Comparative genomic analysis of Pseudomonas aeruginosa strains susceptible and resistant to carbapenems and aztreonam isolated from patients with healthcare-associated infections in a Mexican hospital.

Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that causes different infections on immunocompromised patients. Within PA accessory genome, differences in virulence, antibiotic resistance and biofilm formation have been described between strains, leading to the emergence of multidrug-resistant strains. The genome sequences of 17 strains isolated from patients with healthcare-associated infections in a Mexican hospital were genomically and phylogenetically analyzed and antibiotic resistance genes, virulence genes, and biofilm formation genes were detected. Fifteen of the 17 strains were resistant to at least two of the carbapenems meropenem, imipenem, and the monobactam aztreonam. The antibiotic resistance (mexA, mexB, and oprM) and the biofilm formation (pslA and pslD) genes were detected in all strains. Differences were found between strains in accessory genome size. The strains had different sequence types, and seven strains had sequence types associated with global high risk epidemic PA clones. All strains were represented in two groups among PA global strains. In the 17 strains, horizontally acquired resistance genes to aminoglycosides and beta-lactams were found, mainly, and between 230 and 240 genes that encode virulence factors. The strains under study were variable in terms of their accessory genome, antibiotic resistance, and virulence genes. With these characteristics, we provide information about the genomic diversity of clinically relevant PA strains.

Novas variantes de KPC em isolados do Complexo Klebsiella pneumoniae resistentes à ceftazidima-avibactam / New KPC variants in ceftazidime-avibactam-resistant K. pneumoniae Complex isolates

O Complexo K. pneumoniae (C-Kp) é o principal grupo de bacilos Gram-negativos responsáveis por infecções nosocomiais graves em todo o mundo e o tratamento empírico dessas infecções usualmente inclui os carbapenêmicos. O principal mecanismo de resistência a essa classe de antimicrobianos é a expressão de carbapenemases, e no Brasil, mais frequentemente as do tipo KPC. Em março de 2019 a ceftazidima-avibactam foi disponibilizada para uso clínico no Brasil, sendo amplamente utilizada no tratamento infecções causadas por bacilos gram-negativos produtores de KPC. Diversos países já relataram a presença de K. pneumoniae produtores de KPC resistentes à ceftazidima-avibactam. No entanto, há poucos relatos dessa ocorrência no Brasil. O objetivo deste trabalho foi caracterizar genotipicamente e fenotipicamente isolados do C-Kp produtores de KPC, resistentes à ceftazidima-avibactam. No período de julho/2019 a julho/2021, 46 isolados do C-Kp, um por paciente, foram detectados em diferentes sítios de infecção ou culturas de vigilância de pacientes internados em hospitais privados de seis estados brasileiros. Os isolados tiveram seu genoma completo sequenciado nas plataformas MiSeq e MinION para determinação da variante alélica de blaKPC e avaliação do seu contexto genético. As taxas de resistência ao ertapenem e à ceftazidima-avibactam foram calculadas a partir de banco de dados. A clonalidade dos isolados foi avaliada por PFGE e MLST. A localização plasmidial do gene blaKPC foi confirmada por conjugação e/ou transformação. A concentração inibitória mínima (CIM) para betalactâmicos foi determinada por microdiluição em caldo segundo o BrCAST. Ensaios imunocromatográficos, NG-Test CARBA-5 e O.K.N.V.I. RESIST-5, foram avaliados quanto à sua performance na detecção de variantes KPC. A taxa de resistência ao ertapenem entre isolados do C-Kp aumentou 15,6% em 2019 para 27,3% em 2021. A taxa de resistência à ceftazidima-avibactam entre isolados do C-Kp resistentes ao ertapenem aumentou de 4,2% em 2019 para 17,2% em 2021. Onze isolados apresentaram novas variantes de KPC designadas KPC-103 a KPC-108 e KPC-139 a KPC-143. Os demais isolados apresentavam variantes de KPC já descritas, sendo a KPC-33 a variante mais frequente (36%). Quinze grupos clonais foram identificados, sendo que a maioria dos isolados pertencia ao ST11. O grupo clonal A foi o mais numeroso e pertencia ao ST258. A principal variante detectada nesse grupo foi a KPC-33. Diferentes grupos de incompatibilidade foram identificados em plasmídeos alberguando blaKPC, sendo os grupos IncN (n=12) e IncF, com replicons FII(K)-FIB (n=11), os grupos mais frequentes, seguido de InX3-IncU (n=9) e IncQ1 (n=1). Dos 46 isolados resistentes à ceftazidima-avibactam, 36 foram capazes de transferir o gene blaKPC para cepas receptoras. A maioria dos isolados foi sensível dose padrão ou sensível aumentabdo exposição ao meropenem e apresentaram redução significativa da CIM quando o avibactam foi adicionado ao aztreonam. O NG-Test CARBA-5 detectou 12 das 24 variantes testadas enquanto que o O.K.N.V.I. RESIST-5 detectou apenas nove variantes. A grande diversidade de variantes KPC, e a predominância do grupo clonal ST11, e da KPC- 33 retratam um cenário preocupante no Brasil

The K. pneumoniae Complex (C-Kp) is the main group of gram-negative bacilli responsible for serious nosocomial infections worldwide and the empirical treatment of these infections usually includes carbapenems. The main mechanism of resistance to this class of antimicrobials is the expression of carbapenemases, and in Brazil, more frequently the KPC type. In March 2019, ceftazidime-avibactam was available for clinical use in Brazil, being widely used to treat infections caused by KPC-producing gram-negative bacilli. Several countries have already reported the presence of KPC-producing K. pneumoniae resistant to ceftazidime-avibactam; however, there are few reports of this occurrence in Brazil. This work aimed to genotypically and phenotypically characterize KPC-producing C-Kp isolates resistant to ceftazidimeavibactam. From July 2019 to July 2021, 46 C-Kp isolates, one per patient, were detected in different sites of infection or surveillance cultures from patients admitted to private hospitals in six Brazilian states. The isolates had their complete genome sequenced on the MiSeq and MinION platforms to determine the allelic variant of blaKPC and evaluate its genetic context. Resistance rates to ertapenem and ceftazidime-avibactam were calculated from a database. The clonality of the isolates was evaluated by PFGE and MLST. The plasmid location of the blaKPC gene was confirmed by conjugation and/or transformation. The minimal inhibitory concentrations for beta-lactams were determined by broth microdilution according to BrCAST. Immunochromatographic assays, NG-Test CARBA-5 and O.K.N.V.I. RESIST-5, were evaluated for its performance in detecting KPC variants. The resistance rate to ertapenem among C-Kp isolates increased from 15.6% in 2019 to 27.3% in 2021. The resistance rate to ceftazidime-avibactam among ertapenem-resistant C-Kp isolates increased from 4.2% in 2019 to 17.2% in 2021. Eleven isolates showed new KPC variants designated KPC-103 to KPC-108 and KPC-139 to KPC-143. The remaining isolates presented previously described KPC variants, with KPC-33 being the most common variant (36%). Fifteen clonal groups were identified, with most isolates belonging to ST11. Different incompatibility groups were identified in plasmids harboring blaKPC, with the IncN (n=12) and IncF groups, with FII(K)-FIB(n=11) replicons, being the most frequent groups, followed by InX3-IncU (n= 9) and IncQ1 (n=1). All IncX3-IncU plasmids were approximately 46 kb in size and were identified among isolates belonging to the largest identified clonal group (A) and ST258. The main variant detected in this group was KPC-33. Of the 46 ceftazidime-avibactam-resistant isolates, 36 were able to transfer the blaKPC gene to recipient strains. Most isolates were susceptible standard dose or susceptible increased exposure to meropenem and showed a significant decrease in MIC when avibactam was added to aztreonam. The NG-Test CARBA-5 was able to detect 12 of the 24 variants evaluated while the O.K.N.V.I. RESIST-5 detected only nine variants. The great diversity of KPC variants, the predominance of the ST11 clonal group, and KPC-33 portray a worrying scenario in Brazil

In vitro effectiveness of ceftazidime-avibactam in combination with aztreonam on carbapenemase-producing Enterobacterales.

OBJECTIVE: This work aimed to describe the in vitro performance of the combined activity of ceftazidime-avibactam (CZA) plus aztreonam (ATM) against carbapenemase-producing Enterobacterales (CPE). METHODS: We studied 44 CPE clinical isolates: NDM-1 (31), KPC-2 (5), KPC-3 (3), VIM-2 (2), NDM-1+KPC-2 (2), and OXA-48 (1). The efficacy of CZA in combination with were determined by two methods: (i) Kirby-Bauer's double disk synergy test and; (ii) Determination of the minimum inhibitory concentration to CZA by E-test, in either Mueller-Hinton agar alone or, supplemented with ATM 4 mg/L. Additionally, the Fractional inhibitory concentration index (FICI) was determined; values of ≤ 0.5 were interpreted as synergistic, while FICI > 0.5 were considered indifferent. RESULTS: All isolates were carbapenem-resistant, 14 were resistant to CZA and ATM, 15 were only CZA resistant, 12 were only ATM resistant, and three were susceptible to both. 34/44 isolates presented positive double disk synergy tests between CZA and ATM regardless of their susceptibility profile, the isolates with negative synergy tests were susceptible to at least one of the agents. On the other hand, the 21 isolates selected to compare the MIC to CZA alone and CZA plus 4 mg/L ATM of exhibited FICI values between 0.016 and 0.125, indicating a synergistic effect. CONCLUSIONS: This method is available to clinical laboratories and would provide valuable information to guide the treatment of infections with CZA and ATM. In this sense, the use of CZA together with ATM is a potentially suitable combination for the treatment of carbapenemase-producing microorganisms.

Synergistic combination of aztreonam and ceftazidime/avibactam against resistant Stenotrophomonas maltophilia on pancreatitis.

INTRODUCTION: Stenotrophomonas maltophilia is a Gram-negative, opportunistic pathogen associated with a high morbidity and mortality rate. We report our clinical experience in treating a patient with infected pancreatic necrosis caused by multidrug-resistant (MDR) S. maltophilia with a novel drug combination. CASE REPORT: A 65-year-old male with history of type II diabetes was admitted with acute pancreatitis, voluminous ascites, and signs of sepsis after undergoing an echo-endoscopy procedure with pancreas biopsy to investigate a Wirsung duct dilatation. Retroperitoneal fluid culture revealed S. maltophilia resistant to colistin and with intermediate susceptibility to trimethoprim-sulfamethoxazole and levofloxacin. The synergy between aztreonam (ATM) and ceftazidime/avibactam (CZA) was demonstrated using the combined disk pre-diffusion test. CONCLUSIONS: There are sparse data providing guidance on the optimal regimen against MDR S. maltophilia infections. Although in this case a surgical excision was essential, combination of ATM and CZA provided effective synergistic antimicrobial treatment with clinical cure of severe acute pancreatitis infected with S. maltophilia. The combined disk pre-diffusion test with ATM and CZA requires no special equipment and can be routinely performed in clinical microbiology labs. Combination of ATM with CZA should be considered for cases of MDR S. maltophilia infections with limited treatment options.

Incidence of ESBLs and carbapenemases among Enterobacterales and carbapenemases in Pseudomonas aeruginosa isolates collected globally: results from ATLAS 2017-2019.

OBJECTIVES: To assess the global and regional distribution of ESBLs in Enterobacterales and carbapenemases in Enterobacterales and Pseudomonas aeruginosa. METHODS: Antimicrobial susceptibility of isolates collected from ATLAS (2017-2019) was determined per CLSI guidelines. Enterobacterales exhibiting meropenem MICs ≥2 mg/L and/or ceftazidime/avibactam and/or aztreonam/avibactam MICs ≥16 mg/L, Escherichia coli and Klebsiella pneumoniae with aztreonam and/or ceftazidime MICs ≥2 mg/L, and P. aeruginosa with meropenem MICs ≥4 mg/L were screened for ß-lactamases by PCR and sequencing. RESULTS: Globally, ESBL-positive E. coli (23.7%, 4750/20047) and K. pneumoniae (35.1%, 6055/17229) carried predominantly the CTX-M-15 variant (E. coli: 53.9%; K. pneumoniae: 80.0%) with highest incidence in Africa/Middle East (AfME). Among carbapenem-resistant (CR) E. coli (1.1%, 217/20047) and Enterobacter cloacae (3.8%, 259/6866), NDMs were predominant (E. coli in AfME: 62.5%; E. cloacae in Asia Pacific: 59.7%). CR K. pneumoniae (13.3%, 2299/17 229) and P. aeruginosa (20.3%, 4187/20 643) carried predominantly KPC (30.9%) and VIM (14.7%), respectively, with highest frequency in Latin America. Among ESBL-positive Enterobacterales, susceptibility to ceftazidime/avibactam (>90.0%) and amikacin (>85.0%) was higher than to piperacillin/tazobactam (>45.0%) and ciprofloxacin (>7.4%). In CR Enterobacterales, susceptibility to amikacin (>54.0%) and ceftazidime/avibactam (>31.0%) was higher than to ciprofloxacin (>2.7%) and piperacillin/tazobactam (>0.5%). CR P. aeruginosa similarly demonstrated higher susceptibility to amikacin (63.4%) and ceftazidime/avibactam (61.9%) than to ciprofloxacin (26.2%) and piperacillin/tazobactam (25.3%). CONCLUSIONS: Varied distribution of resistance genotypes across regions among ESBL-positive Enterobacterales and CR Enterobacterales and P. aeruginosa provide crucial insights on major resistance mechanisms and trends observed in recent years. Continued surveillance is warranted for monitoring global dissemination and resistance.

Detecting KPC-2 and NDM-1 Coexpression in Klebsiella pneumoniae Complex from Human and Animal Hosts in South America.

Reports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological diagnosis. We evaluated phenotypic methods for detecting Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-ß-lactamase-1 (NDM-1) coexpression in members of the K. pneumoniae complex (i.e., K. pneumoniae, K. quasipneumoniae, and K. variicola) isolated from human and animal hosts, based on inhibition of ceftazidime-avibactam (CZA) and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, or avibactam (AVI). While the presence of blaKPC-2 and blaNDM-1 genes was confirmed by whole-genome sequencing, PCR, and/or GeneXpert, coexpression was successfully detected based on the following: (i) a ≥5-mm increase in the zone diameter of ATM (30 µg) disks plus AVI (4 or 20 µg) and ≥4-mm and ≥10-mm increases in the zone diameters for "CZA 50" (30 µg ceftazidime [CAZ] and 20 µg AVI) and "CZA 14" (10 µg CAZ and 4 µg AVI) disks, respectively, when we added DPA (1 mg/disk) or EDTA (5 mM) in a combined disk test (CDT); (ii) a positive ghost zone (synergism) between ATM (30 µg) and CZA 50 disks and between CZA 50 and DPA (1 mg) disks, using the double-disk synergy test (DDST) at a disk-disk distance of 2.5 cm; (iii) ≥3-fold MIC reductions of ATM and CZA in the presence of AVI (4 µg/mL), DPA (500 µg/mL), or EDTA (320 µg/mL); and (iv) immunochromatography. Although our results demonstrated that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex, additional studies are necessary to confirm the accuracy of these methodologies by testing other Gram-negative bacterial species and other KPC and NDM variants coexpressed by WHO critical priority pathogens detected worldwide. IMPORTANCE Alerts regarding the emergence and increase of combinations of carbapenemases in Enterobacterales in Latin America and the Caribbean have recently been issued by PAHO and WHO, emphasizing the importance of appropriate microbiological diagnosis and the effective and articulated implementation of infection prevention and control programs. In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, and avibactam (AVI) inhibitors for the identification of KPC-2- and NDM-1-coexpression in members of the K. pneumoniae complex recovered from human and animal hosts. Our results demonstrate that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex.

A simple disk pre-diffusion test to predict in vitro aztreonam/avibactam activity against NDM-producing Klebsiella pneumoniae complex.

OBJECTIVES: The aim of this study was to describe a simple test to predict in vitro efficacy of aztreonam/avibactam (ATM-AVI) combination based on a pre-diffusion assay involving routinely available ceftazidime/avibactam (CAZ-AVI) and aztreonam (ATM) disks. METHODS: A total of 113 non-repetitive NDM-producing Klebsiella had the species identified by multiplex PCR. Minimum inhibitory concentrations (MICs) for ATM and ATM-AVI were determined by broth microdilution. For the combined disk pre-diffusion method, a disk containing ceftazidime 10 µg and avibactam 4 µg (CAZ-AVI) was applied on the surface of an uninoculated Mueller-Hinton agar plate. Following incubation for 2 h at 36°C, the disk was removed, the bacterial suspension was applied and a 30 µg ATM disk was placed precisely in the same position as the removed CAZ-AVI disk. Following incubation for 16-20 h, inhibition zone diameters were measured and correlated with ATM-AVI MICs. RESULTS: The distribution of species among the 113 isolates was 85 Klebsiella pneumoniae (75.2%), 19 Klebsiella quasipneumoniae (16.8%) and 9 Klebsiella variicola (8.0%). A total of 99 isolates had only blaNDM and 14 had both blaNDM and blaKPC genes. Regarding the isolates positive for blaNDM only, 38.4% were susceptible to ATM and 7.1% were susceptible, increased exposure. All isolates had ATM-AVI MICs of ≤1 mg/L, and the smallest inhibition zone diameter observed was 23 mm. CONCLUSION: Modified disk pre-diffusion can be used as a simple test to screen for ATM-AVI in vitro activity against Klebsiella, since ATM-AVI disks, gradient strips or microdilution panels are not commercially available.

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

In vitro synergistic activity of aztreonam (AZT) plus novel and old ß-lactamase inhibitor combinations against metallo-ß-lactamase-producing AZT-resistant Enterobacterales.

The emergence of metallo-ß-lactamase (MBL)-producing Enterobacterales, mainly New Delhi metallo-ß-lactamase (NDM), represents a clinical threat due to the limited therapeutic alternatives. Aztreonam (AZT) is stable to MBLs, but most MBL-producing Enterobacterales isolates usually co-harbour other ß-lactamases that confer resistance to AZT and, consequently, its use is restricted in these isolates. We compared the ability of sulbactam (SUL), tazobactam (TAZ), clavulanic acid (CLA) and avibactam (AVI) to restore the AZT activity in MBL-producing AZT-resistant Enterobacterales isolates. A collection of 64 NDM-producing AZT-resistant Enterobacterales from five hospitals in Buenos Aires city, Argentina, were studied during the period July-December 2020. MICs were determined using the agar dilution method with Mueller-Hinton agar according to Clinical and Laboratory Standards Institute (CLSI) recommendations. AVI, SUL and TAZ were used at a fixed concentration of 4 mg l-1, whereas CLA was used at a fixed concentration of 2 mg l-1. A screening method based on disc diffusion to evaluate this synergy was also conducted. Detection of bla KPC, bla OXA, bla NDM, bla VIM, bla CTXM-1, bla PER-2 and bla CIT was performed by PCR. The AZT-AVI combination restored the AZT activity in 98.4 % of AZT-resistant strains, whereas CLA, TAZ and SUL did so in 70.3, 15.6 and 12.5 %, respectively, in isolates co-harbouring extended-spectrum ß-lactamases, but were inactive in isolates harbouring AmpC-type enzymes and/or KPC. The synergy screening test showed an excellent negative predictive value to confirm the absence of synergy, but positive results should be confirmed by a quantitative method. The excellent in vitro performance of the AZT-CLA combination represents a much more economical alternative to AZT-AVI, which could be of use in the treatment of MBL-producing, AZT-resistant Enterobacterales.

In vitro synergy of ticarcillin/clavulanate in combination with aztreonam and ceftolozane/tazobactam against SPM-1-producing Pseudomonas aeruginosa strains.

Minimal inhibitory concentrations (MICs) of ticarcillin/clavulanic acid (TLc), ceftolozane/tazobactam (C/T), and aztreonam (AT) were determined for 6 SPM-1-producing Pseudomonas aeruginosa (PSA) using Etest® strips and the synergistic effect of such antimicrobials against was evaluated by gradient diffusion strip crossing (GDSC) test. The fraction inhibitory concentration indexes (FICI) were calculated and showed a synergistic (n = 3) and additive (n = 2) effects of TLc + AT against SPM-1 producers, while TLc + C/T combination caused no effect. Average MIC reduction of TLc and AT by GDSC was 3-fold and 2-fold dilutions, respectively. Thus, TLc + AT might be a candidate as a combination therapy to treat SPM-1-producing PSA infections.

Aztreonam plus ceftazidime-avibactam as treatment of NDM-1-producing Klebsiella pneumoniae bacteraemia in a neutropenic patient: Last resort therapy?

OBJECTIVES: We report the successful treatment of a bloodstream infection caused by Klebsiella pneumoniae harbouring NDM-1 using aztreonam-ceftazidime-avibactam in a neutropenic patient in whom colistin and meropenem therapy had previously failed. METHODS: A clinical isolate was evaluated to determine the presence of NDM, TEM, SHV, CTX, and CMY, and the killing kinetics of aztreonam (ATM; 4 µg/mL), aztreonam-avibactam (ATM-AVI; 4/4 µg/mL), and colistin (2 and 4 µg/mL) were tested. RESULTS: ATM-AVI showed in vitro activity against the Klebsiella pneumoniae harbouring NDM-1, whereas colistin allowed re-growth. CONCLUSIONS: This report supports reconsideration of use of colistin for treatment of infections caused by K. pneumoniae harbouring NDM. CZA/ATM use should be kept in mind as a treatment option, perhaps earlier than colistin.

In vitro synergy of ceftolozane/tazobactam in combination with fosfomycin or aztreonam against MDR Pseudomonas aeruginosa.

OBJECTIVES: Carbapenem-resistant Pseudomonas aeruginosa (CR-PSA) imposes great limitations on empirical therapeutic choices, which are further complicated by metallo-ß-lactamase production. This study evaluated in vitro antimicrobial synergy of ceftolozane/tazobactam in combination with aztreonam and fosfomycin against MDR PSA. METHODS: MICs were determined by broth microdilution and gradient strips. The effect of ceftolozane/tazobactam+aztreonam and ceftolozane/tazobactam+fosfomycin combinations were tested against 27 MDR PSA isolates carrying blaSPM-1 (n = 13), blaIMP (n = 4), blaVIM (n = 3), blaGES-1 (n = 2) and blaCTX-M-like (n = 2), and 3 isolates with no acquired ß-lactamase production detected by gradient diffusion strip crossing (GDSC). Six genetically unrelated SPM-1-producing isolates were also evaluated by time-kill analysis (TKA). RESULTS: All CR-PSA isolates harbouring blaSPM-1, blaGES-1 and blaIMP-1 were categorized as resistant to ceftolozane/tazobactam, meropenem and fosfomycin, with 70% being susceptible to aztreonam. Synergism for ceftolozane/tazobactam+fosfomycin and ceftolozane/tazobactam+aztreonam combinations was observed for 88.9% (24/27) and 18.5% (5/27) of the isolates by GDSC, respectively. A 3- to 9-fold reduction in ceftolozane/tazobactam MICs was observed, depending on the combination. Ceftolozane/tazobactam+fosfomycin was synergistic by TKA against one of six SPM-1-producing isolates, with additional non-synergistic bacterial density reduction for another isolate. Aztreonam peak concentrations alone demonstrated a ≥3 log10 cfu/mL reduction against all six isolates, but all strains were within the susceptible range for the drug. No antagonism was observed. CONCLUSIONS: In the context of increasing CR-PSA and the genetic diversity of resistance mechanisms, new combinations and stewardship strategies may need to be explored in the face of increasingly difficult to treat pathogens.

Investigación traslacional en microbioma (InTraMic): un área de interés común y creciente en el IMTIB, Hospital Italiano e Instituto Universitario / Translational research in microbiome (InTraMic): an area of common and growing interest at IMTIB, Hospital Italiano and Instituto Universitario

Se estima que aproximadamente 100 trillones de microorganismos (incluidos bacterias, virus y hongos) residen en el intestino humano adulto y que el total del material genético del microbioma es 100 veces superior al del genoma humano. Esta comunidad, conocida como microbioma se adquiere al momento del nacimiento a través de la flora comensal de la piel, vagina y heces de la madre y se mantiene relativamente estable a partir de los dos años desempeñando un papel crítico tanto en el estado de salud como en la enfermedad. El desarrollo de nuevas tecnologías, como los secuenciadores de próxima generación (NGS), permiten actualmente realizar un estudio mucho más preciso de ella que en décadas pasadas cuando se limitaba a su cultivo. Si bien esto ha llevado a un crecimiento exponencial en las publicaciones, los datos sobre las poblaciones Latinoamérica son casi inexistentes. La investigación traslacional en microbioma (InTraMic) es una de las líneas que se desarrollan en el Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB). Esta se inició en 2018 con la línea de cáncer colorrectal (CCR) en una colaboración con el Colorectal Cancer Research Group del Leeds Institute of Medical Research en el proyecto Large bowel microbiome disease network: Creation of a proof of principle exemplar in colorectal cancer across three continents. A fines de 2019 se cumplió el objetivo de comprobar la factibilidad de la recolección, envío y análisis de muestras de MBF en 5 continentes, incluyendo muestras provenientes de la Argentina, Chile, India y Vietnam. Luego de haber participado de capacitaciones en Inglaterra, se ha cumplido con el objetivo de la etapa piloto, logrando efectivizar la recolección, envío y análisis metagenómico a partir de la secuenciación de la región V4 del ARNr 16S. En 2019, la línea de enfermedad de hígado graso no alcohólico se sumó a la InTraMic iniciando una caracterización piloto en el marco de una colaboración con el laboratorio Novartis. Los resultados de ese estudio, así como el de cáncer colorrectal, están siendo enviados a publicación. En 2020, con la incorporación de la línea de trasplante alogénico de células progenitoras hematopoyéticas, fue presentado un proyecto para un subsidio del CONICET que ha superado la primera etapa de evaluación. En el presente artículo se brinda una actualización sobre la caracterización taxonómica de microbioma y se describen las líneas de investigación en curso. (AU)

It is estimated that approximately 100 trillion microorganisms (including bacteria, viruses, and fungi) reside in the adult human intestine, and that the total genetic material of the microbiome is 100 times greater than that of the human genome. This community, known as the microbiome, is acquired at birth through the commensal flora of the mother's skin, vagina, and feces and remains relatively stable after two years, playing a critical role in both the state of health and in disease. The development of new technologies, such as next-generation sequencers (NGS), currently allow for a much more precise study of it than in past decades when it was limited to cultivation. Although this has led to exponential growth in publications, data on Latin American populations is almost non-existent. Translational research in microbiome (InTraMic) is one of the lines developed at the Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB). This started in 2018 with the Colorectal Cancer Line (CRC) in a collaboration with the Colorectal Cancer Research Group of the Leeds Institute of Medical Research in the project "Large bowel microbiome disease network: Creation of a proof of principle exemplar in colorectal cancer across three continents". At the end of 2019, the objective of verifying the feasibility of collecting, sending and analyzing MBF samples on 5 continents, including samples from Argentina, Chile, India and Vietnam, was met. After having participated in training in England, the objective of the pilot stage has been met, achieving the collection, delivery and metagenomic analysis from the sequencing of the V4 region of the 16S rRNA. In 2019, the non-alcoholic fatty liver disease line joined InTraMic, initiating a pilot characterization in the framework of a collaboration with the Novartis laboratory. The results of that study, as well as that of colorectal cancer, are being published. In 2020, with the incorporation of the allogeneic hematopoietic stem cell transplantation line, a project was presented for a grant from the CONICET that has passed the first stage of evaluation. This article provides an update on the taxonomic characterization of the microbiome and describes the lines of ongoing research. (AU)

Multi-locus sequence typing and antimicrobial susceptibility profile of Cronobacter sakazakii and Cronobacter malonaticus isolated from corn-based farinaceous foods commercialized in Brazil.

The aim of this study was to evaluate the microbiological quality of 45 samples of corn-based farinaceous foods commercialized in Brazil. The bacteriological analysis performed were: detection of Salmonella and Cronobacter, and enumeration of faecal coliforms and Bacillus cereus. The Cronobacter isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene and MLST. No sample presented contamination by Salmonella or B. cereus (<102 UFC/g). Faecal coliforms were detected in two (4.4%) samples but in low concentration (≤23.0 MPN/g), and 20 samples (44.4%) contained Cronobacter. Twenty-nine unique Cronobacter isolates were identified as C. sakazakii (n = 18), C. malonaticus (n = 2); that presented 11 different fusA alleles, including new fusA 183. MLST analysis revealed 17 sequence types (STs), six of which were newly identified (ST687-690, 693, and 694). Resistance or intermediary resistance were found to ceftazidime (15.0%), aztreonam (15.0%), nalidixic acid (15.0%), nitrofurantoin (15.0%), cefepime (10.0%), gentamicin (5.0%), and tetracycline (5.0%). The presence of Cronobacter in corn-based farinaceous foods could be a significant risk to infants as these products are used as alternatives to commercially available infant formula. Strategies to manage the risk of Cronobacter infections due to the consumption of these alternative feeds need to be developed by the regulatory agencies.

Importance of the ß5-ß6 Loop for the Structure, Catalytic Efficiency, and Stability of Carbapenem-Hydrolyzing Class D ß-Lactamase Subfamily OXA-143.

The class D ß-lactamase OXA-143 has been described as an efficient penicillinase, oxacillinase, and carbapenemase. The D224A variant, known as OXA-231, was described in 2012 as exhibiting less activity toward imipenem and increased oxacillinase activity. Additionally, the P227S mutation was reported as a case of convergent evolution for homologous enzymes. To investigate the impact of both mutations (D224A and P227S), we describe in this paper a deep investigation of the enzymatic activities of these three homologues. OXA-143(P227S) presented enhanced catalytic activity against ampicillin, oxacillins, aztreonam, and carbapenems. In addition, OXA-143(P227S) was the only member capable of hydrolyzing ceftazidime. These enhanced activities were due to a combination of a higher affinity (lower Km) and a higher turnover number (higher kcat). We also determined the crystal structure of apo OXA-231. As expected, the structure of this variant is very similar to the published OXA-143 structure, except for the two M223 conformations and the absence of electron density for three solvent-exposed loop segments. Molecular dynamics calculations showed that both mutants experience higher flexibility compared to that of the wild-type form. Therefore, our results illustrate that D224A and P227S act as deleterious and positive mutations, respectively, within the evolutionary path of the OXA-143 subfamily toward a more efficient carbapenemase.

Change in the antimicrobial resistance profile of Pseudomonas aeruginosa from soil after exposure to herbicides.

The extensive use of pesticides represents a risk to human health and to the environment. This study aimed to investigate if the exposure to atrazine and diuron, two herbicides widely used in Brazil, could induce changes in the susceptibility profile to aztreonam, colistin and polymyxin B antimicrobials in isolates of P. aeruginosa obtained from soil samples by using the determination of minimum inhibitory concentration (MIC) test. Three isolates had an increase of MIC to aztreonam after exposure to both herbicides and one isolate did not show any MIC change. The MexAB-OprM efflux pump has already been upregulated in these isolates and the herbicides atrazine and diuron did not increase MexAB-OprM overexpression. Therefore, the decrease in aztreonam susceptibility was not directly related to this pump, suggesting that probably other mechanisms should be involved.

Prevalence, Antimicrobial Susceptibility, and Clonal Diversity of Pseudomonas aeruginosa in Chronic Wounds.

PURPOSE: Our purposes in this study were to (1) identify Pseudomonas aeruginosa strains collected from swabs of chronic wounds, (2) evaluate the susceptibility of P. aeruginosa strains to various antimicrobials, (3) detect the presence of virulence factors exoenzyme S (exoS) and exoenzyme U (exoU) in P. aeruginosa strains, and (4) evaluate wound colonization by P. aeruginosa via pulsed-field gel electrophoresis (PFGE). DESIGN: Descriptive research using a quantitative approach. SAMPLE AND SETTING: Swabs from 43 adults with chronic wounds treated in an outpatient setting in Niterói City, Brazil, were included using convenience sampling. METHODS: Swabs were collected at 2 points during treatment, 30 to 45 days apart. P. aeruginosa isolates were identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed using the disk diffusion method. The presence of exoS and exoU genes was evaluated using polymerase chain reaction. Genotyping diversity was determined through PFGE. RESULTS: Forty-eight P. aeruginosa isolates were detected in chronic wounds, and 3 were multidrug resistant (6%). Resistance to aztreonam and ciprofloxacin was observed in 48% and 27% of isolates, respectively. The presence of the exoS gene was verified in 54% of isolates, and 27% were positive for the exoU gene. In most wounds, P. aeruginosa strains had the same genetic characteristics at the 2 time points analyzed, indicating that the wound beds remained colonized. CONCLUSIONS: P. aeruginosa was present in 75% of tested chronic wound samples, and the same clones persisted for more than 1 month. In addition, most bacteria contained virulence genes that were associated with high potential to establish infection. The use of silver in chronic wounds may be associated with multidrug resistance in P. aeruginosa; therefore, it is important to avoid colonization by these bacteria.