Genetic characterization of the mechanisms of resistance to amoxicillin/clavulanate and third-generation cephalosporins in Salmonella enterica from three Spanish hospitals

The mechanisms of antimicrobial resistance were characterized in 90 Salmonella enterica isolates either resistant or with intermediate resistance to amoxicillin/clavulanate (AMCR/I) or resistant to third-generation cephalosporins (C3GR). These isolates were recovered in three Spanish hospitals during 2007-2009. The C3GR phenotype was expressed by three isolates that carried the following extended-spectrum β-lactamase genes: phage-associated blaCTX M-10 in S. Virchow, blaCTX-M-14a surrounded by ISEcp1 and IS903 in S. Enteritidis, and blaCTX-M-15 linked to ISEcp1 and orf477 in S. Gnesta (first description in this serotype). The AMCR/I phenotype was found in 87 isolates (79 S. Typhimurim, 7 S. Enteritidis, and one S. Thompson). The blaPSE-1 gene, followed by blaOXA-1 was mostly found among S. Typhimurim, and the blaTEM-1 gene among S. Enteritidis. Three different gene combinations [blaPSE-1+floR+aadA2+sul+tet(G); blaOXA-1+catA+aadA1/strA-strB+sul+tet(B) and blaTEM-1+cmlA1+aadA/strA-strB+sul+tet(A)/tet(B) genes] were associated with the ampicillin-chloramphenicol-streptomycin-sulfonamides-tetracycline phenotype in 68 AMCR/I S. enterica isolates. Class 1 integrons were observed in 79% of the isolates and in most of them (45 isolates) two integrons including the aadA2 and blaPSE-1 gene cassettes, respectively, were detected. The blaOXA-1+aadA1 arrangement was detected in 23 isolates, and the aac(6′)-Ib-cr+blaOXA-1+catB3+arr3 in another one. Non-classicclass 1 integrons were found in three isolates: dfrA12+orfF+aadA2+cmlA1+aadA1 (1 isolate), dfrA12+orfF+aadA2+cmlA1+aadA1+qacH+IS440+sul3 (1 isolate) and dfrA12+orfF+aadA2+cmlA1+aadA1+qacH+IS440+ sul3+orf1+mef(B)Δ-IS26 (1 isolate). Taken together, these results underline the need for clinical concern regarding β-lactam resistance in Salmonella and thus for vigilant monitoring

Ten Issues for Updating in Community-Acquired Pneumonia: An Expert Review

Aetiology; Community acquired pneumonia; Radiologic findingsEtiologia; Pneumònia adquirida a la comunitat; Troballes radiològiquesEtiología; Neumonía adquirida en la comunidad; Hallazgos radiológicosCommunity-acquired pneumonia represents the third-highest cause of mortality in industrialized countries and the first due to infection. Although guidelines for the approach to this infection model are widely implemented in international health schemes, information continually emerges that generates controversy or requires updating its management. This paper reviews the most important issues in the approach to this process, such as an aetiologic update using new molecular platforms or imaging techniques, including the diagnostic stewardship in different clinical settings. It also reviews both the Intensive Care Unit admission criteria and those of clinical stability to discharge. An update in antibiotic, in oxygen, or steroidal therapy is presented. It also analyzes the management out-of-hospital in CAP requiring hospitalization, the main factors for readmission, and an approach to therapeutic failure or rescue. Finally, the main strategies for prevention and vaccination in both immunocompetent and immunocompromised hosts are reviewed

Diversity among clinical isolates of penicillin-resistant Streptococcus mitis: indication for a PBP1-dependent way to reach high levels of penicillin resistance

A total of 12 non-epidemiologically related clinical isolates of Streptococcus mitis that showed different levels of resistance to penicillin were studied. Membrane-protein profiles and penicillin-binding protein (PBP) patterns showed a great polymorphism; and patterns of 4—7 PBPs, withsizes that ranged from ~101 kDa to ~40 kDa, were detected in eachstrai n. No association could be found between PBP pattern and resistance level to penicillin among these isolates. Arbitrarily primed PCR confirmed the genetic diversity among this group of streptococci. One of the isolates of intermediate level of resistance to penicillin, which showed a PBP pattern similar to that of the high-resistance strains, was used as a laboratory model to analyse the mechanism underlying high-resistance acquisition by these strains. A 14-fold increase in penicillin resistance was obtained after a single selection step, which resulted in a decrease in penicillin affinity for PBP1. The size of this PBP (92 kDa) and the differences in PBP profiles of the penicillin-resistant clinical isolates suggest the existence in S. mitis of PBP-mediated mechanisms to acquire high-level resistance to penicillin, among which alterations in PBP1 seem to play a main role, in contrast to the PBP2X mediated mechanism described for other streptococci

Reporting antimicrobial susceptibilities and resistance phenotypes in Staphylococcus spp.: a nationwide proficiency study

Objectives To evaluate the proficiency of microbiology laboratories in Spain in antimicrobial susceptibility testing (AST) of Staphylococcus spp. Materials and methods Eight Staphylococcus spp. with different resistance mechanisms were selected: six Staphylococcus aureus (CC-01/mecA, CC-02/mecC, CC-03/BORSA, CC-04/MLSBi, CC-06/blaZ and CC-07/linezolid resistant, cfr); one Staphylococcus epidermidis (CC-05/linezolid resistant, 23S rRNA mutation); and one Staphylococcus capitis (CC-08/daptomycin non-susceptible). Fifty-one laboratories were asked to report: (i) AST system used; (ii) antimicrobial MICs; (iii) breakpoints used (CLSI or EUCAST); and (iv) clinical category. Minor, major and very major errors (mEs, MEs and VMEs, respectively) were determined. Results The greatest MIC discrepancies found were: (i) by AST method: 19.4% (gradient diffusion); (ii) by antimicrobial agent: daptomycin (21.3%) and oxacillin (20.6%); and (iii) by isolate: CC-07/cfr (48.0%). The greatest error rates were: (i) by AST method: gradient diffusion (4.3% and 5.1% VMEs, using EUCAST and CLSI, respectively); (ii) by breakpoint: 3.8% EUCAST and 2.3% CLSI; (iii) by error type: mEs (0.8% EUCAST and 1.0% CLSI), MEs (1.8% EUCAST and 0.7% CLSI) and VMEs (1.2% EUCAST and 0.6% CLSI); (iii) by antimicrobial agent: VMEs (4.7% linezolid and 4.3% oxacillin using EUCAST); MEs (14.3% fosfomycin, 9.1% tobramycin and 5.7% gentamicin using EUCAST); and mEs (22.6% amikacin using EUCAST). Conclusions Clinical microbiology laboratories should improve their ability to determine the susceptibility of Staphylococcus spp. to some antimicrobial agents to avoid reporting false-susceptible or false-resistant results. The greatest discrepancies and errors were associated with gradient diffusion, EUCAST breakpoints and some antimicrobials (mEs for aminoglycosides; MEs for fosfomycin, aminoglycosides and oxacillin; and VMEs for linezolid and oxacillin)

An increase in erythromycin resistance in methicillin-susceptible Staphylococcus aureus from blood correlates with the use of macrolide/lincosamide/streptogramin antibiotics. EARS-Net Spain (2004–2020)

Staphylococcus aureus; Antibiotic resistance; MacrolidesStaphylococcus aureus; Resistència als antibiòtics; MacròlidsStaphylococcus aureus; Resistencia a los antibióticos; MacrólidosObjectives: To describe and analyse erythromycin resistance trends in blood isolates of Staphylococcus aureus (EARS-Net Spain, 2004–2020) and the association of these trends with the consumption of macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. To assess molecular changes that could be involved in erythromycin resistance trends by whole genome analysis of representative isolates. Materials and methods: We collected antibiotic susceptibility data for all first-blood S. aureus isolates in patients from 47 Spanish hospitals according to EARS-Net criteria. MLSB antibiotic consumption was obtained from the Spanish Agency for Medicines and Medical Devices (2008–2020). We sequenced 137 representative isolates for core genome multilocus sequence typing, resistome and virulome analysis. Results: For the 36,612 invasive S. aureus isolates, methicillin resistance decreased from 26.4% in 2004 to 22.4% in 2020. Erythromycin resistance in methicillin-susceptible S. aureus (MSSA) increased from 13.6% in 2004 to 28.9% in 2020 (p < 0.001); however, it decreased from 68.7 to 61.8% (p < 0.0001) in methicillin-resistant S. aureus (MRSA). Total consumption of MLSB antibiotics increased from 2.72 defined daily doses per 1,000 inhabitants per day (DID) in 2014 to 3.24 DID in 2016. By WGS, the macrolide resistance genes detected were erm (59.8%), msrA (46%), and mphC (45.2%). The erm genes were more prevalent in MSSA (44/57, 77.2%) than in MRSA (38/80, 47.5%). Most of the erm genes identified in MSSA after 2013 differed from the predominant ermC gene (17/22, 77.3%), largely because ermT was significantly associated with MSSA after 2013 (11/29, 37.9%). All 13 ermT isolates in this study, except one, belonged to ST398 and came from 10 hospitals and six Spanish provinces. Conclusion: The significant increase in erythromycin resistance in blood MSSA correlated with the consumption of the MLSB antibiotics in Spain. These preliminary data seem support the hypothesis that the human ST398 MSSA clade with ermT-mediated resistance to erythromycin may be involved in this trend.This research was supported by CIBER—Consorcio Centro de Investigación Biomédica en Red (CB21/13/00095, CB21/13/00006, CB21/13/00054, CB21/13/00068, CB21/13/00084, CB21/13/00099 groups of CIBERINFEC; CB06/06/0058 group of CIBERES), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU. This research was also supported by Personalized and precision medicine grant from the Instituto de Salud Carlos III (MePRAM Project, PMP22/00092), and by the Antibiotic Resistance and Staphylococcus aureus Surveillance Programs of the National Center for Microbiology, Instituto de Salud Carlos III

Spread of streptococcus pneumoniae serotype 8-ST63 multidrug-resistant recombinant clone, Spain

Since 2004, a total of 131 isolates of Streptococcus pneumoniae multidrug-resistant invasive serotype 8 have been detected in Spain. These isolates showed resistance to erythromycin, clindamycin, tetracycline, and ciprofloxacin. All isolates were obtained from adult patients and shared a common genotype (sequence type [ST]63; penicillin-binding protein 1a [pbp1a], pbp2b, and pbp2x gene profiles; ermB and tetM genes; and a ParC-S79F change). Sixty-eight isolates that required a ciprofloxacin MIC ≥16 μg/mL had additional gyrA gene changes. Serotype 8-ST63 pbp2x sequences were identical with those of antimicrobial drug-susceptible serotype 8-ST53 isolates. Serotype 8-ST63 pbp2b sequences were identical with those of the multidrug-resistant Sweden 15A-ST63 clone. Recombination between the capsular locus and flanking regions of an ST53 isolate (donor) and an ST63 pneumococcus (recipient) generated the novel 15A-ST63 clone. One recombination point was upstream of pbp2x and another was within pbp1a. A serotype 8-ST63 clone was identified as a cause of invasive disease in Spain

Prevalence and Genetic Characteristics of Staphylococcus aureus CC398 Isolates From Invasive Infections in Spanish Hospitals, Focusing on the Livestock-Independent CC398-MSSA Clade

Background: Livestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported. Objectives: To determine the prevalence of Staphylococcus aureus (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates. Methods: One thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6–12 months in 17 Spanish hospitals (2018–2019) were studied. CC398 lineage identification, detection of spa-types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing. Results: Forty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven spa-types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent spa-types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin–inducible-resistant/erm(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km2) (p = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately spa-t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD (p < 0.005). Conclusion: CC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers.Agencia Estatal de Investigación (AEI) (project SAF2016-76571- R) and by the Fondo Europeo de Desarrollo Regional (FEDER

Decrease of invasive pneumococcal disease (IPD) in adults after introduction of pneumococcal 13-valent conjugate vaccine in Spain

A prospective laboratory-based multicenter study that collected all adult invasive pneumococcal disease (IPD) episodes from 6 Spanish hospitals before (2008-2009) and after (2012-2013). The 13-valent pneumococcal conjugate vaccine (PCV13) licensure was conducted in order to analyze the impact of PCV13 introduction for children on adult IPD. A total of 1558 IPD episodes were detected. The incidence of IPD decreased significantly in the second period by-33.9% (95% CI,-40.3% to-26.8%). IPD due to PCV7 serotypes (-52.7%; 95% CI,-64.2% to-37.5%) and to PCV13 additional serotypes (-55.0% 95% CI, -62.0% to-46.7%) significantly decreased whereas IPD due to non-PCV13 serotypes remained stable (1.0% 95% CI,-12.9% to 17.2%). IPD due to all PCV13 additional serotypes significantly declined with the exception of serotype 3 (-11.3%; 95% CI-35.0% to 21.1%). IPD due to two non-PCV13 serotypes varied: serotype 6C that rose (301.6%; 95% CI, 92.7% to 733.3%, p<0.001), related to the expansion of ST386(6C), and serotype 8 that decreased (-34.9%, 95% CI,-57.1 to-1.2, p = 0.049), related to a decline of the ST63(8). The recombinant clone ST6521(11A) (variant of ST156(9V)) increased in frequency. The decrease of serotype 19A IPD was linked to a fall in those antibiotic susceptible clones. In the last period, rates of penicillin-and cefotaxime-resistance remained under 10% and 4%, respectively. Adult IPD decreased after the PCV13 introduction in Spain due to herd protection. The spread of multidrug resistant clones (ST386(6C), ST6521(11A)) related to non-PCV13 serotypes needs further surveillance

Synthetic inhibitors of bacterial cell division targeting the GTP-binding site of FtsZ

Cell division protein FtsZ is the organizer of the cytokinetic Z-ring in most bacteria and a target for new antibiotics. FtsZ assembles with GTP into filaments that hydrolyze the nucleotide at the association interface between monomers and then disassemble. We have replaced FtsZ's GTP with non-nucleotide synthetic inhibitors of bacterial division. We searched for these small molecules among compounds from the literature, from virtual screening (VS), and from our in-house synthetic library (UCM), employing a fluorescence anisotropy primary assay. From these screens we have identified the polyhydroxy aromatic compound UCM05 and its simplified analogue UCM44 that specifically bind to Bacillus subtilis FtsZ monomers with micromolar affinities and perturb normal assembly, as examined with light scattering, polymer sedimentation, and negative stain electron microscopy. On the other hand, these ligands induce the cooperative assembly of nucleotide-devoid archaeal FtsZ into distinct well-ordered polymers, different from GTP-induced filaments. These FtsZ inhibitors impair localization of FtsZ into the Z-ring and inhibit bacterial cell division. The chlorinated analogue UCM53 inhibits the growth of clinical isolates of antibiotic-resistant Staphylococcus aureus and Enterococcus faecalis. We suggest that these interfacial inhibitors recapitulate binding and some assembly-inducing effects of GTP but impair the correct structural dynamics of FtsZ filaments and thus inhibit bacterial division, possibly by binding to a small fraction of the FtsZ molecules in a bacterial cell, which opens a new approach to FtsZ-based antibacterial drug discovery.This work was supported by grants from Plan Nacional de Investigación BFU 2011-23416 (J.M.A.), BFU2099-09552 (P.C.), and SAF2010-22198 (M.L.L.-R.), grant CM S2010/BMD-2353 (M.L.L.-R, P.C., J.M.A.), and fellowships FPI (L.B.R.-A.), FPU (M.A.) and CSIC-JAE (E.R.-A.)

Inhibitor-resistant TEM- and OXA-1-producing Escherichia coli isolates resistant to amoxicillin-clavulanate are more clonal and possess lower virulence gene content than susceptible clinical isolates

In a previous prospective multicenter study in Spain, we found that OXA-1 and inhibitor-resistant TEM (IRT) β-lactamases constitute the most common plasmid-borne mechanisms of genuine amoxicillin-clavulanate (AMC) resistance in Escherichia coli. In the present study, we investigated the population structure and virulence traits of clinical AMC-resistant E. coli strains expressing OXA-1 or IRT and compared these traits to those in a control group of clinical AMC-susceptible E. coli isolates. All OXA-1-producing (n = 67) and IRT-producing (n = 45) isolates were matched by geographical and temporal origin to the AMC-susceptible control set (n = 56). We performed multilocus sequence typing and phylogenetic group characterization for each isolate and then studied the isolates for the presence of 49 virulence factors (VFs) by PCR and sequencing. The most prevalent clone detected was distinct for each group: group C isolates of sequence type (ST) 88 (C/ST88) were the most common in OXA-1 producers, B2/ST131 isolates were the most common in IRT producers, and B2/ST73 isolates were the most common in AMC-susceptible isolates. The median numbers of isolates per ST were 3.72 in OXA-1 producers, 2.04 in IRT producers, and 1.69 in AMC-susceptible isolates; the proportions of STs represented by one unique isolate in each group were 19.4%, 31.1%, and 48.2%, respectively. The sum of all VFs detected, calculated as a virulence score, was significantly higher in AMC-susceptible isolates than OXA-1 and IRT producers (means, 12.5 versus 8.3 and 8.2, respectively). Our findings suggest that IRT- and OXA-1-producing E. coli isolates resistant to AMC have a different and less diverse population structure than AMC-susceptible clinical E. coli isolates. The AMC-susceptible population also contains more VFs than AMC-resistant isolates.This study was supported by the Plan Nacional de I+D+i 2008-2011 and the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015). The study was cofinanced by the European Development Regional Fund (ERDF; A way to achieve Europe) and the Fondo de Investigación Sanitaria (grant PI09/0917).S