of MDR per isolate. In most isolates, various degrees of MDR were detected to macrolides, quinolones, fluoroquinolones, polymyxins and cephalosporins (third and fourth generations), which are listed, by the World Health Organisation, to be among the highest priority and critically important antimicrobials in human medicine. Our findings underlined that freshwater fish can act as potential source of MDR motile aeromonads. Due to their zoonotic potential, this can pose serious threat to human health

Reduction of food phatogens prevalence in dietary S.cerevisiae-fed poultry orally challenged with S.enteritidis and C. jejuni.

The effect of yeast supplementation in broiler chickens on Salmonella enteritidis and Campylobacter jejuni contamination in faeces, cecum, breast, and neck skin was evaluated. Two groups (12 replicates/group, 20 Hubbard female chickens 1d old/replicate) were fed pre-starter (0-10d), starter (11-20d) and growing (21-38d) diets, and administered (Y) or not (C) Levucell\uae SB20 (Saccharomyces cerevisiae type boulardii I-1079; 106 CFU/g feed through a 0.05% premix). Birds were orally challenged at 10 days of life (S. enteritidis, 1x105 CFU/bird, and C. jejuni, 3x105 CFU/bird). On day 10 and 28 post-infection (PI), 10 animals/replicate were slaughtered and pooled ceca content of 5 birds/replicate was analysed for Salmonella and Campylobacter detection and enumeration together with total yeast count. Neck and breast skin were tested for Salmonella and Campylobacter presence on 1 subject/replicate. Data were analysed by a GLM procedure of SAS considering two experimental periods, from 0 to 20 days and from 20 to 38 days. Growth performance and faecal coliforms content were not affected by treatment. Higher yeast and lactobacilli (P=0.01) faecal count, and a significant decreased Salmonella enumeration and frequency in neck (-41%, P=0.03) and tendency in faeces (-25%; P=0.06), cecum (-25%; P=0.06), and breast skin (-33%; P=0.08) were found in Y group on day 38. No fecal Campylobacter was detected at 10d (P<0.01) or 28d (P=0.06) PI in Y birds, while in neck skin absence of Campylobacter was only recorded on day 10 PI (P=0.01). Campylobacter was significantly lower in Y birds in cecum (-42%; P=0.01), and breast skin (-58%; P=0.04) on 10d PI, while on day 38 it was reduced in breast skin (-42%; P=0.02), and tended to decrease in faeces (-25%; P=0.06). Saccharomyces cerevisiae (CNCM I-1079) significantly controlled Campylobacter carriage in chickens with some positive results also on Salmonella contamination, thus reducing the contamination of carcasses with both food borne pathogens

Sequence types and pleuromutilin susceptibility of Brachyspira hyodysenteriae isolates from Italian pigs with swine dysentery: 2003–2012

Swine dysentery is a mucohaemorrhagic colitis of pigs caused by infection with Brachyspira hyodysenteriae. The disease can be controlled by treatment with antimicrobial agents, with the pleuromutilins tiamulin and valnemulin being widely used. In recent years, the occurrence of B.hyodysenteriae with reduced susceptibility to these drugs has been increasing. The aim of this study was to determine temporal changes in genetic groups and pleuromutilin susceptibility amongst B.hyodysenteriae isolates from Italy. Multilocus sequence typing (MLST) was performed on 108 isolates recovered from 87 farms in different regions of Italy from 2003 to 2012, and their minimum inhibitory concentrations (MICs) for tiamulin and valnemulin were determined. Logistic regression was performed to assess associations between susceptibility to the two antimicrobial agents and genetic group, year and region of isolation. The isolates were allocated to 23 sequence types (STs), with five clonal clusters (Ccs) and seven singletons. More than 50% of isolates were resistant to both pleuromutilins (MIC >2.0 μg/mL for tiamulin and >1.0 μg/mL for valnemulin). All 10 isolates in ST 83 were resistant; these were first isolated in 2011 and came from nine farms, suggesting recent widespread dissemination of a resistant strain. Significant associations were found between the proportion of pleuromutilin susceptible isolates and the genetic group and year of isolation. Although resistant isolates were found in all Ccs, isolates in Ccs 2 and 7 were over five times more likely to be susceptible than those in the other Ccs. A significant trend in the reduction of susceptibility over time also was observed

Indagine sulla patogenicit&#224; di ceppi mobili di aeromonas spp. isolati da specie ittiche

Lo studio della patogenicit\ue0 di Aeromonas spp. \ue8 caratterizzato da notevole complessit\ue0, sviluppandosi in genere su pi\uf9 livelli e comprendendo saggi biologici in vitro (linee cellulari) ed in vivo (modelli animali), esami molecolari ed analisi delle propriet\ue0 fenotipiche degli isolati. Nell\u2019ambito di una tesi di specialit\ue0 (Allevamento, Igiene, Patologia delle Specie Acquatiche e Controllo dei Prodotti Derivati) sulla caratterizzazione in vitro della virulenza di questi microrganismi, sono stati esaminati 101 ceppi di Aeromonas mobili, provenienti da specie ittiche selvatiche (69), allevate (26), ed ornamentali (6). L\u2019isolamento \ue8 avvenuto da pesci setticemici (27) e apparentemente sani (74). Per ciascuno degli isolati, identificati biochimicamente a livello di complex, sono state analizzate le propriet\ue0 citotossiche su tre diverse linee cellulari (EPC, BF2, Vero) ed una serie di caratteristiche colturali (crescita a 37\ub0C, emolisi), biochimiche (test VP, decarbossilazione della lisina, fenomeno del suicidio) ed enzimatiche (lipasi, gelatinasi) indicate in letteratura come potenziali fattori di virulenza di Aeromonas spp. In particolare, si \ue8 indagato sulla correlazione fra questi fattori, la fenospecie dei ceppi (A. hydrophila, A. sobria, A. caviae), la provenienza (selvatica, allevata) ed il quadro clinico delle specie ittiche d\u2019isolamento. In generale, lo studio ha registrato alte prevalenze (54.5-99.0%) dei potenziali indicatori di patogenicit\ue0 all\u2019interno del campione testato. La produzione di lipasi (98.0%) e gelatinasi (99.0%) \ue8 stata il fattore pi\uf9 frequentemente riscontrato. Anche le caratteristiche colturali/biochimiche hanno mostrato un\u2019ampia diffusione fra i ceppi esaminati, con prevalenze variabili dal 70.3% (crescita a 37\ub0C) al 94.1% (fenomeno del suicidio: attivit\ue0 non suicida). L\u2019attivit\ue0 citotossica ha rappresentato il fattore di virulenza meno prevalente (54.5%). Dai risultati \ue8 emersa una correlazione della capacit\ue0 di crescita a 37\ub0C, attivit\ue0 emolitica e citotossica con la fenospecie dei ceppi, in particolare con i complex A. caviae (crescita a 37\ub0C) ed A. hydrophila (attivit\ue0 emolitica e citotossica). Ad esclusione dell\u2019azione decarbossilasica sulla lisina, prevalente negli isolati selvatici, nessuno dei markers di virulenza \ue8 risultato associato all\u2019origine dei ceppi. Solamente la crescita a 37\ub0C \ue8 stata correlata con presenza di patologia nei pesci, indicando una scarsa significativit\ue0 dei fattori fenotipici analizzati nella caratterizzazione in vitro in tal senso della patogenicit\ue0 di Aeromonas spp. Dei sistemi cellulari testati, le cellule Vero si sono dimostrate le pi\uf9 sensibili alle citotossine prodotte da Aeromonas spp. Si ritiene comunque che l\u2019importanza di utilizzare le cellule Vero sia legata, oltre ad una migliore visualizzazione dell\u2019effetto citopatico, anche alla possibilit\ue0 di individuare ceppi in grado di esprimere il loro potenziale patogeno nei mammiferi e nell\u2019uomo, il quale andrebbe per\uf2 confermato mediante l\u2019impiego dei tradizionali modelli animali. Ulteriori studi si rendono necessari per meglio definire il significato clinico dei fattori di virulenza di Aeromonas spp. nei pesci, da eseguirsi su un maggior numero di isolati ed associando l\u2019esame dei caratteri fenotipici dei ceppi alle analisi molecolari e, soprattutto, alle prove biologiche in vivo

Mycoplasma bovis infection in respiratory disease of dairy calves less than one month old

Mycoplasma bovis is an important cause of bovine respiratory disease, especially in young calves where it can also cause arthritis, tenosynovitis and otitis. During 2009 and 2010 a survey was carried out on carcasses of calves less than one month old sent to the Diagnostic Laboratory of the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna in Brescia, regardless of the presence of lung lesions, to detect this pathogen. PCR tests for Mycoplasma spp. and M. bovis were applied. 83 out of 224 (37%) lung tissue samples examined were positive at PCR test for Mycoplasma spp.; in 64 cases of these we observed typical respiratory lesions (P< 0.001). M. bovis was identified in 26 out of 83 (31%) lung tissue samples positive at PCR test for Mycoplasma spp.; in 24 cases of these we observed typical respiratory lesions (P= 0.039). Our data demonstrate that presence of Mycoplasma spp. and M. bovis positively correlates with pneumonic lung lesions in young dairy calve

Experimental induction of Escherichia coli diarrhoea in weaned piglets

Escherichia coli diarrhoea is a multifactorial condition which usually occurs during the post-weaning and is responsible for economic losses in pig production. One approach, to evaluate if substitute of antibiotic in vivo is effective in con- trolling postweaning diarrhea in the gastrointestinal tract ecosystem, is to use an appropriate disease model. However, there are still many criticisms related to the incidence and the severity of the diarrhoea in the experimental conditions. The aim of the study was to set up an Escherichia coli challenge model in order to induce a significant percentage of diarrhoea in weaned piglets for the evaluation of innovative compounds in vivo. A total of 35 piglets, weaned at 33 \uf0b1 2 days were randomized into 3 groups: control (CG), infected 1 (IG1) and infected 2 (IG2). One day after arrival piglets of IG1 and IG2 were orally inoculated with 3.7 7 108 CFU of Escherichia coli O149. All piglets were fed a high protein ration for 3 days. Daily health status and faeces were recorded by a point scale individually. Challenge strains in faecal samples were evaluated by polymerase chain reaction, serotyping and biochemical identification. Diarrhoea was ob- served in 96.67% (58.6% severe; 41.4% mild) of all infected piglets and occurred on average 1.3 days after the chal- lenge. The CG group presented one piglet with a transient mild diarrhoea. The E. coli challenge significantly affected the consistency and color of faeces (P < 0.001). The E. coli O149, mainly hemolytic (88%), was isolated in 56% of fae- cal samples and the 70% of piglets with severe diarrhoea shed E. coli O149 in the faeces. Zootechnical parameters did not show significant differences. The experimental conditions described in this study allowed to effectively induce di- arrhoea in weaned piglets. In conclusion a multifactorial approach (infectious, nutritional and management) is necessary to reproduce in vivo diarrhea in piglets

Severe Escherichia coli O111 septicaemia and polyserositis in hens at the start of lay

Three very severe episodes of Escherichia coli infection in hens from the same farm, at the beginning of laying, are reported. They were characterized by no clinical signs, but sudden mortality, from 5 to 10%, with severe lesions of septicaemia and fibrinous polyserositis. A Gram-negative bacterium was consistently isolated in pure culture from tissues. Isolates were typed biochemically as E. coli, but they were lactose negative and non-motile. The serotyping tests typed the isolates as somatic group O111. The isolates were sensitive to enrofloxacin, amoxycil and colimycin, and partially sensitive to flumequine, all of which were used for therapy. The disease was reproduced experimentally in both specific pathogen free chickens and commercial layers by intramuscular inoculation of the E. coli, but only in some layers when inoculated by the oro-nasal route. The stress of the onset of lay seemed to be the most probable precipitating cause of the disease