Advances in genomics of multi-drug resistant Stenotrophomonas / 生物工程学报

Stenotrophomonas species are non-fermentative Gram-negative bacteria that are widely distributed in environment and are highly resistant to numerous antibiotics. Thus, Stenotrophomonas serves as a reservoir of genes encoding antimicrobial resistance (AMR). The detection rate of Stenotrophomonas is rapidly increasing alongside their strengthening intrinsic ability to tolerate a variety of clinical antibiotics. This review illustrated the current genomics advances of antibiotic resistant Stenotrophomonas, highlighting the importance of precise identification and sequence editing. In addition, AMR diversity and transferability have been assessed by the developed bioinformatics tools. However, the working models of AMR in Stenotrophomonas are cryptic and urgently required to be determined. Comparative genomics is envisioned to facilitate the prevention and control of AMR, as well as to gain insights into bacterial adaptability and drug development.

Antimicrobial resistance and plasmid-mediated colistin resistance mechanism of diarrheagenic Escherichia coli recovered from foods in parts of China in 2020 / 中华预防医学杂志

Objective: To investigate the antimicrobial resistance of food-borne diarrheagenic Escherichia coli (DEC) and the prevalence of mcr genes that mediates mobile colistin resistance in parts of China, 2020. Methods: For 91 DEC isolates recovered from food sources collected from Fujian province, Hebei province, Inner Mongolia Autonomous Region and Shanghai city in 2020, Vitek2 Compact biochemical identification and antimicrobial susceptibility testing platform was used for the detection of antimicrobial susceptibility testing (AST) against to 18 kinds of antimicrobial compounds belonging to 9 categories, and multi-polymerase chain reaction (mPCR) was used to detect the mcr-1-mcr-9 genes, then a further AST, whole genome sequencing (WGS) and bioinformatics analysis were platformed for these DEC isolates which were PCR positive for mcr genes. Results: Seventy in 91 isolates showed different antimicrobial resistance levels to the drugs tested with a resistance rate of 76.92%. The isolates showed the highest antimicrobial resistance rates to ampicillin (69.23%, 63/91) and trimethoprim-sulfamethoxazole (59.34%, 54/91), respectively. The multiple drug-resistant rate was 47.25% (43/91). Two mcr-1 gene and ESBL (extended-spectrum beta-lactamase) positive EAEC (enteroaggregative Escherichia coli) strains were detected. One of them was identified as serotype of O11:H6, which showed a resistance profile to 25 tested drugs referring to 10 classes, and 38 drug resistance genes were predicted by genome analysis. The other one was O16:H48 serotype, which was resistant to 21 tested drugs belonging to 7 classes and carried a new variant of mcr-1 gene (mcr-1.35). Conclusion: An overall high-level antimicrobial resistance was found among foodborne DEC isolates recovered from parts of China in 2020, and so was the MDR (multi-drug resistance) condition. MDR strains carrying multiple resistance genes such as mcr-1 gene were detected, and a new variant of mcr-1 gene was also found. It is necessary to continue with a dynamic monitoring on DEC contamination and an ongoing research into antimicrobial resistance mechanisms.

Clinical distribution and drug resistance characteristics of carbapenem-resistant Klebsiella pneumoniae / 中华预防医学杂志

To explore the clinical distribution and drug resistance characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP), in order to provide reference for the prevention and treatment of CRKP infection. Retrospective analysis was performed on 510 clinical isolates of CRKP from January 2017 to December 2021, and strain identification and drug sensitivity tests were conducted by MALDI-TOF mass spectrometer and VITEK-2 Compact microbial drug sensitivity analyzer. The carbapenemase phenotype of CRKP strain was detected by carbapenemase inhibitor enhancement test. The CRKP strain was further categorized by immunochromogenic method and polymerase chain reaction (PCR) was used for gene detection. The results showed that 302 strains (59.2%) were derived from sputum, 127 strains (24.9%) from urine and 47 strains (9.2%) from blood. 231 (45.3%) were mainly distributed in intensive care, followed by 108 (21.2%) in respiratory medicine and 79 (15.5%) in neurosurgery. Drug susceptibility test result shows that the resistant rate of tigecycline increased from 1.0% in 2017 to 10.1% in 2021, the difference was statistically significant (χ2=14.444,P<0.05). The results of carbapenemase inhibitor enhancement test showed that 461 carbapenemase strains (90.4%) of 510 CRKP strains, including 450 serinase strains (88.2%), 9 metalloenzyme strains (1.8%), and 2 strains (0.4%) produced both serine and metalloenzyme. 49 strains (9.6%) did not produce enzymes. Further typing by immunochromogenic assay showed that 461 CRKP strains were KPC 450 (97.6%) and IMP 2 (0.4%). 7 NDM (1.5%); 2 strains of KPC+NDM (0.4%); PCR results were as follows: 450 strains of blaKPC (97.6%), 2 strains of blaIMP (0.4%), 7 strains of blaNDM (1.5%), and 2 strains of blaKPC+NDM (0.4%). In conclusion, CRKP strains mainly originated from sputum specimens and distributed in intensive care department, and the drug resistance characteristics were mainly KPC type in carbapenemase production. Clinical microbiology laboratory should strengthen the monitoring of CRKP strains, so as to provide reference for preventing CRKP infection and reducing the production of bacterial drug resistance.

Prevention and control of antimicrobial resistance using CRISPR-Cas system: a review / 生物工程学报

Bacterial multi-drug resistance (MDR) is a global challenge in the fields of medicine and health, agriculture and fishery, ecology and environment. The cross-region spread of antibiotic resistance genes (ARGs) among different species is one of the main cause of bacterial MDR. However, there is no effective strategies for addressing the intensifying bacterial MDR. The CRISPR-Cas system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins, can targetedly degrade exogenous nucleic acids, thus exhibiting high application potential in preventing and controlling bacterial MDR caused by ARGs. This review briefly introduced the working mechanism of CRISPR-Cas systems, followed by discussing recent advances in reducing ARGs by CRISPR-Cas systems delivered through mediators (e.g. plasmids, bacteriophages and nanoparticle). Moreover, the trends of this research field were envisioned, providing a new perspective on preventing and controlling MDR.

Characterization of Enterococcus spp. isolated from a fish farming environment in southern Brazil / Caracterização de Enterococcus spp. isolado de um ambiente de piscicultura no sul do Brasil

Abstract The aim of present study is to characterize the resistance and virulence profile of enterococci isolated from aquaculture excavated ponds and masonry tanks (6 samples) in southern Brazil. Samples were cultured in selective medium, 10 colonies were randomly selected from each sample, which were identified by MALDI-TOF and tested against 13 antimicrobials. The presence of resistance (tetL, tetM, tetS, ermB and msrC) and virulence (ace, esp, agg, cylA and gelE) genes were determined by PCR. A total of 79 enterococci were identified, and Entecococcus faecalis (44.3%) and E. casseliflavus (36.7%) were the most prevalent species isolated. Sixty-five strains (82.3%) were resistant to at least one of the antimicrobials tested, whereas 27 (34.2%) strains were multiresistant. The overall percentages of antimicrobial resistant isolates were: 58.2% to rifampicin, 40.5% to fluoroquinolones, 36.7% to erythromycin and 30.4% to tetracycline. The tetL and tetM genes were found in 57.7% of the tetracycline-resistant strains; and msrC in 31.01% of erythromycin-resistant strains. The most frequently detected virulence factors were ace and gelE genes. Although limited to a single farm, these data suggest that aquaculture may be a reservoir of resistant and virulent enterococci. This study is the first step towards enhancing our understandingof distribution, resistance and virulence profile in enterococci isolated from fish farming environments in the south Brazil.

Resumo O objetivo do estudo apresentado é caracterizar o perfil de resistência e virulência de enterococos isolados de viveiros escavados e tanques de alvenaria (6 amostras) de uma pisicultura no Sul do Brasil. As amostras foram cultivadas em meio seletivo, 10 colônias foram selecionadas aleatoriamente de cada amostra, que foram identificadas por MALDI-TOF e testadas contra 13 antimicrobianos. A presença de genes de resistência (tetL, tetM, tetS, ermB e msrC) e virulência (ace, esp, agg, cylA e gelE) foi determinada por PCR. Foram identificados 79 enterococos, sendo Entecococcus faecalis (44,3%) e E. casseliflavus (36,7%) as espécies mais frequentes isoladas. Sessenta e cinco cepas (82,3%) eram resistentes a pelo menos um dos antimicrobianos testados, enquanto 27 (34,2%) eram multirresistentes. As porcentagens gerais de isolados resistentes a antimicrobianos foram: 58,2% para rifampicina, 40,5% para fluoroquinolonas, 36,7% para eritromicina e 30,4% para tetraciclina. Os genes tetL e tetM foram encontrados em 57,7% das cepas resistentes à tetraciclina; e msrC em 31,01% das cepas resistentes à eritromicina. Os fatores de virulência mais comumente detectados foram ace e gelE. Embora limitados a uma única fazenda, esses dados indicam que a aquicultura pode ser uma fonte de enterococos resistentes e virulentos. Este estudo é o primeiro passo para melhorar nosso entendimento da distribuição, resistência e perfil de virulência em enterococos isolados de ambientes de piscicultura no sul do Brasil.

Genotypic evaluation of antimicrobial resistance in Staphylococcus spp. isolated from bovine clinical mastitis / [Avaliação genotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite clínica bovina]

Bovine clinical mastitis caused by Staphylococcus spp. is a serious and widespread disease in the world of dairy farming. Antimicrobial therapy is of fundamental importance in the prevention and treatment of infectious mastitis, but the indiscriminate use of antimicrobials acts as a determining factor for the spread of the disease. The present study evaluated the resistance profiles of 57 Staphylococcus spp. isolated from bovine clinical mastitis to beta-lactams and gentamicin, relating characteristics of phenotype (in vitro susceptibility tests) and genotype (detection and expression of genes encoding resistance - mecA, mecALGA251, blaZ, femA, femB, and aacA-aphD - using PCR and RT-PCR, respectively). One or more genes coding for resistance to different antimicrobials were detected in 50 Staphylococcus spp. isolates. The femA and femB genes were the most frequent (75.4% for both). The observed expression of the genes was as follows: blaZ (60%), femA (39.5%), aacA-aphD (50%), femB (32.6%), mecA (8.3%), and mecALGA251 (0%). Considering the relevance of the genus Staphylococcus to bovine mastitis, this study aimed to elucidate aspects regarding the genotypic and phenotypic profiles of these microorganisms so as to contribute to the development of effective strategies for mastitis control.(AU)

A mastite clínica bovina causada por Staphylococcus spp. é uma doença grave e generalizada no mundo da pecuária leiteira. A terapia antimicrobiana é de fundamental importância na prevenção e no tratamento da mastite infecciosa, mas o uso indiscriminado de antimicrobianos atua como fator determinante para a disseminação da doença. O presente estudo avaliou os perfis de resistência de 57 Staphylococcus spp. isolados de mastite clínica bovina em relação ao uso de betalactâmicos e gentamicina, relacionando características do fenótipo (testes de suscetibilidade in vitro) e genótipo (detecção e expressão de genes que codificam resistência - mecA, mecALGA251, blaZ, femA, femB, e aacA-aphD - usando PCR e RT-PCR, respectivamente). Um ou mais genes que codificam resistência a diferentes antimicrobianos foram detectados em 50 Staphylococcus spp. isolados. Os genes femA e femB foram os mais frequentes (75,4% para ambos). A expressão observada dos genes foi a seguinte: blaZ (60%), femA (39,5%), aacA-aphD (50%), femB (32,6%), mecA (8,3%) e mecALGA251 (0%). Considerando-se a relevância do gênero Staphylococcus para a mastite bovina, este estudo teve como objetivo elucidar aspectos referentes aos perfis genotípico e fenotípico desses microrganismos, a fim de contribuir para o desenvolvimento de estratégias eficazes para o controle da mastite.(AU)

Virulence genotyping and antimicrobial resistance profiles of Yersinia enterocolitica isolated from meat and meat products in Egypt / Genotipagem de virulência e perfis de resistência antimicrobiana de Yersinia enterocolitica isolados de carne e derivados no Egito

Pathogenic Yersinia enterocolitica (Y. enterocolitica) is one of the food-borne entero-pathogen responsible for yersiniosis in humans. The purpose of this research was to survey the prevalence, virulence-associated genes, and antimicrobial resistance of Y. enterocolitica isolated from meat and meat product samples in Egypt. Forty-one (5.9%) out of 700- samples of chicken meat, beef, ground beef, and sausage were positive Y. enterocolitica with a high prevalence in chicken meat (12%). Five virulence genes (ail, inv, ystA, ystB, and yadA) were characterized among 41 Y. enterocolitica isolates with variable frequencies. Among the strains tested, the ystB gene was detected with a high percentage (78.1%), followed by inv gene (70.7%), ail gene (14.6%), ystA gene (12.2%), and yadA gene (2.4%). A high resistance rate was estimated to amoxicillin-clavulanic acid (100%), followed by cefazolin (95%), ampicillin (65.9%), and doxycycline (51.2%), whilst a high sensitivity rate was observed to gentamicin and ciprofloxacin (97.6% each). Interestingly, the multidrug resistance was specified in the 70.7% of strains and showing 13 resistance patterns. Based on nucleotide sequence analysis of the 16s rRNA gene, the phylogenetic tree showed the genetic relatedness amongst Y. enterocolitica isolates. These findings highlighted the emergence of virulent and multidrug-resistant pathogenic Y. entrocolitica in retailed meat and meat products in Egypt.

A Yersinia enterocolitica patogênica (Y. enterocolitica) é um dos enteropatógenos de origem alimentar responsáveis pela yersiniose no ser humano. O objetivo desta pesquisa foi avaliar a prevalência, genes associados à virulência e resistência antimicrobiana de Y. enterocolitica isolada de amostras de carne e produtos à base de carne no Egito. Quarenta e um (5,9%) de 700 amostras de carne de frango, carne bovina, moída e linguiça foram Y. enterocolitica positivas, com alta prevalência em carne de frango (12%). Cinco genes de virulência (ail, inv, ystA, ystB e yadA) foram caracterizados entre 41 isolados de Y. enterocolitica com frequências variáveis. Entre as cepas testadas, o gene ystB foi detectado com uma alta porcentagem (78,1%), seguido pelo gene inv (70,7%), ail genes (14,6%), gene ystA (12,2%) e gene yadA (2,4%). Foi estimada uma alta taxa de resistência ao ácido amoxicilina-clavulânico (100%), seguida de cefazolina (95%), ampicilina (65,9%) e doxiciclina (51,2%), enquanto uma alta taxa de sensibilidade foi observada para gentamicina e ciprofloxacina (97,6% cada). Curiosamente, a resistência a múltiplas drogas foi especificada em 70,7% das cepas e mostrando 13 padrões de resistência. Com base na análise da sequência nucleotídica do gene rRNA 16s, a árvore filogenética mostrou a relação genética entre isolados de Y. enterocolitica. Esses achados destacaram o surgimento de Y. entrocolitica patogênica virulenta e multirresistente em carnes e produtos à base de carne no Egito.

Detection of drug-resistance genes of / 中国当代儿科杂志

OBJECTIVE@#To study the drug resistance of @*METHODS@#BALF specimens were collected from 245 children with RMPP who were admitted to the Children's Hospital Affiliated to Zhengzhou University from March 2016 to December 2020. A rapid cultured drug sensitivity assay was used to detect the resistance of MP isolates to nine commonly used antimicrobial drugs. The real-time PCR was used to measure MP DNA. The direct sequencing was used to detect gene mutations in MP 23SrRNA V region central ring.@*RESULTS@#Among the 245 BALF specimens, 207 tested positive for MP DNA, with a positive rate of 84.5%. The results of drug susceptibility test showed that the children with RMPP had a resistance rate of > 70% to macrolide antimicrobial drugs, with the highest resistance rate to clarithromycin, followed by roxithromycin, clindamycin, acetylspiramycin, erythromycin, and azithromycin, and these children had a resistance rate of < 5% to quinolone antimicrobial drugs. Among the 207 MP DNA-positive specimens, 41 (19.8%) had no drug-resistance gene mutations and 166 (80.2%) had drug-resistance gene mutations, among which 154 (74.4%) had an A→G mutation at 2063 locus of 23SrRNA V region central ring, 7 (3.4%) had an A→G mutation at 2064 locus, and 5 (2.4%) had mutations in both 2063 and 2064 loci. Among the 166 specimens with point mutations of the MP 23SrRNA gene, 159 (95.8%) had point mutations at 2063 locus. The A→G point mutation at 2063 locus of 23SrRNA V region central ring had a great impact on resistance to macrolide antimicrobial drugs. There was a significant difference in the distribution of alleles at 2063 locus between the children with resistance to clarithromycin, roxithromycin, clindamycin, acetylspiramycin, erythromycin, and azithromycin (@*CONCLUSIONS@#MP in the BALF of children with RMPP has a relatively high resistance rate to macrolide antimicrobial drugs. Resistance to macrolide antimicrobial drugs is closely associated with the A→G point mutation in the 23SrRNA gene, and the point mutation at 2063 locus of 23SrRNA V region central ring may affect the drug-resistance mechanism of MP.

Development of molecular markers of Mycobacterium tuberculosis rifampicin resistance gene rpoB by PARMS technology / 生物工程学报

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

Comparative Study of the Genetic Diversity, Antimicrobial Resistance, and Pathogenicity of / 生物医学与环境科学（英文）

Objective@#This study was performed to compare the genetic diversity, virulence, and antimicrobial resistance of @*Methods@#A total of 38 clinical strains and 19 strains from healthy individuals were isolated from the samples collected in Ma'anshan City, Anhui Province. Their taxonomy was investigated using concatenated @*Results@#The 57 @*Conclusions@#The taxonomy, virulence properties, and antibiotic resistance of

Role of aminoglycoside-modifying enzymes and 16S rRNA methylase (ArmA) in resistance of Acinetobacter baumannii clinical isolates against aminoglycosides

Abstract INTRODUCTION: This study aimed to determine the role of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase (ArmA) in Acinetobacter baumannii clinical isolates. METHODS: We collected 100 clinical isolates of A. baumannii and identified and confirmed them using microbiological tests and assessment of the OXA-51 gene. Antibiotic susceptibility testing was carried out using disk agar diffusion and micro-broth dilution methods. The presence of AME genes and ArmA was detected by PCR and multiplex PCR. RESULTS: The most and least effective antibiotics in this study were netilmicin and ciprofloxacin with 68% and 100% resistance rates, respectively. According to the minimum inhibitory concentration test, 94% of the isolates were resistant to gentamicin, tobramycin, and streptomycin, while the highest susceptibility (20%) was observed against netilmicin. The proportion of strains harboring the aminoglycoside resistance genes was as follows: APH(3′)-VIa (aphA6) (77%), ANT(2")-Ia (aadB) (73%), ANT(3")-Ia (aadA1) (33%), AAC(6′)-Ib (aacA4) (33%), ArmA (22%), and AAC(3)-IIa (aacC2) (19%). Among the 22 gene profiles detected in this study, the most prevalent profiles included APH(3′)-VIa + ANT(2")-Ia (39 isolates, 100% of which were kanamycin-resistant), and AAC(3)-IIa + AAC(6′)-Ib + ANT(3")-Ia + APH(3′)-VIa + ANT(2")-Ia (14 isolates, all of which were resistant to gentamicin, kanamycin, and streptomycin). CONCLUSIONS: High minimum inhibitory concentration of aminoglycosides in isolates with the simultaneous presence of AME- and ArmA-encoding genes indicated the importance of these genes in resistance to aminoglycosides. However, control of their spread could be effective in the treatment of infections caused by A. baumannii.

Coagulase-negative staphylococci: a 20-year study on the antimicrobial resistance profile of blood culture isolates from a teaching hospital

ABSTRACT The increasing rates of nosocomial infection associated with coagulase-negative staphylococci (CoNS) were the rationale for this study, aiming to categorize oxacillin-resistant CoNS species recovered from blood culture specimens of inpatients at the UNESP Hospital das Clínicas in Botucatu, Brazil, over a 20-year period, and determine their sensitivity to other antimicrobial agents. The mecA gene was detected in 222 (74%) CoNS samples, and the four types of staphylococcal chromosomal cassette mec (SCCmec) were characterized in 19.4%, 3.6%, 54.5%, and 14.4% of specimens, respectively, for types I, II, III, and IV. Minimal inhibitory concentration (MIC) values to inhibit 50% (MIC50) and 90% (MIC90) of specimens were, respectively, 2 and >256 µL/mL for oxacillin, 1.5 and 2 µL/mL for vancomycin, 0.25 and 0.5 µL/mL for linezolid, 0.094 and 0.19 µL/mL for daptomycin, 0.19 and 0.5 µL/mL for quinupristin/dalfopristin, and 0.125 and 0.38 µL/mL for tigecycline. Resistance to oxacillin and tigecycline and intermediate resistance to quinupristin/dalfopristin were observed. Eight (2.7%) of all 300 CoNS specimens studied showed reduced susceptibility to vancomycin. Results from this study show high resistance rates of CoNS to antimicrobial agents, reflecting the necessity of using these drugs judiciously and controlling nosocomial dissemination of these pathogens.

Antibiotic susceptibility patterns and prevalence of streptococcus agalactiae rectovaginal colonization among pregnant women in iran

Abstract Objective Streptococcus agalactiae is an important pathogen in neonates and pregnant women. Neonatal invasive infections due to S. agalactiae are life-threatening and preventive strategies for this challenge of human have become a concern. The aim of the present study was to determine the prevalence of rectovaginal colonization, related risk factors and antibiotic resistance pattern of S. agalactiae among pregnant women in Iran. Methods The present study was performed on 240 pregnant women. Vaginal and rectal swabs were obtained from all of the women and then were transferred to the laboratory. The isolation and identification of S. agalactiae was performed by standard microbiological tests and polymerase chain reaction (PCR) assay. The antimicrobial susceptibility patterns of the isolates were determined by the Kirby-Bauer disk diffusion. Polymerase chain reaction was used to detect ermB and mefA genes in erythromycin-nonsusceptible isolates. Results Out of 240 pregnant women, 16 cases (6.7%) were colonized by S. agalactiae. There is no significant association between demographic-obstetric factors and maternal S. agalactiae colonization in the pregnant women. Linezolid, vancomycin and ampicillin were the most effective antibiotics against S. agalactiae. The ermB gene was present in 6 (35.29%) S. agalactiae isolates. However, the mefA gene was not detected in any of the isolates. Conclusion Given the relatively significant prevalence of S. agalactiae colonization in the pregnant women in the present study and the risk of serious neonatal infections, the screening of pregnant mothers for the bacteria seems necessary. Our findings highlight the importance of appropriate antibiotic prophylaxis during pregnancy for the prevention of early onset S. agalactiae-neonatal infection and comorbidity.

Overview of antibiotic resistance genes database / 生物工程学报

The discovery of antibiotics is a big revolution in human history, and its clinical application has saved countless lives. However, with the widespread and abuse of antibiotics, many pathogens have developed resistance, and even "Super Bacteria" resistance to multiple drugs have evolved. In the arms race between humans and pathogens, humans are about to face a situation where no medicine is available. Research on microbial antibiotic resistance genes, resistance mechanisms, and the spread of resistance has attracted the attention of many scientific researchers, and various antibiotic resistance gene databases and analysis tools have emerged. In this review, we collect the current databases that focus on antibiotics resistance genes, and discuss these databases in terms of database types, data characteristics, antibiotics resistance gene prediction models and the types of analyzable sequences. In addition, a few gene databases of anti-metal ions and anti-biocides are also involved. It is believed that this summary will provide a reference for how to select and use antibiotic resistance gene databases.

Antibiotic resistance of Enterococcus faecalis from persistent endodontic infections / Resistencia antibiótica de Enterococcus faecalis provenientes de infecciones endodónticas persistentes

Enterococci are important nosocomial pathogens due to their intrinsic multiresistance and the acquisition of new antibiotic resistance genes (ARG). Enterococcus faecalis has been shown to be one of the main pathogens in persistent endodontic infections, therefore, the main objective of this study was to evaluate the phenotype and resistance genotype of strains of E. faecalis isolated from teeth with persistent endodontic lesions, to the most commonly prescribed antibiotics in dentistry. Thirteen strains of E. faecalis of different pulsotype were analyzed to evaluate the susceptibility to antibiotics, amoxicillin, amoxicillin/clavulanic acid, tetracycline, erythromycin and metronidazole, using the Epsilometer test (E- test) and the presence of beta-lactamases with nitrocefin test. Finally, the detection of ARG was performed with a molecular polymerase chain reaction (PCR) technique and confirmed by the sequencing of the amplification products. Fisher's exact test was used, using 95 % confidence. Regarding the phenotype of resistance, the evaluated strains, independent of the pulsotype, were totally resistant to the action of metronidazole. Antibiotics with higher minimum inhibitory concentration (MIC) after metronidazole include tetracycline and erythromycin. In contrast, lower MIC are applied to the combination of amoxicillin with clavulanic acid. The nitrocefin test was positive only in one strain. Genotypically, two genetically distant strains isolated from a single patient, presented a genotype of resistance to erythromycin, determined by the presence of the ermB gene. No statistically significant relationship was found between phenotypic resistance and the presence of ARG in relation to erythromycin (p> 0.05). It was concluded that isolates of E. faecalis from persistent endodontic infections showed phenotypes of resistance to several antimicrobial agents, all of which were susceptible to amoxicillin/clavulanic acid. Periodic evaluation of susceptibility to antibiotics is suggested as an important practice for the surveillance of antibiotic resistance in oral strains.

Los enterococos son importantes patógenos nosocomiales debido a su multi resistencia intrínseca y la adquisición de nuevos genes de resistencia a los antibióticos (ARG). Enterococcus faecalis es uno de los principales patógenos en infecciones endodónticas persistentes, por lo tanto, el objetivo principal de este estudio fue evaluar el fenotipo y el genotipo de resistencia de cepas de E. faecalis aisladas de dientes con lesiones endodóncicas persistentes, a los antibióticos comúnmente recetados en odontología. Se analizaron 13 cepas de E. faecalis de diferentes pulsotipos para evaluar la susceptibilidad a los antibióticos, amoxicilina, amoxicilina / ácido clavulánico, tetraciclina, eritromicina y metronidazol, utilizando la prueba de Epsilometría (E-test) y la presencia de beta-lactamasas con prueba de nitrocefina. Finalmente, la detección de ARG se realizó con una técnica molecular de reacción en cadena de la polimerasa (PCR) y se confirmó mediante la secuenciación de los productos de amplificación. Se utilizó la prueba exacta de Fisher, con un 95 % de confianza. En cuanto al fenotipo de resistencia, las cepas evaluadas, independientes del pulsotipo, fueron totalmente resistentes a la acción del metronidazol. Los antibióticos con los valores más altos de concentración mínima inibitoria (CMI) después del metronidazol incluyen tetraciclina y eritromicina. En contraste, las CMI mas bajas se aplican a la combinación de amoxicilina con ácido clavulánico. La prueba de nitrocefina fue positiva solo en una cepa. Genotípicamente, dos cepas distantes genéticamente, aisladas de un mismo paciente fueron positivas para el gen ermB. No se encontró una relación estadísticamente significativa entre la resistencia fenotípica y la presencia de ARG en relación con la eritromicina (p> 0,05). Se concluyó que los aislamientos de E. faecalis de infecciones endodónticas persistentes mostraron fenotipos de resistencia a varios agentes antimicrobianos, todos los cuales fueron susceptibles a amoxicilina / ácido clavulánico. Se sugiere una evaluación periódica de la susceptibilidad a los antibióticos como una práctica importante para la vigilancia de la resistencia a los antibióticos en las cepas orales.

Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

Mutaciones que confieren resistencia a fármacos antituberculosis de primera línea en Perú: una revisión sistemática de la literatura / Mutations conferring resistance to first-line anti-tuberculosis drugs in Peru: a systematic review of the literature

RESUMEN Objetivos. Sistematizar la información disponible referente a las mutaciones que confieren resistencia a los fármacos antituberculosis de primera línea. Materiales y métodos. Se realizó una revisión sistemática de la literatura científica para identificar artículos que reportaron mutaciones que confieren resistencia a fármacos antituberculosis de primera línea. Esta búsqueda hizo énfasis en la resistencia a los fármacos de isoniazida y rifampicina en cepas de M. tuberculosis de pacientes peruanos. La búsqueda fue realizada en PubMed y LILACS (Literatura Latinoamericana y del Caribe en Ciencias de la Salud). Resultados. Se incluyeron 14 artículos de los cuales tres reportaron mutaciones asociadas con resistencia a isoniazida, seis a rifampicina, ocho a pirazinamida y uno a etambutol. Todas las mutaciones a isoniazida o rifampicina fueron identificadas directa o indirectamente mediante la prueba de diagnóstico molecular GenoType MTBDRplus® v2.0. La mayor variabilidad de mutaciones fue determinada en la resistencia a pirazinamida. Conclusiones. Existe una gran variabilidad de mutaciones asociadas con resistencia a fármacos antituberculosis que han sido reportadas en Perú, y se sistematizan en el presente reporte. Estas mutaciones deben de ser tomadas en cuenta para el desarrollo de dispositivos diagnósticos o selección de pruebas diagnósticas a ser aplicadas en nuestro país.

ABSTRACT Objective. To systematize available information regarding mutations that confer resistance to first-line anti-tuberculosis drugs. Materials and Methods. A systematic review of the scientific literature was conducted to identify articles that reported mutations conferring resistance to first-line anti-tuberculosis drugs. This search emphasized resistance to isoniazid and rifampicin drugs in M. tuberculosis strains of Peruvian patients. The search was performed on PubMed and LILACS (Latin American and Caribbean Health Sciences Literature). Results. Fourteen (14) articles were included, of which three reported mutations associated with resistance to isoniazid, six to rifampicin, eight to pyrazinamide and one to ethambutol. All mutations to isoniazid or rifampicin were identified directly or indirectly by the molecular diagnostic test GenoType MTBDRplus® v2.0. The greatest variability of mutations was determined in resistance to pyrazinamide. Conclusions. There is a great variability of mutations associated with resistance to anti-tuberculosis drugs that have been reported in Peru, and they are systematized in this report. These mutations must be taken into account for the development of diagnostic devices or selection of diagnostic tests to be applied in our country.

Mutaciones del gen ARN ribosómico 23S de Helicobacter pylori asociadas con resistencia a claritromicina en pacientes atendidos en una unidad de endoscopia de Medellín, Colombia / Mutations in the Helicobacter pylori 23S rRNA gene associated with clarithromycin resistance in patients at an endoscopy unit in Medellín, Colombia

Resumen Introducción. La claritromicina es el antibiótico de primera línea para el tratamiento de la infección por Helicobacter pylori. La resistencia bacteriana se produce principalmente por mutaciones puntuales del gen ARN ribosómico 23S (ARNr 23S). Objetivo. Determinar la frecuencia de las mutaciones puntuales A2143G y A2142G del gen ARNr 23S asociadas con la resistencia de H. pylori a la claritromicina en muestras de pacientes con manifestaciones dispépticas en Medellín, región noroccidental de Colombia. Materiales y métodos. Se extrajo ADN a partir de muestras de biopsia gástrica obtenidas de pacientes con manifestaciones dispépticas atendidos en una unidad de endoscopia entre el 2016 y el 2017. Mediante reacción en cadena de la polimerasa (PCR), se amplificaron las regiones s y m del gen vacA y una región del gen ARNr 23S bacteriano. La presencia de las mutaciones A2142G y A2143G se determinó por la técnica de polimorfismos de longitud de fragmentos de restricción (RFLP) con las enzimas BbsI y BsaI, respectivamente. Resultados. Se encontró una prevalencia de infección de 44,2 % (175/396), según el informe de histopatología. En 143 de estas 175 muestras positivas se amplificaron las tres regiones del genoma bacteriano. Se identificaron las mutaciones A2143G y A2142G en 27 muestras (18,8 %; 27/143), la mutación más frecuente fue la A2143G (81,5 %; 22/27). Conclusiones. Hubo una gran prevalencia de mutaciones asociadas con la resistencia de H. pylori a la claritromicina en la población de estudio. Se requieren estudios adicionales para establecer la resistencia bacteriana en la población colombiana y, así, determinar los tratamientos de primera línea y de rescate.

Abstract Introduction: Clarithromycin is the first-line antibiotic for the treatment of Helicobacter pylori infection. Bacterial resistance is mainly due to the presence of specific mutations in the 23S ribosomal RNA (rRNA) gene. Objective: To determine the frequency of A2143G and A2142G specific mutations in the 23S rRNA gene associated with clarithromycin resistance of H. pylori in samples from patients with dyspeptic manifestations in Medellín, northwestern Colombia. Materials and methods: DNA was extracted from gastric biopsy samples of patients with dyspeptic manifestations seen at an endoscopy unit in Medellín between 2016 and 2017. PCR was performed to amplify the bacterial s and m vacA regions, and a region in the 23S rRNA gene. The presence of the A2142G and A2143G mutations was determined using the restriction fragment length polymorphism (RFLP) technique with the BbsI and BsaI enzymes, respectively. Results: The prevalence of infection was 44.2% (175/396), according to the histopathology report. The positive samples were analyzed and the three regions of the bacterial genome were amplified in 143 of the 175 samples. The A2143G and A2142G mutations were identified in 27 samples (18.8%, 27/143). The most frequent mutation was A2143G (81.5%, 22/27). Conclusions: We found a high prevalence of H. pylori mutations associated with clarithromycin resistance in the study population. Further studies are required to determine the bacterial resistance in the Colombian population in order to define first line and rescue treatments.

Resistencia inducible a clindamicina en Staphylococcus aureus resistentes a meticilina aislados de pacientes pediátricos en Paraguay / Inducible resistance to clindamycin in methicillin-resistant Staphylococcus aureus isolated from Paraguayan children

Resumen Introducción: El método de difusión de doble disco se presenta como una alternativa diagnóstica que permite identificar aislados de Staphylococcus aureus susceptibles a clindamicina, ante el aumento de resistencia a meticilina, reduciendo así la posibilidad de fallo en el tratamiento. Objetivo: Determinar la frecuencia de resistencia a clindamicina inducida por eritromicina en S. aureus resistentes a meticilina (SARM) aislados de niños paraguayos. Materiales y Métodos: Estudio observacional, descriptivo, de corte transversal. Se colectaron 145 aislados S. aureus que causaron infecciones de piel y tejidos blandos y osteo-articulares en pacientes pediátricos del Hospital Central del Instituto de Previsión Social en el período de diciembre-2012 a noviembre-2013. La resistencia a clindamicina se determinó por métodos automatizados y de difusión de doble disco. Se realizó reacción de polimerasa en cadena para genes ermA, ermB, ermC y msrA de aislados representativos. Resultados: La resistencia global a meticilina y clindamicina fue de 67 y 13%, respectivamente (11% atribuible al mecanismo de resistencia a clindamicina inducible). Los genes ermC y msrA fueron detectados individualmente en 25 y 17% de los aislados, respectivamente, mientras que un aislado presentó ambos genes en simultáneo. Discusión: La frecuencia de mecanismo de resistencia inducible a clindamicina señala la importancia de los métodos de difusión de doble disco en la práctica microbiológica, así como se encuentran en los límites de puntos de cortes considerados como aceptables para el uso de este antimicrobiano para infecciones cutáneas y osteo-articulares causadas por SARM.

Background: The double disc diffusion method is an alternative diagnostic that allows the identification of Staphylococcus aureus isolates apparently susceptible to clindamycin but that may develop resistance due to an induction phenomena, mainly asociated to the increase in resistance to methicillin, thus increasing the possibility of failure in the treatment. Aim: To determine the frequency of induced clindamycin resistance in methicillin-resistant S. aureus (MRSA) isolated from Paraguayan children. Materials and Methods: In this cross sectional study, we collected 145 S. aureus isolates that caused skin and soft tissue and osteoarticular infections in pediatric patients of the Central Hospital I.P.S. in the period from December-2012 to November-2013. Resistance to clindamycin was determined by automated methods and double disc diffusion. PCR was performed for ermA, ermB, ermC and msrA genes from representative isolates. Results: The global resistance to methicillin and clindamycin was 67 and 13%, respectively (11% attributable to the inducible mechanism). The ermC and msrA genes were detected individually in 25 and 17% of the isolates respectively while an isolate presented both genes simultaneously. Discussion: The frequency of inducible resistance to clindamycin indicates the importance of double disc diffusion methods in microbiological practice, as well as being within the cut off points considered acceptable for the use of this antibiotic for skin infections. and osteoarticular caused by MRSA.

Marcadores de resistencia plasmídica a quinolonas qnr en aislamientos clínicos de enterobacterias productoras de betalactamasas CTX-M en Lima, Perú / Markers of plasmid resistance to qnr quinolones in clinical isolates of CTX-M beta-lactamases-producing enterobacteria in Lima, Peru

RESUMEN Con el objetivo de reportar marcadores de resistencia plasmídica a quinolonas qnr en aislamientos clínicos de enterobacterias productoras de betalactamasas CTX-M, se realizó un estudio descriptivo, con aislamientos del cepario del proyecto TO-06/09 del Instituto Nacional de Salud del Niño. Se recuperaron 138 aislamientos. La susceptibilidad antimicrobiana se determinó por el método de disco difusión y la identificación de genes por reacción en cadena de la polimerasa. De los 138 aislados, 67 (48,5%) fueron positivos para proteínas qnr por el método genotípico. De los cuales 38 (56,7%) presentaron determinantes qnrB y 48 (71,6%) determinantes qnrS. Ningún aislado presentó determinantes qnrA. Se detectó determinantes qnr en aislamientos que presentaban betalactamasas CTX-M en una población no expuesta.

ABSTRACT Aimed at reporting markers of plasmid resistance to qnr quinolones in clinical isolates of CTX-M beta-lactamase-producing enterobacteria, a descriptive study was conducted with isolates from the strain repository of TO-06/09 project of the National Children´s Health Institute. 138 isolates were recovered. Antimicrobial susceptibility was determined by the diffusion disk method, and gene identification by polymerase chain reaction. Of the 138 isolates, 67 (48.5%) were genotypically positive for qnr proteins; of these, 38 (56.7%) had qnrB determinants and 48 (71.6%) had qnrS determinants. No isolate presented qnrA determinants. qnr determinants were detected in isolates containing CTX-M beta-lactamases in a non-exposed population.