Caracterização molecular e fenotípica de Klebsiella spp. produtoras de NDM / Molecular and phenotypic characterization of Klebsiella spp. NDM producers

O perfil de resistência, que algumas das espécies do complexo Klebsiella pneumoniae podem expressar, representa uma grande ameaça à saúde humana, particularmente quando resistentes aos carbapenêmicos, que são amplamente utilizados no tratamento de infecções graves em pacientes hospitalizados. O principal mecanismo de resistência aos carbapenêmicos é a produção de carbapenemases, particularmente dos tipos KPC e NDM. Um dos compostos desenvolvidos para o tratamento de infecções causadas por cepas produtoras de KPC é a combinação ceftazidimaavibactam (CAZ-AVI), mas que não tem atividade inibitória sobre metalo-betalactamases, a exemplo das NDMs. Os objetivos deste trabalho foram determinar a frequência das espécies do complexo K. pneumoniae e da coprodução de KPC, avaliar a clonalidade dos isolados, a sensibilidade ao aztreonam-avibactam (ATM-AVI), o desempenho do disco de meropenem (MEM) com inibidores para detecção de coprodução de NDM e KPC e desenvolver um teste de triagem para prever a sensibilidade ao ATM-AVI. Um total de 113 isolados do complexo K. pneumoniae produtoras de NDM ou coprodutoras de NDM e KPC, provenientes da coleção de bactérias do Grupo Fleury, coletadas períodos pré e pós início do uso de CAZ-AVI no Brasil, foram utilizadas neste estudo. A identificação da espécie e a presença dos genes blaNDM e blaKPC foi confirmada por PCR multiplex. A clonalidade dos isolados foi avaliada por eletroforese em campos pulsados (PFGE) após clivagem com XbaI. A produção de carbapenemases foi confirmada utilizando-se o teste Blue Carba. O desempenho dos discos de meropenem e CAZ-AVI contendo um ou mais inibidores de carbapenemases foi comparado com o teste molecular. A pré-difusão combinada foi realizada pré-incubando-se o ágar não inoculado com disco de CAZ-AVI, e a seguir aplicando-se o inóculo bacteriano e um disco de ATM após remover o disco de CAZ-AVI. Após incubação, os halos foram aferidos e correlacionados com a concentração inibitória mínima para ATM-AVI. As CIMs para ATM e ATM-AVI foram determinadas segundo o EUCAST. A identificação das espécies por PCR evidenciou as seguintes frequências: K. pneumoniae 75,2% (n=85); K. quasipneumoniae 16,8% (n=19), e K. variicola 8% (n=9). Uma fração de 12,4% (n=14) dos isolados apresentaram os genes blaNDM e blaKPC e 87,6% (n=99) apenas blaNDM. A análise dos perfis de PFGE de K. pneumoniae evidenciou a presença de cinco grupos clonais predominantes. Isolados do principal grupo clonal Ap (n=15) foram detectados nas cidades de São Paulo e Porto Alegre durante todo o período analisado. O grupo clonal Lp foi detectado nas cidades de São Paulo e Recife em 2019. Os dois principais grupos clonais no período pré-CAZ-AVI continham maior número de isolados do que aqueles no período de uso do CAZ-AVI. Os perfis de PFGE de K. quasipneumoniae evidenciaram quatro grupos clonais predominantes, e presentes apenas no estado de São Paulo, com persistência do grupo clonal Aq desde 2017. Quanto à K. variicola, foram observados dois grupos clonais predominantes Av e Bv, o primeiro presente apenas em São Paulo desde 2018 e o segundo em Porto Alegre apenas em 2019. Calculando-se a diferença entre os diâmetros de halo do disco MEM contendo EDTA e ácido fenilborônico (AFB) e o maior dos halos obtidos para MEM com EDTA ou AFB, observou-se que todos os isolados com coexpressão de KPC e NDM apresentaram diferença ≥ 5 mm. Uma fração de 42,3% dos isolados positivos apenas para blaNDM apresentaram sensibilidade para ATM (CIM ≤ 4 mg/L). Todos os isolados testados apresentaram CIM para ATM-AVI ≤ 1/4 mg/L, sendo a CIM90 0,125/4 mg/l. No teste de pré-difusão combinada, o menor diâmetro de halo obtido foi de 23 mm. A espécie predominante na amostragem foi K. pneumoniae. A disseminação clonal, observada neste estudo, contrasta com a diversidade clonal descrita em outros locais do mundo para produtores de NDM, exceto Grécia e China. Considerando os pontos de corte atuais para ATM, é provável que haja resposta clínica adequada no uso de ATM-AVI no tratamento de infecções causadas por isolados produtores de NDM e coprodutores de KPC e NDM. Utilizando-se o valor de corte de ≤ 5 mm para a diferença entre halos de inibição, de MEM com AFB e EDTA e o segundo maior halo com inibidor, a sensibilidade foi de 100% e a especificidade foi de 96,1,0%. O método de pré-difusão com CAZ-AVI e ATM é um método simples e o diâmetro ≥ 23 mm tem excelente correlação com a CIM para ATM-AVI ≤ 1/4 mg/L

The resistance profile, which some species of the Klebsiella pneumoniae complex may express, represent a great threat to human health, particularly when resistant to carbapenems, which are widely used in the treatment of severe infections in hospitalized patients. The main mechanism of resistance to carbapenems is the production of carbapenemases, particularly KPCs and NDMs. One of the compounds developed for the treatment of infections caused by KPC-producing strains is the combination ceftazidime-avibactam (CAZ-AVI), but which has no inhibitory activity on metallobetalactamases, as is the case for NDMs. The objectives of this work were to determine the frequency of K. pneumoniae complex species and KPC co-production, evaluate the clonality of isolates, the susceptibility to aztreonam-avibactam (ATM-AVI), the performance of meropenem (MEM) disks with inhibitors for detecting NDM co-production and KPC and develop a screening test to predict sensitivity to ATM-AVI. A total of 113 NDM-producing or NDM and KPC co-producing K. pneumoniae complexes, from the Fleury Group's bacteria collection, collected in the pre- and post-starting periods of CAZ-AVI use in Brazil, were used in this study. Species identification and the presence of the blaNDM and blaKPC genes were confirmed by multiplex PCR. The clonality of the isolates was evaluated by pulsed field electrophoresis (PFGE) after cleavage with XbaI. Carbapenemase production was confirmed using the Blue Carba test. The performance of MEM and CAZ-AVI disks containing one or more carbapenemase inhibitors was compared with the molecular test. Combined pre-diffusion was performed by preincubating the uninoculated agar with a CAZ-AVI disk, and then applying the bacterial inoculum and na ATM disk after removal of the CAZ-AVI disk. After incubation, halos were measured and correlated with the minimum inhibitory concentration (MIC) for ATM-AVI. ATM and ATM-AVI MICs were determined according to EUCAST. The identification of species by PCR evidenced the following frequencies: K. pneumoniae 75.2% (n=85); K. quasipneumoniae 16.8% (n=19), and K. variicola 8% (n=9). A fraction of 12.4% (n=14) of the isolates had the blaNDM and blaKPC genes and 87.6% (n=99) had only blaNDM. The analysis of the PFGE profiles of K. pneumoniae evidenced the presence of five predominant clonal groups. Isolates from the main clonal group Ap (n=16) were detected in the cities of São Paulo and Porto Alegre throughout the analyzed period. The clonal group Lp was detected in the cities of São Paulo and Recife 2019. The PFGE profiles of K. quasipneumoniae showed four predominant clonal groups, present only in the state of São Paulo, with persistence of the clonal group Aq since 2017. As for K. variicola, two predominant clonal groups Av and Bv were observed, the first present only in São Paulo since 2018 and the second in Porto Alegre only in 2019. Calculating the difference between the inhibition zone diameters of the MEM disk containing EDTA and phenylboronic acid (AFB) and the largest of the inhibition zone diameters obtained for MEM with EDTA or AFB, it was observed that all isolates with co-expression of KPC and NDM showed a difference 5 ≥mm. A fraction of 42.3% of isolates positive only for blaNDM showed sensitivity to ATM (MIC ≤ 4 mg/L). All tested isolates presented MIC for ATM-AVI ≤ 1/4 mg/L, being the MIC90 0.125/4 mg/l. In the combined pre-diffusion test, the smallest inhibition zone diameter obtained was 23 mm. The predominant species in the sample was K. pneumoniae, but a significant fraction of the other species in the complex was also observed in the sample. The clonal spread observed in this study contrasts with the clonal diversity described elsewhere in the world for NDM-producing isolates, except Greece and China. Considering the current cut-off points for ATM, it is likely that there is an adequate clinical response in the use of ATM-AVI in infections caused by NDM-producing and KPC-NDM co-producing isolates in Brazil. Using the cutoff value of 5 mm for the difference between inhibition zones, of MEM with AFB and EDTA and the second largest zone of MEM with inhibitor, the sensitivity was 100% and the specificity was 96.1%. The pre-diffusion method with CAZ-AVI and ATM is a simple method and the diameter ≥ 23 mm has excellent correlation with the MIC for ATM-AVI ≤ 1/4 mg/L

Analysis of drug resistance genes and molecular typing of carbapenem-resistant Klebsiella variicola strains / 中华微生物学和免疫学杂志

Objective To investigate the prevalence and drug resistance of clinical Klebsiella vari-icola ( K. variicola ) isolates and to illuminate the mechanism of drug resistance in carbapenem-resistant strains. Methods Clinical K. variicola isolates were identified with matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry ( MALDI-TOF MS ) . The antimicrobial susceptibility profile of these strains was determined using broth microdilution. Resistance genes carried by carbapenem-resistant K. vari-icola strains were detected by PCR with specific primers. Multilocus sequence typing ( MLST) was used for molecular typing. A pan-drug resistant strain which was isolated from cerebrospinal fluid sample was ana-lyzed with whole genome sequencing ( WGS) . Results Twenty-six isolates were identified as K. variicola by MALDI-TOF MS. Results of the antimicrobial susceptibility test showed that there were 15. 4％ (4/26) re-sistant to carbapenem and 11. 5％ (3/26) unsusceptible to tigecycline. These strains were highly suscepti-ble to amikacin and gentamicin, which accounted for 96. 2％ (25/26). As for the third-and fourth-genera-tion cephalosporins, the resistance rate was 23. 1％ (6/26). All of the four carbapenem-resistant isolates carried the resistance genes of blaIMP-4 , qnrA/B and blaTEM , and one of them was also positive for blaNDM-1 gene. The fosfomycin resistance gene, fosA, was detected in three of them. Molecular typing analysis indica-ted these isolates belonged to two sequence types ( ST) of ST357 ( three strains) and ST1737 ( one strain) . Two plasmids were obtained from the pan-drug resistant strain by WGS, including IncFⅡ/FIB( k) type plas-mid (160 kb) that was highly homologous to LMG 23571 plasmid (GenBank: CP013986. 1) and IncHⅠ1B/FIB type plasmid (260 kb) sharing high homology with pIMP4 LL34 (GenBank: CP025964. 1). Be-sides the resistance genes mentioned above, the two plasmids also carried a variety of other genes that media-ted the resistance to aminoglycosides (strB, strA, armA, aac3-Ⅱd, aadA2), macrolides (msrE, mphE), chloramphenicol (catA2), sulfonamides (sulⅠ) tigecycline (tetA variant) and trimethoprim (dfrA16). However, no virulence genes were detected. Conclusions In general, the resistance profile of K. variicola was similar to that of Klebsiella pneumoniae, but the differences were that carbapenem-resistant K. variicola strains mainly belonged to ST357 and the leading causes of resistance were carrying the genes encoding IMP-4 and NDM-1 metalβ-lactamases. WGC analysis revealed that the pan-drug resistant K. variicola strain carried multiple drug resistance genes without virulence determinants, which might be resulted from the evo-lution of drug resistance.

Whole genome analysis of Klebsiella: Unique genes associated with isolates from Indonesian tempeh

Aims: Our previous study demonstrated that Klebsiella IIEMP-3 associated with tempeh was genetically different from those of medical isolates. In addition to the whole genome sequence of Klebsiella IIEMP-3, the draft genome sequence of another isolate, i.e. IWJB-6 was employed for comparison. In this study, the details of the virulence genes and unique gene in both Klebsiella isolates were compared employing in silico and in vitro analysis. Methodology and results: Whole genome of Klebsiella IIEMP-3 and IWJB-6 were annotated to investigate the virulence factor. Klebsiella IIEMP-3 and IWJB-6 were obtained from tempeh producers in Bogor, West Java - Indonesia. Genome sequences were analyzed employing BLAST Ring Image Generator (BRIG) software. The results showed that all of the samples, including isolates IIEMP-3 and IWJB-6 did not harbor rmpA, i.e. DNA sequence for K. pneumoniae virulence factor. Conclusion, significance and impact of study: Klebsiella could be found in almost all tempeh samples from Indonesia and could be harmless for human due to the absence of rmpA and other virulence-associated genes. The significance of this study showed that IIEMP-3 and IWJB-6 isolates were more closely related to K. variicola. However, K. variicola At22 harbored sdsA gene which is lacking in those two tempeh isolates. Combined with PCR analysis for specific gene/s; our study suggested that isolates from Indonesian tempeh were closely related to K. variicola, and proposed to be designated as K. variicola subsp. tempehensis.

Emergence of novel variants of gyrA and parC genes in a group of multiple resistant Klebsiella / 中华临床感染病杂志

Objective To investigate the distribution and variety of quinolone-resistance genes in a group of multiple resistant Klebsiella.Methods A total of 20 strains of multiple resistant Klebsiella were collected from inpatients in Huai' an First People' s Hospital of Nanjing Medical University during February 2010 and March 2012.Strains were identified by molecular identification,and quinolone target genes (gyrA,ParC) and quinolone-resistance genes mediated by mobile genetic elements (qnrA,qnrB,qnrS,aac (6′)-Ⅰ b-Cr,qepA) were analyzed with polymerase chain reaction (PCR).Results In 20 strains of Klebsiella,19 were Klebsiella pneumonia,and 1 was Klebsiella variicola.gyrA and parC genes were found in all 20 strains,and gyrA mutation was found in 11 strains (55.0％),andparC mutation was also found in 11strains (55.0％).aac(6′)-Ⅰ b-cr was positive in 10 strains (50.0％),qnrA was positive in 1 strain (5.0％),and qnrB was positive in 3 strains (15.0％).gyrA and parC in strain No.6 and No.10 were both novel variants (GenBank registration number:JX123016,JX123017,JX144393,JX144394).Conclusion Quinolone-resistance-determining region plays a key role in resistance to quinolones in this group of Klebsiella,and it is the first report on the emergence of novel variants of gyrA and parC in one Klebsiella pneumonia in China.