Review of the impact of MALDI-TOF MS in public health and hospital hygiene, 2018.

IntroductionMALDI-TOF MS represents a new technological era for microbiology laboratories. Improved sample processing and expanded databases have facilitated rapid and direct identification of microorganisms from some clinical samples. Automated analysis of protein spectra from different microbial populations is emerging as a potential tool for epidemiological studies and is expected to impact public health.AimTo demonstrate how implementation of MALDI-TOF MS has changed the way microorganisms are identified, how its applications keep increasing and its impact on public health and hospital hygiene.MethodsA review of the available literature in PubMED, published between 2009 and 2018, was carried out.ResultsOf 9,709 articles retrieved, 108 were included in the review. They show that rapid identification of a growing number of microorganisms using MALDI-TOF MS has allowed for optimisation of patient management through prompt initiation of directed antimicrobial treatment. The diagnosis of Gram-negative bacteraemia directly from blood culture pellets has positively impacted antibiotic streamlining, length of hospital stay and costs per patient. The flexibility of MALDI-TOF MS has encouraged new forms of use, such as detecting antibiotic resistance mechanisms (e.g. carbapenemases), which provides valuable information in a reduced turnaround time. MALDI-TOF MS has also been successfully applied to bacterial typing.ConclusionsMALDI-TOF MS is a powerful method for protein analysis. The increase in speed of pathogen detection enables improvement of antimicrobial therapy, infection prevention and control measures leading to positive impact on public health. For antibiotic susceptibility testing and bacterial typing, it represents a rapid alternative to time-consuming conventional techniques

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

Monitoring the antimicrobial susceptibility of Gramnegative organisms involved in intraabdominal and urinary tract infections recovered during the SMART study (Spain, 2016 and 2017)

OBJECTIVE: Continuous antimicrobial resistance surveillance is recommended by Public Health authorities. We up-dated data from the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. METHODS: The antimicrobial susceptibility data and extended-spectrum beta-lactamase (ESBL) production in isolates recovered from intra-abdominal (IAI) (n=1,429) and urinary tract (UTI) (n=937) infections during the 2016- 2017 SMART study in 10 Spanish hospitals were analysed. RESULTS: Escherichia coli was the most frequently microorganism isolated (48.3% and 53.7%) followed by Klebsiella spp. (11.5% and 21.9%) in IAIs and UTIs, respectively. Figures for Pseudomonas aeruginosa were 9.0% and 6.1%, being more frequently recovered from patients with nosocomial infections. Overall, 9.9% (IAI) and 14.0% (UTI) of E. coli, Klebsiella spp. and Proteus mirabilis isolates were ESBL-producers, being Klebsiella pneumoniae (34.5%) from UTI of nosocomial origin the most frequent. ESBL-producers were higher in patients >60 years in both IAIs and UTIs. As in previous years, amikacin (96.3%-100% susceptibility), ertapenem (84.2%-100%) and imipenem (70.3%- 100%) were the most active antimicrobials tested among Enterobacterales species. The activity of amoxicillin-clavulanic, piperacillin-tazobactam, and ciprofloxacin susceptibility was lower, particularly among ESBL-producers. Ertapenem susceptibility (88.9%-100%) was retained in ESBL-E. coli isolates that were resistant to these antimicrobials but decreased (28.6%-100%) in similar isolates of K. pneumoniae. CONCLUSIONS: Continuous antimicrobial resistance surveillance from the SMART study reveals overall maintenance of ESBL-producers in Spain, although with higher presence in isolates from UTIs than from IAIs. Moreover, ertapenem activity was high in E. coli irrespective of ESBL production but decreased in K. pneumoniae, particularly among ESBL-producers

Penicillin susceptibility among invasive MSSA infections: a multicentre study in 16 Spanish hospitals

Objectives: To determine the prevalence of penicillin susceptibility among MSSA causing bloodstream infections (BSIs) in 16 Spanish hospitals and to characterize the penicillin-susceptible MSSA (MSSA-PENS) isolates. Methods: A total of 1011 Staphylococcus aureus isolates were collected from blood cultures in 16 Spanish hospitals during 2018–19 (6–12 months) and their susceptibility to 18 antimicrobials was determined. The MSSA-PENS isolates were selected and examined by PCR to determine the presence of the blaZ gene, other resistance genes and the genes lukF/lukS-PV, eta, etb and tst. The immune evasion cluster (IEC) type was also analysed. All the MSSA-PENS isolates were submitted to S. aureus protein A (spa) typing and the clonal complexes (CCs) were assigned according to their spa type. Results: The prevalence of MSSA was 74.6% (754/1011) and 14.9% (151/1011) were MSSA-PENS-blaZnegative. MSSA-PENS-blaZnegative isolates (n = 151) were ascribed to 88 spa types and 11 CCs. The most frequent CCs were CC5 (35/151) and CC398 (25/151), with t002-CC5 and t571-CC398 being the most common lineages. Pan-susceptibility was identified in 117 of the 151 MSSA-PENS-blaZnegative isolates (77.5%). In the remaining isolates, erythromycin and clindamycin resistance was the most frequent resistance found, although tobramycin, ciprofloxacin, fusidic acid, mupirocin and/or tetracycline resistance was also detected. Thirty-eight MSSA-PENS-blaZnegative isolates were IEC negative and four isolates were Panton–Valentine leucocidin (‘PVL’) positive. Conclusions: A high penicillin susceptibility rate was detected among MSSA, opening therapeutic opportunities for BSIs. The emergence of new successful MSSA-PENS clones could be responsible for these data. The detection among MSSA-PENS-blaZnegative isolates of the clonal lineage CC398 or the absence of an IEC raises questions about their possible animal origin, requiring further analysis

Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems

Automated antimicrobial susceptibility testing devices are widely implemented in clinical microbiology laboratories in Spain, mainly using EUCAST (European Committee on Antimicrobial Susceptibility Testing) breakpoints. In 2007, a group of experts published recommendations for including antimicrobial agents and selecting concentrations in these systems. Under the patronage of the Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) and the Study Group on Mechanisms of Action and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), and aligned with the Spanish National Plan against Antimicrobial Resistance (PRAN), a group of experts have updated this document. The main modifications from the previous version comprise the inclusion of new antimicrobial agents, adaptation of the ranges of concentrations to cover the EUCAST breakpoints and epidemiological cut-off values (ECOFFs), and the inference of new resistance mechanisms. This proposal should be considered by different manufacturers and users when designing new panels or cards. In addition, recommendations for selective reporting are also included. With this approach, the implementation of EUCAST breakpoints will be easier, increasing the quality of antimicrobial susceptibility testing data and their microbiological interpretation. It will also benefit epidemiological surveillance studies as well as the clinical use of antimicrobials aligned with antimicrobial stewardship programs

Sensibilidad de microorganismos gramnegativos de infecciones intraabdominales y evolución de los aislados con ß-lactamasas de espectro extendido en el estudio smart en españa (2002-2010)

Introducción. El estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) tiene como objetivo monitorizar la sensibilidad a los antimicrobianos de los microorganismos gramnegativos aislados en la infección intraabdominal, con especial seguimiento de los que producen β-lactamasas de espectro extendido (BLEE). Material y métodos. Se han analizado por microdulución los datos de sensibilidad de 8.869 aislados recogidos en el estudio SMART en España entre 2002 y 2010 en el que han participado 16 centros. Resultados. Escherichia coli fue el patógeno más frecuente (60,9% en la infección intraabdominal adquirida en la comunidad y 49,9% en la nosocomial) seguido de Klebsiella pneumoniae (8,9% vs 9,2%). Pseudomonas aeruginosa fue más habitual en la infección nosocomial (5,6% comunitaria y 8,6% nosocomial). La frecuencia de aislados con BLEE fue: E. coli 8,7%, K. pneumoniae 8,4%, Klebsiella oxytoca 1,4% y Proteus mirabilis 1,6%. En los pacientes de mayor edad aumentó la proporción global de aislados con BLEE (6,8% en pacientes >60 años). Ertapenem y meropenem fueron los antimicrobianos más activos en el conjunto de las enterobacterias (rango de sensibilidad con criterios EUCAST, 89-100%) y también entre los aislados con BLEE (95,5-100%). La actividad de amoxicilina/ácido clavulánico y piperacilina/tazobactam fue considerablemente inferior, en particular en los aislados con BLEE. Ertapenem mantuvo una buena actividad (sensibilidad >95%) en los productores de BLEE resistentes a amoxicilina/ácido clavulánico, piperacilina/tazobactam o fluoroquinolonas. Conclusiones. Los datos de sensibilidad del estudio SMART en España avalan las guías terapéuticas actuales de infección intraabdominal que sitúan al ertapenem como tratamiento empírico de elección, teniendo en cuenta sobre todo la elevada frecuencia de aislados con BLEE en nuestro medio. Introduction. The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study records the antimicrobial susceptibility of Gram-negative bacilli obtain from intraabdominal infections with special focus in isolates with extended spectrum ß-lactamases (ESBLs). Material and Methods. The antimicrobial susceptibility of 8,869 isolates was analyzed by microdilution during the SMART study performed in Spain from 2002 to 2010. Isolates were recovered in 16 centres. Results. Escherichia coli was the most prevalent pathogen (60.9% from intraabdominal infections acquired in the community and 49.9% in those from nosocomial origin) followed by Klebsiella pneumoniae (8.9% vs 9.2%). Pseudomonas aeruginosa was more common in intraabdominal infections from nosocomial origin (5.6% community and 8.6% nosocomial). Frequency of ESBL-producing isolates was: E. coli, 8.7%; K. pneumoniae, 8.4%; Klebsiella oxytoca, 1.4%; and Proteus mirabilis, 1.6%. Overall, ESBL-producing isolates were more frequently isolated from elderly patients (6.8% >60 years). Ertapenem and meropenem were the most active antimicrobials (susceptibility range with EUCAST criteria, 89.0-100%) when considering all Enterobacteriaceae isolates and also against ESBL producers (95.5-100%). Susceptibility of amoxicillin/clavulanic acid and piperacillin/tazobactam was lower, particularly among ESBL-producing isolates. Nevertheless, ertapenem maintained a good activity (susceptibility >95%) in ESBL-producers that were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam or fluoroquinolones. Conclusions. Antimicrobial susceptibility data from the SMART-Spain study reinforce current therapeutic guidelines of intraabdominal infections that include ertapenem as the empirical choice for treatment. This is also supported by the high frequency of ESBL-producers in our geographic area

Phenotypic and molecular characterization of IMP-producing Enterobacterales in Spain: Predominance of IMP-8 in Klebsiella pneumoniae and IMP-22 in Enterobacter roggenkampii

ObjectivesLittle is known about IMP-producing Enterobacterales (IMP-Ent) in Europe. We analyzed at genomic and phenotypic level IMP-Ent isolates circulating in Spain in a 9-year period.Materials and methodsIMP-Ent isolates submitted to our reference laboratory were included. Antibiotic susceptibility was performed using microdilution method (EUCAST), and IMP-carbapenemase activity was measured with carbapenemase inhibitors, the β-CARBA method, the modified Hodge test (MHT), and the modified carbapenemase inhibition method (mCIM). All isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsFifty IMP-Ent isolates, collected from 19 hospitals in 13 Spanish provinces, were detected: Klebsiella pneumoniae (IMP-Kpn) (24; 48%), Enterobacter roggenkampii (13; 26%), Enterobacter hormaechei (8, 16%), Klebsiella oxytoca (two; 4%), Enterobacter asburiae (one, 2%), Serratia marcescens (one; 2%) and Escherichia coli (one; 2%). All isolates were positive by the MHT and β-CARBA tests; 48 (96%) were mCIM positive; 12 (24%) and 26 (52%) displayed positive inhibition with dipicolinic (meropenem) and EDTA (ertapenem), respectively. Five IMP-carbapenemase types were identified: IMP-8 (22; 44%), IMP-22 (17; 34%), IMP-13 (7; 14%), IMP-28 (two; 4%), and IMP-15 (two; 4%), predominating IMP-8 in K. pneumoniae and IMP-22 in E. roggenkampii. IMP-28 was exclusively identified in K. oxytoca and IMP-15 in E. hormaechei. Predominant STs were ST405 (29.2%), ST15 (25%) and ST464 (20.8%) in IMP-Kpn; ST96 (100%) in E. roggenkampii and ST182 (62.5%) in E. hormachei. Colistin and amikacin were the most active non-carbapenem antibiotics against IMP-Ent.ConclusionIMP-Ent isolates remain infrequent in Spain, although in recent years have been circulating causing nosocomial outbreaks, being IMP-8-producing K. pneumoniae and IMP-22-producing E. roggenkampii the most frequently detected in this study. Inhibition with EDTA or dipicolinic acid presented false negative results in some IMP-producing strains. Active microbiological and molecular surveillance is essential for a better comprehension and control of IMP-Ent dissemination

Investigation of Staphylococcus strains with heterogeneous resistance to glycopeptides in a Turkish university hospital

BACKGROUND: The hetero-glycopeptide intermediate staphylococci is considered to be the precursor of glycopeptide intermediate staphylococci especially vancomycin intermediate Staphylococcus aureus (VISA). For this purpose, we aimed to investigate the heterogeneous resistance to glycopeptide and their frequencies in 135 Staphylococcus strains. METHODS: Heterogeneous resistance of Staphylococcus strains was detected by inoculating the strains onto Brain Heart Infusion agar supplemented with 4 mg/L of vancomycin (BHA-V4). Agar dilution method was used for determining MICs of glycopeptides and population analysis profile was performed for detecting frequency of heterogeneous resistance for the parents of selected strains on BHA-4. RESULTS: Eight (6%) out of 135 Staphylococcus strains were exhibited heterogeneous resistance to at least one glycopeptide. One (1.2%) out of 81 S. aureus was found intermediate resistance to teicoplanin (MIC 16 mg/L). Other seven strains were Staphylococcus haemolyticus (13%) out of 54 coagulase negative staphylococci (CoNS). Six of the seven strains were detected heterogeneously reducing susceptibility to vancomycin (MICs ranged between 5–8 mg/L) and teicoplanin (MICs ranged between 32–64 mg/L), and one S. haemolyticus was found heterogeneous resistance to teicoplanin (MIC 32 mg/L). Frequencies of heterogeneous resistance were measured being one in 10(6 )– 10(7 )cfu/ml. MICs of vancomycin and teicoplanin for hetero-staphylococci were determined as 2–6 folds and 3–16 folds higher than their parents, respectively. These strains were isolated from six patients (7%) and two (4%) of health care wokers hands. Hetero-VISA strain was not detected. CONCLUSION: Heterogeneous resistance to glycopeptide in CoNS strains was observed to be significantly more emergent than those of S. aureus strains (vancomycin P 0.001, teicoplanin, P 0.007). The increase MICs of glycopeptide resistance for subpopulations of staphylococci comparing with their parents could be an important clue for recognizing the early steps in the appearance of VISA strains. We suggested to screen clinical S. aureus and CoNS strains, systematically, for the presence of heterogeneously resistance to glycopeptide

Carbapenem-resistant Citrobacter spp. isolated in Spain from 2013 to 2015 produced a variety of carbapenemases including VIM-1, OXA-48, KPC-2, NDM-1 and VIM-2

Objectives: There is little information about carbapenemase-producing (CP) Citrobacter spp.We studied the molecular epidemiology and microbiological features of CP Citrobacter spp. isolates collected in Spain (2013-15). Methods: In total, 119 isolates suspected of being CP by the EUCAST screening cut-off values were analysed. Carbapenemases and ESBLs were characterized using PCR and sequencing. The genetic relationship among Citrobacter freundii isolates was studied by PFGE. Results: Of the 119 isolates, 63 (52.9%) produced carbapenemases, of which 37 (58.7%) produced VIM-1, 20 (31.7%) produced OXA-48, 12 (19%) produced KPC-2, 2 (3.2%) produced NDM-1 and 1 (1.6%) produced VIM- 2; 9 C. freundii isolates co-produced VIM-1 plus OXA-48. Fourteen isolates (22.2%) also carried ESBLs: 8 CTX-M-9 plus SHV-12, 2 CTX-M-9, 2 SHV-12 and 2 CTX-M-15. Fifty-seven isolates (90.5%) were C. freundii, 4 (6.3%) were Citrobacter koseri, 1 (1.6%) was Citrobacter amalonaticus and 1 (1.6%) was Citrobacter braakii. By EUCAST breakpoints, eight (12.7%) of the CP isolates were susceptible to the four carbapenems tested. In the 53 CP C. freundii analysed by PFGE, a total of 44 different band patterns were observed. Four PFGE clusters were identified: cluster 1 included eight isolates co-producing VIM-1 and OXA-48; blaVIM-1 was carried in a class 1 integron (intI-blaVIM-1 - aacA4-dfrB1-aadA1-catB2-qacE¿1/sul1) and blaOXA-48 was carried in a Tn1999.2 transposon. Conclusions: We observed the clonal and polyclonal spread of CP Citrobacter spp. across several Spanish geographical areas. Four species of Citrobacter spp. produced up to five carbapenemase types, including coproduction of VIM-1 plus OXA-48. Some CP Citrobacter spp. isolates were susceptible to the four carbapenems tested, a finding with potential clinical implications