Prophage association of mef(A) elements encoding efflux-mediated erythromycin resistance in Streptococcus pyogenes.

OBJECTIVES: To compare different mef(A) elements of Streptococcus pyogenes for a possible chimeric genetic nature, i.e. a transposon inserted into a prophage. METHODS: Eleven S. pyogenes isolates with efflux-mediated erythromycin resistance were used. The isolates were typed using several genotypic approaches. Gene detection was performed by PCR using specific primer pairs. The mef(A) elements of the test strains were induced with mitomycin C and phage DNA was extracted. Induction was monitored by PCR using primers targeting mef(A). RESULTS: Six tetracycline-susceptible isolates had PCR evidence of all of the eight open reading frames (ORFs) of the Tn1207.1 element; their mef(A) element was consistent with the Tn1207.3 element in four isolates and with the 58.8 kb chimeric element in two. Five tetracycline-resistant isolates had no PCR evidence of orf1 and orf2 and showed variable patterns as to orf3, orf7, and orf8. Three ORFs placed along the conserved region downstream of Tn1207.1 in the 58.8 kb mef(A) chimeric element were detected in the six tetracycline-susceptible, but not in the five tetracycline-resistant isolates. Induction assays with mitomycin C demonstrated that the mef(A) elements of all strains tested were present in culture supernatants in a DNAse-resistant form, such as a phage capsid. CONCLUSIONS: All recognized mef(A) elements of S. pyogenes appear to be prophage-associated. Whereas the two elements detected in tetracycline-susceptible isolates (Tn1207.3 and the 58.8 kb one) were apparently inserted into the same prophage, the tet(O)-mef(A) element was inserted into a different prophage. Phage transfer is likely to play a critical role in the dissemination of erythromycin resistance in S. pyogenes populations

Clinical and microbiological features of ceftolozane/tazobactam-resistant Pseudomonas aeruginosa isolates in a university hospital in central Italy

Objectives: Ceftolozane/tazobactam (C/T) is a novel cephalosporin and β-lactamase inhibitor combination with great activity against Pseudomonas aeruginosa. To assess P. aeruginosa susceptibility to C/T, a surveil- lance study was conducted from October 2018 to March 2019 at the University Hospital ‘Ospedali Riuniti’ in Ancona, Italy. Methods: Minimum inhibitory concentrations (MICs) to C/T were determined by Etest strip. Resistant iso- lates were characterized by phenotypic (broth microdilution antimicrobial susceptibility testing and mod- ified Carbapenem Inactivation Method [mCIM]) and genotypic (Polymerase Chain Reaction [PCR], Pulsed Field Gel Electrophoresis [PFGE], and whole-genome sequencing [WGS]) methods. Clinical variables of patients infected by C/T-resistant P. aeruginosa were collected from medical records. Results: Fifteen of 317 P. aeruginosa collected showed resistance to C/T (4.7%). Ten strains demonstrated carbapenemase activity by mCIM method, and PCR confirmed that eight strains harbored a blaVIM gene while the other two were positive for blaIMP. Additionally, three isolates carried acquired extended spec- trum β-lactamase genes (two isolates carried blaPER and one carried blaGES). Eight strains were strictly related by PFGE and WGS analysis confirmed that they belonged to sequence type (ST)111. The other STs found were ST175 (two isolates), ST235 (two isolates), ST70 (one isolate), ST621 (one isolate), and the new ST3354 (one isolate). Most patients had received previous antibiotic therapies, carried invasive devices, and experienced prolonged hospitalization. Conclusion: This study demonstrated the presence of C/T-resistant P. aeruginosa isolates in a regional hospital carrying a number of resistance mechanisms acquired by different high-risk clone

Characterization of a Multiresistance Plasmid Carrying the optrA and cfr Resistance Genes From an Enterococcus faecium Clinical Isolate

open13noEnterococcus faecium E35048, a bloodstream isolate from Italy, was the first strain where the oxazolidinone resistance gene optrA was detected outside China. The strain was also positive for the oxazolidinone resistance gene cfr. WGS analysis revealed that the two genes were linked (23.1 kb apart), being co-carried by a 41,816-bp plasmid that was named pE35048-oc. This plasmid also carried the macrolide resistance gene erm(B) and a backbone related to that of the well-known Enterococcus faecalis plasmid pRE25 (identity 96%, coverage 65%). The optrA gene context was original, optrA being part of a composite transposon, named Tn6628, which was integrated into the gene encoding for the ζ toxin protein (orf19 of pRE25). The cfr gene was flanked by two ISEnfa5 insertion sequences and the element was inserted into an lnu(E) gene. Both optrA and cfr contexts were excisable. pE35048-oc could not be transferred to enterococcal recipients by conjugation or transformation. A plasmid-cured derivative of E. faecium E35048 was obtained following growth at 42°C, and the complete loss of pE35048-oc was confirmed by WGS. pE35048-oc exhibited some similarity but also notable differences from pEF12-0805, a recently described enterococcal plasmid from human E. faecium also co-carrying optrA and cfr; conversely it was completely unrelated to other optrA- and cfr-carrying plasmids from Staphylococcus sciuri. The optrA-cfr linkage is a matter of concern since it could herald the possibility of a co-spread of the two genes, both involved in resistance to last resort agents such as the oxazolidinones.openMorroni, Gianluca; Brenciani, Andrea; Antonelli, Alberto; Maria D’Andrea, Marco; Di Pilato, Vincenzo; Fioriti, Simona; Mingoia, Marina; Vignaroli, Carla; Cirioni, Oscar; Biavasco, Francesca; Varaldo, Pietro E.; Rossolini, Gian Maria; Giovanetti, EleonoraMorroni, Gianluca; Brenciani, Andrea; Antonelli, Alberto; Maria D’Andrea, Marco; Di Pilato, Vincenzo; Fioriti, Simona; Mingoia, Marina; Vignaroli, Carla; Cirioni, Oscar; Biavasco, Francesca; Varaldo, Pietro E.; Rossolini, Gian Maria; Giovanetti, Eleonor

Oxazolidinones: mechanisms of resistance and mobile genetic elements involved

The oxazolidinones (linezolid and tedizolid) are last-resort antimicrobial agents used for the treatment of severe infections in humans caused by MDR Gram-positive bacteria. They bind to the peptidyl transferase centre of the bacterial ribosome inhibiting protein synthesis. Even if the majority of Gram-positive bacteria remain susceptible to oxazolidinones, resistant isolates have been reported worldwide. Apart from mutations, affecting mostly the 23S rDNA genes and selected ribosomal proteins, acquisition of resistance genes (cfr and cfr-like, optrA and poxtA), often associated with mobile genetic elements [such as non-conjugative and conjugative plasmids, transposons, integrative and conjugative elements (ICEs), prophages and translocatable units], plays a critical role in oxazolidinone resistance. In this review, we briefly summarize the current knowledge on oxazolidinone resistance mechanisms and provide an overview on the diversity of the mobile genetic elements carrying oxazolidinone resistance genes in Gram-positive and Gram-negative bacteria

A Novel Efflux System in Inducibly Erythromycin-Resistant Strains of Streptococcus pyogenes

Streptococcus pyogenes strains inducibly resistant (iMLS phenotype) to macrolide, lincosamide, and streptogramin B (MLS) antibiotics can be subdivided into three phenotypes: iMLS-A, iMLS-B, and iMLS-C. This study focused on inducibly erythromycin-resistant S. pyogenes strains of the iMLS-B and iMLS-C types, which are very similar and virtually indistinguishable in a number of phenotypic and genotypic features but differ clearly in their degree of resistance to MLS antibiotics (high in the iMLS-B type and low in the iMLS-C type). As expected, the iMLS-B and iMLS-C test strains had the erm(A) methylase gene; the iMLS-A and the constitutively resistant (cMLS) isolates had the erm(B) methylase gene; and a control M isolate had the mef(A) efflux gene. mre(A) and msr(A), i.e., other macrolide efflux genes described in gram-positive cocci, were not detected in any test strain. With a radiolabeled erythromycin method for determination of the intracellular accumulation of the drug in the absence or presence of an efflux pump inhibitor, active efflux of erythromycin was observed in the iMLS-B isolates as well as in the M isolate, whereas no efflux was demonstrated in the iMLS-C isolates. By the triple-disk (erythromycin plus clindamycin and josamycin) test, performed both in normal test medium and in the same medium supplemented with the efflux pump inhibitor, under the latter conditions iMLS-B and iMLS-C strains were no longer distinguishable, all exhibiting an iMLS-C phenotype. In conjugation experiments with an iMLS-B isolate as the donor and a Rif(r) Fus(r) derivative of an iMLS-C isolate as the recipient, transconjugants which shared the iMLS-B type of the donor under all respects, including the presence of an efflux pump, were obtained. These results indicate the existence of a novel, transferable efflux system, not associated with mef(A) or with other known macrolide efflux genes, that is peculiar to iMLS-B strains. Whereas the low-level resistance of iMLS-C strains to MLS antibiotics is apparently due to erm(A)-encoded methylase activity, the high-level resistance of iMLS-B strains appears to depend on the same methylase activity plus the new efflux system

Genetic basis of the association of resistance genes mef(I) (macrolides) and catQ (chloramphenicol) in streptococci

In streptococci mef(I) and catQ, two relatively uncommon macrolide and chloramphenicol resistance genes, respectively, are typically linked in a genetic module designated IQ module. Though variable, the module consistently encompasses, and is sometimes reduced to, a conserved ∼5.8-kb mef(I)-catQ fragment. The prototype IQ module was described in Streptococcus pneumoniae. IQ-like modules have subsequently been detected in Streptococcus pyogenes and in different species of viridans group streptococci, where mef(E) maybe found instead of mef(I). Three genetic elements, one carrying the prototype IQ module from S. pneumoniae and two carrying different, defective IQ modules from S. pyogenes, have recently been characterized. All are integrative and conjugative elements (ICEs) belonging to the Tn5253 family, and have been designated ICESpn529IQ, ICESpy029IQ and ICESpy005IQ, respectively. ICESpy029IQ and ICESpy005IQ were the first Tn5253 family ICEs to be described in S. pyogenes. A wealth of new information has been obtained by comparing their genetic organization, chromosomal integration, and transferability. The origin of the IQ module is unknown. The mechanism by which it spreads in streptococci is discussed

Different genetic elements carrying the tet(W) gene in two human clinical isolates of Streptococcus suis.

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

Stability of the cargo regions of the cfr-carrying, multiresistance plasmid pSP01 from Staphylococcus epidermidis

This study investigated the stability or instability — i.e. the ability or inability to undergo excision in circular form — of the four cargo regions (cr1 to cr4) of the novel cfr-carrying, multiresistance plasmid pSP01, arboured by a clinical Staphylococcus epidermidis isolate. Only cr4 proved unstable. The stability of cr1 and cr2 was substantially expected. Insertion sequences (ISs) played an important role in the stability of cr3 (the cfr gene context) and in the instability of cr4. Whereas the stability of cfr genetic contexts is associated with the presence of a single IS copy (istAS-istBS in cr3), their instability is associated with two identical, flanking ISs with the same orientation. cr4 is bracketed between two identical IS257 elements, and appears to behave as a composite transposon. Its instability is of interest because of the existence of a closely related cfr plasmid from S. epidermidis (pSP01.1) that differs from pSP01 only by the lack of cr4. An integration/recombination mechanism is suggested to explain how cr4 may have moved to pSP01.1 to form pSP01

Commentary: Nationwide Surveillance of Novel Oxazolidinone Resistance Gene optrA in Enterococcus Isolates in China from 2004 to 2014

none6noopenMorroni, Gianluca; Brenciani, Andrea; Simoni, Serena; Vignaroli, Carla; Mingoia, Marina; Giovanetti, EleonoraMorroni, Gianluca; Brenciani, Andrea; Simoni, Serena; Vignaroli, Carla; Mingoia, Marina; Giovanetti, Eleonor