Genome Mining for Antimicrobial Compounds in Wild Marine Animals-Associated Enterococci

New ecosystems are being actively mined for new bioactive compounds. Because of the large amount of unexplored biodiversity, bacteria from marine environments are especially promising. Further, host-associated microbes are of special interest because of their low toxicity and compatibility with host health. Here, we identified and characterized biosynthetic gene clusters encoding antimicrobial compounds in host-associated enterococci recovered from fecal samples of wild marine animals remote from human-affected ecosystems. Putative biosynthetic gene clusters in the genomes of 22 Enterococcus strains of marine origin were predicted using antiSMASH5 and Bagel4 bioinformatic software. At least one gene cluster encoding a putative bioactive compound precursor was identified in each genome. Collectively, 73 putative antimicrobial compounds were identified, including 61 bacteriocins (83.56%), 10 terpenes (13.70%), and 2 (2.74%) related to putative nonribosomal peptides (NRPs). Two of the species studied, Enterococcus avium and Enterococcus mundtti, are rare causes of human disease and were found to lack any known pathogenic determinants but yet possessed bacteriocin biosynthetic genes, suggesting possible additional utility as probiotics. Wild marine animal-associated enterococci from human-remote ecosystems provide a potentially rich source for new antimicrobial compounds of therapeutic and industrial value and potential probiotic application.     

桑蚕来源肠球菌的溶血性

从健康桑蚕消化道来源的肠球菌（Ｅｎｔｅｒｏｃｏｃｕｓ）中发现了α、β和γ（非）溶血性的菌株都有，而且β－溶血性菌株的分离比例较高（２２．５％）。这些β－溶血性菌株只在厌氧条件下得到好的表达。在ＡＰＩ２０ＳＴＰＥＰ（Ｖ．５．０）系统中这些菌株主要是一些Ｅｎｔ．ｄｕｒａｎｃｅ菌株和不能认定菌株。     

Possibility of vancomycin-resistant enterococci transmission from human to broilers, and possibility of using the vancomycin-resistant gram-positive cocci as a model in a screening study of vancomycin-resistant enterococci infection in the broiler chick

In this study, vancomycin‐resistant enterococci (VRE) from humans and vancomycin‐resistant gram‐positive cocci (VRPC) from pigs were examined for their ability to transmit in the chick intestine (Experiment 1). A model study on the spread speed of VRPC was also estimated from chick to chick under semi‐production conditions with different administration routes (not inoculated, oral administration to a chick, sprayed on the floor) (Experiment 2). Furthermore, the disappearance of VRPC from their litter with composting processes was examined (Experiment 3). Each of six chicks was inoculated with VRPC or VRE at 1 day old in Experiment 1. All the chicks had VRPC or VRE in their glandular stomach at 22 days of age. In Experiment 2, 6 floor pens covered with sawdust were prepared and 20 chicks were allotted to each pen. The chicks were inoculated with VRPC at 1 day of age. The VRPC were detected in each group in cloacal swabs at 2 days of age (detection rate; 20–80%). And they were also detected in the not‐inoculated group. The VRPC detection rate gradually decreased, and detection was rare (0–10%) in the packing chicks (50 days old). VRPC were detected in the litter of each pen in Experiment 2. A composting process was effectively used to eliminate VRPC by the 6th week (Experiment 3).     

Bacteriocin Activity of Enterococci from Rabbits

The antimicrobial spectrum of bacteriocin-producing enterococcal isolates from rabbits faeces, the biochemical characterization of bacteriocins, and their molecular mass and the presence of structural genes for bacteriocin production were investigated. Among enterococci selected from rabbit faeces, six strains of Enterococcus faecium (EF2019, EF1819, EF2119, EF1839, EF529, EF24/10) showed inhibitory activity against the indicators E. avium EA5, Listeria innocua LMG13568 and Listeria monocytogenes CCM4699 and against other enterococci and staphylococci tested. The molecular mass of bacteriocin-like substances ranged from 3 to 10 kDa. The presence of the structural genes for enterocins (ent) A, P and L50B was detected in all enterococci tested. However, no strain possessed the gene for ent B. E. faecium EF2019 showed the broadest inhibitory activity. A proteinaceous substance produced by the EF2019 strain was partially purified. This is a thermostable substance that is stable at pH 4.0, 7.0 and 9.0. Its production starts in the early logarithmic growth phase and culminates in the late logarithmic growth phase of the EF2019 strain. Partially purified bacteriocin (PPB) EF2019 added to the growing strain L. innocua LMG13568 (after 4 h) inhibited its growth as early as 1 h after addition with a decrease of 1.5 log cycles (5 h of cultivation). This effect was extended up to 24 h. The bacteriocinogenic E. faecium EF2019 strain and/or bacteriocin EF2019 could be utilized against contaminant bacteria, e.g. in rabbitries. More detailed studies under in vitro and in vivo conditions are in progress.     

Adhesion Properties of Enterococci to Intestinal Mucus of Different Hosts

The adhesive capacity of selected enterococci to human, canine and porcine intestinal mucus was investigated in order to select for potential probiotic strains with good adhesive properties for human or animal use. The adhesion to the human intestinal mucus of the tested strains was found to range from log10 3.8 to log10 8.6 cfu per microtitre plate well. The highest adhesion to the human intestinal mucus was found among strains from horse faeces, dog faeces and dog feed. The adhesion to canine mucus was observed to range from log10 3.8 to log10 8.3 cfu/well, with the highest adhesive capacity among strains from dog faeces, horse faeces and dog's feed; on average log10 7.9, 7.3 and 7.0 cfu/well, respectively. Isolates from dogs did not bind at higher levels to canine mucus than to human mucus. A strong correlation was observed for the adhesion to human and canine intestinal mucus (p < 0.0001) and also between porcine and canine or human mucus (p < 0.05 for both). When comparing the adhesion of Enterococcus faecium and E. faecalis, no statistical significant differences were observed for any of the tested mucus types. The tested Enterococcus strains were found to exhibit a strain dependent on in vitro adhesion to human, canine and porcine intestinal mucus and did not exhibit host specificity in their adhesion, though some sources appeared to contain more adhesive strains than others. To our knowledge, this is the first report on the in vivo adhesion to intestinal mucus of a large number of enterococci from different sources.     

The Zoonotic Potential of Daptomycin Non‐susceptible Enterococci

Daptomycin non‐susceptible Enterococcus (DNSE) is an emerging clinical problem. Little is known about how de novo DNSE infections develop or the risk factors associated with them. Determining risk factors associated with de novo DNSE infections will aid in understanding the mechanisms of daptomycin non‐susceptibility. Humans in contact with animals worldwide are at risk of carriage of multidrug‐resistant bacteria. Herein, I review the scientific evidence that supports the hypothesis that transport of daptomycin non‐susceptibility genes between animals and humans may be a possible mechanism for development of de novo daptomycin non‐susceptibility in enterococci.     

Concentrations of faecal coliforms,Escherichia coli,enterococci and Campylobacter spp. in equine faeces

Abstract

AIMS: To determine the concentration of Campylobacter spp. as well as faecal indicator bacteria; faecal coliforms, Escherichia coli and enterococci in the faeces of healthy adult horses in a sample of properties in the Canterbury region of New Zealand.

METHODS: The faeces of healthy adult horses (n=59), including ponies, pleasure horses and Thoroughbreds, were collected from eight properties around Christchurch, New Zealand. The faeces were analysed for concentrations of Campylobacter spp and faecal indicator bacteria; faecal coliforms, Escherichia coli and enterococci. The presence of other animals on the properties sampled as well as the age, feed and health of the horses at the time of sampling was recorded.

RESULTS: Enterococci and faecal coliforms were isolated from all samples, and E. coli was isolated from 58/59 samples. Mean concentrations of faecal coliforms and E. coli did not differ between properties, but there was a significant difference in mean concentration of enterococci between properties. Campylobacter spp. were detected in two faecal samples with one isolate being determined by PCR analysis to be a thermotolerant Campylobacter species, the other C. jejuni.

CONCLUSIONS: This is the first known report quantifying the concentration of Campylobacter spp. present in healthy adult horses in New Zealand. The presence of equine faecal material in water could elevate concentrations of faecal bacteria and therefore needs to be considered as a source of water contamination. The access of horses to waterways and coastal environments may also need to be restricted to prevent transmission of faecal indicator bacteria and potentially zoonotic agents.     

Study of Vancomycin Resistance in Faecal Enterococci from Healthy Humans and Dogs in Spain a Decade after the Avoparcin Ban in Europe

One hundred and 26 faecal samples from healthy dogs (2009) and 157 faecal samples from healthy humans (2007) from La Rioja region (Spain) were tested to know the carriage of vancomycin‐resistant enterococci (VRE). VRE with intrinsic resistance (vanC) were found in 12% of healthy dogs and humans (29 Enterococcus gallinarum and four Enterococcus casseliflavus). Nevertheless, VRE with acquired mechanism of resistance were not detected among these samples. Four Enterococcus faecalis isolates with vancomycin MIC of 8‐16 mg L^?1 were recovered in human samples, but no single organism with known mechanism of acquired resistance could be identified. These 37 VRE isolates (33 E. gallinarum/E. casseliflavus and four E. faecalis) of dog and human origin were further characterized (PCR detection of antibiotic resistance, virulence and bacteriocin genes). High prevalence of tetracycline resistance was identified (70%), especially among dog isolates harbouring tet(M) ± tet(L) genes; erythromycin resistance was also higher among isolates from dogs and they harboured the erm(B) gene, associated with erm(A) gene in one case. Virulence genes were only identified among E. faecalis isolates of human origin (agg, cpd and/or gelE) and never among E. gallinarum/E. casseliflavus of human or dog origin. Five E. gallinarum isolates of dog and three E. faecalis of human origin expressed bacteriocin activity; among them, only one E. faecalis presented activity against Listeria monocytogenes. The bacteriocin structural gene ef1097 was identified in 3 bacteriocin‐producing E. faecalis isolates, associated with ent1071 in one of them.     

Isolation and Molecular Characterization of Vancomycin‐Resistant Enterococcus faecium from Swine in Michigan,USA

In 2008, we identified vancomycin‐resistant enterococci (VRE) in Michigan swine, which was the first report of VRE in livestock from North America. Continued sampling in 2009 and 2010 was conducted to determine whether VRE persisted in Michigan. In 2009, swine faecal and feed samples (n = 56), county fair pig barn manure samples (n = 9) and pooled Michigan State Fair pig barn manure samples (n = 18) were screened for VRE. In 2010, swine faecal samples were collected from 26 county fairs (n = 73) and nine commercial swine farms in six states (n = 28). Recovered VRE isolates were molecularly evaluated by polymerase chain reaction, restriction fragment length polymorphism, pulsed‐field gel electrophoresis (PFGE), S1 nuclease digestion and multilocus sequence typing (MLST). Six VRE isolates were identified in 2009 from the State Fair, and another six (8.2%) were recovered from the five county fairs in 2010. All 12 isolates were highly related to the first‐reported VRE from Michigan swine: all were confirmed to be vancomycin‐resistant Enterococcus faecium (VREf) carrying vanA gene on Tn1546 (Type D), were negative for IS1251, hyl and esp gene, carried a 150–160 kb megaplasmid, and have closely similar PFGE patterns with >80% similarity. Classified as ST5, ST6 or ST185 by MLST, all belong to the clonal complex 5, a strain recognized to be circulating among European pigs. This study reveals that VREf are widespread in Michigan swine and persist in the historical absence of the use of agricultural glycopeptides.