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Horizontal gene transfer was long thought to be marginal in Mollicutes, but the capacity of some of these wall-less bacteria to exchange large chromosomal regions has been recently documented. Mycoplasma chromosomal transfer (MCT) is an unconventional mechanism that relies on the presence of a functional integrative conjugative element (ICE) in at least one partner and involves the horizontal acquisition of small and large chromosomal fragments from any part of the donor genome, which results in progenies composed of an infinite variety of mosaic genomes. The present study focuses on Mycoplasma bovis, an important pathogen of cattle responsible for major economic losses worldwide. By combining phylogenetic tree reconstructions and detailed comparative genome analyses of 36 isolates collected in Spain (2016 to 2018), we confirmed the mosaic nature of 16 field isolates and mapped chromosomal transfers exchanged between their hypothetical ancestors. This study provides evidence that MCT can take place in the field, most likely during coinfections by multiple strains. Because mobile genetic elements (MGEs) are classical contributors of genome plasticity, the presence of phages, insertion sequences (ISs), and ICEs was also investigated. Data revealed that these elements are widespread within the M. bovis species and evidenced classical horizontal transfer of phages and ICEs in addition to MCT. These events contribute to wide-genome diversity and reorganization within this species and may have a tremendous impact on diagnostic and disease control. [ABSTRACT FROM AUTHOR]

Objectives: To phenotypically and genetically characterize the antibiotic resistance determinants and associated mobile genetic elements (MGEs) among macrolide-resistant (MR) Streptococcus pyogenes [Group A streptococci (GAS)] clinical isolates collected in Barcelona, Spain.Methods: Antibiotic susceptibility testing was performed by microdilution. Isolates were emm and MLST typed and 55 were whole-genome sequenced to determine the nature of the macrolide resistance (MR) determinants and their larger MGE and chromosomal context.Results: Between 1998 and 2018, 142 of 1028 GAS (13.8%) were MR. Among 108 isolates available for molecular characterization, 41.7% had cMLSB, 30.5% iMLSB and 27.8% M phenotype. Eight erm(B)-containing strains were notable in having an MDR phenotype conferred by an MGE encoding several antibiotic resistance genes. MR isolates were comprised of several distinct genetic lineages as defined by the combination of emm and ST. Although most lineages were only transiently present, the emm11/ST403 clone persisted throughout the period. Two lineages, emm9/ST75 with erm(B) and emm77/ST63 with erm(TR), emerged in 2016-18. The erm(B) was predominantly encoded on the Tn916 family of transposons (21/31) with different genetic contexts, and in other MGEs (Tn6263, ICESpHKU372 and one harbouring an MDR cluster called ICESp1070HUB). The erm(TR) was found in ICESp2905 (8/17), ICESp1108-like (4/17), ICESpHKU165 (3/17) and two structures described in this study (IMESp316HUB and ICESp3729HUB). The M phenotype [mef(A)-msr(D)] was linked to phage φ1207.3. Eight integrative conjugative element/integrative mobilizable element (ICE/IME) cluster groups were classified on the basis of gene content within conjugation modules. These groups were found among MGEs, which corresponded with the MR-containing element or the site of integration.Conclusions: We detected several different MGEs harbouring erm(B) or erm(TR). This is the first known description of Tn6263 in GAS and three MGEs [IMESp316HUB, ICESp3729HUB and ICESp1070HUB] associated with MR. Periods of high MR rates in our area were mainly associated with the expansion of certain predominant lineages, while in low MR periods different sporadic and low prevalence lineages were more frequent. [ABSTRACT FROM AUTHOR]

Copper resistance mechanisms provide an important adaptive advantage to plant-pathogenic bacteria under exposure to copper treatments. Copper resistance determinants have been described in Pseudomonas syringae pv. syringae (Pss) strains isolated from mango intimately associated with 62-kb plasmids belonging to the pPT23A family (PFP). It has been previously described that the indiscriminate use of copper-based compounds promotes the selection of copper-resistant bacterial strains and constitutes a selective pressure in the evolution of copper resistance determinants. Hence, we have explored in this study the copper resistance evolution and the distribution of specific genetic determinants in two different Pss mango populations isolated from the same geographical regions, mainly from southern Spain, with an average of 20 years of difference. The total content of plasmids, in particular the 62-kb plasmids, and the number of copper-resistant Pss strains were maintained at similar levels over time. Interestingly, the phylogenetic analysis indicated the presence of a phylogenetic subgroup (PSG) in the Pss mango phylotype mostly composed of the recent Pss population analyzed in this study that was strongly associated with a hyperresistant phenotype to copper. Genome sequencing of two selected Pss strains from this PSG revealed the presence of a large Tn7-like transposon of chromosomal location, which harbored putative copper and arsenic resistance genes (COARS Tn7-like). Transformation of the copper-sensitive Pss UMAF0158 strain with some putative copper resistance genes and reverse transcription-quantitative PCR experiments brought to light the role of COARS Tn7-like transposon in the hyperresistant phenotype to copper in Pss. [ABSTRACT FROM AUTHOR]

This study aimed at determining the mechanisms of linezolid resistance and the molecular characteristics of clinical Staphylococcus aureus (n = 2) and coagulase-negative staphylococci (n = 15) isolates obtained from four Spanish hospitals. The detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. The antimicrobial resistance and virulence profile was determined, and the isolates were typed by different molecular techniques. Moreover, the genetic environment of the cfr gene was determined by whole-genome sequencing. The cfr gene was detected in one methicillin-resistant S. aureus (MRSA) that also displayed the amino acid change Val118Ala in the ribosomal protein L4. The second S. aureus isolate was methicillin susceptible and showed different alterations in the ribosomal protein L4. All remaining linezolid-resistant Staphylococcus epidermidis (n = 14) and Staphylococcus hominis isolates (n = 1) showed the mutation G2576T (n = 14) or C2534T (n = 1) in the 23S rRNA. Moreover, different amino acid changes were detected in the ribosomal proteins L3 and L4 in S. epidermidis isolates. All S. epidermidis isolates belonged to the multilocus sequence type ST2. Linezolid-resistant staphylococci (LRS) showed a multiresistance phenotype, including methicillin resistance that was detected in all isolates but one, and was mediated by the mecA gene. The cfr gene in the MRSA isolate was located together with the fexA gene on a conjugative 38,864 bp plasmid. Linezolid- and methicillin-resistant S. epidermidis ST2 showing mutations in the 23S rRNA and in the ribosomal proteins L3 and L4 are spread among Spanish hospitals, whereas LRS carrying acquired linezolid resistance genes are rarely detected. [ABSTRACT FROM AUTHOR]

The antimicrobial resistance (AMR) genes of 268 non-duplicated coagulase-negative staphylococci (CoNS) previously obtained from nasotracheal cavities of nestling storks were characterized. They included S. sciuri isolates (n = 191), and non- sciuri -CoNS isolates (NSc-CoNS, n = 77). All S. sciuri carried the intrinsic salA gene (for clindamycin-resistance) and so, clindamycin was not considered for general analysis in this species. About 71.7%/41.6% of the S. sciuri /NSc-CoNS isolates were susceptible to all antibiotics tested; moreover, 14.1%/16.9% and 3.1%/20.8% of S. sciuri /NSc-CoNS showed single antibiotic resistance and multidrug resistance (MDR) phenotype, respectively. Of the ten mecA -positive CoNS isolates, six were associated with SCC mec types-III, -IV or -V elements. Remarkably was the detection of one MDR- S. lentus isolate carrying both mecA and mecC genes, as well as the SCC mec type-XI element. MDR-CoNS was relatively higher in nestlings of parent storks foraging in landfills (21.3%) than those in natural areas (9.7%) (χ 2 = 3.421, df=1, p = 0.064). AMR phenotypes (and genes detected) include penicillin (blaZ, bla ARL), erythromycin-clindamycin-constitutive (ermA, ermC, ermT), clindamycin (lnuA, salA, vgaA), erythromycin (msrA, mphC), tetracycline (tetK, tetL, tetM), tobramycin (ant4′), tobramycin-gentamicin (aac6′-aph2″), sulfamethoxazole-trimethoprim (dfrA, dfrG, dfrK), chloramphenicol (fexA, fexB, catPC221), and mupirocin (mupA). Interestingly, one S. epidermidis isolate carried the ermT gene. About 29.9% of nestlings harboured more than one non-duplicated CoNS (with varied 2–5 AMR profiles). This study demonstrated that most of the CoNS isolates were susceptible to all the antibiotics tested (63.1%). However, AMR genes of public health importance were found, including the mecC -mediated methicillin resistance trait. • Nestling storks could be vectors of AMR genes from or to other ecological niches. • About 63.1% of CoNS from the nestlings were susceptible to all antibiotics tested. • A mecA/mecC - S. lentus and ermT - S. epidermidis isolates were detected. • salA gene in all S. sciuri isolates mediated varied clindamycin resistance levels. • These highlight the need for inclusion of CoNS & wild animals in AMR surveillance. [ABSTRACT FROM AUTHOR]

Objectives: The global emergence of OXA-48-producing Klebsiella pneumoniae clones is a significant threat to public health. We used WGS and phylogenetic analysis of Spanish isolates to investigate the population structure of blaOXA-48-like-expressing K. pneumoniae ST11 and ST405 and to determine the distribution of resistance genes and plasmids encoding blaOXA-48-like carbapenemases.Methods: SNPs identified in whole-genome sequences were used to reconstruct phylogenetic trees, identify resistance determinants and de novo assemble the genomes of 105 blaOXA-48-like-expressing K. pneumoniae isolates.Results: Genome variation was generally lower in outbreak-associated isolates compared with those associated with sporadic infections. The relatively limited variation observed within the outbreak-associated isolates was on average 7-10 SNPs per outbreak. Of 24 isolates from suspected sporadic infections, 7 were very closely related to isolates causing hospital outbreaks and 17 were more diverse and therefore probably true sporadic cases. On average, 14 resistance genes were identified per isolate. The 17 ST405 isolates from sporadic cases of infection had four distinct resistance gene profiles, while the resistance gene profile differed in all ST11 isolates from sporadic cases. Sequence analysis of 94 IncL/M plasmids carrying blaOXA-48-like genes revealed an average of two SNP differences, indicating a conserved plasmid clade.Conclusions: Whole-genome sequence analysis enabled the discrimination of outbreak and sporadic isolates. Significant inter-regional spread within Spain of highly related isolates was evident for both ST11 and ST405 K. pneumoniae. IncL/M plasmids carrying blaOXA-48-like carbapenemase genes were highly conserved geographically and across the outbreaks, sporadic cases and clones. [ABSTRACT FROM AUTHOR]

The emergence of 16S rRNA methyltransferases (RMTs) in Gram-negative pathogens bearing other clinically relevant resistance mechanisms, such as carbapenemase-producing Enterobacterales (CPE), is becoming an alarming concern. We investigated the prevalence, antimicrobial susceptibility, resistance mechanisms, molecular epidemiology and genetic support of RMTs in CPE isolates from Spain. This study included a collection of 468 CPE isolates recovered during 2018 from 32 participating Spanish hospitals. MICs were determined using the broth microdilution method, the agar dilution method (fosfomycin) or MIC gradient strips (plazomicin). All isolates were subjected to hybrid whole-genome sequencing (WGS). Sequence types (STs), core genome phylogenetic relatedness, horizontally acquired resistance mechanisms, plasmid analysis and the genetic environment of RMTs were determined in silico from WGS data in all RMT-positive isolates. Among the 468 CPE isolates evaluated, 24 isolates (5.1%) recovered from nine different hospitals spanning five Spanish regions showed resistance to all aminoglycosides and were positive for an RMT (21 RmtF, 2 ArmA and 1 RmtC). All RMT-producers showed high-level resistance to all aminoglycosides, including plazomicin, and in most cases exhibited an extensively drug-resistant susceptibility profile. The RMT-positive isolates showed low genetic diversity and were global clones of Klebsiella pneumoniae (ST147, ST101, ST395) and Enterobacter cloacae (ST93) bearing bla OXA-48 , bla NDM-1 or bla VIM-1 carbapenemase genes. RMTs were harboured in five different multidrug resistance plasmids and linked to efficient mobile genetic elements. Our findings highlight that RMTs are emerging among clinical CPE isolates from Spain and their spread should be monitored to preserve the future clinical utility of aminoglycosides and plazomicin. [ABSTRACT FROM AUTHOR]

We studied the usefulness of the restriction-modification (RM) test, staphylococcal cassette chromosome (SCC) mec types, and Panton-Valentine leukocidin (PVL)-encoding phages to identify Staphylococcus aureus methicillin-resistant lineages and to differentiate clones that belong to the same lineage. A total of 108 methicillin-resistant S. aureus (MRSA) strains were characterized by pulsed-field gel electrophoresis (PFGE)-multi-locus sequence typing (MLST)- spa-typing. The RM correctly identified the lineages CC5, CC8, CC30, and CC398, but not CC25 and CC72. The SCC mec and RM combined analysis allowed differentiation between MLST types within the same lineage. Only 5 MRSA strains were PVL-positive. Four PVL-positive USA300 isolates carried elongated-head type PVL-encoding phages, while the sequence type (ST)-30 strain carried an icosahedral-head phage. The combination of the RM test method, SCC mec types, and PVL phage identification could be useful for MRSA typing purposes. [ABSTRACT FROM AUTHOR]

Salmonellosis is a common subclinical infection in pigs and therefore apparently healthy animals may represent a reservoir of antibiotic-resistant Salmonella for humans. This study estimates and characterizes resistance to two classes of antimicrobials considered of the highest priority within the critically important antimicrobials for humans, i.e. colistin (CR) and 3rd generation cephalosporins (3GC), on a collection of Salmonella isolates from pigs from two periods: between 2008 and 09, when colistin was massively used; and in 2018, after three years under a National Plan against Antibiotic Resistance. Prevalence of CR was low (6 out of 625; 0.96%; 95%CI: 0.44–2.1) in 2008–09 and associated mostly to the mcr-1 gene, which was detected in four S. 4,5,12:i:- isolates. Polymorphisms in the pmrAB genes were detected in a S. 9,12:-:- isolate. No CR was detected in 2018 out of 59 isolates tested. Among 270 Salmonella isolates considered for the assessment of resistance to 3GC in the 2008–2009 sampling, only one Salmonella Bredeney (0.37%; 95%CI: 0.07–2.1) showed resistance to 3GC, which was associated with the bla CMY-2 gene (AmpC producer). In 2018, six isolates out of 59 (10.2%; 95%CI: 4.7–20.5) showed resistance to 3GC, but only two different strains were identified (S. 4,12:i:- and S. Rissen), both confirmed as extended-spectrum β-lactamases (ESBL) producers. The bla CTX-M-3 and bla TEM-1b genes in S. 4,12:i:- and the bla TEM-1b gene in S. Rissen seemed to be associated with this resistance. Overall, the prevalence of CR in Salmonella appeared to be very low in 2008–2009 despite the considerable use of colistin in pigs at that time, and seemed to remain so in 2018. Resistance to 3GC was even lower in 2008–2009 but somewhat higher in 2018. Resistance was mostly coded by genes associated with mobile genetic elements. Most serotypes involved in these antimicrobial resistances displayed a multidrug resistance pattern and were considered zoonotic. • Colistin-resistant Salmonella in pigs appears to be low in Spain. • Colistin-resistance was mostly driven by the presence of the mcr-1 gene. • 3rd generation cephalosporins resistance would show a slightly increase in pig Salmonella. • Resistance involved zoonotic Salmonella serotypes with multidrug resistant patterns. [ABSTRACT FROM AUTHOR]