Maria Scrascia

SXT-related integrating conjugative element and IncC plasmids in Vibrio cholerae O1 strains in Eastern Africa

OBJECTIVES The objective of this study was to investigate the extent of resistance patterns and associated mobile genetic elements in epidemic V. cholerae O1 El Tor strains isolated from Eastern Africa in the late 1990s. METHODS Self-transmissible genetic elements and associated clusters of genes encoding resistance were detected by conjugation experiments. Detection of SXT-related integrating conjugative elements (ICEs) and associated antibiotic resistance genes was performed by PCR to amplify the SXT element-integrase gene (int), right SXT element-chromosome junction (attP-prfC) and genes conferring resistance to chloramphenicol (floR), sulfamethoxazole (sulII), streptomycin (strA) and trimethoprim (dfrA1). Genomic relatedness was established by random amplified polymorphic DNA patterns. RESULTS Of 224 strains analysed, 200 isolates exhibited resistance to four or more antimicrobials. An IncC plasmid, encoding resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and trimethoprim, conferred multidrug resistance to 113 strains isolated from Somalia and Ethiopia, whereas an SXT-related ICE, encoding resistance to chloramphenicol, streptomycin, sulfamethoxazole and trimethoprim, conferred multidrug resistance to 74 strains isolated from Sudan, Kenya and Tanzania. CONCLUSIONS This study has shown the spread of SXT-related ICEs among V. cholerae O1 African isolates. It has also highlighted the role of two distinct genetic elements in conferring multiple resistance to the two distinct groups of V. cholerae O1 strains that, in the late 1990s, spread through Eastern Africa, a critical geographic region for the persistence and transmission of cholera to the entire continent.

Cholera in Ethiopia in the 1990s: epidemiologic patterns, clonal analysis, and antimicrobial resistance.

In 1993, after 6 years of absence, cholera re-emerged in the Horn of Africa. Following its introduction to Djibouti, the disease spread to the central and southern areas of Ethiopia reaching Somalia in 1994. Cholera outbreaks persisted in Ethiopia with a recrudescence of cases in 1998. Twenty-two Vibrio cholerae O1 strains, selected to represent the 1998 history of cholera in Ethiopia, were characterized by random amplified polymorphic DNA patterns, BglI ribotyping and antimicrobial susceptibility. All isolates showed a unique amplified DNA pattern and a prevalent ribotype B8a. All strains were multidrug-resistant and harboured an IncC plasmid which conferred resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and trimethoprim. These findings indicate that a group of closely related V. cholerae O1 strains was responsible for the cholera epidemic in Ethiopia in 1998.

Clonal relationship among Vibrio cholerae O1 El Tor strains isolated in Somalia

One hundred and three Vibrio cholerae O1 strains, selected to represent the cholera outbreaks which occurred in Somalia in 1998-1999, were characterized by random amplified polymorphic DNA patterns, ribotyping, and antimicrobial susceptibility. All strains showed a unique amplified DNA pattern and 2 closely related ribotypes (B5a and B8a), among which B5a was the more frequently identified. Ninety-one strains were resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim, conferred, except for spectinomycin, by a conjugative plasmid IncC. These findings indicated that the group of strains active in Somalia in the late 1990s had a clonal origin.

A novel group of IncQ1 plasmids conferring multidrug resistance

The IncQ is a group of non-conjugative but mobilisable plasmids that are found and stably maintained in a wide range of bacteria contributing to the spread of antimicrobial resistance genes and to the insurgence of multidrug resistant bacteria. Here we report the identification, in clinical Salmonella Typhimurium strains, of an IncQ1 plasmid (pNUC) which confers resistance to sulfamethoxazole, streptomycin and tetracycline through the presence of sul2, strAB and tetA genes, respectively. pNUC was detected in five multidrug resistant S. Typhimurium strains collected in Southern Italy from various hospitals and years of isolation. Bioinformatics analyses highlighted the presence of pNUC-like plasmids in pathogenic bacteria of various Enterobacteriaceae genera or species. Taken as a whole, these plasmids constitute a novel group of IncQ1 plasmids that might have originated through recombination events between a tetR-tetA gene cluster (possibly derived from a Tn1721) and a recipient IncQ1 plasmid related to RSF1010. Our findings raise concerns regarding the possible contribution of the newly identified group of IncQ1 plasmids to the spread of tetracycline resistance.

Identification of pigmented Serratia marcescens symbiotically associated with Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae)

To characterize red pigment‐producing bacteria (RPPB) regularly released during oviposition by red palm weevil (RPW), RPPB were recovered from eggs deposited in apples supplied as substrate for oviposition. The presence of RPPB was also detected from gut, the reproductive apparatus of dissected adult and virgin insects and from pupal cases collected within infested palms. RPPB were also identified all along the tissue of these palms. Analysis of the 16S rDNA, gyrB, rpoB, recA, and groEL sequences assigned RPPB to the species Serratia marcescens. RPPB exhibited an antimicrobial activity assessed by the agar well diffusion method against a number of gram‐positive and gram‐negative bacteria. In this study, we first report the identification of a red pigment‐producing S. marcescens as extracellular symbiont of RPW. Route of transmission, detection within different organs, and a wide spread along the infested palm tissue, suggested S. marcescens is present as extracellular symbiont in different developmental stages of the RPW. Additionally, the antimicrobial activity exhibited versus Bacillus spp., Paenibacillus spp., and Lysinibacillus spp., reported as insect pathogens and potential candidates for biocontrol agents, could ascribe for S. marcescens a potential protective role.

Carbapenemases-producing Klebsiella pneumoniae in hospitals of two regions of Southern Italy

Carbapenem‐resistant Klebsiella pneumoniae infections are reported with increasing frequency elsewhere in the world, representing a worrying phenomenon for global health. In Italy, there are hotspot data on the diffusion and type of carbapenemase‐producing Enterobacteriaceae and K. pneumoniae in particular, with very few data coming from Apulia and Basilicata, two regions of Southern Italy. This study was aimed at characterizing by phenotypic and genotypic methods carbapenem‐resistant K. pneumoniae isolated from several Hospitals of Apulia and Basilicata, Southern Italy. Antibiotic susceptibility was also evaluated. The relatedness of carbapenemase‐producing K. pneumoniae strains was established by pulsed‐field gel electrophoresis (PFGE). Among the 150 K. pneumoniae carbapenemase producers, KPC‐3 genotype was the most predominant (95%), followed by VIM‐1 (5%). No other genotypes were found and no co‐presence of two carbapenemase genes was found. A full concordance between results obtained by both the phenotypic and the genotypic tests was observed. All strains were resistant to β‐lactam antibiotics including carbapenems, and among antibiotics tested, only tetracycline and gentamycin showed low percentage of resistance (18% and 15%, respectively). Resistance to colistin was detected in 17.3% of strains studied. The analysis of PFGE profiles of the carbapenemases‐positive strains shows that one group (B) of the five (A to E) main groups identified was the most prevalent and detected in almost all the hospitals considered, while the other groups were randomly distributed. Three different sequence types (ST 307, ST 258, and ST 512) were detected with the majority of isolates belonging to the ST 512. Our results demonstrated the wide diffusion of K. pneumoniae KPC‐3 in the area considered, the good concordance between phenotypic and genotypic tests. Gentamicin and colistin had a good activity against these strains.

IS26 mediated antimicrobial resistance gene shuffling from the chromosome to a mosaic conjugative FII plasmid

In the present study we report the identification of a sul3-associated class 1 integron containing the dfrA12-orfF-aadA2-cmlA1-aadA1-qacH array embedded in a Tn21-derived element that is part of a conjugative FII plasmid named pST1007-1A. The plasmid was identified in the Salmonella Typhimurium strain ST1007, a member of a clinically relevant clonal MDR lineage diffuse in Italy. ST1007 exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulphamethoxazole, tetracycline and trimethoprim encoded by blaTEM-1, cmlA1, (aadA1, aadA2, strAB), (sul2, sul3), tet(B) and dfrA12 genes, respectively. Apart from pST1007-1A, ST1007 also harbours two chromosome-integrated resistance units RU1 (blaTEM-1-sul2-strAB) and RU2 (tet(B)), flanked by IS26 elements. RU1 and RU2 were able to move as translocatable units, respectively TU1 and TU2, and integrate via IS26 mediated recombination into pST1007-1A. A family of conjugative plasmids, harbouring different sets of antimicrobial resistance genes (ARG) was then generated: pST1007-1B (dfrA12-aadA2-cmlA1-aadA1-sul3- tet(B)), pST1007-1C (dfrA12-aadA2-cmlA1-aadA1-sul3-blaTEM-1-sul2-strAB), pST1007-1D (blaTEM-1-sul2-strAB), pST1007-1E (tet(B)) and pST1007-1F (dfrA12-aadA2-cmlA1-aadA1-sul3- tet(B) -blaTEM-1-sul2-strAB). pST1007-1A is also a mosaic plasmid containing two distinct DNA fragments acquired from I1 plasmids through recombination within the repA4, rfsF and repeat-3 sites. This study further highlights the role played by IS26 in intracellular ARGs shuffling. Moreover, attention has been focused on recombination hot spots that might play a key role in generating mosaic plasmids.

Does Unaspis euonymi (Comstock) (Hemiptera: Diaspididae) host Serratia symbiotica Moran (Bacteria: Enterobacteriaceae)?

The euonymus scale Unaspis euonymi (Comstock) (Hemiptera: Diaspididae) is a pest of spindle that exhibits a strong preference for Euonymus, although it has been detected on at least 18 genera in 13 plant families (Buxus, Camellia, Celastrus, Daphne, Eugenia, Euonymus, Hibiscus, Ilex, Jasminum, Ligustrum, Lonicera, Olea, Pachistima, Pachysandra, Perychmenum, Prunus and Syringa) (Salisbury et al., 2013). Heavy infestation by this pest may lead to the death of the host plant and consequential loss of income from the cultivation of ornamental plants (Kaygin et al., 2008). U. euonymi is an armored scale insect originally from mild Eastern Asia and probably introduced into Europe in the 20th century (Pellizzari & Germain, 2010). Its lifecycle, depending on climate conditions, comprises two-three generations a year and the control measures to limit its diffusion mainly rely on the use of insecticides or the growing of resistant cultivars. The insects can engage mutualistic interactions or symbioses with a variety of bacteria that can profoundly affect the host’s biology. Apart from obligate symbionts (maternally transmitted), a growing number of facultative or secondary symbionts (that can be horizontally transmitted) have been identified (Sandstrom et al., 2001, Moran et al., 2008). Despite not being essential for the host’s life cycle, this last type of symbiont can strongly influence their fitness (Oliver et al., 2003, Jaenike & Brekke, 2011). Additionally, the mutualistic association between insects and bacteria may play a role in the evolution of the latter as described for some groups of Entereobacteriaceae (Moran et al., 2005). A number of genomic and phylogenetic studies on mutualistic associations between Enterobacteriaceae and aphids, psyllids, scale insects, whiteflies, weevils and other insects have been reported (Lefevre et al., 2004, Thao & Baumann, 2004). Here we report the identification of Serratia symbiotica (strain UESS2016) in U. euonymi adult females collected from Sofia (Bulgaria) in 2013.