Claudio Palmieri

Dysbiosis contributes to fibrogenesis in the course of chronic liver injury in mice

Nonalcoholic fatty liver disease (NAFLD) may lead to hepatic fibrosis. Dietary habits affect gut microbiota composition, whereas endotoxins produced by Gram‐negative bacteria stimulate hepatic fibrogenesis. However, the mechanisms of action and the potential effect of microbiota in the liver are still unknown. Thus, we sought to analyze whether microbiota may interfere with liver fibrogenesis. Mice fed control (CTRL) or high‐fat diet (HFD) were subjected to either bile duct ligation (BDL) or CCl4 treatment. Previously gut‐sterilized mice were subjected to microbiota transplantation by oral gavage of cecum content obtained from donor CTRL‐ or HFD‐treated mice. Fibrosis, intestinal permeability, bacterial translocation, and serum endotoxemia were measured. Inflammasome components were evaluated in gut and liver. Microbiota composition (dysbiosis) was evaluated by Pyrosequencing. Fibrosis degree was increased in HFD+BDL versus CTRL+BDL mice, whereas no differences were observed between CTRL+CCl4 and HFD+CCl4 mice. Culture of mesenteric lymph nodes showed higher density of infection in HFD+BDL mice versus CTRL+BDL mice, suggesting higher bacterial translocation rate. Pyrosequencing revealed an increase in percentage of Gram‐negative versus Gram‐postive bacteria, a reduced ratio between Bacteroidetes and Firmicutes, as well as a dramatic increase of Gram‐negative Proteobacteria in HFD+BDL versus CTRL+BDL mice. Inflammasome expression was increased in liver of fibrotic mice, but significantly reduced in gut. Furthermore, microbiota transplantation revealed more liver damage in chimeric mice fed CTRL diet, but receiving the microbiota of HFD‐treated mice; liver damage was further enhanced by transplantation of selected Gram‐negative bacteria obtained from cecum content of HFD+BDL‐treated mice. Conclusions: Dietary habits, by increasing the percentage of intestinal Gram‐negative endotoxin producers, may accelerate liver fibrogenesis, introducing dysbiosis as a cofactor contributing to chronic liver injury in NAFLD. (Hepatology 2014;59:1738–1749)

Streptococcus suis, an Emerging Drug-Resistant Animal and Human Pathogen

Streptococcus suis, a major porcine pathogen, has been receiving growing attention not only for its role in severe and increasingly reported infections in humans, but also for its involvement in drug resistance. Recent studies and the analysis of sequenced genomes have been providing important insights into the S. suis resistome, and have resulted in the identification of resistance determinants for tetracyclines, macrolides, aminoglycosides, chloramphenicol, antifolate drugs, streptothricin, and cadmium salts. Resistance gene-carrying genetic elements described so far include integrative and conjugative elements, transposons, genomic islands, phages, and chimeric elements. Some of these elements are similar to those reported in major streptococcal pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae and share the same chromosomal insertion sites. The available information strongly suggests that S. suis is an important antibiotic resistance reservoir that can contribute to the spread of resistance genes to the above-mentioned streptococci. S. suis is thus a paradigmatic example of possible intersections between animal and human resistomes.

Characterization of a Streptococcus suis tet(O/W/32/O)-Carrying Element Transferable to Major Streptococcal Pathogens

ABSTRACT Mosaic tetracycline resistance determinants are a recently discovered class of hybrids of ribosomal protection tet genes. They may show different patterns of mosaicism, but their final size has remained unaltered. Initially thought to be confined to a small group of anaerobic bacteria, mosaic tet genes were then found to be widespread. In the genus Streptococcus, a mosaic tet gene [tet(O/W/32/O)] was first discovered in Streptococcus suis, an emerging drug-resistant pig and human pathogen. In this study, we report the molecular characterization of a tet(O/W/32/O) gene-carrying mobile element from an S. suis isolate. tet(O/W/32/O) was detected, in tandem with tet(40), in a circular 14,741-bp genetic element (39.1% G+C; 17 open reading frames [ORFs] identified). The novel element, which we designated 15K, also carried the macrolide resistance determinant erm(B) and an aminoglycoside resistance four-gene cluster including aadE (streptomycin) and aphA (kanamycin). 15K appeared to be an unstable genetic element that, in the absence of recombinases, is capable of undergoing spontaneous excision under standard growth conditions. In the integrated form, 15K was found inside a 54,879-bp integrative and conjugative element (ICE) (50.5% G+C; 55 ORFs), which we designated ICESsu32457. An ∼1.3-kb segment that apparently served as the att site for excision of the unstable 15K element was identified. The novel ICE was transferable at high frequency to recipients from pathogenic Streptococcus species (S. suis, Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae), suggesting that the multiresistance 15K element can successfully spread within streptococcal populations.

Interspecies mobilization of an erm(T)-carrying plasmid of Streptococcus dysgalactiae subsp. equisimilis by a coresident ICE of the ICESa2603 family

OBJECTIVES The recently documented presence of almost identical, small, non-self-transmissible, erm(T)-carrying plasmids in clonally unrelated erythromycin-resistant isolates of Streptococcus pyogenes and Streptococcus agalactiae suggests that these plasmids somehow circulate in the streptococcal population. The objective of this study was to characterize the erm(T)-carrying genetic element in a clinical isolate of Streptococcus dysgalactiae subsp. equisimilis (Sde5580) and to provide a possible explanation for the spread of erm(T)-carrying plasmids in streptococci. METHODS The erm(T)-carrying element of Sde5580 was investigated by plasmid analysis, PCR experiments and sequencing. Transfer and retransfer experiments were performed using S. pyogenes, S. agalactiae and Streptococcus suis strains as recipients and by selection in the presence of suitable drug concentrations. Transconjugants were analysed by SmaI-macrorestriction analysis. Genetic studies also included PCR-restriction fragment length polymorphism analysis using HindIII endonuclease. RESULTS Sde5580 contained two mobile genetic elements: a 4950 bp erm(T)-carrying plasmid (p5580) almost identical to the non-self-transmissible erm(T)-carrying plasmids of S. pyogenes and S. agalactiae mentioned above, and an ~63 kb cadC/cadA-carrying integrative and conjugative element (ICESde3396-like) of the ICESa2603 family. p5580 was transferable at high frequency to the recipients of all three species through in trans mobilization by the coresident ICESde3396-like element. p5580 and ICESde3396-like were able to be transferred either separately or together. CONCLUSIONS This is the first evidence of horizontal transfer of an erm(T)-carrying plasmid between streptococci. In trans mobilization by coresident ICEs may be one mechanism for the spread of erm(T)-carrying plasmids in the streptococcal population.

Different Genetic Elements Carrying the tet(W) Gene in Two Human Clinical Isolates of Streptococcus suis

ABSTRACT The genetic support for tet(W), an emerging tetracycline resistance determinant, was studied in two strains of Streptococcus suis, SsCA and SsUD, both isolated in Italy from patients with meningitis. Two completely different tet(W)-carrying genetic elements, sharing only a tet(W)-containing segment barely larger than the gene, were found in the two strains. The one from strain SsCA was nontransferable, and aside from an erm(B)-containing insertion, it closely resembled a genomic island recently described in an S. suis Chinese human isolate in sequence, organization, and chromosomal location. The tet(W)-carrying genetic element from strain SsUD was transferable (at a low frequency) and, though apparently noninducible following mitomycin C treatment, displayed a typical phage organization and was named ΦSsUD.1. Its full sequence was determined (60,711 bp), the highest BLASTN score being Streptococcus pyogenes Φm46.1. ΦSsUD.1 exhibited a unique combination of antibiotic and heavy metal resistance genes. Besides tet(W), it contained a MAS (macrolide-aminoglycoside-streptothricin) fragment with an erm(B) gene having a deleted leader peptide and a cadC/cadA cadmium efflux cassette. The MAS fragment closely resembled the one recently described in pneumococcal transposons Tn6003 and Tn1545. These resistance genes found in the ΦSsUD.1 phage scaffold differed from, but were in the same position as, cargo genes carried by other streptococcal phages. The chromosome integration site of ΦSsUD.1 was at the 3′ end of a conserved tRNA uracil methyltransferase (rum) gene. This site, known to be an insertional hot spot for mobile elements in S. pyogenes, might play a similar role in S. suis.

Genetic determinants and elements associated with antibiotic resistance in viridans group streptococci

OBJECTIVES To investigate the distribution of erythromycin, tetracycline and chloramphenicol resistance mechanisms and determinants and the relevant genetic environments and elements in viridans group streptococci (VGS). METHODS A total of 263 VGS collected from routine throat swabs in 2010-12 and identified to the species level were studied. Antibiotic resistance determinants and the relevant genetic contexts and elements were determined using amplification and sequencing assays and restriction analysis. RESULTS The investigation provided original information on the distribution of resistance mechanisms, determinants and genetic elements in VGS. Erythromycin-resistant isolates totalled 148 (56.3%; 37 belonging to the cMLS phenotype and 111 belonging to the M phenotype); there were 72 (27.4%) and 7 (2.7%) tetracycline- and chloramphenicol-resistant isolates, respectively. A number of variants of known genetic contexts and elements carrying determinants of resistance to these antibiotics were detected, including the mega element, Φ10394.4, Tn2009, Tn2010, the IQ element, Tn917, Tn3872, Tn6002, Tn916, Tn5801, a tet(O) fragment from ICE2096-RD.2 and ICESp23FST81. CONCLUSIONS These findings shed new light on the distribution of antibiotic resistance mechanisms and determinants and their genetic environments in VGS, for which very few such data are currently available. The high frequency and broad variety of such elements supports the notion that VGS may be important reservoirs of resistance genes for the more pathogenic streptococci. The high rates of macrolide resistance confirm the persistence of a marked prevalence of resistant VGS in Europe, where macrolide resistance is, conversely, declining among the major streptococcal pathogens.

Genetic diversity and virulence properties of Streptococcus dysgalactiae subsp. equisimilis from different sources.

A recent increase in virulence of pathogenic Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been widely proposed. Such an increase may be partly explained by the acquisition of new virulence traits by horizontal gene transfer from related streptococci such as Streptococcus pyogenes (GAS) and Streptococcus agalactiae (GBS). A collection of 54 SDSE strains isolated in Italy in the years 2000-2010 from different sources (paediatric throat carriage, invasive and non-invasive diseases) was characterized by emm typing and pulsed-field gel electrophoresis (PFGE) analysis. The virulence repertoire was evaluated by PCR for the presence of GAS superantigen (spe) genes, the streptolysin S (sagA) gene, the group G fibronectin-binding protein (gfbA) gene and GAS-GBS alpha-like protein family (alp) genes; moreover, the ability to invade human epithelial cells was investigated. Resistance to tetracycline, erythromycin and clindamycin was assessed. The combined use of emm typing and PFGE proved to be a reliable strategy for the epidemiological analysis of SDSE isolates. The most frequent emm types were the same as those more frequently reported in other studies, thus indicating the diffusion of a limited number of a few successful emm types fit to disseminate in humans. The speG gene was detected in SDSE strains of different genetic backgrounds. Erythromycin resistance determined by the erm(T) gene, and the unusual, foggy MLSB phenotype, observed in one and seven strains, respectively, have never previously, to our knowledge, been reported in SDSE. Moreover, a new member of the alp family was identified. The identification of new antibiotic and virulence determinants, despite the small size of the sample analysed, shows the importance of constant attention to monitoring the extent of lateral gene transfer in this emerging pathogen.

Recombination between Streptococcus suis ICESsu32457 and Streptococcus agalactiae ICESa2603 yields a hybrid ICE transferable to Streptococcus pyogenes.

Integrative conjugative elements (ICEs) are mobile genetic elements that reside in the chromosome but retain the ability to undergo excision and to transfer by conjugation. Genes involved in drug resistance, virulence, or niche adaptation are often found among backbone genes as cargo DNA. We recently characterized in Streptococcus suis an ICE (ICESsu32457) carrying resistance genes [tet(O/W/32/O), tet(40), erm(B), aphA, and aadE] in the 15K unstable genetic element, which is flanked by two ∼1.3kb direct repeats. Remarkably, ∼1.3-kb sequences are conserved in ICESa2603 of Streptococcus agalactiae 2603V/R, which carry heavy metal resistance genes cadC/cadA and mer. In matings between S. suis 32457 (donor) and S. agalactiae 2603V/R (recipient), transconjugants were obtained. PCR experiments, PFGE, and sequence analysis of transconjugants demonstrated a tandem array between ICESsu32457 and ICESa2603. Matings between tandem array-containing S. agalactiae 2603V/R (donor) and Streptococcus pyogenes RF12 (recipient) yielded a single transconjugant containing a hybrid ICE, here named ICESa2603/ICESsu32457. The hybrid formed by recombination of the left ∼1.3-kb sequence of ICESsu32457 and the ∼1.3-kb sequence of ICESa2603. Interestingly, the hybrid ICE was transferable between S. pyogenes strains, thus demonstrating that it behaves as a conventional ICE. These findings suggest that both tandem arrays and hybrid ICEs may contribute to the evolution of antibiotic resistance in streptococci, creating novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.

Unconventional Circularizable Bacterial Genetic Structures Carrying Antibiotic Resistance Determinants

Particular genetic structures which—though they lack their own recombinase genes— can be excised in circular form thanks to extensive direct repeats (DRs) flanking the DNA segment undergoing excision have recently been described in both Gram-negative and Gram-positive bacteria (1–6). They carry mostly antibiotic resistance genes. The earliest and the latest three of the above-noted studies were published in Antimicrobial Agents and Chemotherapy in 2006 (1) and 2012 (4–6). Although it is probably too early to consider such structures a new group of mobile elements, they are positively unlike conventional mobile genetic elements (MGEs) (plasmids, bacteriophages, integrative and conjugative elements [ICEs], or transposons) (7) and are here tentatively referred to as unconventional circularizable structures (UCSs). Reported UCSs and some putative UCSs are shown in Table 1. Besides excision, UCS integration in the repaired genetic context has been demonstrated experimentally, suggesting that, once excised, the DNA fragment can not only be lost but also undergo transposition (3). A recent study of eukaryotic genomes (Arabidopsis) hypothesized that intrachromosomal recombination of DRs having nontransposon sequences and subsequent insertion of the circular product may be the predominant mechanism of gene transposition (8). UCSs occurring in bacteria may play a similar role, which their frequent carriage of antibiotic resistance determinants makes even more intriguing. On the other hand, the resulting resistant phenotype could make those UCSs easier to find than UCSs devoid of resistance genes. The DRs acting in UCSs are usually long— up to more than 100 times longer than the well-established att sites acting in conventional MGEs (7)—and imperfect, and they may contain genes (of course, genes not involved in transposition). The encompassed DNA segments vary in length and often carry niche adaptation determinants. The recA gene has been shown to be dispensable for UCS excision/integration (2, 3, 6). A parasitic mobilization strategy via site-specific recombination and exploitation of the host trans-acting functions has been hypothesized (3), although the possibility of alternative homologous recombination pathways cannot be excluded, nor can the possibility that different UCSs have different mechanisms of excision/integration. An early report of a genetic structure apparently representing a UCS involved a circular minielement carrying the tetracycline resistance determinant tet(W) in the conjugative transposon TnB1230 of Butyrivibrio fibrisolvens (1). Remarkably, the minielement was detected in the transconjugants but not in the donor, suggesting that excision was dependent on host functions. Afterwards, two UCSs were characterized in enteric bacteria, one representing a defective prophage (2) and the other a microcin-encoding genomic island (3). Very recent studies of Gram-positive bacteria described UCSs consistently carrying antibiotic resistance genes. One, bearing the multidrug resistance gene cfr and containing the macrolide resistance gene erm(B) in the DRs, was reported in a methicillin-resistant Staphylococcus aureus isolate (4). Two more UCSs were described in streptococci, namely, Streptococcus suis and Streptococcus pneumoniae. The former (5), carried on an ICE, contains a number of antibiotic resistance genes: tet(O/W/ 32/O) and tet(40) (tetracycline), erm(B) (erythromycin), aadE (streptomycin), and aphA (kanamycin). The latter (6) is the wellknown MAS (macrolide-aminoglycoside-streptothricin) element, whose insertion distinguishes Tn1545/Tn6003 from Tn6002 (9, 10); again, the DRs contain the erm(B) gene. Of special interest is the involvement of erm(B) in recombination events concerning some UCSs. Besides the two erm(B)-containing DRs mentioned above (4, 6), erm(B)-containing DRs are likely to account for a deleted form (11) of Tn5398, the best-known erm(B)carrying element of Clostridium difficile (12). erm(B), one of the most prevalent and best-conserved antibiotic resistance genes in bacteria (http://faculty.washington.edu/marilynr/), may enable those UCSs that exploit it for integration to attain diverse, even phylogenetically distant, bacterial genomes. The antibiotic resistance determinants carried by UCSs are often freshly acquired genes for the host. This is true of tet(W) in B. fibrisolvens (13), of chromosomally located cfr in S. aureus (14), of tet(O/W/32/O) and tet(40) in S. suis (5, 15), and of aphA and sat4 (streptothricin) in the MAS element, when the clinical pneumococcus carrying it was originally isolated (16). It also applies to other instances where a UCS is suspected but was not expressly investigated. For example, when tet(W) was first described in Rothia, it was found in a region flanked by DRs containing a mef (macrolide efflux) gene (17). The cat (chloramphenicol acetyltransferase) gene was found in a spontaneously curable cargo DNA region flanked by DRs containing toxin/antitoxin genes when detected in Tn5253 of S. pneumoniae (18–20). In addition, erm(43), a new erm gene lately identified in Staphylococcus lentus, was found in an acquired DNA fragment flanked by DRs (21). The inherent instability of UCSs makes them unlikely to persist long as such in a given genetic context; rather, they will tend either to become stable (e.g., by sequence divergence between DRs or deletion of either DR) or to be lost (and possibly move to another genetic context). It is reasonable to assume that several resistance determinants have been acquired via UCSs and have later stabilized. The fact that UCSs are often carried by conventional MGEs might entail a mutual benefit, with UCSs contributing to prompt

Streptococcus pneumoniae Transposon Tn1545/Tn6003 Changes to Tn6002 Due to Spontaneous Excision in Circular Form of the erm(B)- and aphA3-Containing Macrolide-Aminoglycoside-Streptothricin (MAS) Element

ABSTRACT The macrolide-aminoglycoside-streptothricin (MAS) element, an ∼4.2-kb insertion containing erm(B) and aphA3 resistance determinants, distinguishes Streptococcus pneumoniae transposon Tn1545/Tn6003 from Tn6002. Here, it is shown to be an unstable genetic element that, although it lacks recombinase genes, can exploit long, erm(B)-containing direct repeats acting as att sites for spontaneous excision that may result in loss. Consequent to excision, which is RecA independent, Tn1545/Tn6003 changes to Tn6002. In pneumococcal populations harboring Tn1545/Tn6003, the latter appears to coexist with Tn6002.