Dynamics of the MRSA Population in a Chilean Hospital: a Phylogenomic Analysis (2000-2016).

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P < 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or more limited typing techniques that lack the resolution to represent an accurate description of the genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000 and 2016 in Chile providing the largest and most detailed study of clonal dynamics of MRSA in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing in frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

Evaluación de la Susceptibilidad Antimicrobiana de Porphyromonas gingivalis Aisladas de Pacientes Periodontales en Población Chilena

RESUMEN: Porphyromonas gingivalis (P. gingivalis) es un microorganismo frecuentemente aislado en pacientes con periodontitis, en los que también se incluye los de la población chilena. El modelo de "Keystone bacteria" demostró que P. gingivalis induce la disbiosis del biofilm subgingival y permite el desarrollo de algunas especies sobre otras, modulando la patogenicidad de la comunidad microbiológica completa. El tratamiento de la periodontitis es principalmente mecánico, pero en condiciones específicas es necesario el complemento con antibioterapia. Los estudios globales de antibióticos evaluados en ensayos clínicos y estudios in vitro han mostrado resultados mixtos en cuanto a eficacia y susceptibilidad. Este estudio descriptivo tuvo como objetivo evaluar el perfil de susceptibilidad antimicrobiana in vitro a metronidazol, clindamicina, amoxicilina más ácido clavulánico, moxifloxacino y azitromicina de P. gingivalis aisladas de pacientes periodontales chilenos. Se obtuvieron muestras microbiológicas de pacientes con diagnóstico de periodontitis estadíos III y IV generalizada, las que se sometieron a procesos de identificación mediante un espectrómetro de masas (MALDI-TOF MS). Posteriormente, a cada muestra positiva a P. gingivalis se aplicó el protocolo gold standard de susceptibilidad para los cinco antimicrobianos evaluados (Dilución en Agar Sangre Brucella- McFarland 0.5). Se seleccionaron 50 pacientes (25 mujeres, 25 hombres) entre 34-69 años. Finalmente, se recuperaron 25 cepas de P. gingivalis (50 %) para el análisis de susceptibilidad y todas ellas fueron sensibles a todos los antibióticos evaluados (100 % susceptibilidad). Las cepas de P. gingivalis fueron altamente sensibles a los cinco antibióticos evaluados en esta población, lo que podría implicar contar con diferentes alternativas de tratamiento farmacológico antimicrobi ano como complemento al tratamiento mecánico convencional en pacientes específicos.

ABSTRACT: Porphyromonas gingivalis (P. gingivalis) is a microorganism frequently isolated in patients with periodontitis, which also include those of Chilean population. The "Keystone bacteria" model demonstrated that P. gingivalis induces dysbiosis of the subgingival biofilm and allows the development of some species over others, modulating the pathogenicity of the entire microbiological community. The treatment of periodontitis is mainly mechanical; nevertheless, under specific conditions the complement with antibiotherapy is needed. Global studies of antibiotics evaluated in clinical trials and in vitro studies have shown mixed results in terms of efficacy and susceptibility. This descriptive study aimed to evaluate antimicrobial susceptibility profile in vitro to metronidazole, clindamycin, amoxicillin plus clavulanic acid, moxifloxacin and azithromycin of P. gingivalis isolated from Chilean periodontal patients. Microbiological samples were obtained from patients with a diagnosis of generalized periodontitis stages III and IV, which were exposed to identification processes by a mass spectrometer (MALDI-TOF MS). Subsequently, the gold standard susceptibility protocol for the five antimicrobials evaluated was applied to each P. gingivalis- positive sample (Dilution in Brucella-McFarland Blood Agar 0.5). 50 patients (25 women, 25 men) between 34-69 years old were selected. Finally, 25 P. gingivalis strains (50 %) were recovered for susceptibility testing and all of them were susceptible to all antibiotics tested (100 % susceptibility). P. gingivalis strains were highly susceptible to the five antibiotics evaluated in this population, which could implies counting different antimicrobial pharmacological treatment alternatives as a complement to conventional mechanical treatment in specific patients.

A Multicenter Study To Evaluate Ceftaroline Breakpoints: Performance in an Area with High Prevalence of Methicillin-Resistant Staphylococcus aureus Sequence Type 5 Lineage.

Ceftaroline (CPT) is a broad-spectrum agent with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). The sequence type 5 (ST5) Chilean-Cordobés clone, associated with CPT nonsusceptibility, is dominant in Chile, a region with high rates of MRSA infections. Here, we assessed the in vitro activity of CPT against a collection of MRSA isolates collected between 1999 and 2018 from nine hospitals (n = 320) and community settings (n = 41) in Santiago, Chile, and evaluated performance across testing methodologies. We found that our hospital-associated isolates exhibited higher CPT MIC distributions (MIC50 and MIC90 of 2 mg/liter) than the community isolates (MIC50 and MIC90 of 0.5 mg/liter), a finding that was consistent across time and independent of the culture source. High proportions (64%) of isolates were CPT nonsusceptible despite the absence of CPT use in Chile. Across methodologies, the Etest underestimated the MIC relative to the gold standard broth microdilution (BMD) test (MIC50 and MIC90 of 1 and 1.5 mg/liter, respectively). There was low (∼51%) categorical agreement (CA) between Etest and BMD results across CLSI and EUCAST breakpoints. The recent revision of CLSI guidelines abolished "very major error" (VME) from the previous guidelines (81%), which perform similarly to the EUCAST guidelines. The level of concordance between CLSI and EUCAST for BMD testing and Etest was >95%. Disk diffusion performed poorly relative to BMD under CLSI (CA, 55%) and EUCAST (CA, 36%) guidelines. Comparison of EUCAST to CLSI for disk diffusion (with EUCAST used as the reference) showed low agreement (CA, 25%; VME, 70%). In summary, CPT-nonsusceptible MRSA are dominant in clinical settings in Chile. Our results provide data to support the reevaluation of CPT breakpoints and to improve agreement across methodologies and agencies.

[Impact of mass spectrometry by MALDI-TOF MS for the rapid identification of aerobic and anaerobic bacteria of clinical importance]. / Impacto de la espectrometría de masas por MALDI-TOF MS en la identificación rápida de bacterias aeróbicas y anaeróbicas de importancia clínica.

BACKGROUND: MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry) technology, recently introduced in the microbiology laboratory has proven to be a precise and rapid method for bacterial identification. OBJECTIVE: To evaluate the performance, costs associated and turnaround time of MALDI-TOF in a routine laboratory. MATERIAL AND METHOD: Five hundred and sixty one clinical isolates (281 aerobes and 280 anaerobes) previously identified by conventional methods were evaluated. Discordances were resolved by means of 16S rRNA sequencing. RESULTS: MALDI-TOF identified 95, 7% of the aerobes isolates and 86, 4% of the anaerobes. The groups with better performance were the enterobacteriacea and Bacteroides spp with 95% and 100% identification at the species level. The error rate of MALDI-TOF and conventional methods compared to sequencing was 0, 39% and 9, 4% respectively. The costs associated were 8 times lower with a turnaround time of 6 hours. CONCLUSION: MALDI-TOF proved to be simple, precise and less expensive technology compared to the traditional methods.

Impacto de la espectrometría de masas por MALDI-TOF MS en la identificación rápida de bacterias aeróbicas y anaeróbicas de importancia clínica / Impact of mass spectrometry by MALDI-TOF MS for the rapid identification of aerobic and anaerobic bacteria of clinical importance

Background: MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry) technology, recently introduced in the microbiology laboratory has proven to be a precise and rapid method for bacterial identification. Objective: To evaluate the performance, costs associated and turnaround time of MALDI-TOF in a routine laboratory. Material and Method: Five hundred and sixty one clinical isolates (281 aerobes and 280 anaerobes) previously identified by conventional methods were evaluated. Discordances were resolved by means of 16S rRNA sequencing. Results: MALDI-TOF identified 95, 7% of the aerobes isolates and 86, 4% of the anaerobes. The groups with better performance were the enterobacteriacea and Bacteroides spp with 95% and 100% identification at the species level. The error rate of MALDI-TOF and conventional methods compared to sequencing was 0, 39% and 9, 4% respectively. The costs associated were 8 times lower with a turnaround time of 6 hours. Conclusion: MALDI-TOF proved to be simple, precise and less expensive technology compared to the traditional methods.

Introducción: La tecnología MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) incorporada recientemente en el laboratorio de microbiología ha demostrando ser un método rápido y preciso para la identificación bacteriana. Objetivo: Evaluar el desempeño de MALDI-TOF para la identificación de aislados clínicos, comparar los costos asociados y el tiempo en la entrega de resultados en un laboratorio de rutina. Material y Método: Se evaluaron un total de 561 aislados de pacientes (281 aeróbicos y 280 anaeróbicos estrictos) identificados previamente por métodos convencionales, los que fueron identificados por MALDI-TOF. Las discordancias fueron resueltas mediante secuenciación del 16S ARNr. Resultados: MALDI-TOF identificó adecuadamente a 95,7% de los aislados aeróbi-cos y 86,4% de los anaeróbicos estrictos, observándose el mayor porcentajes de identificación a nivel de especie en los grupos de enterobacterias y Bacteroides spp (95 y 100% respectivamente). La tasa de error de MALDI-TOF y métodos convencionales vs secuenciación fue de 0,39 y 9,4%, respectivamente. El costo asociado por identificación fue ocho veces menor que el de los métodos tradicionales con una demora promedio de seis horas en la entrega de resultados. Conclusión: MALDI-TOF mostró ser una tecnología simple, precisa y de menor costo que los métodos tradicionales.

Antimicrobial susceptibility and genetic characteristics of Propionibacterium acnes isolated from patients with acne.

BACKGROUND: Propionibacterium acnes is an important target in acne management. Antibiotic resistance has increased, reducing its clinical efficiency. OBJECTIVE: To study the prevalence, antimicrobial susceptibility patterns, and resistance mechanisms of P. acnes isolated from patients with acne. METHODS: Skin swabs were collected from 83 patients. Agar dilution determined the minimum inhibitory concentrations of five antibiotics. Polymerase chain reaction and DNA sequencing were used to identify mutations. Results P. acnes was isolated in 80 of 83 patients (96%), and 27 patients had resistance to antibiotics (33.7%). The mean age was older in the antibiotic-resistant group (20.8 ± 5.8 vs. 18.3 ± 3.7, P = 0.02). Resistance to trimethoprim-sulfamethoxazole was 26.3%, erythromycin 12.5%, and clindamycin 7.5%. All clindamycin-resistant strains had cross-resistance to erythromycin, and 40% erythromycin-resistant strains had cross-resistance to trimethoprim-sulfamethoxazole. All strains were sensitive to tetracycline and doxycycline. The use of topical erythromycin or clindamycin was a risk factor to carry resistant strains (P = 0.02, P = 0.04, respectively). Resistance to trimethoprim-sulfamethoxazole was associated with acne severity (P = 0.02). Six of the 10 erythromycin-resistant strains had a mutation in the peptidyl transferase region of the 23S rRNA gene: one A2058G and five A2059G. No strain carrying mutation G2057A was found. CONCLUSIONS: Resistance to trimethoprim-sulfamethoxazole was the most common pattern found, and further studies are required to clarify its resistance mechanism. A certain tetracycline resistance was expected, but interestingly all strains remained sensitive. Resistance to erythromycin and clindamycin were influenced using topical formulations. Mutation A2059G was related to high resistance to erythromycin. Antibiotic resistance is increasing, and new strategies are needed.

Coagulase-negative staphylococci: clinical, microbiological and molecular features to predict true bacteraemia.

Coagulase-negative staphylococci (CNS) are frequently isolated from blood cultures, where they may be only a contaminant or the cause of bacteraemia. Determining whether an isolate of CNS represents a true CNS bacteraemia is difficult, and there is no single criterion with sufficient specificity. The aim of this study was to assess those clinical, microbiological, pathogenic and genotypic features that characterize true CNS bacteraemia. Twenty patients having two or more blood cultures positive for CNS and 20 patients with only one positive blood culture were studied. Significant bacteraemia was defined according to clinical and laboratory criteria. Incubation time for blood cultures to become positive, macroscopic appearance of colonies, species determination, biotype, susceptibility to antimicrobials, PFGE pattern and adherence capacity were all studied. Clinical bacteraemia was present in 16/20 patients with two or more positive blood cultures and in 2/20 patients with only one positive blood culture. A significant difference was seen in the median time to positivity between the 18 clinical bacteraemias and 22 contaminations (23.6 versus 29.2 h; P = 0.04, Wilcoxon). There was also a significant difference between the two groups in the median absorbance of the slime test (1.36 versus 0.58; P = 0.005). All significant bacteraemias with two or more positive blood cultures had the same species identified, the same antimicrobial susceptibility pattern and the same PFGE pattern. In two patients with true bacteraemia with only one positive blood culture, the incubation time for the culture to turn positive was <24 h and the slime production absorbance was >2.5. The most useful parameters for the diagnosis of true CNS bacteraemia for patients with two positive blood cultures were incubation time until positive, species identification, antimicrobial susceptibility pattern, slime production and PFGE pattern. For patients with only one blood culture positive for CNS, the useful parameters for prediction of true bacteraemia were incubation time until positive and slime production, both of which are simple, low-cost tests.